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    PESTICIDE RESIDUES IN FOOD - 1997


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    with the support of the International Programme
    on Chemical Safety (IPCS)




    TOXICOLOGICAL AND ENVIRONMENTAL
    EVALUATIONS 1994 1997




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group 

    Lyon 22 September - 1 October 1997



    The summaries and evaluations contained in this book are, in most
    cases, based on unpublished proprietary data submitted for the purpose
    of the JMPR assessment. A registration authority should not grant a
    registration on the basis of an evaluation unless it has first
    received authorization for such use from the owner who submitted the
    data for JMPR review or has received the data on which the summaries
    are based, either from the owner of the data or from a second party
    that has obtained permission from the owner of the data for this
    purpose.



    2,4-Dichlorophenoxyacetic acid (2,4-D), salts and esters

    First draft prepared by
    Dr P.J. Campbell
    Ministry of Agriculture, Fisheries, and Food
    York, United Kingdom

         Environmental transport, distribution, and transformation
              Volatilization
              Water
                   Hydrolysis
                   Photolysis
                   Biodegradation
                   Bioaccumulation and biomagnification
              Soil
                   Hydrolysis
                   Photolysis
                   Adsorption and desorption
                   Mobility and leaching
                   Degradation in soil under laboratory conditions
                   Degradation in soil under field conditions
                   Uptake by plants
              Environmental levels
                   Air
                   Water
                   Soil
                   Plants
              Effects on organisms in the laboratory and the field
                   Microorganisms
                   Aquatic organisms
                        Plants
                             Toxicity
                             Other effects on plants
                        Invertebrates 
                             Toxicity
                             Other effects on invertebrates
                        Vertebrates
                             Toxicity
                             Other effects on vertebrates
                   Terrestrial organisms
                        Plants
                        Invertebrates
                             Toxicity to arthropods
                             Toxicity to earthworms
                             Other effects on invertebrates
                        Vertebrates
                             Toxicity to birds 
                             Toxicity to birds' eggs
                             Effects on mammals
                             Effects on amphibia

              Risk assessment based on agricultural use 
                   Microorganisms 
                   Aquatic organisms
                        Acute risk to freshwater pelagic organisms
                        Long-term risk to freshwater pelagic organisms 
                        Risk to sediment-dwelling invertebrates
                        Risk to amphibia
                        Bioaccumulation
                   Terrestrial organisms
                        Plants
                        Invertebrates
                        Vertebrates
                             Birds
                             Mammals
                   Evaluation of effects on the environment
                        Risk assessment
                             Aquatic environment
                             Terrestrial environment
                   References

    1.  Environmental transport, distribution, and transformation

    1.1  Volatilization

    Certain ester formulations (butyl, ethyl, isopropyl) of 2,4-D are more
    volatile than amine salt formulations, such that vapour drift can be
    virtually eliminated by use of salts of 2,4-D (Que Hee & Sutherland,
    1974), and use of the more volatile esters is being discontinued in
    most countries owing to potential drift of either droplets or vapour.
    Use of the highly volatile esters may result in drifting of as much as
    25-30% of the applied 2,4-D off target crops (Grover et al., 1972;
    Maas & Kerssen, 1973; Grover, 1974, 1976; Maybank et al., 1978).

    In an evaluation of the use of silica gel and XAD-4 as adsorbents for
    the removal of the  n-butyl and isooctyl esters of 2,4-D, their
    overall efficiencies for both trapping and ease of extraction were 85-
    100% (Grover & Kerr, 1978). The amount of the dimethylamine salt of
    2,4-D that volatilized after 48 h at 38°C under light emitted by a
    100-watt incandescent bulb was < 10%, irrespective of the surface
    (Que Hee & Sutherland, 1974; Que Hee et al., 1975).

    The volatilization of 14C-2,4-D ethylhexyl ester (as Esteron 99TM
    concentrate) under laboratory conditions, according to the guidelines
    of the US Environmental Protection Agency, was characterized by
    applying it to Galstown sandy loam at an equivalent rate of 13.5-17.7
    lb acid equivalent per acre (15-20 kg/ha). The volatility was
    maintained by passing air over the soil at 25°C and 95-100% relative
    humidity at air flow rates of 100-300 ml/min for 15 days; the pressure
    in the chamber was held at 1 atm (101.3 kPa). The volatile gases were
    then trapped. The losses from the soil were only 0.05-0.23%.
    Volatilization was most rapid immediately after application, declined
    with time up to day 7, and then remained essentially constant up to

    day 15. The volatility increased directly with the air flow rate
    (Doyle, 1991).

    In an extensive review of the literature on the volatility of salts
    and esters of 2,4-D, Que Hee and Sutherland (1981) concluded that the
    effective partial pressure for deposits on plants, in water, and on
    soils may be different from that expected in comparison with glass
    surfaces. The most important factor in assessing the risk for vapour
    drift is the volatility of the compound itself. Thus, the predicted
    rates  of loss from 1 ha were 0.2 kg/h for the  n-butyl ester of
    2,4-D and 0.25 kg/day for the isooctyl ester (Elliott & Wilson, 1983).
    In a test to rate the volatility of the methyl, ethyl, isobutyl,
    butyl, 2-butoxyethyl, and 2-ethylhexyl esters of 2,4-D in tomato
    plants, the esters and formulations with a vapour pressure > 3.3 mPa
    (at 25°C) were rated as highly volatile while those with a vapour
    pressure <: 0.6 mPa were considered to have low volatility (Noble &
    Hamilton, 1990).

    In a study of the rate of dissipation of the propylene glycolbutyl
    ether ester of 2,4-D and vapour loss in air from soil in chambers at
    temperatures up to 35°C, 87-97% had dissipated by degradation and
    0.1-2.6% by volatilization in air at all temperatures after 154 days
    (Nash, 1989a). An empirical model was used to estimate dissipation
    when volatilization is the predominant pathway for loss (Nash, 1989b).

    1.2  Water

    1.2.1  Hydrolysis

    A comparison of the kinetics of hydrolysis and the half-lives for many
    salts and esters of 2,4-D, based on an extensive review of the
    literature, showed that the hydrocarbon esters hydrolyse much more
    slowly than the alkyl ether esters, with the exception of the methyl
    ester (Que Hee & Sutherland, 1981). In a comparison of the hydrolysis
    of the methyl and butoxyethyl esters and the butyl and octyl esters of
    2,4-D in aqueous solution, esters with ether linkages near the carboxy
    group were generally hydrolysed more rapidly than the hydrocarbon-
    chain esters. The rapid rates observed in basic water, considerably
    longer than those in acidic or neutral water, suggest that hydrolysis
    is the major pathway for degradation of 2,4-D esters in natural water.
    The half-lives for hydrolysis of 2,4-D esters in aqueous solution at
    28°C and pH 9 were from a low of 0.6 h for the 2-butoxyethyl ester to
    37 h for the 2-octyl ester (Zepp et al., 1975, 1976).

    In studies conducted according to the guidelines of the US
    Environmental Protection Agency, 14C-2,4-D acid (at 10-4mol/L) was
    not hydrolysed at pH 5, 7, or 9 after 30 days at 24.9°C in sterile
    aqueous solution in the dark (Creeger, 1989a). The hydrolysis of
    14C-2,4-D ethylhexyl ester in sterile aqueous solutions containing 1%
    acetonitrile, in the dark, at 25°C for up to 30 days was slow at pH 5
    and moderate at pH 7. At the end of the study, 77.7 % of the applied
    radiolabel was present as the parent ester at pH 5, while only 59.3%
    remained at pH 7. At pH 9, 2,4-D ester degraded rapidly and

    represented 14.2% of the applied dose after 144 h. The half-life of
    14C-2,4-D ethylhexyl ester at pH 5 was found by extrapolation to be
    99.7 days, with a half-life at pH 7 calculated to be 48.3 days; the
    half-life at pH 9 was calculated to be 52.2 h. 14C-2,4-D represented
    81.4% of the applied radiolabel at the end of the study (Concha et
    al., 1993a). Hydrolysis of 14C-2,4-D ethylhexyl ester was evaluated
    in natural river water containing 1% acetonitrile, for up to 24 h in
    the dark at 25°C. The ester degraded rapidly, only 7.2% of the applied
    radiolabel remaining as parent compound after 24 h. 14C-2,4-D
    represented 93.8% of the applied dose. The half-life was calculated to
    be 6.2 h (Concha et al., 1993b).

    1.2.2  Photolysis

    The final oxidation product of 2,4-D in aqueous solutions irradiated
    with a polychromatic lamp was carbon dioxide (Boval & Smith, 1973).
    The kinetics of photodegradation and the photolysis products of 2,4-D
    at various pH values have been described after photolysis by
    ultraviolet radiation (Chamarro & Esplugas, 1993) and ultraviolet
    radiation with ozone (Prado et al., 1994). The photoproducts of the
    ethyl, butyl, and 2-methylheptyl esters of 2,4-D under Pyrex-filtered
    light included hydrochloric acid and esters of 2- and 4-
    chlorophenoxyacetic acid (Binkley & Oakes, 1974). The photoproducts of
    the butyl ester of 2,4-D under ultraviolet light included 2,4-
    dichloro-phenol, the methyl ester of 2,4-D, the  n-butyl ester of
    5-chloro-2-hydroxyphenylacetic acid, and hydrochloric acid (Que Hee &
    Sutherland, 1979).

    Two studies were conducted according to the guidelines of the US
    Environmental Protection Agency. In one, 14C-2,4-D was irradiated for
    30 days under simulated sunlight (one-half day light, one-half day
    dark) at 24.8°C in a filter-sterilized aqueous solution buffered at pH
    7. The decay was first-order over more than two half-lives, with a
    half-life of 12.98 days (7.57 days of constant exposure to light).
    Only 1,2,4-benzenetriol was present at > 10% of the initial
    concentration of 14C-2,4-D. Production of 14C-carbon dioxide had
    reached 25% by the end of the study. No photodegradation occurred in
    the dark (Creeger, 1989b). In the second study, 14C-2,4-D ethylhexyl
    ester was irradiated with natural sunlight for up to 31 days in
    aqueous solution at 25.3°C and pH 5; control samples were tested
    concurrently in the dark. The ester degraded slowly when exposed to
    natural sunlight, accounting for 80.3% of the applied dose after 31
    days of exposure, with 84.8% in the controls. The three major
    degradates were 2,4-D (5.0%), 2,4-dichlorophenol (2.1%), and
    2-ethylhexyl-4-chlorophenoxyacetate (1.1%). The ester degraded more
    slowly in the dark, only 6.5% 2,4-D being observed after 31 days. The
    half-life for degradation of 14C-2,4-D ethylhexyl ester was
    calculated to be 128.2 days in light and 252.5 days in the dark
    (Concha & Shepler, 1993a). The same photoproducts were reported by
    Zepp et al. (1975). The quantum yields for photoalteration and the
    photolysis half-lives were also reported for several other esters of
    2,4-D (Zepp et al., 1976).

    In a study of the photodynamics of the methyl and butoxyethyl esters
    of 2,4-D in surface waters, the major photoproducts at concentrations
    exceeding the solubilities of the esters in water (> 300 ppm) were
    the dehalogenated 2-and 4-chlorophenoxy acetic acid esters (Zepp et
    al., 1975). No significant phototransformation of 2,4-D was seen in
    water after irradiation for up to 168 h at 304 nm. The upper limit of
    the quantum efficiency,  F, was found to be < 0.014 (Klopffer,
    1991).

    After irradiation with ultraviolet light of the pure  n-butyl, mixed
     n-butyl, and isobutyl esters of 2,4-D in aqueous and hydrocarbon
    solutions, reductive dechlorination was preferential at the  ortho 
    position at 300 nm, irrespective of the solvent, wheres
    photodecomposition was negligible at 350 nm (Que Hee et al., 1979).
    Addition of 0.85% (v/v) acetophenone to aqueous solutions of 2,4-D
    containing 0.15% (v/v) oxysorbic strongly sensitized photodegradation
    of 2,4-D (Harrison & Wax, 1985). A review of the photodecomposition of
    2,4-D includes the photoproducts of 2,4-D irradiated in alcohol, the
    rates of degradation of 2,4-D esters irradiated in different solvents,
    and a discussion of the photodegradation factors for esters and salts
    (Que Hee & Sutherland, 1981).

    1.2.3  Biodegradation

    The dissociation of 2,4-D and its dimethylamine salt in an aqueous
    solution were studied by conductometry according to the guidelines of
    the US Environmental Protection Agency. 2,4-D dimethylamine
    dissociated within 1 min when added to stirred water, whereas 2,4-D
    acid dissociated within > 120 min. These results confirm the
    complete, rapid dissociation of 2,4-D dimethylamine in aqueous
    solution, forming dimethylammonium ion and the conjugate base of 2,4-D
    (Reim, 1989).

    In a study of anaerobic aquatic metabolism conducted according to the
    guidelines of the US Environmental Protection Agency,  14C-2,4-D was
    tested in viable pond sediment and water at 25.1°C for up to one year.
    Most of the applied radiolabel (24.1-85.5%) was found in the aqueous
    phase and a moderate amount (11.2-42.3%) in organic sediment. The
    amount of bound radiolabel increased with increasing degradation, so
    that after 240 days 34.7% of the applied radiolabel remained
    unextracted. Partitioning of the extracted residue on day 240 showed
    2.4% in the humic acid fraction and 14.9% in the fulvic acid fraction,
    23.5% remaining unextracted. The level of 14C-carbon dioxide reached
    22.1% after 365 days. 14C-2,4-D accounted for 25.9% of the applied
    dose in the aqueous phase and 13.2% in the sediment after one year.
    The two maj or metabolites were 2,4-dichlorophenol, accounting for
    21.6% at day 30 and 4.2% after one year, and carbon dioxide. The
    volatile compounds seen after one year were 4-chlorophenol (1.9%),
    2,4-dichloroanisole (0.7%), and 2,4-dichlorophenol (0.7%). The
    half-life for anaerobic degradation of 14C-2,4-D in water was
    calculated to be 312 days (Concha & Shepler, 1994a).

    In a similar study of aerobic metabolism, conducted at 25°C for up to
    46 days, 14C-2,4-D degraded slowly for the first 25 days,
    representing > 75% of the applied dose, but degraded rapidly within
    the next 10 days, accounting for 0.5% of the applied dose at the end
    of the study (Concha & Shepler, 1993b). An initial lag in the
    degradation rate had been observed previously (McCall et al., 1981)
    and was postulated to be the lag time necessary to degrade 2,4-D, to
    mutate in order to produce this specific enzyme (Loos, 1975), or to
    allow enough time for a small microbial population to degrade enough
    2,4-D to be detectable (Chen & Alexander, 1989). In a plot of time of
    exposure against percent 14C-2,4-D, the concentration reached 50% at
    about 29 days after application. The half-life was determined to be
    4.5 days. The major metabolite under aerobic conditions was carbon
    dioxide, which comprised 63.9% of the applied radiolabel at the end of
    the study. The three metabolites found were 2,4-dichlorophenol
    (representing 1.1% of the applied dose on day 35 and 0.1% at the end
    of the study), 4-chlorophenoxyacetic acid (1.1% on day 14 but
    insignificant amounts at the end of the study), and 4-chlorophenol
    (1.4% radiolabel after 20 days and undetectable at the end of the
    study). The unextractable residue increased with time and comprised
    15.6% of the applied dose at the end of the study. During the lag time
    of 25 days, > 64% of the radiolabel was found in the aqueous phase.
    About 10-14% could be extracted from the sediment with an alkaline
    solvent, and about 4% could be extracted with acidic acetone. After
    the lag time, the distribution of radiolabel shifted markedly as the
    2,4-D acid degraded, and by the end of the study, 3% of the applied
    dose was in the aqueous phase, 1% in basic solvents, and 0.6% in
    acidic solvents (Concha & Shepler, 1993a).

    Three trials were conducted in the United States according to the
    guidelines of the US Environmental Protection Agency. In Louisiana,
    the formulated dimethylamine salt of 2,4-D was applied aerially to
    rice in the green-ring stage of growth with a canopy height of 25-30
    in (64-76 cm) at a target application rate of 1.5 lb/acre (1.68
    kg/ha). The concentration of 2,4-D acid residue in water was highest
    on day 0 (mean, 1.372 ppm) and then declined to a mean of 0.194 ppm
    three days after application. No 2,4-D acid was detected (limit of
    quantification, 0.01 ppm) in water samples on day 7, 15, or 30 of
    sampling. The half-life in water was calculated to be 1.1 days.
    Residues of 2,4-dichlorophenol and 4-chlorophenoxyacetic acid were
    detected in samples taken on days 0, 1, and 3, but were below the
    limit of quantification (Barney, 1994). Two further trials were
    conducted during 1994 in small ponds in North Carolina and North
    Dakota, with two applications of the dimethylamine salt (Hatfield,
    1995a,b). In North Carolina, a subsurface application of 40.38 lb/acre
    (45 kg/ha) was followed by an application of 45.01 lb/acre (50 kg/ha)
    30 days later. In North Dakota, 41.8 lb/acre (47 kg/ha) were applied
    twice. In North Carolina, 2,4-dichlorophenol and 2,4-dichloroanisole
    were found immediately after the first application, but no residues
    were detected 21 days after the second application. The half-lives in
    pond water were 19.7 days after the first application and 2.7 days
    after the second. In North Dakota, 2,4-dichlorophenol and
    4-chlorophenol were detected in water samples immediately after the

    first application, but none were detected 60 days after the last
    application. The half-lives were 13.9 days after the first application
    and 6.5 days after the second.

    Mineralization of 14C-2,4-D by microbial communities in two streams
    in southwestern Ohio, USA, was very limited (Palmisano et al., 1991).
    In an investigation of the degradation rate of 2,4-D in river water in
    relation to the nutrient levels, sediment load, and dissolved organic
    carbon content of the water, the limiting factor was found to be not
    the numbers of organisms capable of degrading 2,4-D but rather the
    nutrient status of the river (Nesbitt & Watson, 1980a).

    The dissipation of the isooctyl ester of 2,4-D (60% acid equivalent)
    at nominal rates of 1.0 or 2.5 kg/ha was measured in outdoor
    enclosures constructed in a typical bog lake in sandy soil in
    northeastern Ontario, Canada. The rate of dissipation of 50% of the
    initial concentration of 2,4-D (DT50) from the lake water was 4.5-7.8
    days, depending on the application rate. Within 15 days, < 5% of the
    applied dose remained in the water, but up to 25% was adsorbed on the
    sides of the enclosure (Solomon et al., 1988). The average half-lives
    of 2,4-D in samples of groundwater from three locations in eastern
    Arkansas, USA, were > 800 days, although the absence of aquifer
    materials may have resulted in unrealistically slow degradation rates
    (Cavalier et al., 1991).

    1.2.4  Bioaccumulation and biomagnification

    The dissipation of residues of 2,4-D w as evaluated after application
    to ponds in Missouri, Georgia, and Florida, USA. When the
    dimethylamine salt was applied at 2.24, 4.48, or 8.96 kg/ha, the
    residues in water were negligible (< 5 µg/L) within two weeks, and
    those in silt and fish were < 200 µg/kg. Of 307 fish samples
    analysed, only 45 contained detectable residues. The highest level in
    pond water was 692 µg/L three days after application of 8.96 kg/ha,
    the highest in silt was 170 µg/kg three days after application of 8.86
    kg/ha, and the highest in fish was 102 µg/kg four days after
    application of 8.86 kg/ha (Schultz & Harman, 1974; Schultz & Gangstad,
    1976). In a study of the fate of the butoxyethanol ester of 2,4-D
    applied at 23 kg ai/ha, as granules, to outdoor, artificial,
    polyethylene ponds infested with Eurasian watermilfoil, the water
    temperature dropped from 25 to 0°C after 56 days. The levels of the
    ester were low and fell to < 1.0 µg/L within 15 days, the rapid
    disappearance being attributed to hydrolysis in the alkaline pond
    water, which had a pH of 9.5 at the time of initiation, the low
    aqueous solubility of the ester, its uptake by the water weeds, and to
    its sorption to the plastic lining of the tanks (Birmingham & Colman,
    1985).

    Four 10-ha areas of Lake Seminole, Georgia, USA, with dense beds of
    water milfoil were treated with either 2,4-D dimethylamine salt at
    22.5 kg/ha or 2,4-D butoxyethanol ester at 45 kg/ha. Both formulations
    were converted to free 2,4-D acid within 24 h after application. The
    maximum water concentrations found were 3.6 mg/L for the dimethylamine

    salt and 0.68 mg/L for the butoxyethanol ester. No fish sampled 13
    days after application contained detectable levels of 2,4-D (Hoeppel &
    Westerdahl, 1983).

    After application of the dodecyl-tetradecyl amine salt of 2,4-D at 4.5
    kg/ha followed by spot treatments of this amine and/or dimethylamine
    salt to 7000 acres of canal in Florida, USA, the highest level of
    2,4-D found in water was 27 µg/L after one day and the highest level
    in silt was 5 µg/kg after 3-15 days. Three of 60 fish analysed
    contained > 10 µg/kg 2,4-D, while 16 of 60 had levels < 10 µg/kg
    (Schultz & Whitney, 1974).

    The mean residues of 2,4-D in 353 individual fish of eight species
    from lakes in Canada after treatment with 2,4-D in 1977-80 ranged from
    < 5 to 60 µg/kg (Frank et al., 1987).

    1.3  Soil

    1.3.1  Hydrolysis

    No reliable data exist on the hydrolysis of salts or esters in sterile
    soils.

    1.3.2  Photolysis

    In a study conducted according to the guidelines of the US
    Environmental Protection Agency, 14C-2,4-D was irradiated for 30 days
    under simulated sunlight (one-half day light, one-half day dark) at
    24.9°C on air-dried, sieved, autoclave-sterilized loam. Aliquots were
    also treated in the dark. 2,4-D was not photodegraded substantially.
    The photolysis half-life was found by extrapolation to be 68 days. The
    levels of metabolites other than 2,4-D found in the were not > 10%
    of the initially applied dose. 14C-Carbon dioxide production had
    reached 5.05% by the end of the study (Creeger, 1989c).

    1.3.3  Adsorption and desorption

    Organic matter, soil pH, and exchangeable aluminium are the major
    factors that determine adsorption of 2,4-D acid and its dimethylamine
    salt in soils (Liu & Cibes-Viade, 1973; Grover & Smith, 1974). In a
    study of the sorption of [1-14C]-2,4-D on 42 samples of topsoil and
    subsoil from 21 sites in Belgium (pH, 3.25-6.91), most sorption
    occurred on the soil with the lowest pH (3.25) and least on a subsoil
    with pH of 6.4. The soil adsorption coefficients based on organic
    carbon (KOC) were 31.2-470.9 for 18 of the soils. After application
    of 2.0 kg/ha of 14C-2,4-D, about 70% of the initial amount was
    recovered in the first 5 cm of a loamy sand and 1.4% at 10-15 cm after
    48 mm of percolating water, whereas in a Zolder sand 73% of the 2,4-D
    was found at 10-15 cm. The mobility of 2,4-D was inversely related to
    the amount adsorbed. After application of 2,4-D at 4.4 kg/ha to a 1-m
    Podzol soil column (Zolder sand) with 800 mm of 0.025 mol/L CaCl2, no
    2,4-D was found in the soil solution at the bottom of the column. The
    maximum depth of penetration was 50 cm, and only 7% of the applied

    2,4-D was recovered in the entire soil profile after 62 days (Moreale
    & Van Bladel, 1980). These findings are consistent with those of
    Grover (1977), who found that the adsorption of 2,4-D correlated with
    the organic content of seven Canadian soils and not with the clay
    content. When the rate constants, activation energy, heats of
    activation, and entropies of activation for the adsorption of
    analytically pure 2,4-D on humic acid from a black chernozem soil from
    Canada were calculated, the adsorption was found to follow the
    Freundlich-type isotherm. The rate of adsorption of 2,4-D on humic
    acid at both 5 and 25°C was initially rapid but was slower
    subsequently (Khan, 1973, 1975a).

    The Freundlich coefficients for the sorption of 2,4-D on seven
    Canadian prairie soils ranged from 0.09 to 1.3, with KOC values of
    8.7-21. Sorption of 1-butyl and 2-octyl esters could not be measured
    because of rapid hydrolysis but is apparently similar to that of 2,4-D
    acid (Grover, 1973). Significantly more 2,4-D was adsorbed by the top
    0-5 cm than in the lower layers, which correlates well with the
    organic content of silt loam. The Freundlich sorption coefficients
    were 0.67 µg/g of soil (or 20.9 µg/g of organic matter) for  k and
    0.82 for  1/n. As the adsorption-desorption process is the main
    mechanism that affects the availability, mobility, and degradation of
    the herbicide in soils, it is not surprising that 2,4-D binds more
    strongly to soils with a high content of organic matter than to those
    with a low content. In a study of the degradation of [carboxyl-14C]-2
    4-D and the dimethylamine and isooctyl ester formulations in runoff,
    over 80% of the applied compound decomposed to 14C-carbon dioxide
    within five weeks, during the active degradation phase, but only 3%
    more decomposed during an additional five weeks. An average of 0.26%
    of the applied radiolabel could be identified as 2,4-D, with 0.05% in
    sediment and 0.21% in water. The amount of 2,4-D applied was thus
    affected by the concentration in the runoff (Wilson & Cheng, 1978).

    The adsorption properties of 14C-radiolabelled 2,4-D and its
    2-ethylhexyl and butyl esters were evaluated at 24°C in a silt loam, a
    sandy loam, a loam, and a clay (organic carbon, 0.22-3.08; pH 5.9-7.5)
    at initial concentrations of 0.2-5.0 mg/L. The supernatant was by
    analysed by radio-high-performance liquid chromatography (HPLC). No
    degradation of 2,4-D was observed. The soil adsorption coefficient
    (Kd) was 0.08-1.11 ml/g, with an average of 0.78 ml/g. The KOC was
    34-79 ml/g, with an average of 48 ml/g. The KOC calculated from the
    Freundlich equation was 31-74 ml/g, with an average of 45 ml/g. The
    weakest sorption was that in a sandy loam, which had the highest pH
    (7.5) and the lowest organic carbon content (0.22%). Sorption of the
    esters was not measured owing to rapid hydrolysis (half-life = 79 min
    for the 2-ethylhexyl ester and 26 min for the butyl ester) (McCoy &
    Lehmann, 1988).

    The role of chemical structure, the nature of soil constituents, and
    physico-chemical factors such as pH were analysed to evaluate the
    adsorption of 2,4-D in soils, expressed as Freundlich isotherms. Nine
    soils with organic contents varying from 0.9 to 14.1% were
    investigated. The sorption (KOC) varied from 21-33 in a calcic

    cambisol to 196-767 in an acidic ferralsol. The Kd values for 2,4-D
    ranged from 0.3 for the calcic cambisol to 26.6 for an andosol
    (Barriuso & Calvet, 1992). The adsorption of 2,4-D was examined in two
    Brazilian oxysols, with plots under natural vegetation and with plots
    that had borne crops for up to 56 years after clearing. The adsorption
    of 2,4-D was always greater in soils with natural vegetation and
    increased strongly with decreasing soil pH (Barriuso et al., 1992).

    Sorption coefficients were developed in a study of degradation in
    silty clay loam, silty clay, loamy sand, and a clay. The soil-water
    distribution ranged from 0 in clay to 9.05 mg/L in the silty clay
    (Ogram et al., 1985). 2,4-D was not removed from domestic water by
    activated sludge treatment with conventional methods for treating
    potable water (Hill et al., 1986).

    The adsorption of 2,4-D by 19 soil-sediment materials, in which the
    clay, sand, silt, and organic matter contents varied greatly, was
    investigated with a batch equilibrium technique. The organic content
    was the single most important factor. The adsorption coefficient (K)
    ranged from a low of 0.38 to a high of 39.1 after 24-h equilibrium,
    and the KOC ranged from 9 to 330 for 2-h adsorption and from 40 to
    415 for 24-h adsorption (Reddy & Gambrell, 1987). The soil adsorption
    coefficient (log KOC) based on HPLC capacity factors was 2.59, which
    compared well with values in the literature ranging from 1.70 to 2.73
    (Hodson & Williams, 1988). The adsorption coefficient K for three
    soils, determined by HPLC, was 0.6-4.3 and the KOC was 112-145, in
    comparison with the KOC in model adsorbents for 2,4-D based on OECD
    guidelines, which was 20 (Rippen et al., 1982). In a similar study of
    10 Danish soils, adsorption correlated significantly with the organic
    content of the soils (Lokke, 1984).

    In batch and column experiments with a sandy loam under saturated and
    unsaturated conditions, the sorption of 2,4-D had a slight but
    significant effect on its transport under either saturated
    (retardation factor, 1.8) or unsaturated conditions (retardation
    factor, 3.4). Biodegradation was extensive. In batch experiments,
    2,4-D (100 mg/kg) was completely mineralized under either saturated or
    unsaturated conditions over a four-day period after a three-day lag
    phase (Estrella et al., 1993). In a model of the competitive
    adsorption and desorption of 2,4-D on a volcanic soil with a high
    organic content (8.7%), the agreement with experimental data was
    excellent (Susarla et al., 1992).

    Freundlich constants and equilibrium desorption values were determined
    for the adsorption of 2,4-D and biodegradation of the adsorbed 2,4-D
    in soil columns containing a New Zealand silt loam soil. The
    equilibrium desorption values ranged from 80% at a soil concentration
    of 1% (w/v) to 67.8% at a concentration of 50% (Bhamidimarri & Petrie,
    1992). In a study of the effects of long-term conventional and
    long-term low-input farming on the adsorption of 14C-2,4-D and the
    soil properties that control adsorption at various slope positions,
    samples were characterized for soil organic carbon and clay content,

    soil pH, and linear adsorption partition coefficients (K d) The K d
    values were higher for the low-input farms ( 1.27-0.54, bottom to top
    slope positon) than for the conventional farms (0.77-0.31)
    (Mallawatantri & Mulla, 1992).

    In a study conducted according to the guidelines of the US
    Environmental Protction Agency, the adsorption and desorption of
    unaged 14C-2,4-D was evaluated in non-sterilized Louisiana rice paddy
    sediment (clay soil), at concentrations of 0.10, 0.51, 1.00, 2.47, and
    5.02 ppm at 22°C. Partitioning between the sediment and 0.01 mol/L
    CaCl2 was determined. The Kd value for was 1.22 (KOC = 58.1), which
    suggests that the acid was adsorbed onto sediment from the water
    within 24 h. In the desorptive phase, the K1value was 1.64 (K1OC=
    78.1), which suggests that the acid is moderately to highly mobile in
    rice paddy sediment (Cohen, 1991).

    It has been suggested that subsoil horizons be included in models
    designed to predict the mobility of herbicides in soils. The sorption
    distribution coefficient (Kdm) for surface and subsoil horizons was
    determined for 14C-2,4-D in six Atlantic coastal plain soils in the
    USA. The mean distribution coefficient, averaged over all soils and
    horizons, was 0.65 L/kg. A model was developed involving only organic
    matter and exchangeable acidity, which predicted the sorption of 2,4-D
    (R2 = 0.68) (Johnson & Sims, 1993).

    Supercritical carbon dioxide, with and without methanol as a modifier,
    was used to extract bound residues of 2,4-D from soil, plant, and
    grain samples. Three months after application of 14C-2,4-D, at .226
    µCi/g (10ppm) to soil, 14% was found to be bound in the soil. The
    recovery of 2,4-D on mineral soil was only 57.4%. After addition of
    methanol to the process, the recovery improved significantly, to 80.7%
    (Khan, 1995). 1995).

    1.3.4  Mobility and leaching

    In a study conducted according to the guidelines of the US
    Environmental Protection Agency, the mobility of soil-aged residues of
    14C-2,4-D was determined in a sandy soil. The soil was first treated
    with 1.4 mg/kg 14C-2,4-D and aged aerobically for 28 days at 20°C, at
    a 263 moisture content of 75% field capacity. After 28 days, an
    aliquot of the treated soil was placed on 28 cm of untreated soil of
    the same type in a column. In order to evaluate the leaching of 2,4-D
    in this soil, it was percolated with 0.01 N CaCL. After 28 days, 69.1%
    of the applied radioactivity was released as 14C-carbon dioxide,
    5.39% being extractable. Of the extractable residue, 23.9% was
    recovered as 2,4-D. Fully 98.8% of the radiolabel applied to the top
    of the column was retained after addition of the CaCl2, 96.73% being
    retained in the top 5 cm. Analysis of the radiolabel in the top 5 cm
    showed that 8.14% was on 2,4-D and 72.98% on unidentified, highly
    polar metabolites (Zohner, 1990a). In a similar study, conducted
    according to a different guideline, the same soil was treated with
    14C-2,4-D at a rate of 2.19 mg/kg and aged aerobically for 30 days at

    20°C, when 100 gm dry weight of the aged soil was placed on top of an
    untreated 28-cm column of the same soil type. It was then percolated
    with distilled water. After 30 days, the total radiolabel in the soil
    had decreased to 16.27% of the applied dose, of which 4.08% was
    extractable. 14C-Carbon dioxide released during aging represented
    71.57% of the applied radiolabel; and the extractable residues were
    unidentified, highly polar metabolites. A total of 2.15% of the
    radiolabel applied to the column was recovered in the leachate, of
    which 0.015 µg was identified as 2,4-D (Zohner, 1990b).

    The mobility and degradation of 14C-2,4-D dimethylamine salt on a
    sandy soil was evaluated in two lysimeters for two years. A total of
    2025 mm water was added to the soil during this period from rainfall
    and irrigation. The results were consistent with those of studies in
    the laboratory, indicating rapid degradation of 2,4-D. Extremely small
    amounts of residues of 14C-2,4-D (0.133 and 0.070 µg/L of parent
    equivalents) were found. Apart from 14C-carbon dioxide, no typical
    soil metabolites were detected in the leachate. Only 0.26-0.29% of the
    applied radiolabel was found on 2,4-D in extracts from the upper 17 cm
    of soil. Thus, although some sorption experiments in the laboratory
    indicate that 2,4-D is mobile, its rapid degradation in soil prevents
    significant downward movement under normal agricultural conditions
    (Burgener, 1993).

    In studies of the mobility of 2,4-D on thin-layer plates and its
    dissipation in tobacco soils in Ontario, Canada, 2,4-D moved with the
    water front 'as expected' from its solubility in water (Sharom &
    Edgington, 1986). The mobility of 14C-2,4-D acid was compared with
    that of triclopyr and picloram on thin-layer chromatographic plates
    prepared from several agricultural and forestry soils in Ontario (a
    luvisol, a gleysol, a podsol, and an organic soil). The mobilities
    were similar for herbicides of similar structure. In a comparison of
    the relative adsorptions of concentrations of 5, 10, 25, and 50 mg/L
    of the three herbicides on the basis of adsorption coefficients (Kd)
    in at least one horizon, the adsorption coefficients for triclopyr and
    2,4-D were similar in all soils examined (Jotcham et al., 1989).

    The mobility of 14C-2,4-D and its hydrolysis product (by both
    chemical and microbial degradation), 14C-2,4-dichlorophenol, was
    studied in six soils with clay contents of 8.0-34.6% and organic
    matter contents of 0.7-6.1% by thin-layer chromatography. The  Rf
    value for the parent compound was 0.56-1.00, and that for the
    hydrolysis product was 0.11-0.58, indicating that the hydrolysis
    product was less mobile than the parent. The mobility of the parent
    acid was pH dependent (Somasundaram et al., 1991).

    The dimethylamine and isooctyl ester formulations of 2,4-D were
    applied at a high rate (8.9 or 0.9 kg/ha) to a silt loam field plot
    under both winter wheat and fallow cropping schemes, and simulated
    rainfall (16 mm) was applied one day later. 2,4-D was found at 24 cm
    three days after application, at 40 cm after five days, and moved
    downwards for 30 days; there was some evidence of movement below the
    40-cm sampling depth during the growing season. After 191 days, the

    concentrations at 40 cm were similar (0.02-0.04 ppm) for the winter
    wheat and fallow cropping plots at high and low application rates;
    however, the concentration of residues was extremely low (0.05 ppm)
    after 45 days at the highest rate of application (8.9 kg/ha) (Wilson &
    Cheng, 1976). In two scenarios of the chemical transport and fate of
    2,4-D in soil and groundwater--no water flowing and steady-state water
    flow on a sandy soil in which 2,4-D was located in a 10-cm layer--2,4-
    D degraded to the same extent (36.5 and 37%) with and without water,
    and the same amount remained in the soil (58.5 and 63%, respectively).
    It was concluded that the model assumptions should be modified on a
    site-specific basis (Jury, 1992).

    Polyethylene columns, 4.8 cm in diameter and 50 cm high, were filled
    with sandy loam, and 2,4-D was applied at 8 kg/ha. The columns were
    watered with 55.8 ml/day for 30 days, equivalent to four years of
    rainfall in the United Kingdom. The sorption and degradation were KOC
    = 108 and half-life = 73 h at 1 µg/g and 213 h at 10 µg/g. 2,4-D was
    virtually completely eluted from the column, and 39-47% was recovered
    in the eluate. The compound began to leave the column on day 9, whe
    there was an equivalent of 28 cm water, and the maximum concentration
    left the column on days 10-12 (Lopez-Avila et al., 1986).

    The leaching of 2,4-D from home lawns was monitored on ceramic plates
    placed under a sandy loam after application at 3.3 or 1.1 kg/ha per
    year, with an irrigation scheme. The mean concentrations of 2,4-D in
    the soil water percolate were only 0.87 - 0.55 µg/L, suggesting
    excellent degradation in the root zone and no threat to groundwater
    from these application rates (Gold et al., 1988). The annual geometric
    mean concentration of 2,4-D in leachate from home lawns characterized
    by silt loam or a sandy loam mantle was less than 1 µg/L (Gold &
    Groffman, 1993). The maximum concentration of 2,4-D in drainage water
    after application to two clay soils at a rate of 0.40 kg ai/ha
    intermittently, between spring 1985 and autumn 1990, was only 0.235
    µg/L at one site at one sampling interval (Felding, 1995).

    1.3.5  Degradation in soil under laboratory conditions

    The hydrolysis of 14C-2,4-D ethylhexyl ester was evaluated in one
    silty clay and one sandy loam soil slurry in the dark in an incubator
    for 4 h at 25°C. The ester degraded rapidly in both slurries, only
    9.6% remaining in the silty clay and 12.6% in the sandy loam at the
    end of the study. The half-life of the ester was calculated to be 1.25
    h in the silty clay and 1.45 h in the sandy loam (Concha et al.,
    1993a).

    The half-life for hydrolysis of the isopropyl,  n-butyl, and isooctyl
    esters of 2,4-D was < 1 min in aqueous 0.1 N NaOH solution. After 24
    h in a sandy loam, a heavy clay, and a loam from Saskatchewan, Canada,
    the isopropyl and n-butyl esters had completely hydrolysed, while
    20-30% of the isooctyl ester remained (Smith, 1972a).

    The rate constant,  k, for the linear regression plot of
    de-esterification of 14C-2,4-D isopropyl ester in a sandy loam
    maintained at 25°C for 8 h was 0.752 h-1 and the half-life was 0.9 h
    The application rate was based on a dry weight in soil of 9.66 ppm.
    The k value for de-esterification in a water/sediment mixture
    maintained at 25°C for 24 h was 0.053 h-1 after application at a rate
    of 9.67 ppm based on the total weight of water and sediment, and the
    half-life was 13.1 h.

    In an extensive review of the literature on the degradation, fate, and
    persistence of phenoxyalkanoic acid herbicides in soil, 2,4-D was
    reported to degrade rapidly in the presence of microorganisms. In the
    tables presented, the half-life for degradation of 2,4-D ranged from
    two days in a silt loam to about 40 days in a sandy clay, and the
    percent carbon dioxide evolution on several soils was 10-95% (Smith,
    1989). High concentrations of 2,4-D break down slowly, with an initial
    lag phase followed by a greatly increased rate; the first-order rate
    constants for the fast phases of degradation were independent of the
    concentration, but the duration of the slow phase increased linearly
    from 11 to 28 days as the concentration of 2,4-D increased from 1.3 to
    25 µg/gm of soil. Soils treated with large amounts of 2,4-D retained
    their ability to degrade additional applications of 2,4-D rapidly
    (Parker & Doxtader, 1982). Hydrolysis of the isooctyl ester of 2,4-D
    in a Naff silt loam and aqueous solution at either 10 or 30°C for up
    to 192 h was rapid, with > 80% degraded within 72 h at 30°C. A lag
    phase for breakdown was again seen (Wilson & Cheng, 1978). It was
    reported that the kinetics of the degradation of 2,4-D in soil are
    affected by the effects of temperature and moisture on the microbial
    population. An initial lag phase was seen only at temperatures above
    the optimum (27°C), and no fast phase was observed. Further, the rate
    of decomposition of 2,4-D decreased with soil moisture tension at
    temperatures between 20 and 35°C, which corresponded to reduced
    activity of 2,4-D-degrading microorganisms. The soils used in this
    study had been stored for three years after air drying (Parker &
    Doxtader, 1983). When a flooded Finnish sandy clay (pH 4.7) was
    incubated after application at a rate of 1000 ppm, only 159 ppm were
    recovered after 72 weeks. At an application rate of 10 ppm, 1.7 ppm
    2,4-D were recovered (Sattar & Paasivirta, 1980).

    The effects of soil properties and soil-degrading microorganisms on
    the degradation of 2,4-D have been studied widely (Torstensson, 1978;
    Loos et al., 1979; Fournier et al., 1981; Ou, 1984; Sinton et al.,
    1986; Scheunert et al., 1987; Kuwatsuka & Miwa, 1989; Rothmel &
    Chakrabarty, 1990; Somasundaram & Coats, 1990; Oh, 1991; Masson et
    al., 1993; Han & New, 1994; Ka et al., 1994; Myers et al., 1994;
    Robertson & Alexander, 1994; Smith et al., 1994; Veeh et al., 1996).
    The environmental factors that are significant in determining
    degradation rates are pH, temperature, moisture, supplemental
    nutrients, substrate concentration, and aeration. Rapid degradation
    correlated well with increased microbial concentrations. 
     Pseudomonas, Arthrobacter, Mycoplana, and  Xanthobacter spp. and
     Flavobacterium peregilum have been shown to cleave the ether linkage

    between the oxygen and the aliphatic side chain of 2,4-D to form
    2,4-dichlorophenol.

    After an investigation of the effect of inoculum preparation and
    density on the efficiency of remediation of 2,4-D in a  Pseudomonas 
     cepacia strain (BRI6001) after full-scale application, it was
    recommended that diffusion first be minimized by ensuring proper soil
    fragmentation, water content, and aeration, and then use of a
    bioaugmentation level of 106 to 108 bacteria/g. At the highest level
    of bioaugmentation, the rate of degradation of 2,4-D was less than two
    days (Greer et al., 1980; Comeau et al., 1993).

    Stoichiometric equations were found to be useful in predicting oxygen
    consumption and heat production during the aerobic degradation of
    14C-ring- and carboxy-labelled 2,4-D by  Pseudomonas cepacia 
    (Fradette et al., 1994a). Thermograms of the rate of heat evolved
    versus time derived by microcalorimetry indicated the time required
    for primary biodegradation of 2,4-D by  Pseudomonas cepacia in a
    liquid medium and sterilized sand under aerobic conditions (Fradette
    et al., 1994b).

    Another soil bacterium,  Pseudomonas testosteroni, isolated from
    field soils, used 2,4-D amine residues from farm operations and
    herbicide containers as a carbon energy source and degraded the
    herbicide over a range of temperature and concentrations. The
    water-soluble amine formulations were found to degrade rapidly but the
    ester formulations degraded very slowly (Smith & Mortenson, 1991).

    Pemberton and Fisher (1977) and Fisher et al. (1978) isolated plasmids
    that code for degradation of 2,4-D from a strain of  Alcaligenes 
     paradoxus. Subsequently, the ability of 70 environmental strains of
     Pseudomonas to use 2,4-D as a sole source of carbon and energy was
    shown to be controlled by plasmids (Pierce et al., 1981, 1982).

    Of six microroganisms isolated from herbicide-treated soils
    ( Flavobacterium peregrinum, Pseudomonas fluorescens, Arthrobacter 
     globiformis, Brevibacterium sp.,  Streptomyces viridochromogenes, 
    and an unidentified  Streptomyces sp.),  Flavobacterium was the most
    active in degrading 2,4-D, 20 mg/kg being completely degraded within
    20-30 days. In a liquid medium,  Flavobacterium degraded 93.5% of
    added 2,4-D within 80 h (Le Van To, 1984).

    More microorganisms capable of degrading 2,4-D were found in soils in
    Natal under sugar cane  (Saccharum officinarum) with rhizospheres
    than under African clover  (Trifolium africanum), suggesting that the
    rapid degradation in the rhizospheres is an additional mechanism for
    the protection of certain plants against herbicides applied to soils
    (Sandmann & Loos, 1984). Thermophilic microorganisms mineralized 18%
    of 14C-2,4-D in composting yard trimmings after 50 days at a
    temperature of 55°C (Michel et al., 1995).

    Melanic fungi incorporate significant amounts of the ring portion of
    2,4-D into the relatively resistant humic acid-type polymers they
    form, while the side-chain portion of 2,4-D is used for synthesis of
    general cell-wall components and products (Wolf & Martin, 1976). In a
    study of the biodegradation, incorporation into biomass, and
    stabilization in humus of labelled 2,4-D carbons and decomposition
    products from four soils, the side-chain carbon in particular was
    rapidly biodegraded at rates comparable to that of glucose at low
    concentrations. After one year, up to 8% of the residual activity was
    present in the biomass in an unextractable form (Stott et al., 1983).
    Concentrations of dissolved oxygen below 1.0 mg/L were rate limiting
    for the biodegradation of 2,4-D at 25°C by a bacterial culture from
    sewage sludge, suggesting that 2,4-D requires dissolved oxygen as a
    cosubstrate for metabolism (Shaler & Klecka, 1986).

    Degradation of 2,4-D ethyl ester on a wetland farm soil containing 42%
    clay was evaluated for up to 14 days. Degradation was rapid, less than
    0.22 ppm remaining after 14 days at various moisture contents
    (Bhanumurthy et al., 1989).

    In a laboratory experiment with technical-grade 2,4-D (purity, 97%)
    and its dimethylamine salt, 14C-2,4-D was introduced into a Webster
    silty clay loam, a sandy loam, and a muck soil. Degradation was
    measured by the evolution of carbon dioxide. All three soils degraded
    2,4-D at application rates of 500 ppm, but at 5000 ppm degradation
    ranged from 59% of formulated 2,4-D in the organic soil to a very low
    percentage of the technical-grade product in Cecil soil (Ou et al.,
    1978).

    Samples of chernozem soil were enriched with various nutrients and
    2,4-D to determine whether the presence of available metabolic
    substrates alters the decomposition of 2,4-D. Overall proliferation of
    bacteria and an increased relative proportion of bacterial strains
    capable of mineralizing 2,4-D were seen in the enriched samples (Kunc
    & Rybarova, 1984). Samples of a Cecil sandy loam were analysed for
    2,4-D for up to 14 days after the addition of lime and sulfur. Neither
    compound altered the degradation of 2,4-D in sterile soil; the most
    rapid degradation occurred in nonsterile soils that had been limed to
    adjust the pH to 7-7.4 (Smith, 1972b).

    2,4-D was added to municipal sewage processed such that 2,4-D was the
    sole carbon source, and biological oxygen depletion was monitored as a
    measure of degradation. Less than 5% of the available oxygen was
    depleted, indicating poor biodegradation due to the low numbers of
    degrading microorganisms (Lieberman & Alexander, 1981). Degradation of
    2,4-D was enhanced in clay, sandy loam, and fine sandy loam from
    southwestern USA that was either freshly amended or preconditioned for
    two months with sewage sludge. Sludge additions had no effect on 2,4-D
    degradation in a soil that had been treated previously with 2,4-D
    (O'Connor et al., 1981). After incubation of 2,4-D with 52 bacteria
    isolated from soil and sewage, 41 of the isolates metabolized 2,4-D
    but none grew by using 2,4-D as a carbon source. Nearly all of the

    2,4-D had been metabolized within seven days (Rosenberg & Alexander,
    1980).

    In a study of aerobic soil metabolism conducted according to the
    guidelines of the US Environmental Protection Agency, 14C-2,4-D was
    added to Catlin silty clay at 25°C for up to 16 days. 14C-2,4-D
    degraded rapidly, representing 0.5% of the applied dose after 16 days
    of exposure. The calculated half-life was 1.7 days, with pseudo-first-
    order kinetics. The major metabolite under aerobic conditions was
    carbon dioxide, which accounted for 51.2% of the applied dose at the
    end of the study. Two other metabolites were found:
    2,4-dichlorophenol, accounting for 3.5% at day 2 and 0.4% at the end
    of the study, and 2,4-dichloroanisole, accounting for 2.5% at day 9
    and 1.5% at the end of the study. After partitioning of fulvic acid
    and humic acid in an extracted residue sample from day 5, 16.1% of the
    applied dose was in the fulvic acid fraction and 11.1% in the humic
    fraction. HPLC of the fulvic fraction showed that 6.1% of the applied
    dose was 2,4-D (Concha & Shepler, 1994b).

    Half-lives of 10.8-31.4 days were observed for [1- or 2-14C]-2,4-D in
    six soils from Saskatchewan, Canada, under aerobic conditions at field
    capacity moisture at 26°C, in an aerobic soil metabolism study.
    Initial degradation occurred by cleavage of the ether linkage and not
    by decarboxylation (Foster & McKercher, 1973). The half-life for
    mineralization of [1- or 2-14C]-2,4-D to carbon dioxide in a
    chernozem soil incubated at 28°C was about 90 h, with a lag phase of
    less than three days (Kunc & Rybarova, 1983). In a study of the
    mineralization of [1-14C]-2,4-D in nine Belgian soils at 22°C, the
    more alkaline soils (pH > 6) mineralized 2,4-D rapidly, 80-95% being
    degraded after 30 days, with a lag phase of 10-15 days in the
    evolution of 14C-carbon dioxide. Mineralization was slow in acidic
    soils (pH < 6), < 10% of the applied dose being degraded after 30
    days (Moreale & Van Bladel, 1980). Aerobic soil degradation kinetics
    were determined for 14C-2,4-D on a loam, a silty clay loam, clay, clay
    loam, silt loam, and a sandy loam in the dark at 25°C for up to 230
    days. The average half-life was four days, and the main metabolite was
    carbon dioxide (McCall et al., 1981).

    In three sandy soils at 18°C in the laboratory, the average half-life
    for 2,4-D was again four days, and degradation closely followed
    first-order kinetics for five half-lives (Altom & Stritzke, 1973).
    After application of 2,4-D to a heavy clay, clay loam, and sandy loam
    from the prairies in Saskatchewan, Canada, at 20°C for up to 35 days,
    the half-life was less than seven days (Smith, 1978a). The half-life
    of 14C-2,4-D was not affected by the addition of other herbicides in
    the laboratory at 20°C for up to 35 days, and the half-life in a heavy
    clay and a sandy loam was again less than seven days (Smith, 1979). In
    investigations on the persistence of 14C-2,4-D in heavy clay, sandy
    loam, and clay loam, the half-lives were similar regardless of whether
    the soils had been pretreated with other herbicides or insecticides,
    being three to six days in the clay loam, three to eight days in the
    sandy loam, and three to 11 days in the heavy clay soil (Smith, 1980).

    The aerobic metabolism of 14C-2,4-D was evaluated in four Canadian
    soils, in the dark, under laboratory conditions at 20°C for up to 24
    days. In soils that had not received herbicides recently, > 89% of
    the applied 14C-2,4-D was metabolized, 25-31% being released as carbon
    dioxide, 2-10% recovered as 2,4-dichloroanisole, and 39-43% being
    unextractable. In soils that had received treatment with 2,4-D, about
    50% of the applied 14C-2,4-D was metabolized to carbon dioxide, 1-4%
    to 2,4-dichlorophenol, 2-5% to 2,4-dichloroanisole, and 22-30% being
    unextractable (Smith & Aubin, 1991a). Degradation of 14C-2,4-D under
    laboratory conditions was faster in Canadian soil that had received 43
    annual applications than in soils from untreated control plots (Smith
    & Aubin, 1991b).

    As the pKa of 2,4-dichlorophenol is 7.89 ( Dictionary of Organic 
     Compounds, 1996), it is present in most soils in the volatile
    phenolic form rather than in the phenolate anion form. Smith (1985)
    postulated that failure to isolate 2,4-dichlorophenol and
    2,4-dichloroanisole from soils treated with 2,4-D may be due to prior
    volatilization or losses during sample preparation. 14C-2,4-D was
    broken down rapidly in a clay, a clay loam, and a sandy loam, with
    > 70% degradation within 10 days. Carbon dioxide was the main
    degradation product, representing 30-42% of the applied radiolabel.
    The presence of 2,4-dichlorophenol and 2,4-dichloroanisole was
    confirmed by gas chromatography. These metabolites are generated
    initially by cleavage of the oxygen-carbon bond at the 2 position on
    the side-chain. Methylation in the soils then accounts for the
    formation of 2,4-dichloroanisole. Several other studies have indicated
    that 2,4-dichlorophenol in soil is rapidly dissipated by both
    biological and nonbiological mechanisms (Baker & Mayfield, 1980;
    Bollag et al., 1980; Soulas & Fournier, 1987), so that such residues
    are not likely to accumulate under field conditions.

    In the report of a study on the effect of sulfate on the anaerobic
    dechlorination of 27 ppm 2,4-dichlorophenol in freshwater sediments,
    few experimental details were provided, but the compound was
    completely dechlorinated to 4-chlorophenol when samples from the
    sulfate-amended sediments were incubated at 19-40°C (Kohring et al.,
    1989). Identical dechlorination of 2,4-D to 4-chlorophenol by cleavage
    of the ether linkage and loss of the 2-chloro atom was found in
    anaerobically digested municipal sewage (Mikesell & Boyd, 1985).

    Estuarine sediment treated with 2,4-D at 24 µmol/L at 13.5°C under
    anaerobic conditions formed 2,4-dichlorophenol, at 0.5% of the applied
    dose, after 33 days (Eder, 1980).

    In a stable, sulfate-reducing model ecosystem capable of reproducing
    biological, chemical, and hydraulic conditions under anaerobic
    conditions, 2,4-D was almost resistant to biodegradation (Kuhlmann &
    Kaczmarzcyk, 1995).

    The phytotoxicity and detoxification of 2,4-D in 11 soils from spruce
    and pine forests in Sweden with pH values of 3.5-5.1 were compared
    with its degradation in three agricultural soils from Sweden with pH
    values of 6.3-7.6. In one experiment in which the soils were
    inoculated with a 2% salt solution containing 100 ppm 2,4-D, the
    degradation time corresponded to a residual amount of 3 ppm of the
    2,4-D left in the flasks and ranged from one to four weeks in the
    forestry soils and only one week in the agricultural soils
    (Torstensson, 1975).

    The distribution and effects of 14C-2,4-D butyl ester were evaluated
    in a ryegrass ecosystem which consisted of sandy loam soil, annual
    ryegrass, numerous invertebrates, and a vole, all housed in a
    terrestrial microcosm chamber. Thirty days after application at 1.0
    kg/ha, all of the radiolabel detected in the soil was in unextractable
    residues in the top 1.0 cm, while the plant material contained an
    average of 8.9 mg/kg, which was identified primarily as
    2,5-dichloro-4-hydroxyphenoxyacetic acid (Gile, 1983).

    A vehicle-portable analytical system for on-site analyses developed by
    the Emergencies Science Division of Environment Canada was used to
    analyse soil contaminated from burst barrels of chemicals at the scene
    of a fire. 2,4-D was found on the site. The recoveries with the
    microwave-assisted process and with Soxhlet extraction were acceptable
    (Li et al., 1995).

    1.3.6  Degradation in soil under field conditions

    The dissipation of 2,4-D has been evaluated in many soils in many
    areas of the world, and analytical methods have been developed for a
    wide range of formulations and soils, typically involving hydrolysis
    of the 2,4-D ester to the acid, extraction of the samples with various
    solvents, chemical derivatization, and identification by gas
    chromatography or capillary column gas chromatography and mass
    spectroscopy (Khan, 1975b; Smith, 1976, 1978b; Kan et al., 1981, 1982;
    Ahmed et al., 1989; Bruns et al., 1991). A quantitative method has
    been developed for the determination of 2,4-D in soils, involving use
    of anion-exchange membranes and detection by gas chromatography, which
    was used successfully to determine 2,4-D in a commercial formulation
    on the surfaces of a loam and a heavy clay. A linear relationship was
    observed between the amount of 2,4-D amine detected and the level
    spiked onto the soil surface (Szmigielska & Schonenau, 1995).

    After application of an  n-butyl-isobutyl ester formulation of 2,4-D
    to wheat at the three-leaf stage planted in a Canadian black chernozem
    soil of unknown composition at a rate of 420g acid equivalent per
    hectare, no residues of 2,4-D acid were found in the straw or grain
    collected after 80 days. No butyl ester residues were found in the
    soil, and < 0.01 ppm 2,4-D was found within 22 days of application
    (Cochrane & Russell, 1975). Field experiments were conducted on a Naff
    silt loam soil with a split-plot design and two replicates in
    Washington State, USA, for up to 175 or 191 days in 1973 and 1974; one
    plot was planted with winter wheat and one was under fallow cropping

    conditions. The applications consisted of formulated dimethylamine
    salt or the isooctyl ester of 2,4-D at 1.1 and 11.2 kg/ha in 1973 and
    0.9 and 8.9 kg/ha in 1974, respectively. Considerably more 2,4-D was
    recovered from the plots treated with the amine than from those
    treated with the ester at the high rate of application for both years.
    The average residue concentrations of amine and ester were very low,
    regardless of the application rate, after 175 days (0.02-0.12 ppm in
    winter wheat and 0.01-0.11 ppm in fallow) in 1973 or 191 days
    (0.024).04 ppm in winter wheat or fallow) in 1974 (Wilson & Cheng,
    1976). The residues of 2,4-D dimethylamine salt in wheat 35-45 days
    after application as a tank mixture during two consecutive growing
    seasons had dissipated to < 0.1 mg/kg. No residues were detected in
    straw or seeds at maturity in either year (Cessna & Hunter, 1993).

    Applications of the propylene glycol butyl ether ester of 2,4-D at 3.4
    kg acid equivalent per hectare under conditions of low rainfall on
    moderately coarse soils with relatively low organic matter disappeared
    rapidly. A maximum concentration of 95.2 mg/kg was found in samples of
    vegetation collected within 15 min of application. After 12 months,
    the mean residue level had declined to 3.8 mg/kg (Plumb et al., 1977).
    The persistence of 2,4-D was also evaluated in a chaparral
    environment. Chamise  (Adenostoma fasciculatum), grass and forbs,
    soil surface litter, and soil were sampled for up to 360 days after
    treatment at 4.5 kg/ha. The residues on foliage and litter decreased
    rapidly up to 93% within 30 days after treatment and remained constant
    until the winter rainfall. No residues were found below 0-5 cm of the
    soil surface (Radosevich & Winterlin, 1977). Liquid and granular
    formulations of the dimethylamine salt of 2,4-D were applied to turf
    at 1.0 or 2.24 kg acid equivalent per hectare in both laboratory and
    field experiments. In all of the field experiments, there was a rapid,
    significant decrease in dislodgeable residues over time, and by day 7
    the residue levels were about 0.02% of the total applied (Thompson et
    al., 1984a).

    Dissipation of the isooctyl ester of 2,4-D and its acid metabolite
    were measured in air, wheat, and soil components in Canada for up to
    35 days after application. The acid was detected immediately after
    application of a rate of 450 g/ha as the isooctyl ester. Its
    hydrolysis to the acid and subsequent degradation of the acid in the
    soil were dependent on the availability of water. The acid levels
    increased from 156 g/ha on day 1 to 185-210 g/ha over the next 14 days
    (Grover et al., 1985).

    The dissipation of 2,4-D in soil and water associated with rice
    production in three cultural systems--paddy rice, rainfed lowland
    rice, and bare ground--in Arkansas, USA, was rapid up to 49 days after
    application, with a DT50 in all three systems of 10 days or less
    (Johnson et al., 1995).

    The degradation of 14C-2,4-D was evaluated after application at a
    rate of 1.0 kg/ha on small sandy loam plots for up to 95 weeks. Less
    than 2% of the applied radiolabel was recovered from 2,4-D after 45
    weeks and < 1% after 95 weeks (Smith & Muir, 1984).

    The persistence of the dimethylamine salt was evaluated in four
    agricultural soils (loam, clay, fine sand, and sandy clay loam planted
    with barley) in southern Ontario and two forestry soils (sand and clay
    containing a variety of grasses and conifers) in northern Ontario,
    Canada. The application rates were 0.56 kg/ha in the agricultural
    fields and 2.24 kg/ha in the forestry areas. The levels of residues on
    the agricultural soils were close to the levels of detection (about 5
    µg/kg) at the end of the growing season. The degradation patterns in
    forest soils were similar to those in the agricultural soils but
    higher at the end of the growing season, probably due to the fact that
    four applications were made to the forest soils (Thompson et al.,
    1984b). Persistence of 2,4-D in forest soil was also evaluated for
    brash control in areas of conifer reforestation in five sites in
    southern Sweden and six in northern Sweden. 2,4-D disappeared rapidly
    from all sites, but low concentrations remained on leaves and branches
    for up to two years in the northern sites, ranging from 0.2 to 0.7 µg
    2,4-D per soil core (Torstensson et al., 1989).

    Soils treated with 2,4-D in the laboratory can develop enhanced
    ability to degrade the chemical, seemingly by metabolism by increased
    numbers of microorganisms (Roeth, 1986; Smith & Lafond, 1990; Smith et
    al., 1991). Increased rates of 2,4-D degradation after repeated use
    have also been reported in field studies (Torstensson et al., 1975;
    Smith et al., 1989). Significantly more 2,4-D-degrading microorganisms
    were found in soils in Saskatchewan, Canada, after 32 years of 2,4-D
    treatment than in soils from control plots (Cullimore, 1981). When the
    same field plots had received 40-43 annual applications of 2,4-D
    formulations, they retained greater degradation of 2,4-D than
    untreated control plots for at least 48 weeks after the last
    treatment. The levels of residues were below the level of detection
    (< 0.02 mg/kg), with no evidence of leaching from the topsoil. The
    residues degraded rapidly, 6-9% of the applied 2,4-D remaining after
    eight days (Smith et al., 1989; Smith & Aubin, 1991 a). No
    agronomically significant effects of these annual treatments were seen
    on soil biochemical processes, such as microbial biomass, respiration,
    and soil enzyme activity, or on soil fertility (Biederbeck et al.,
    1987). After cessation of 2,4-D applications to a Canadian clay soil,
    the microflora maintained their ability to degrade 2,4-D rapidly for
    at least 204 weeks (Smith & Aubin, 1994). In a study of the effects of
    2,4-D and glyphosate on microbial activity in a chernozem soil under
    zero-tillage chemical fallow conditions, 2,4-D was applied as the
    amine salt at field rates and at 10 times the field rate. No effect
    was seen on microbial biomass, mineralization, or nitrification in the
    field; however, in the laboratory, application at rates of 2 and 100
    times the field rate reduced nitrification by 1 and 79%, respectively
    (Olson & Lindwall, 1991).

    2,4-D was applied as the propylene glycol and butyl ether esters by
    helicopter to brushfields on shallow, rocky, clay loam in three
    mountainous areas of southwestern Oregon, USA. The estimated
    half-times (overestimates according to the authors) ranged from 18.9
    days in the litter, at an application rate of 2.2 kg/ha, to 234.7 days
    in the crown, with an application rate of 3.3 kg/ha. The

    concentrations of 2,4-D in the surface soil layer decreased rapidly
    initially and then more slowly throughout the winter, rarely exceeding
    0.02 mg/kg at 45-60 cm, the lowest depth sampled. Less than 30% of the
    applied 2,4-D was recovered 37 days after application, and no residues
    was recovered 15 cm below the soil surface (Newton et al., 1990). The
    soil persistence and lateral movement of 2,4-D after application as a
    stem-foliage spray for brush control on two power line rights-of-way
    were measured by analysing runoff water and soil samples for up to 48
    weeks after treatment. No residue of 2,4-D was found in soil or water
    15 weeks after spraying (Meru et al., 1990). After application of
    2,4-D amine salt to a power line right-of-way in Ontario, Canada,
    which had recently been cleared, < 0.1% of the applied salt was lost
    laterally during seven rainfalls (the average rainfall during one day
    was > 30 mm), indicating that rain falling even 24 h after spraying
    does not cause the release of significant concentrations of 2,4-D
    (Suffling et al., 1974). Residues of 2,4-D were detected in soil
    samples from a forest watershed adjacent to a treated right-of-way in
    eastern Ontario, Canada, at least 36 m from the right-of-way 12 days
    and two and eight months after application. It was not clear whether
    the 2,4-D found was actually derived from the application site or was
    from another source (McKinley & Arron, 1988).

    Water, sediment, and samples from the wall of an outdoor enclosure
    located in a typical bog lake in a sandy soil area of northeastern
    Ontario, Canada, were analysed for residues of the isooctyl ester of
    2,4-D for more than 100 days. Less 2,4-D was adsorbed to the sediments
    than to the sides of the enclosure, where < 25% was found (Solomon
    et al., 1988).

    Thirty-five studies on terrestrial, aquatic, and forestry field
    dissipation were conducted in the USA in 1993 and 1994 according to
    the guidelines of the US Environmental Protection Agency. The
    half-lives and other data are given in Table 1. Fourteen studies of
    terrestrial dissipation were conducted in 1993 to compare the
    disappearance of the dimethylamine salt and the 2-ethylhexyl ester of
    2,4-D when applied in a spray solution to pasture and turf in
    Colorado, North Carolina, and Texas with grass growing on the plots
    and to bare soil in the same area. Four trials on cultured wheat were
    established in North Carolina and four in Colorado. Twelve similar
    studies were conducted in 1994 in California, North Dakota, Nebraska,
    and Ohio on pasture and turf with grass present and on bare soil
    according to the timing for use on turf, wheat, and corn. Four further
    studies were conducted in 1994 to compare the disappearance of
    granules of the dimethylamine salt and 2-ethylhexyl ester, the former
    in North Dakota and the latter in Ohio. The materials were applied as
    for the treatment of turf, with turf present and on bare soil.

    An aquatic trial was conducted in 1993 in Louisiana on rice, in which
    the dimethylamine salt was applied aerially to a water-filled rice
    paddy at a target application rate of 1.5 lb acid equivalent per acre
    (1.68 kg/ha). The highest 2,4-D acid residue in soil (mean total of
    three depths) was found one day after application, the residues
    declined to 0.013 ppm three days after application. Metabolites were


        Table 1. Dissipation of 2,4-D after application at various sites in the USA

                                                                                                                                               

    Site  Type         2,4-D   No. of        Soil type         Crop             Rate        Metabolite   Half-life           Reference
                       form    applications                                     (kg ae/ba)               (days)
                                                                                                                                               

    CA    Terrestrial  DMA     2, ground     Sandy loam        Pasture          2.24        DCA          5.7 (1st)           Hatfield (1995c)
                                                                                            DCP          30.5 (2nd)

    CA    Terrestrial  2-EHE   2, ground     Sandy loam        Pasture          2.24        DCA          1.8 ester (1st)     Hatfield (1995d)
                                                                                            DCP          4.7 ester (2nd)
                                                                                                         10.6 acid (1st)
                                                                                                         27.3 acid (2nd)

    CA    Terrestrial  DMA     2, ground     Sandy loam        Turf             2.24        DCA          29.1 (1st)          Hatfield (1995e)
                                                                                                         7.5 (2nd)

    CA    Terrestrial  2-EHE   2, ground     Loamy sand        Turf, soil       2.24        DCP          < 2 ester (both)    Hatfield (1995f)
                                                               Soil                                      7.9 acid (1st)
                                                                                                         9.7 acid (2nd)
                                                               Grass                                     5.7 acid (1st)
                                                                                                         9.3 acid (2nd)
                                                               Thatch                                    9.8 acid (1st)
                                                                                                         12.9 acid (2nd)

    CA    Terrestrial  DMA     2, ground     Sandy loam        Bare soil        2.24        DCA          7.3 (1st)           Hatfield (1995g)
                                                                                                         7.6 (2nd)

    CA    Terrestrial  2-EHE   2, ground     Loamy sand        Bare soil        2.24        DCA          < 3 ester (both)    Hatfield (1995h)
                                                                                            DCP          4.4 acid (1st)
                                                                                                         15.0 acid (2nd)

    CO    Terrestrial  2-EHE   2, broadcast  Sandy clay loam   Wheat            1.4         DCP          2.2 ester (1st)     Silvoy (1995a)
                                                                                                         2.1 ester (2nd)
                                                                                                         4.8 acid (1st)
                                                                                                         2.5 acid (2nd)

    Table 1. (continued)

                                                                                                                                               

    Site  Type         2,4-D   No. of        Soil type         Crop             Rate        Metabolite   Half-life           Reference
                       form    applications                                     (kg ae/ba)               (days)
                                                                                                                                               

    CO    Terrestrial  2-EHE   2, broadcast  Sandy clay loam   Bare soil        1.4         DCP          1.4-1.7 ester(both) Silvoy (1995b)
                                                                                                         6.0 acid (1st)
                                                                                                         2.0 acid (2nd)

    CO    Terrestrial  DMA     2             Sandy clay loam   Bare soil        1.4         DCA          5.1 (1st)           Silvoy (1994a)

    CO    Terrestrial  DMA     2             Sandy clay loam   Wheat            1.4         DCA          5.1 (1st)           Silvoy (1994b)

    NC    Terrestrial  DMA     2, broadcast  Sand              Wheat            1.4         DCP          5.5 (1st)           Barney (1995a)
                                                                                            DCA          2.7 (2nd)

    NC    Terrestrial  2-EHE   2, broadcast  Sand              Wheat            1.4         DCP          4.0 ester (1st)     Barney (1995b)
                                                                                            DCA          1.8 ester (2nd)
                                                                                                         9.3 acid (1st)
                                                                                                         6.2 acid (2nd)

    NC    Tenestrial   DMA     2, broadcast  Sand              Turf, soil       2.24        DCP          3.3 (1st)           Barney (1995c)
                                                                                            DCA          2.3 (2nd)
                                                               Grass                                     6.4 (1st)
                                                                                                         7.7 (2nd)

    NC    Terrestrial  DMA     2, broadcast  Sand              Bare soil        2.24        DCP          3.4 (1st)           Barney (1995d)
                                                               (turf rates)                 DCA          2.5 (2nd)

    NC    Terrestrial  2-EHE   2, broadcast  Sand              Turf             2.24        DCP          0.34 ester (1st)    Barney (1995e)
                                                                                            DCA          4.4 acid (1st)
                                                                                                         2.2 acid (2nd)

    NC    Terrestrial  DMA     2, broadcast  Sand              Bare soil        1.4         DCP          2.9 (1st)           Barney (1995f)
                                                               (wheat rates)                DCA          2.6 (2nd)

    Table 1. (continued)

                                                                                                                                               

    Site  Type         2,4-D   No. of        Soil type         Crop             Rate        Metabolite   Half-life           Reference
                       form    applications                                     (kg ae/ba)               (days)
                                                                                                                                               

    NC    Terrestrial  2-EHE   2, broadcast  Sand              Bare soil        1.4         DCA          12.9 ester (1st)    Barney (1995g)
                                                               (wheat rates)                DCP          5.2 ester (2nd)
                                                                                                         6.0 acid (1st)
                                                                                                         3.9 acid (2nd)

    NC    Terrestrial  2-EHE   2, broadcast  Sand              Bare soil        2.24                                         Barney (1995h)
                                                               (turf rates)

    ND    Terrestrial  DMA     2, ground     Sandy loam        Turf, soil       2.24        DCP          10.3 (1st)          Hatfield (1995i)
                                                                                            DCA          5.1 (2nd)
                                                               Grass and thatch                          2.5-6.4 (both)

    ND    Terrestrial  DMA     2, ground     Loam              Bare soil        2.24        DCP          19.6 (1st)          Hatfield (1995j)
                                                                                            DCA          18.4 (2nd)

    ND    Terrestrial  DMA     2, ground                       Bare soil                    DCP          3.9 (1st)           Hatfield (1995k)
                                                                                            DCA          4.5 (2nd)

    ND    Terrestrial  2-EHE   2, ground                       Bare soil                    DCP          4.4 ester (1st)     Hatfield (1995l)
                                                                                            DCA          3.6 ester (2nd)
                                                                                                         6.1 acid (1st)
                                                                                                         6.4 acid (2nd)

    NE    Terrestrial  DMA     4, ground     Silt loam         Bare soil        2.24        DCA          8.6 (1st)           Hatfield (1995m)
                                                                                1.12        DCP          3.9 (2nd)
                                                                                0.86                     1.1 (3rd)
                                                                                1.68                     2.8 (4th)

    NE    Terrestrial  2-EHE   4, ground     Silt loam         Bare soil        2.24        DCP          2,5-4.1 ester       Hatfield (1995n)
                                                                                1.12        DCA          All applications
                                                                                0.86                     46.5 acid (1st)
                                                                                1.68                     3.5 acid (2nd)
                                                                                                         4.4 acid (4th)

    Table 1. (continued)

                                                                                                                                               

    Site  Type         2,4-D   No. of        Soil type         Crop             Rate        Metabolite   Half-life           Reference
                       form    applications                                     (kg ae/ba)               (days)
                                                                                                                                               

    OH    Terrestrial  DMA     4, ground     Silt clay loam    Bare soil        2.24        DCA          23.5 (1st)          Hatfield (1995o)
                                                               (corn site)      1.12        DCP          5.9 (2nd)
                                                                                0.56                     0.9 (3rd)
                                                                                1.68                     10.4 (4th)

    OH    Terrestrial  2-EHE   4, ground     Clay loam         Bare soil        2.24        DCP          10.9 ester (1st)    Hatfield (1995p)
                                                                                1.12                     4.6 ester (2nd)
                                                                                0.68                     1.2 ester (3rd)
                                                                                1.68                     2.9 ester (3rd)
                                                                                                         5.5 acid (2nd)
                                                                                                         1.2 acid (3rd)
                                                                                                         7.0 acid (4th)

    OH    Terrestrial  2-EHE   2, ground     Silt loam         Turf             2.24        DCP          14.3 ester (1st)    Hatfield (1995q)
                               (granule)                                                    DCA          10.9 ester (2nd)
                                                               Soil                                      13 acid (2nd)
                                                               Grass and thatch                          9.3-4.0 acid

    OH    Terrestrial  2-EHE   2, ground     Silt loam         Bare soil        2.24        DCP          7.4 ester (1st)     Hatfield (1995a)
                               (granule)                       (Turf site)                  DCA          6.3 ester (2nd)
                                                                                                         -10.7 acid (1st)
                                                                                                         6.6 acid (2nd)

    TX    Terrestrial  DMA     2, broadcast  Sandy loam        Pasture          2.24        DCP          7.9 (1st)           Barney (1995i)
                                                                                                         10.2 (2nd)

    TX    Terrestrial  2-EHE   2, broadcast  Silt loam         Pasture          2.24        DCP          1.4 ester (1st)     Barney (1995j)
                                                                                                         0.5 ester (2nd)
                                                                                                         4.2 acid (1st)
                                                                                                         13.1 acid (2nd)

    Table 1. (continued)
                                                                                                                                               
    Site  Type         2,4-D   No. of        Soil type         Crop             Rate        Metabolite   Half-life           Reference
                       form    applications                                     (kg ae/ba)               (days)
                                                                                                                                               
    LA    Aquatic      DMA     1, aerial     Silt loam         Rice (water)     1.68        DCP          1.1                 Barney (1994)
                                                               Rice (soil)                  4-CPA        1.5

    NC    Aquatic      DMA     2, subsurface                   Pond (water)     46.81       DCP          19.7 (1st)          Hatfield (1995b)
                               injection                       Pond (soil)                  DCA          2.7 (2nd)
                                                                                            DCP          7.6 (1st)
                                                                                                         2.0 (2nd)

    ND    Aquatic      DMA     2, subsurface                   Pond (water)     46.81       DCP          13.9 (1st)          Hatfield (1995r)
                               injection                       Pond (soil)                  4-CPA        6.5 (2nd)
                                                                                            DCP          -17.4 (1st)
                                                                                                         29.5 (2nd)

    GA    Forest       2-EHE   1, aerial     Sandy clay loam   Exposed soil     4.48        DCP          1.0 ester           Barney (1996)
                               broadcast                       Protected soil               DCA          1.7 ester
                                                               Exposed soil                              4.0 acid
                                                               Protected soil                            3.6 acid
                                                               Foliage                                   7.2 2-EHE
                                                                                                         23.5 2,4-D
                                                                                                         44.0 DCP
                                                               Leaf litter                               51.0 2-EHE
                                                                                                         52.2 2,4-D
                                                                                                         68.3 DCP
                                                                                                         84.7 DCA

    OR    Forest       DMA     1, aerial     Loam              Exposed soil     4.48        DCP          38.7                Barney (1995k)
                               broadcast                       Protected soil               DCA          50.8
                                                               Foliage                                   37.4 2,4-D
                                                                                                         80.8 DCP
                                                               Leaf litter                               65.7 2,4-D
                                                                                                         111.3 DCP
                                                                                                                                               

    CA, California; CO, Colorado; NC, North Carolina; ND, North Dakota; NE, Nebraska; OH, Ohio; TX, Texas; LA, Louisiana; GA, Georgia; 
    OR, Oregon;
    DMA, dimethylamine samt; 2-EHE, 2-ethylhexyl ester; DCA, 2,4-dichloroanisole; DCP, 2,4-dichlorophenol, 4-CPA, 4-chlorophenoxyacetate
    

    not detected above the limit of quantification at any sampling
    interval. Two aquatic trials were conducted during 1994, in North
    Carolina and in North Dakota, in which two applications of the
    dimethylamine salt at approximately 4 ppm were made. At both sites,
    the half-life was much shorter for the second application than the
    first, especially in water, due perhaps to the build-up of
    microorganisms capable of degrading 2,4-D. The half-life of 2,4-D at
    the North Carolina site was shorter than that in North Dakota. The
    effects of 2,4-D in the water phase in these studies are discussed in
    section 4.2.4.

    Two studies of forestry dissipation were conducted in 1993, one in
    Georgia with a 2-ethylhexyl ester formulation on a sandy clay loam and
    one in Oregon with a dimethylamine salt formulation on a loam. Each
    site received one broadcast aerial application at 4.0 lb acid
    equivalent per acre (4.48 kg/ha). Soil, leaf litter, foliage,
    sediment, and water were collected and analysed. The half-lives are
    given in Table 1. The dimethylamine salt of 2,4-D disassociates
    rapidly, leaving free 2,4-D, which can undergo further degradation.
    The 2-ethylhexyl ester undergoes de-esterification on contact with a
    microbially active substrate. Since the ester first undergoes
    de-esterification to yield 2,4-D, the amount of 2,4-D increases during
    the first few days, while the amount of the ester decreases. The
    conversion from ester to acid is not complete before some of the 2,4-D
    has degraded. Degradation of the ester is determined by adding the
    amount of ester (converted to 2,4-D equivalent) and the amount of
    2,4-D found. The data for the 1994 study of field spraying indicate
    average half-lives of 4.3 days for the dimethylamine salt and 5.3 days
    for the 2-ethylhexyl ester, after removal of a few outliers. The data
    for 1993 indicate average half-lives of 4.5 days for the dimethylamine
    salt and 5.1 days for the ester. These results do not indicate any
    difference between the two forms of 2,4-D in the rate of degradation
    or dissipation. The data are also consistent with those for field
    dissipation over 40 years.

    1.3.7  Uptake by plants

    The effects of 2,4-D acid, dimethylamine salt, diethanolamine salt,
    and 2-ethylhexyl ester on the vegetative vigour, seed germination, and
    seedling emergence of 10 non-target crops (buckwheat, corn, cucumber,
    mustard, oats, onion, radish, sorghum, soya bean, and tomato) was
    determined in studies conducted according to the guidelines of the US
    Environmental Protection Agency.

    When 2,4-D acid was applied at concentrations equivalent to 4.2, 2.1,
    1.05, 0.53, 0.26, 0.13, 0.065, 0.03, 0.015, and 0.0075 lb ai/acre
    (4.7, 2.4, 1.2, 0.59, 0.29, 0.15, 0.07, 0.03, 0.017, and 0.0084
    kg/ha), onion was the most sensitive monocot, with an NOEL and an
    EC25 value of < 0.0075 lb ai/acre (< 0.0084 kg/ha); the EC50
    was 0.089 lb ai/acre (0.1 kg/ha). Neither corn nor oats was sensitive
    to 2,4-D acid. Cucumber, mustard, and tomato were the most sensitive
    dicot species, with calculated NOELs lower than the lowest dose
    tested. The response of radish at 0.0075 lb acid equivalent/acre

    (0.0084 kg/ha) was 93% that of the controls and appeared to be the
    NOEL (Bakus, 1992a). In a continuation of the study, cucumber,
    mustard, onion, and tomato were tested at concentrations of 0.0075,
    0.004, 0.002, and 0.001 lb ai/acre (0.0084, 0.004, 0.002, and 0.001
    kg/ha). The NOEL for cucumber and tomato was 0.002 and the that for
    mustard and onion, 0.0075 lb ai/acre (0.0084 kg/ha) (Bakus, 1993a). In
    studies in which 2,4-D acid was applied in petri dish assays at
    0.75-3.8 lb ai/acre (0.84-4.2 kg/ha) and to seeds planted in field
    soil at 0.75-4.2 lb ai/acre (0.84-4.2 kg/ha), 2,4-D had little effect
    on seed germination in petri dishes. Tomato was the most sensitive
    species, with NOEL, EC25 and EC50 values of < 0.12 lb ai/acre
    (0.84 - 4.2 kg/ha). Slight distortion was observed on tomatoes in the
    seedling emergence phase, and stunting was noted on onion, cucumber,
    sorghum, soya bean, and buckwheat (slight). Buckwheat, corn, cucumber,
    oats, sorghum, and tomato had NOEL, EC25, and EC50 values > 4.2 lb
    ai/acre (< 0.13 kg/ha), while the NOEL values for mustard, onion,
    radish, and soya bean were > 1.05 lb ai/acre (> 1.18 kg/ha)
    (Bakus, 1992b). When tomato was treated at concentrations of 0.0075,
    0.004, 0.002, or 0.001 lb acid equivalent/acre (0.0084, 0.004, 0.002,
    and 0.001 kg/ha), no unusual growth or inhibition of radicle, roots,
    hypocotyl, coleoptile, cotyledon, or leaf was observed, as compared
    with controls. For all the species tested except radish, the percent
    emergence NOEL in the soil was higher than the percent germination
    NOEL in the petri dishes. The plant system is not likely to have
    direct exposure to the test material in a soil substrate (Bakus,
    1993b).

    The effect of the dimethylamine salt on vegetative vigour was studied
    at concentrations of 0.96, 0.48, 0.24, 0.12, 0.06, 0.03, and 0.015 lb
    acid equivalent/acre 1.08, 0.54, 0.27, 0.13, 0.07, 0.03, and 0.0017
    kg/ha). Onion was the most sensitive species, with an EC50 of 0.142
    lb/acre (0.16 kg/ha). The NOEL for sorgham was 0.06 (0.07), and that
    for oats, the least sensitive monocot, was > 0.96 lb acid
    equivalent/acre (> 1.08 kg/ha). The most sensitive dicot was
    mustard, with an EC50 of 0.061 lb acid equivalent/acre (0.07 kg/ha).
    Cucumber was the least sensitive dicot, with an EC50 of 0.96 lb acid
    equivalent/acre (1.08 kg/ha) (Bakus & Crosby, 1992a). When oats were
    treated at 3.8 and 1.9 lb acid equivalent/acre (4.3 and 2.1 kg/ha),
    the symptoms were slight, with a projected NOEL of 1.9 lb acid
    equivalent/acre (2.1 kg/ha) (Bakus, 1993c).

    The effect of 2,4-D dimethylamine salt on seed germination and
    seedling emergence studies was evaluated in petri dishes and in field
    soil. The doses evaluated were 0.96, 0.48, 0.24, 0.12, 0.06, 0.03, and
    0.015 lb acid equivalent/acre (1.08, 0.54, 0.27, 0.13, 0.07, 0.03, and
    0.017 kg/ha) in the soil phase and 0.0075, 0.004, 0.002, 0.001, and
    0.0005 lb acid equivalent/acre (0.0084, 0.004, 0.002, 0.001, and
    0.0006 kg/ha) in the petri dishes. In the petri dish studies, the
    NOEL, EC25, and EC50 values for corn, cucumber, oats, onion, sorghum,
    and soya bean were > 0.96 lb acid equivalent/acre (> 1.08
    kg/ha). A dose-response relationship was seen for tomato, with
    calculated NOEL, EC25 and EC50 values of 0.24, 0.26, and 0.56 lb/acre
    (0.27, 0.29, and 0.63 kg/ha), respectively. No effect was seen on the

    seedling emergence phase of corn and oats at 0.96 lb acid
    equivalent/acre (1.08 kg/ha). The remaining eight species showed some
    stunting, but only cucumber showed symptoms at the lowest rate.
    Mustard, onion, and radish showed significant effects at emergence; no
    significant effect of treatment was seen on buckwheat, corn, cucumber,
    oats, sorghum, soya bean, or tomato. The most sensitive species was
    mustard; radish and onion were less sensitive, with an NOEL of 0.48 lb
    acid equivalent/acre (0.54 kg/ha). All species except tomato that
    showed no effect in the soil bioassay also showed no effect in the
    petri dish assay. The fresh weight of emerging seedlings in soil
    differed from the percent seedling emergence, the NOEL, EC25 and EC50
    values for inhibition of fresh weight of buckwheat, cucumber, mustard,
    radish, and sorghum being lower than the percent emergence. This
    indicates that 2,4-D dimethylamine salt can inhibit the growth of
    seedlings that emerge from treated soil (Bakus & Crosby, 1992b). In
    nine of the same plant species (no buckwheat), concentrations of 3.8,
    1.9, 0.96, and 0.48 lb acid equivalent/acre (4.3, 2.1, 1.08, and 0.54
    kg/ha) were evaluated in both the petri dish assay and seeds planted
    in field soil. The NOEL for corn and soya bean was 3.8 lb acid
    equivalent/acre (4.3 kg/ha), but that for cucumber and oats was 1.9
    (2.1) and that for onions and sorghum was < 1.9 lb acid
    equivalent/acre (< 2.1 kg/ha). The NOEL for mustard and radish was
    reported to be > 0.06 but < 0.48 lb acid equivalent/acre (> 0.07
    but < 0.54 kg/ha). The NOEL for tomato was > 0.96 but < 1.9 lb acid
    equivalent/acre (> 1.08 but < 2.1 kg/ha) (Bakus, 1993d).

    2,4-D 2-ethylhexyl ester was tested at concentrations of 0.96, 0.48,
    0.24, 0.12, 0.06, 0.03, 0.015, and 0.0075 lb acid equivalent/acre
    (1.08, 0.54, 0.27, 0.13, 0.07, 0.03, 0.017, and 0.0084 kg/ha). Onion
    was the most sensitive species, but the data were variable, with an
    NOEL of 0.24 lb acid equivalent/acre (0.27 kg/ha). Corn and oats were
    not affected. The most sensitive dicot was tomato. Cucumber was the
    least sensitive species, with NOEL, EC25, and EC50 values of 0.015,
    0.192, and 0.78 lb acid equivalent/acre (0.017, 0.22, and 0.87 kg/ha),
    respectively (Bakus & Crosby, 1992c). The NOEL in a further study was
    3.8 acid equivalent/acre (4.3 kg/ha) for corn and > 0.96 but < 1.9
    lb acid equivalent/acre (> 1.08 but  < 2.1 kg/ha) for oats (Bakus,
    1993e).

    In studies of the effect of 2,4-D 2-ethylhexyl ester on seed
    germination and seedling emergence in petri dishes and in field soil,
    concentrations of 0.96, 0.48, 0.24, 0.12, 0.06, 0.03, 0.015, 0.0075,
    and 0.004 lb acid equivalent/acre (1.08, 0.54, 0.27, 0.13, 0.07, 0.03,
    0.017, 0.0084, and 0.0045 kg/ha) were evaluated, with additional rates
    of 0.002 and 0.001 lb acid equivalent/acre (0.002 and 0.004 kg/ha) in
    the petri dishes. None of the species examined was very sensitive to
    the ester during germination, and no effects were noted on buckwheat,
    corn, oats, sorghum, soya bean, or tomato in the emergence phase. When
    mustard, onion, radish, and tomato were tested at the lower rates in
    petri dishes, only mustard was more sensitive in the soil bioassay.
    The most sensitive monocots were onion and sorghum, and radish was the
    most sensitive dicot. The percent emergence of cucumber, mustard,
    radish, and tomato was affected (Bakus & Crosby, 1992d). In a similar

    study, concentrations of 3.8, 1.9, 0.96, 0.48, and 0.24 lb acid
    equivalent/acre (4.3, 2.1, 1.08, 0.54, and 0.27 kg/ha) of the ethyl
    ester were evaluated. The NOEL was 3.8 lb acid equivalent/acre (4.3
    kg/ha) for corn, cucumber, oats, onion, sorghum, soya bean, and tomato
    in petri-dish assays. In the emergence bioassay, the NOEL value for
    onion was 0.24 lb acid equivalent/acre (0.27 kg/ha) (Bakus, 1993f). In
    another evaluation of cucumber and onion in the seedling emergence
    bioassay at doses of 0.96, 0.48, 0.24, 0.12, 0.06, 0.03, 0.015,
    0.0075, 0.00375, 0.001875, and 0.0009375 lb acid equivalent/acre
    (1.08, 0.54, 0.27, 0.13, 0.07, 0.03, 0.017, 0.0084, 0.0042, 0.0021,
    and 0.0011 kg/ha), onions were the most sensitive species, with an
    NOEL of 0.24 lb acid equivalent/acre (0.27 kg/ha); the EC25 and EC50
    values were 0.445 and > 0.96 lb acid equivalent/acre (0.5 and > 1.08
    kg/ha), respectively. The NOEL, EC25, and EC50 values for cucumber
    were > 0.96 lb acid equivalent/acre (> 1.08 kg/ha) (Bakus, 1995).

    The effects of 2,4-D diethanolamine salt on vegetative vigour were
    studied at concentrations of 1.5, 0.75, 0.375, 0.1875, 0.09, 0.045,
    0.0225, 0.01, 0.005, and 0.0025 lb acid equivalent/acre (1.7, 0.84,
    0.42, 0.21, 0.1, 0.05, 0.03, 0.01, 0.006, and 0.0028 kg/ha). At the
    two highest rates, only sorghum appeared to be unaffected four days
    after treatment; all of the other species showed stunting, distortion,
    or both. At the two lowest rates, effects were seen only in tomato
    seven days after treatment, the uppermost leaves being distorted.
    Tomato, cucumber, buckwheat, and radish were the most sensitive dicot
    species: the statistically generated NOEL values for tomato and
    cucumber were < 0.0025 lb acid equivalent/acre (< 0.0028 kg/ha),
    the lowest dose tested. The EC25 and the EC50 values were 0.00462 and
    0.1545 lb acid equivalent/acre (0.005 and 0.17 kg/ha for tomato and
    0.00801 and 0.07971 lb acid equivalent/acre (0.009 and 0.089 kg/ha)
    for cucumber, respectively. Onion was the most sensitive monocot
    species tested, with a statistically generated NOEL of 0.0225 lb acid
    equivalent/acre (0.025 kg/ha) and EC25 and the EC50 values of 0.03668
    and 0.12868 lb acid equivalent/acre (0.04 and 0.14 kg/ha). Oats were
    the least sensitive species, with NOEL, EC25, and EC50 all > 1.5
    lb acid equivalent/acre (> 1.7 kg/ha) (Bakus, 1992c).

    The effect of 2,4-D diethanolamine salt on seed germination and
    seedling emergence was studied in petri dishes and field soil at
    concentrations of 1.5, 0.75, 0.375, 0.1875, 0.09, and 0.045 lb acid
    equivalent/acre (1.7, 0.84, 0.42, 0.21, 0.1, and 0.05 kg/ha). In the
    petri dishes, the least sensitive species was cucumber, with an NOEL
    of > 1.5 lb acid equivalent/acre (> 1.7 kg/ha). Buckwheat, corn,
    onion, and radish had an NOEL value of 0.75 lb acid equivalent/acre
    (0.84 kg/ha). Emergence after 14 days in the soil bioassay was lowest
    for onion, being, 30% of that in controls. Buckwheat, corn, oats, and
    soya beans had NOEL, EC25, and EC50 values > 1.5 lb acid
    equivalent/acre (>. 1.7 kg/ha). Mustard and radish were fairly
    sensitive, but onion was the most sensitive, with NOEL, EC25, and
    EC50 values < 0.045 lb acid equivalent/acre (< 0.05 kg/ha). Corn
    was the least sensitive species in fresh weight determinations, with
    an NOEL of 0.75 lb acid equivalent/acre (0.84 kg/ha). Sorghum was
    highly sensitive to the test material, with an NOEL and EC25 value

    < 0.045 lb acid equivalent/acre (< 0.05 kg/ha). The NOEL values
    in the petri dish assays showed that the diethanolamine salt had
    little effect on the germination of seeds, except for tomato and soya
    bean. The percent results for emergence in the soil bioassay generally
    correlate with the results for germination, with two exceptions: the
    percent emergence of tomato and soya bean was higher in this phase of
    the study than the percent germination in the petri dish test (Bakus,
    1992d).

    The effect of 2,4-D diethanolamine salt on seedling emergence and
    early seedling growth of onion was tested at concentrations of 1.5,
    0.75, 0.375, 0.1875, 0.09, 0.045, 0.0225, 0.011, 0.0056, and 0.0028 lb
    acid equivalent/acre (1.7, 0.84, 0.42, 0.21, 0.1, 0.05, 0.03, 0.01,
    0.006, and 0.003 kg/ha). Plant dry weight at the end of the test, 32
    days after treatment, was the most sensitive indicator of effects. The
    lowest values 32 days after planting were 0.1875 lb dry weight of acid
    equivalent/acre (0.21 kg/ha) for the no-observed-effect concentration
    (NOEC), 0.296 lb (0.33 kg/ha) dry weight for the EC25 value, and
    0.713 lb (0.8 kg/ha) dry weight for the EC50 value (Crosby, 1996).

    The effects of 2,4-D isopropyl ester on vegetative vigour, seed
    germination, and seedling emergence were studied in 10 plant species
    (cabbage, corn, cucumber, lettuce, oat, onion, perennial ryegrass,
    soya bean, tomato, turnip) at several concentrations. The measured
    concentrations varied from a high of 0.12 lb ai/acre (0.13 kg/ha) to a
    low of 0.0000073 lb ai/acre (0.0000081 kg/ha) (nominal rate for
    turnip). The vegetative vigor of lettuce and cabbage was most heavily
    affected, with NOEC values of 0.0073 and 0.015 lb ai/acre (0.0082 and
    0.017 kg/ha), respectively. Corn, cucumber, oat, onion, perennial
    ryegrass, and soya bean were the least affected, with NOEC, EC25, and
    EC50 values > 0.12 lb ai/acre (> 0.13 kg/ha). Seed germination
    was most affected for lettuce, with NOEC, EC25, and EC50 values of
    0.00050, 0.0022, and 0.0065lb ai/acre (0.00056, 0.0025, and 0.0073
    kg/ha), respectively. Soya bean, oat, perennial ryegrass, and corn
    were the least affected, with NOEC, EC25, and EC50 values of >
    0.11-0.12 lb ai/acre (> 0.12-0.13 kg/ha). Lettuce seedling
    emergence was the most heavily affected, with NOEC, EC25, and EC50
    values of 0.00056, 0.0011, and 0.0024 lb ai/acre (0.00063, 0.0012, and
    0.0027 kg/ha); the emergence of cabbage, turnip, onion, and soya bean
    seedlings was also affected, with NOEC values ranging from 0.0017
    (0.0019) for cabbage to 0.0070 lb ai/acre (0.008 kg/ha) for turnip.
    Corn, oat, and perennial ryegrass were the least affected, with NOEC,
    EC25, and EC50 values of > 0.11 lb ai/acre (> 0.12 kg/ha) (Hoberg,
    1996).

    An extensive review of the chemical and environmental factors that
    affect the foliar absorption and translocation of 2,4-D is available
    (Richardson, 1977), and another summarizes more than 10 studies of the
    processes, correlations, and models of the mechanisms of uptake of
    organic chemicals, including 2,4-D, by citrus trees and barley, soya
    beans, and vegetables from soil and the atmosphere (Paterson & Mackay,
    1990). A steady-state model for equilibrium partitioning of organic
    chemicals, including 2,4-D, in multi-compartment plants has been

    described, and a dynamic model of the time course of chemical uptake
    and distribution was proposed (Paterson et al., 1991). This was
    expanded into a three-compartment (root, stem, and foliage) mass
    balance model of plants, in which the uptake of organic chemicals,
    including 2,4-D, from soil and the atmosphere was quantified. The
    results for 2,4-D show that an appreciable fraction of the chemical in
    soil is transpired into plant foliage within 72 h, and the compartment
    response times are short. Most of the chemical reaches the foliage
    (Paterson et al., 1994).

    A series of experiments was performed on the accumulation, permeation,
    and desorption of [2-14C]-2,4-D in and across adaxial cuticles on the
    leaves of rubber plants, bitter oranges, tomatoes, and green peppers.
    Accumulation of radiolabel from the cuticles of the four species was
    predicted by determining the partition coefficients for the plant
    cuticle:water system. The agreement between the measured partition
    coefficients ( Kexp) and the coefficients ( Kcal) predicted from log
    octanol:water partition coefficient ( Ko:w) were good, with log
     Kexp = 2.606 and log  Kcal = 2.478 (Kerler & Schönherr, 1988a). A
    study was conducted to determine whether the permeation of [2-14C]-
    2,4-D into astomatous circular membranes of  Citrus auranthium L.
    leaves can be predicted from the  Ko:w. Various equations were used
    for comparisons with measured values. Lipid solubility was the most
    important determinant of the permeation of chemicals from leaf
    surfaces. The experimental permeability ( Pexp) in the [2-14C]-2,4-D
    system was 2.8, which agreed with the calculated permeability ( Pcal)
    of 2.7 when the partition coefficient and the molar volume of the
    solute were considered (Kerler & Schönherr, 1988b). The desorption of
    [2-14C]-2,4-D from cuticular and polymer matrix membranes isolated
    from four plant species ( Citrus, Ficus, Lycopersicon, and 
     Capsicum) was studied by preloading the cuticles and membranes with
    radiolabelled 2,4-D by sorption from solutions and then subjecting
    them to simultaneous bilateral desorption. Only 2-3% of the 2,4-D
    initially contained in the cuticles was desorbed from the outer
    surface, while 86-92% was desorbed from the inner surfaces within 6 h.
    The initial rates of desorption from the inner surfaces were 50-80
    times greater than those from the outer surfaces. The authors
    maintained that asymmetrical desorption is due mainly to the presence
    of apolar and crystalline soluble lipids (waxes) on the surface and in
    the outer layers of the cuticles, which drastically decrease the
    mobility of 2,4-D in the outer layers of the leaf. In fruit cuticles,
    extensive cutinization of the anticlinal and periclinal walls
    increases the inner surface area and thus contributes significantly to
    asymmetry. The fate of chemicals sorbed in cuticles depends on the
    presence or absence of a sink (Schönherr & Riederer, 1988).

    The rate of uptake of 14C-2,4-D in aqueous solution was studied in
    needles from five conifer species. The uptake was biphasic in all
    species, with a rapid first phase, which was complete within about 30
    min, and a slower, constant second phase lasting 30-360 min. The rates
    of uptake were proportional to specific surface areas between the
    solutions and the needles and the partition coefficients. The mean

    permeance,  P, for 2,4-D was 3.07 × 10-11 (Schreiber & Schönherr,
    1992).

    An investigation was carried out to determine the metabolites formed
    when soya bean cotyledon callus cultures were grown with
    [1-14C]-2,4-D as an auxin. ß-Glucosidase treatment of the
    water-soluble fractions from the tissue yielded eight aglycones after
    eight days. The metabolite 4-hydroxy-2,5-dichlorophenoxyacetic acid
    was the most abundant aglycone produced during the 32-day growth
    period, 4-hydroxy-2,3-dichlorophenoxyacetic acid being a minor
    metabolite. Seven ether-soluble components were detected; 2,4-glutamic
    acid was detected in large amounts after 24 h, while 2,4-D aspartic
    acid was the most abundant metabolite after a longer period. It was
    concluded that 2,4-D amino-acid conjugates were actively metabolized
    by the tissue to free 2,4-D and water-soluble metabolites (Feung et
    al., 1972). Similar metabolic patterns were noted in callus tissue
    from soya bean cultures and several other plants, with the addition of
    several new amino-acid conjugates (Feung et al., 1973, 1975), and the
    presence of the amino-acid conjugates was confirmed in a similar study
    (Davidonis et al., 1980).

    An 11-fold increase in uptake of 2,4-D by the roots of barley plants
    was seen as the pH of the nutrient solution dropped from 6.5 to 4.0.
    At pH 4, the uptake of 2,4-D seemed to be influenced by metabolism
    (Shone & Wood, 1974). A similar association with pH was seen in
    excised wheat roots (Zsoldos & Haunold, 1979). The uptake of
    14C-2,4-D in solution into barley roots and subsequent translocation
    into shoots increased as the pH of the solution decreased from 4.0 to
    8.0 (Briggs et al., 1987). In a study of the absorption,
    translocation, and metabolism of 14C-2,4-D in hemp dogbane seedlings
    grown in nutrient solution, the plants were removed from the solution
    at various intervals up to 12 days after treatment and sampled for
    analysis. After 12 days, 34-55% of the radiolabel was found in 2,4-D,
    and negligible amounts were lost as volatile compounds. A temperature
    of 30°C instead of 25°C did not affect translocation of 2,4-D (Schultz
    & Burnside, 1980).

    Experiments were performed to determine whether the absorption of
    14C-2,4-D from culture solutions by excised barley ( Hordeum 
     vulgate L.) roots was passive or active. Absorption was followed at
    0.5°C and at 21°C, over 30 min under anaerobic conditions and in the
    presence of metabolic inhibitors. 2,4-D was concentrated within the
    roots to several times its external concentration, indicating that
    2,4-D is taken up by roots by an adsorption mechanism and that energy
    is required to maintain the integrity of the adsorbing surfaces of the
    cell (Donaldson et al., 1973). Uptake of 14C-2,4-D was similar in
    heterotrophic cell suspensions of soya bean ( Glycine max. L.) and
    four perennial  Glycine accessions, but the metabolism differed
    considerably: soya bean metabolized only 7% of the absorbed radiolabel
    at concentrations of 2 and 10 µmol/L, while the four perennials
    metabolized at least 79% at 10 µmol/L and 64% at 50 µmol/L of 2,4-D.
    The main metabolite identified by HPLC was the glycoside conjugate of
    4-hydroxy-2,5-dichlorophenoxyacetic acid (White et al., 1990).

    In a study of the effects of spray application parameters on foliar
    uptake and translocation of the triethanolamine salt of 2,4-D in the
    second leaf of  Vicia faba after 24 h, the 2,4-D salt was applied to
    provide doses of 4-420 g acid equivalent/ha. The quantity of the
    triethanolamine salt taken up increased with increasing dose, with an
    average uptake of 16 ± 5.5% and 70 ± 8.0% being translocated (Stevens
    & Bukovac, 1987). The absorption, translocation, and metabolism of
    14C-2,4-D was studied after application alone and with 14C-picloram
    to leafy spurge in a greenhouse. Leafy spurge absorbed 34% of the
    14C-2,4-D alone or with 14C-picloram. Eight minor metabolites were
    observed, but only two were present at > 0.1% of the total radiolabel
    recovered, and no attempt was made to identify them (Lym & Moxness,
    1989). In the presence of 0.01 mmol/L 2,4-D, the uptake of potassium
    by maize was reduced, and the effect increased at lower pH (Haunold &
    Zsoldos, 1984).

    The persistence of 2,4-D, as a formulation of mixed amine salts
    containing 500 g acid equivalent/L formulation, as 400 g/L 2,4-D butyl
    ester, and in other formulations, was examined after spraying at rates
    of 1.1-112 g/ha on soya beans, tomatoes, and turnips in a growth
    chamber for up to 35 days. When 2,4-D was applied to tomatoes at a
    simulated drift level of 11.2 g/ha, the residues declined from 0.51 to
    0.05 µg/g within 21 days. Similar results were seen for soya beans
    when 2,4-D was applied at a rate of 0.05 g/ha, but at the highest
    application rate (50 g/ha) the residue level was 0.01 µg/g after 35
    days (Sirons et al., 1982). In similar experiments on  Silene 
     vulgaris with [2-14C] -2,4-D, 65% of the dose, applied by
    microsyringe to the youngest fully expanded leaf as eight to 10
    droplets on either side of the midvein, was absorbed within 72h. More
    than 30% of the absorbed radiolabel had translocated from the treated
    leaf within 24 h after treatment. Metabolism did not appear to be an
    important mechanism in conferring tolerance to 2,4-D (Wall et al.,
    1991).

    The uptake and phytotoxicity of vapours of 2,4-D butyl ester labelled
    at the 2 position and in the ring, and also as a formulated product,
    was studied in tomato, lettuce, and barley leaves. The relationship
    between uptake, measured as the amount of radiolabel in the plant, and
    vapour concentration was linear and independent of the duration of
    exposure for both species. Twenty-four hours after exposure, the
    leaves of tomatoes contained 63-93% of the total 2,4-D. The results
    indicate that about 30% of the dose of 2,4-D butyl ester sprayed on
    barley leaves would evaporate, while 70% would remain in the plant
    (van Rensburg & Breeze, 1990; Breeze, 1990; Breeze et al., 1992).

    Formulated 2,4-D ester markedly increased the penetration of labelled
    difenzoquat into wild oat, while an amine formulation had no effect
    (Sharma et al., 1976). A gas chromatographic method was developed for
    analysis of 2,4-D in triticale over a growing season at two sites.
    2,4-D dimethylamine was applied at 0.56 kg/ha after emergence, and
    2,4-D was derivatized to its methyl ester. On the day after
    application, residues on the order of 30 mg/kg were seen, which

    decreased to undetectable levels in mature straw and seed (Cessna,
    1990).

    Leaf washings were used to determine the relative rate of uptake of
    2,4-D dimethylamine salt from wheat in the field at a post-emergence
    application rate of 487 g acid equivalent/ha (119 g acid equivalent/ha
    as dicamba and 375 g acid equivalent/ha as 2,4-D) at the four- to
    five-leaf growth stage. Samples for residue analysis were taken on the
    day of application and two and seven days after. Leaf washings on the
    day of application indicated that the canopy had intercepted 51% of
    the application, while 65% was taken up by the crop (Cessna, 1993).

    Simultaneous application of 2,4-D sodium salt at 0.33 kg ai/ha with
    asulam had no significant effect on the absorption, translocation, and
    biochemical action of asulam on bracken fern in the field or in the
    laboratory with respect to frond density or the reduction of folate
    levels, although there was noted antagonism with respect to protein
    synthesis (Hinshalwood & Kirkwood, 1988).

    Incubation of cultures of  Daucus carota or  Lactuca sativa cells
    with 2,4-D showed that the toxicity of methyl mercury is partly
    hormone-mediated and light-sensitive (Czuba, 1987, 1991).

    The properties of leaf surfaces and their interactions with spray
    droplets affect the foliar absorption and redistribution of 2,4-D.
    Eleven plant species were treated with 14C-2,4-D in aqueous solutions
    with and without surfactant. Uptake of 2,4-D did not correlate with
    the presence of specialized leaf surface structures, cuticular
    morphology, or distribution within dried deposits. Regression analysis
    indicated that the epicuticular wax and cuticular membranes were the
    major sinks for 2,4-D. Uptake of 2,4-D by apple, field bean, and maize
    was significantly reduced in the presence of surfactant (Stevens &
    Baker, 1987). In another study of the effect of physicochemical
    properties and the role of surfactant on the uptake into and local
    translocation within leaves sprayed with 14C-2,4-D, its movement into
    rape and strawberry leaves was reduced with the addition of
    surfactant; however, uptake was markedly increased in rape but little
    changed in strawberry leaves. It was speculated that a higher
    proportion of the chemical remains associated with the surfactant in
    the leaf cuticle 24 h after application and would not be available for
    movement. The surfactant may also alter the distribution of 2,4-D
    between leaf cells, phloem tissue, and transpiration stream (Stevens
    et al., 1988).

    Seven days after application of 14C-2,4-D to three-week-old seedling
    peas, alfalfa, and grapes as an aqueous solution or in treated soil,
    significant amounts of 2,4-D had been absorbed into alfalfa (63.6%),
    grapes (69.5%), and peas (56.7%) from the aqueous solutions, but the
    comparable rates of uptake from treated soil were very much lower,
    being 3.1% for alfalfa, 1.4% for grapes, and 2.9% for peas. 2,4-D was
    absorbed at a higher rate by grapes grown in a greenhouse (61.4%) than
    in the field (45.4%) (Al-Khatib et al., 1992).

    In a study of the effects of different adjuvants on the foliar uptake
    of 2,4-D sodium salt in wild oat and field bean, 14C-2,4-D was
    applied as approximately 0.2-µl droplets containing about 0.5 g/L ai
    with adjuvants at concentrations of 0.05-5.0 g/L. Concentrations of
    surfactants > 0.05 g/L were necessary to increase the uptake of 2,4-D
    into either plant substantially (Holloway & Edgerton, 1992).

    The leaves of three-year old aspen  (Populus tremens) in the
    greenhouse were treated with the butoxyethanol ester of 2,4-D at 0.5
    kg acid equivalent/L, a very high rate for this application, and the
    residues of 2,4-D were measured for one year. The average residue one
    day after treatment was 2300 mg/kg fresh weight but had fallen to 1300
    mg/kg after 37 days; the average residue after one year was 870 mg/kg
    (Eliasson, 1973). The 2,4-D amine salt and picloram potassium salt
    were applied to the base of aspen saplings with a roller applicator as
    a 7.3:1 w/w mixture (acid equivalent) at concentrations of 0.72, 2.14,
    6.42, or 19.25 g/L. Measurements were made 39 and 84 days after
    treatment. About 86% of the 2,4-D and picloram residues in the average
    poplar sapling were located in the leaves, with 4.5% in the twigs and
    9.1% in the stems (Cessna et al., 1989).

    The effect of simulated rainfall on the phytotoxicity of foliar
    applications of 0.2-0.6 kg/ha of the alkanolamine salt or the
    butyoxyethanol ester of 2,4-D, depending upon the plant being treated,
    was studied in the greenhouse. Simulated rainfall at 1, 5, 10, and 15
    mm within 1 min of 2,4-D treatment reduced the phytotoxicity of the
    alkanolamine salt of 2,4-D to a much greater extent than that of the
    butoxyethanol ester. The effects ranged from complete elimination of
    the phytotoxicity of the alkanolamine salt to soya beans to no
    reduction in the phytotoxicity of the butoxyethanol ester to common
    lambsquarters (Behrens & Elakkad, 1981).

    In a study of the effect of 2,4-D on the cell number, fresh weight,
    dry weight, and stored starch content of three species of
    heterotrophic algae  in vitro, increased concentrations of 2,4-D
    resulted in decreased numbers and decreased starch content of
     Polytoma uvella and  Polytomella papillata cells, but 
     Prototheca chlorelloides was less sensitive (Pelekis et al., 1987).

    Application of 2,4-D dimethylamine salt and 2,4-D butoxyethyl ester to
    man-made freshwater ponds did not affect the numbers of moulds,
    yeasts, or total fungi of  Myriophyllum spicatum in the water column.
    The mean differences between the control and treated ponds were
    erratic and variable (Sherry, 1994). A formulated product of 2,4-D
    used in silvicide practices significantly reduced the radial growth of
    each of three species of ectomycorrhizal fungus that infect forest
    trees in Canada at concentrations > 1000 ppm, and growth was
    completely inhibited at concentrations < 5000 ppm (Estok et al.,
    1989).

    2.  ENVIRONMENTAL LEVELS

    2.1  Air

    2,4-D acid has extremely low volatility, with a vapour pressure
    ranging between 1.05 × 10-2 mm Hg (Grover & Kerr, 1978) and 1.4 ×
    10-7 mm Hg (Chakrabarti & Gennrich, 1987) and with a Henry's law
    constant of 1.3 × l0-10 atm.m3/mol at 25°C. Therefore, the likelihood
    of 2,4-D occurring in air is remote.

    In a study of the dissipation of the isooctyl ester of 2,4-D and its
    acid metabolite in air, wheat, and soil components for up to 35 days
    after application in Canada, the cumulative loss of the isooctyl ester
    over the first five days was 20.8% of the amount applied, apparently
    by volatilization. Measurements of airborne drift after ground
    application demonstrated that only 3-8% of the applied herbicide
    drifts as spray droplets when preparations with low volatility are
    applied as large droplets; however, ultra-low-volume aerial
    applications or use of more volatile esters may result in as much as
    25-30% of the 2,4-D drifting off target. Drift can be reduced by
    accurately following manufacturers' directions under proper
    environmental conditions (Grover et al., 1985).

    The levels of applied liquid and granular 2,4-D taken up by home
    gardeners and household members who did not apply 2,4-D were monitored
    in air samples inside homes and downwind of the application site. The
    'unprotected' group had the highest exposures, but these were
    consistently associated with spills and other contact. Residues of
    2,4-D were detected in five of 76 air samples. A level of 0.006 
    mg/m3 was found inside a house after use of liquid 2,4-D and 0.01
    mg/m3 in outside air after use of the granular preparation (Harris et
    al.,1992).

    Atmospheric deposition samples were collected weekly from early May to
    early September 1984-87 in a small agricultural watershed near Regina,
    Saskatchewan, Canada. As expected, maximum deposits occurred during
    application, followed by a rapid decline. The amount of 2,4-D
    deposited during the period of investigation ranged from 0.08 to 0.28%
    of the amount applied (Waite et al. (1995).

    2.2  Water

    In a study reported as an abstract, 2,4-D was applied at 2.24 or 4.48
    kg/ha to a watershed with an average declivity of 40% in North
    Carolina, USA. The largest proportion of the applied dose that moved
    from plots in surface runoff over eight months was 0.049%, and the
    maximum possible losses of herbicide through the flume of the
    watershed during the 160-day period was < 0.12% (Sheets et al.,
    1972). 2,4-D was not detected in water associated with rice production
    in Arkansas, USA, 28 days after application of 1.1 kg/ha (Johnson et
    al., 1995). In a study of the soil persistence and lateral movement of
    2,4-D after application as a stem-foliage spray for brush control on
    two power-line rights-of-way, runoff water was analysed for up to 48

    weeks after treatment. By week 4, residues of 0.3-1.9 mg/m3 2,4-D
    were found in the treated areas, but no residues were found in water
    8-11 weeks after spraying. Little lateral movement was detected (Meru
    et al., 1990).

    A review of the movement, persistence, and fate of phenoxy herbicides
    in forests included the results of analyses in streams that drain
    forest areas. It was suggested that the concentration of 2,4-D in
    streams would be low after application in forest and rangeland,
    consistent with the short persistence of 2,4-D (Norris, 1981). In a
    study of the ability of several rivers in Western Australia to degrade
    2,4-D, clear seasonal differences in both the concentrations of 2,4-D
    and the degrading capacity of the water were seen, which correlated
    with the amount of agricultural runoff, the sediment content of the
    water, river flow, and the temperature of the water. Rivers receiving
    agricultural runoff degraded 2,4-D faster than those receiving runoff
    principally from forests (Watson, 1977).

    The levels of 2,4-D in well water, watersheds, and streams draining
    watersheds in Ontario, Canada, have been reported (Frank et al., 1979;
    Frank & Sirons, 1980; Frank et al., 1982, 1987), and similar
    evaluations of groundwater, surface water, and spring runoff were
    reported in a watershed in Saskatchewan (Waite et al., 1992a,b).
    Several sources of well-water contamination were investigated,
    including spills from mixing, loading, and application and from
    back-siphoning of spray solutions in 1979-84. The highest level of
    2,4-D found after back-siphoning of spray solutions was 29 µg/L, which
    decreased to < 0.1 µg/L after 17 days. 2,4-D was found in 19 of 255
    wells investigated (Frank et al., 1987). The levels of 2,4-D were
    measured in water samples taken from the mouth of the Grand, Saugeen,
    and Thames rivers in Ontario, Canada, in 1981-90. The mean levels
    never exceeded 0.7 µg/L in any given year in 1981-85 in either of the
    three rivers. During the period 1986-90, no 2,4-D was found in the
    Thames River, while an annual loading of 181 kg (said to be from an
    unusual discharge during 1988) was found in the Grand River (Frank &
    Logan, 1988; Frank et al., 1991). When 2,4-D was incubated in the
    waters of four rivers in China for 56 days, an average of 76.1%
    remained in the waters after application of the equivalent of 100 mg/L
    of 14C-2,4-D, while 83.5% remained after application of 100 µg/L
    (Wang et al., 1994).

    In a small watershed in Saskatchewan, Canada, in 1985-87, 2,4-D was
    found in pond water at a maximum level of 0.51 ppb, with a mean of
    0.08 ppb over the three-year sampling period. The mean levels of 2,4-D
    in spring runoff were 0.17 ppb during 1985 and 0.15 ppb in 1987 (Waite
    et al., 1992b).

    Water and bottom sediment were collected from eight stations in the
    wetlands of the delta of the Axios, Loudias, and Aliakmon rivers in
    Greece during 1992 and 1993. Samples were reported to have been
    analysed within two to three days after sampling after storage at 4°C
    before extraction. The levels of 2,4-D were 0.02-0.46 µg/L (Albanis et
    al., 1994). In an extension of this work to include other products

    from 1985-89, 2,4-D was found in about 1% of the samples of both
    surface (modal range, < 0.2 - 0.3 mg/L; maximum, 2.1 mg/L) and
    groundwater (0.11 - 0.2 mg/L) (Croll, 1991). Extensive analyses of
    groundwater samples in the European Union showed that the levels
    rarely exceeded 0.1 mg/L. The rare levels > 0.1 mg/L are likely to be
    due to point sources such as spills or direct drainage from surface
    water, rather than leaching through the soil profile. Further, point
    sources are major contributors of residues due to poor handling
    (mixing, loading, and clean-up) and poor application practices;
    non-agricultural uses of 2,4-D are primarily responsible for residues
    > 0.1 mg/L. In England, the National Rivers Authority has established
    a maximum allowable concentration of 200 mg/L for non-ester forms of
    2,4-D in freshwater and saltwater and an annual average concentration
    of 40 mg/L. For esters of 2,4-D, the maximum allowable concentration
    is 10 mg/L and the annual average concentration 1.0 mg/L. The vast
    majority of the documented level of residues of 2,4-D salts and esters
    are far below these values (Lewis et al., 1996; Isenbeck-Schroter et
    al., 1997).

    The maximum and mean levels of 2,4-D detected in 10 permanent and nine
    semipermanent lakes in Saskatchewan, Canada, after the severe drought
    of 1988 were 0.43 and 0.10 µg/L. 2,4-D was detected in < 10% of the
    samples of sediment analysed. The frequency of detection was
    significantly greater in brackish than in saline lakes (Donald &
    Syrgiannis, 1995).

    In a literature review on losses in runoff waters from agricultural
    fields, the loss of 2,4-D from runoff from corn fields in Georgia,
    USA, was 0.007-1.0% (Wauchope, 1978). The various processes for
    removing 2,4-D from wastewaters have been reviewed (Cloutier, 1983).
    One means that has been investigated is absorption onto peat. The
    factors that affect the results include the concentration of 2,4-D,
    pH, peat particle size, temperature, and the concentration of peat.
    More than 90% of the 2,4-D was removed by peat under optimal
    conditions, and the adsorption of 2,4-D was adequately described by
    the Freundlich isotherm (Cloutier et al., 1985).

    2.3  Soil

    Samples of dykeland (silt loam) in Canada were collected from depths
    of 20-30 cm and analysed for up to 385 days after surface application
    of mixed amines or mixed esters of 2,4-D over 55 weeks. 2,4-D was
    rapidly degraded from the amine formulation within 14-42 days after
    application, and < 5% remained after 70 days. No residues of 2,4-D
    from the amine application were found below 20-30 cm. At the maximum
    recommended application rate (5.6 kg/ha), the concentration of 2,4-D
    peaked at 1.84 mg/kg soil (top 10 cm) after 14 days and fell to 0.04
    mg/kg after 70 days (Stewart & Gaul, 1977).

    The dissipation of 2,4-D after application at 1 or 2 mg/kg of soil at
    15, 40 and 90 cm was determined for up to 41 months in soya beans in
    the field. Degradation of 2,4-D was rapid under aerobic conditions,

    virtually all having dissipated within the first five months (Lavy et
    al., 1973).

    Residue were determined in soil and shallow groundwater after
    long-term application of 2,4-D in southern Alberta, Canada, at rates
    of 104-915g ai/ha to one site between 1976 to 1989 and to another from
    1980 to 1989. Even with this extensive use, no residues of 2,4-D were
    detected in soil sampled in 1991 (Miller et al., 1995). This finding
    is consistent with the low persistence of 2,4-D in soil (e.g. Foster &
    McKercher, 1973; Stewart & Gaul, 1977).

    2.4  Plants

    Studies conducted in North Carolina and California, USA, in which the
    ethylhexyl ester and dimethylamine salt of 2,4-D were applied to grass
    at a rate of 2.24 kg acid equivalent/ha, showed residues of up to 120
    mg equivalent/kg wet weight for the ester and 153 mg equivalent/kg wet
    weight for the salt. The levels declined after seven days to 28 and 60
    mg acid equivalent/kg wet weight for the salt and ester, respectively
    (Barney, 1995c,e; Hatfield, 1995e,f).

    3.  EFFECTS ON ORGANISMS IN THE LABORATORY AND THE FIELD

    3.1  Microorganisms

    The effect of 2,4-D on microorganisms has been evaluated in several
    studies (Torstensson, 1978; Kuwatsuka & Miwa, 1989; Narain Rai, 1992;
    Masson et al., 1993). Generally, the numbers of aerobic bacteria,
    actinomycetes, and fungi in soils were not affected by 2,4-D applied
    at rates corresponding to 25 ppm. The population of 2,4-D-degrading
    microrganisms increased during the observed lag period in most soils
    (Kuwatsuka & Miwa, 1989). After application of the dimethylamine salt
    and the isooctyl ester at 0.95 kg/ha, the populations of fungi,
    bacteria, or actinomycetes were not significantly affected by sampling
    times but were affected by the form of 2,4-D applied: the ester
    reduced bacterial population by 26.3%, the fungal population by 19.5%,
    and and that of actinomycetes by 30%, while the dimethylamine salt
    reduced the populations by only 10.1, 11.4, and 16%, respectively
    (Narain Rai, 1992).

    In studies of the effects of 2,4-D on the activities of invertase and
    amylase and the respiration of various microorganisms in a sandy soil,
    the extent of oxygen consumption increased with the concentration of
    2,4-D (Tu, 1988). In a sandy loam in southwestern Ontario, Canada,
    2,4-D did not affect microbial ammonification of organic nitrogen
    indigenous to soil and was not toxic to various denitrifying
    microorganisms (Tu, 1994).

    3.2  Aquatic organisms

    3.2.1  Plants

    3.2.1.1  Toxicity

    The effect of 2,4-D acid on various aquatic plants was evaluated in
    screening studies conducted according to the guidelines of the US
    Environmental Protection Agency. The concentrations evaluated were
    1.91-2.13 mg/L, which approximated the maximum aquatic application
    rate of 38 lb acid equivalent/acre (42.6 kg/ha). Exposure of
     Skeletonema costatum (Hughes et al., 1994a) to 2.08 mg/L resulted in
    10% stimulation relative to controls, while exposure of  Navicula 
     pelliculosa (Hughes et al., 1994b),  Anabaena flos-aquae (Hughes et
    al., 1994c), and  Lemna gibba (Hughes et al., 1994d) resulted in
    inhibition of 24.3% (2.13 mg/L), 0.488% (2.02 mg/L), and 75% (1.91
    mg/L), respectively. The isopropyl ester at 0.13 mg/L stimulated
     Selenastrum costatum by 11% (Hughes et al., 1995).

    In most of the studies summarized in Table 2, the acute toxicity of
    2,4-D to aquatic plants was determined according to the guidelines of
    the US Environmental Protection Agency for studies of nontarget
    plants. The five-day EC50, EC25, and NOEC values were determined for
    the ethylhexyl, butoxyethyl, and isopropyl esters ( Selenastrum only)
    and the dimethylamine, diethanolamine, and triisopropanolamine salts
    of 2,4-D. The EC50 for the ethylhexyl ester (chemically identical to
    the isooctyl ester) ranged from a low of 0.23 mg/L for  Skeletonema 
     costatum to a high of > 30 mg/L for  Anabaena flos-aquae (Hughes,
    1990a,b), the EC50 for the butoxyethyl ester was 1.66 mg/L for
     Skeletonema costatum to 24.9 mg/L for  Selenastrum capricornutum 
    (Hughes, 1989, 1990c), the EC50 for the dimethylamine salt ranged
    from 5.28 mg/L for  Navicula pelliculosa to 153 mg/L for  Anabaena 
     flos-aquae (Hughes, 1990b,d), the EC50 for the diethanolamine salt
    ranged from 11 mg/L for  Selenastrum capricornutum to > 97 mg/L for
     Navicula pelliculosa (Thompson & Swigert, 1993a,b), and the EC50
    for the triisopropanolamine salt ranged from 82.4 mg/L for a marine
    diatom to 133 mg/L for  Anabaena flow-aquae (Hughes et al., 1994e,f).
    The EC50 values for the higher aquatic plant  Lemna gibba varied
    with the form of 2,4-D, ranging from 0.5 mg/L for the ethylhexyl ester
    to 3.3 mg/L for 2,4-D acid (Hughes 1990e, Hughes et. al., 1997).

    3.2.1.2  Other effects on plants

    Outdoor artificial ponds planted with  Myriophyllum spicatum were
    treated with the butoxyethanol ester of 2,4-D at a rate of 25 kg
    ai/ha.  M. spicatum had completely collapsed five days after
    application. Significant decreases in the level of dissolved oxygen
    and pH and an increased level of dissolved organic carbon was seen
    seven days after treatment (Birmingham et al., 1983). The effect of
    2,4-D was investigated on total chlorophyll production by  Chlorella 
     vulgaris, Chlorococcum hypnosporum, Stigeoclonium tenue, Tribonoma 
    sp.,  Vaucheria geminata, and  Oscillatoria lutea. At concentrations
    of 0.001, 0.01, 0.05, 1, 10, and 100 mg/L, 2,4-D had no effect on


        Table 2. Toxicity of 2,4-D to aquatic plants

                                                                                                                                        

    Organism                        Flow/    Temperature   Active ingredient         Exposure      2,4-D conc.    Reference
                                    Static   (°C)                                    parameter     (mg ai/L)
                                                                                                                                        

    Green algae (Chlorella fusca)   Static                 Acid                      24-h EC50     88.9           Faust et al. (1994)

    Algae (Skeletonema costatum)    Static                 Ethylhexyl ester          5-d EC25      0.10a          Hughes (1990a)b
                                                                                     5-d EC50      0.23
                                                                                     5-d NOE       0.1875

                                                           Butaxyethyl ester         5-d EC25      1.09           Hughes (1990c)b
                                                                                     5-d EC50      1.66
                                                                                     5-d NOEC      0.785

    Marine diatom                   Static                 Dimethylamine salt        5-d EC25      15.79a         Hughes (1990f)b
                                                                                     5-d EC50      36.60
                                                                                     5-d NOEC      96.25

                                    Static                 Diethanolamine salt       5-d EC50      > 95a          Thompson & Swigert 
                                                                                     5-d NOEC      95                  (1993c)

                                    Static                 Triisopropanolamine salt  5-d EC25      60.1a          Hughes et. al. (1994e)b
                                                                                     5-d EC50      82.4
                                                                                     5-d NOEC      50.4

    Algae (Navicula pelliculosa)    Static                 Ethylhexyl ester          5-d EC25      1.9a           Hughes (1990g)b
                                                                                     5-d EC50      4.1
                                                                                     5-d NOEC      3.75

    Freshwater diatom               Static                 Butoxyethyl ester         5-d EC25      0.957          Hughes (1990h)b
                                                                                     5-d EC50      1.86
                                                                                     5-d NOEC      1.76

    Table 2. (continued)

                                                                                                                                        

    Organism                        Flow/    Temperature   Active ingredient         Exposure      2,4-D conc.    Reference
                                    Static   (°C)                                    parameter     (mg ai/L)
                                                                                                                                        

                                                           Dimethylamine salt        5-d EC25      2.21a          Hughes (1990d)b
                                                                                     5-d EC50      5.28
                                                                                     5-d NOEC      1.70

                                    Static                 Diathanolamine salt       5-d EC50      > 97a          Thompson & Swigert 
                                                                                     5-d NOEC      97             (1993b)

                                    Static                 Triisopropanolamine salt  5-d EC25      86.5a          Hughes et al. (1994g)b
                                                                                     5-d EC50      124.0
                                                                                     5-d NOEC      < 36.6

    Algae (Anabaena flos-aquae)     Static                 Ethylhexyl ester          5-d EC25      > 30a          Hughes (1990b)b
                                                                                     5-d EC50      > 30
                                                                                     5-d NOEC      > 30

                                                           Butoxyethyl ester         5-d EC25      5.96           Hughes (1990i)b
                                                                                     5-d EC50      6.37
                                                                                     5-d NOEC      3.14

    Blue-green algae                Static                 Dimethylamine salt        5-d EC25      38.5a          Hughes (1990j)b
                                                                                     5-d EC50      153.0
                                                                                     5-d NOEC      67.86

    Static                                                 Diethanolamine salt       5-d EC50      > 96a          Thompson & Swigert 
                                                                                     5-d NOEC      96             (1993d)

                                    Static                 Triisopropanolamine salt  5-d EC25      77.4a          Hughes et al. (1994h)b
                                                                                     5-d EC50      133
                                                                                     5-d NOEC      < 99.6

    Table 2. (continued)

                                                                                                                                        

    Organism                        Flow/    Temperature   Active ingredient         Exposure      2,4-D conc.    Reference
                                    Static   (°C)                                    parameter     (mg ai/L)
                                                                                                                                        

    Lemna gibba G3                  Static                 Acid                      14-d EC25     1.721          Hughes et al. (1997)b
                                                                                     14-d EC50     3.30
                                                                                     14-d NOEC     2.029

                                                           Ethylhexyl ester          14-d EC25     0.15a          Hughes (1990d)b
                                                                                     14-d EC50     0.50
                                                                                     14-d NOEC     0.1875

                                                           Butoxyethyl ester         14-d EC25     0.169          Hughes (1990e)b
                                                                                     14-d EC50     0.576
                                                                                     14-d NOEC     0.204

    Duckweed                        Static                 Dimethylamine salt        14-d EC25     0.19a          Hughes (19901)b
                                                                                     14-d EC50     0.58
                                                                                     14-d NOEC     0.27

                                    Static                 Diethanolamine salt       14-d LC50     0.60a          Thompson & Swigert 
                                                                                     14-d NOEC     < 0.079       (1993e)

                                    Static                 Triisopropanolamine salt  14-d EC25     0.794a         Hughes et al. (1994f)b
                                                                                     14-d EC50     2.37
                                                                                     14-d NOEC     0.354

    Algae (Selenastrum              Static   22-26         Acid                      5-d EC25      29.0a          Hughes (1990m)b
    capricornutum)                                                                   5-d EC50      33.2
                                                                                     5-d NOEC      26.4

                                                           Acid                      96-h EC50     25.9           St Laurent et al. 
                                                                                     96-h EC50     24.2       (1992)

    Table 2. (continued)

                                                                                                                                        

    Organism                        Flow/    Temperature   Active ingredient         Exposure      2,4-D conc.    Reference
                                    Static   (°C)                                    parameter     (mg ai/L)
                                                                                                                                        

    Green algae                     Static                 Ethylhexyl ester          5-d EC25      > 22.7a        Hughes (1990n)b
                                                                                     5-d EC50      > 22.7
                                                                                     5-d NOEC      15.0

                                                           Butoxyethyl ester         5-d EC25      10.5           Hughes (1989)b
                                                                                     5-d EC50      24.9
                                                                                     5-d NOEC      12.5

                                    Static                 Dimethylamine salt        5-d EC25      25.9a          Hughes (1990o)b
                                                                                     5-d EC50      66.5
                                                                                     5-d NOEC      19.2

                                    Static                 Isopropyl ester           5-d EC50      > 0.13a        Hughes et al. (1995)
                                                                                     (tier I)

                                    Static                 Diethanolamine salt       5-d EC50      11.0a          Thompson & Swigert 
                                                                                     5-d NOEC      0.5            (1993a)

                                    Static                 Triisopropanolamine salt  5-d EC25      66.8a          Hughes (1994)b
                                                                                     5-d EC50      103.0
                                                                                     5-d NOEC      55.4
                                                                                                                                        

    a Reliable data based on measured concentrations
    b Study conducted for compliance with FIFRA registration by the US Environmental Protection Agency

    Table 3. Toxicity of 2,4-D to freshwater invertebrates

                                                                                                                                               

    Organism             Flow/    Temp       Alkalinity  Hardness   pH        Active              Exposure    2,4-D conc.  Reference
                         Static   (°C)                                        ingredient          parameter   (mg ai/L)
                                                                                                                                               

    Oligochaete worm     Flow     20         30          30         7.8       Free acid           48-h LC50   122.2        Bailey & Liu (1980)
    (Lumbriculus                                                                                  96-h LC50   122.2
    variegatus)                                                                                   96-h LC0    86.7

    Water flea (Daphnia  Static   19-21      51          78         8.4       Isooctyl ester      48-h LC50   5.2          Alexander et al. 
    magna)                                                                                                                 (1983a)a
                         Static   20                                7.0-8.2   Acid                48-h LC50   36.4         Alexander et al. 
                                                                                                                           (1983b)a
                         Static   20         77-84       90-108     7.8-8.0   Dimethylamine salt  48-h LC50   184          Alexander et al. 
                                                                                                                           (1983c)a
                         Static   25         85          100        7.6       Acid                48-h LC50   247.2        McCarty & Batchelder
                                                                                                                           (1977)a

                         Flow     19-21      170-175     160-180    4.0-8.5   Acid                21-d NOEL   79b          Ward & Boeri (1991a)a
                                                                                                  21-d LOEL   151
                                                                                                  21-d MATC   109
                                                                                                  21-d EC50   235

                         Flow     18.9-24.5  22-29       65-68      6.8-7.6   Dimethylamine salt  21-d NOEL   27.5b        Ward (1991a)a
                                                                                                  21-d LOEL   59.6
                                                                                                  21-d MATC   40.5
                                                                                                  3-d LC50    130-243

                         Flow     19-21      160-185     180        7.8-8.6   Ethylhexyl ester    21-d NOEL   0.015b       Ward & Boeri (1991b)a
                                                                                                  21-d LOEL   0.027
                                                                                                  21-d MATC   0.020
                                                                                                  21-d EC50   0.13
                                                                                                  2-d LC50    > 0.2

    Table 3. (continued)

                                                                                                                                               

    Organism             Flow/    Temp       Alkalinity  Hardness   pH        Active              Exposure    2,4-D conc.  Reference
                         Static   (°C)                                        ingredient          parameter   (mg ai/L)
                                                                                                                                               

                         Flow     20                                7.6       Acid                24-h EC50   1.124        Lilius et al. (1995)
                         Static   17                     39         7.2       Dimethylamine salt  48-h LC50   > 100        Mayer & Ellersieck 
                                                                                                                           (1986)
                         Static   20                                8.4-8.6   Free acid           96-h LC50   417.8        Presing (1981)
                         Static   20                                8.4-8.6   Sodium salt         96-h LC50   932.1        Presing (1981)
                         Flow                                                 Diethanolamine salt 48-h EC50   > 100b       Graves & Peters 
                                                                                                  21-d NOEC   23.6         (1991a)

                         Flow                                                 Isopropylamine salt 48-h LC50   583          Alexander et al. 
                                                                                                                           (1983d)
                         Flow                                                 Triisopropanolamine 48-h LC50   748          Mayes (1989)

                         Flow                                                 Isopropyl esterc    48-h EC50   2.6b         Drottar & Swigert 
                                                                                                                           (1996a)

                         Flow                                                 Butoxyethyl ester   48-h LC50   7.2          Alexander et al. 
                                                                                                                           (1983e)

    Water flea           Flow     25         81          57.07      8.18      Acid                48-h LC50   236          Oris et al. (1991)
    (Ceriodaphnia dubia) 

    Water flea (Daphnia  Flow     20                                7.6       Acid                24-h EC50   1.47         Lilius et al. (1995)
    pulex)

    Copepod (nauplius    Static   20         31.6        70         6.7       Free acid           96-h LC50   8.72         Robertson (1975)
    larva)

    (Cyclops varnalis)   Static   20         31.6        70         6.7       Alkanolamine        96-h LC50   54.8         Robertson (1975)

    Scud (Gammarus       Static   15                     272        7.4       Dimethylamine salt  24-h LC50   > 100        Mayer & Ellersieck 
    fasciatas)           Static   15                     272        7.4       Dimethylamine salt  96-h LC50   > 100        (1986)

    Table 3. (continued)

                                                                                                                                               

    Organism             Flow/    Temp       Alkalinity  Hardness   pH        Active              Exposure    2,4-D conc.  Reference
                         Static   (°C)                                        ingredient          parameter   (mg ai/L)
                                                                                                                                               

    Freshwater prawn     Static   27                     113.9      7.5       Sodium salt         24-h LC50   2342         Omkar & Shukla (1984)
    (Macrobranchium      Static   27                     113.9      7.5       Sodium salt         48-h LC50   2309
    lamerrei)            Static   27                     113.9      7.5       Sodium salt         72-h LC50   2267
                         Static   27                     113.9      7.5       Sodium salt         96-h LC50   2224

    Freshwater prawn     Static   27                     112.7      7.5       Sodium salt         24-h LC50   2644         Omkar & Shukla (1984)
    (Macrobranchium      Static   27                     112.7      7.5       Sodium salt         48-h LC50   2536
    naso)                Static   27                     112.7      7.5       Sodium salt         72-h LC50   2435
                         Static   27                     112.7      7.5       Sodium salt         96-h LC50   2397

    Freshwater prawn     Static   28                     112.7      7.5       Sodium salt         24-h LC50   2474         Omkar & Shukla (1984)
    (Macrobranchium      Static   28                     112.7      7.5       Sodium salt         48-h LC50   2381
    dayanum)             Static   28                     112.7      7.5       Sodium salt         72-h LC50   2333
                         Static   28                     112.7      7.5       Sodium salt         96-h LC50   2275

    Red swamp crayfish   Static   20                     100        8.4       Alkanolamine salt   96-h LC50   1389         Cheah et al. (1980)
    (immature) 
    (Procambarus clarki)

    Midge (larva)                 15         78-95       55         7.3-7.8   Dimethylamine salt  24-h IC50   1490         Bunting & Robertson 
    (Chaoborus                    15         78-95       55         7.3-7.8   Dimethylamine salt  96-h IC50   890          (1975)
    punctipennis)
                                                                                                                                               

    a Study conducted in accordance with international guidelines and good laboratory practice
    b Reliable data based on measured concentrations
    c Formulated product
    

    chlorophyll production by these algae (Ramirez Torres & O'Flaherty,
    1976).

    A concentration of 10 µg/ml of the sodium salt of 2,4-D (80% ai) in
    liquid culture medium stimulated the growth and nitrogen fixation of
    the heterocystous bloom-forming blue-green alga  Anabaenopsis 
     raciborskii. The alga tolerated up to 800 µg/ml (Das & Singh, 1977).
    Technical-grade 2,4-D increased the growth and nitrogen assimilation
    of  Azolla mexicana and its phycobiont  Anabaena azollae when 2,4-D
    was added at 1 ppm to the nitrate-containing medium, whereas all of
    the plants died within 10 days at 10 ppm 2,4-D (Holst et al., 1982).

    The phytotoxicity of 2,4-D to  Selenastrum capricornutum was compared
    by the microplate and flask bioassay methods. The EC50 values (95%
    confidence interval) found with the two methods were similar, being
    25.9 (23.8-28.3), r2 = 0.60 and 24.2 (23.7-24.7), r2= 0.93;
    respectively (St Laurent et al., 1992). The phytotoxicity of the
    'expected environmental concentration' (2.197 mg/L) of 14C-2,4-D acid
    to 10 kinds of algae was low, and it caused < 50% inhibition of the
    growth of  Lemna (Peterson et al., 1994).

    3.2.2  Invertebrates

    3.2.2.1  Toxicity

    The toxicity of 2,4-D and its salts and esters to freshwater
    invertebrates and marine organisms is summarized in tables 3 and 4.
    Numerous studies have been conducted of the toxicity in freshwater and
    estuarine aquatic invertebrates. Studies conducted according to the
    guidelines of the US Environmental Protection Agency by the 'Industry
    Task Force II on 2,4-D Research Data' are also summarized in the
    tables.

    The 21-day EC50 of 2,4-D in the water flea,  Daphnia magna, in a
    flow-through system ranged from 0.13 mg/E for the isooctyl ester (Ward
    & Boeri,1991b) to 235 mg/L for the acid (Ward & Boeri, 1991 a). No
    EC50 was available for the dimethylamine salt. Comparable 21-day NOEL
    values were found for the 2-ethylhexyl ester (0.015 mg/L; Ward &
    Boeri, 1991b), the dimethylamine salt (27.5 mg/L; Ward, 1991a), and
    the acid (79 mg/L; Ward & Boeri, 1991a).

    The 48-h LC50 ranged from > 100 mg/L for the dimethylamine salt
    (Mayer & Ellersieck, 1986) to 932.1 mg/L for the sodium salt (Presing,
    1981 ). The 48-h LC50 values for the isopropylamine and
    triisopropanolamine salts fall within this range; the 48-h EC50 for
    the isopropyl ester was 2.6 mg/L (Drottar & Swigert, 1996b) and that
    for the butoxyethyl ester was 7.2 mg/L (Alexander et al., 1983e).
    Clearly, the isooctyl ester, which is chemically identical to the
    ethylhexyl ester, is more toxic to invertebrates than the acid or the
    amine salt. The results of the studies of the Industry Task Force II
    on 2,4-D Research are consistent with those in the literature.

    The toxicity of 2,4-D to the freshwater invertebrates  Oligochaete 
    worms,  Ceriodaphnia dubia, Daphnia pulex, copepods, scuds, and other
    species was generally similar to that in  Daphnia magna, i.e. 10->
    100 mg/L, except for the sodium salt. The 24-, 48- and 72-h LC50
    values for the sodium salt of 2,4-D in static systems were 2200-2700
    mg/L for the freshwater prawn  Macrobranchium lamerrei. The
    dimethylamine salt was also relatively non-toxic to midge larvae
     (Chaoborus punctipennis), with values ranging ranging from 890 mg/L
    for the 96-h LC50 to 1490 mg/L for the 24-h LC50 (Omkar & Shukla,
    1984).

    A considerable body of data is available on the toxicity (shell
    deposition for oysters) of 2,4-D in estuarine aquatic invertebrates
    (Table 4). The 96-h EC50 for eastern oysters  (Crassostrea 
     virginica) ranged from > 0.21 mg/L for the 2-ethylhexyl ester (Ward
    & Boeri, 1991f) to 179 mg/L for the triisopropanolamine salt (Dionne,
    1990b), and the values for the acid, diethanolamine salt,
    dimethylamine salt, and isopropylamine salt fall within this range
    (Wade & Overman, 1991; Graves & Peters, 1991c; Ward, 1991c; Ward et
    al., 1993); the EC50 for formulated Esteron 99 containing 66% 2-
    ethylhexyl ester is also within this range (Ward & Boeri, 1991e). The
    48-h EC50 for the dimethylamine salt, > 210-< 320 mg/L, indicates
    considerably lower toxicity (Heitmuller, 1975).

    The esters of 2,4-D are clearly more toxic to invertebrate species
    such as the tidewater silverside  (Menidia beryllina), Atlantic
    silverside  (Menidia menidia), grass shrimp  (Palaemonetes puqio), 
    pink shrimp  (Panaeus duorarum), and Dungeness crab  (Cancer 
     magister) than is the dimethylamine salt or the acid (Table 4). The
    same is true for formulated 2-ethylhexyl ester (Ward & Boeri,
    1991c,h).

    3.2.2.2  Other effects on invertebrates

    Grass shrimp  (Palaemonetes pugio) showed avoidance reactions to
    water containing 2,4-D butoxyethanol ester at 1 or 10 mg/L (Hansen et
    al., 1973). The phototactic behaviour of larval estuarine grass
    shrimps exposed to 49% 2,4-D amine was reduced at all stages
    evaluated. Adult shrimp were not affected by doses up to 5000 mg/L. No
    correlation with dose was seen for egg hatchability (Moyer, 1975).

    The toxicity of the diethanolamine salt of 2,4-D to  Daphnia magna 
    was examined in a life-cycle toxicity test under flow-through
    conditions conducted according to the guidelines of the US
    Enviromnental Protection Agency. The daphnia were less than 24h old at
    the start. The NOEC was 23.6 mg ai/L (Holmes & Peters, 1991). In a
    similar study, the toxicity of the butoxyethyl ester of 2,4-D to
     Daphnia magna was tested under flow-through conditions for 21 days.
    The maximum acceptable toxicant concentration was 0.70-0.29 mg/L, and
    the NOEC was 0.29 mg/L (Gersich et al., 1989). The effect of 2,4-D and
    its dimethylamine salt on the reproduction of  Daphnia magna was
    tested in a study conducted according to the European guidelines.
    Parental survival was not affected at 21.5, 46.2, or 100 mg/L, but

    reproduction, expressed as the number of young per surviving adult,
    was significantly reduced at 100 and 215 mg/L and in the control
    dimethylamine group (Mark & Hantink-de Rooy, 1989).

    3.2.3  Vertebrates

    3.2.3.1  Toxicity

    Studies of the effects of the potassium or sodium salt on early
    life-stages, embryos, larvae, and fish four days after hatching were
    conducted on several species in flow-through systems (Table 5). The
    12-, 24-, 36-, and 48-h LC50 values for the sodium salt in embryonic
    bleak  (Alburnus alburnus) were 12.9-159.4 mg/L (Biro, 1979). The
    LC50 values for the potassium salt of 2,4-D in embryos and four-day-
    old fish of various species in flow-through systems ranged from 4.2
    mg/L for the 27-day LC50 to > 201 mg/L at four days (Birge et al.,
    1979).

    In studies conducted according to the guidelines of the US
    Environmental Protection Agency, 2,4-D acid and ethylhexyl ester had
    no effect on the early life stages, embryo hatch, larval weight, or
    larval length of the fathead minnow  (Pimephales promelas) at
    concentrations of 12.6-102 mg/L for up to 32 days (acid). The 32-day
    NOEC for the acid was 63.4 mg/L (Mayes et al., 1990a,b), comparable to
    the 33-day NOEC for the diethanolamine salt of 29.1 mg/L (Graves &
    Peters, 1991e). The ethylhexyl ester was more toxic, with a 32-day
    NOEC of 0.12 mg/L (Mayes et al., 1990a), and the 96-h LC50 of the
    butoxyethyl ester was 0.93 mg/L (Mayes et al., 1989a).

    Many data are available on the acute toxicity of 2,4-D and its various
    formulations to fish. Rainbow trout  (Oncorhynchus mykiss) and
    bluegills  (Lepomis machrochirus) are the species most often tested
    for the US Environmental Protection Agency, and the results are
    summarized in Table 6 and by Mayer and Ellersieck (1986).

    In static systems, the dimethylamine salt and the acid had similar
    96-h LC50 values in rainbow trout of 250 and 358 mg/L, respectively
    (Alexander et al., 1983b,c). The 24-, 48-, and 96-h LC50 values for
    the amine salt to grass carp  (Ctenopharyngodon idella) ranged from
    1313 to 3080 mg/L (Tooby et al., 1980) and those for mosquito fish
     (Gambusia affinis) from 405 to 500 mg/L (Johnson, 1978).

    The esters of 2,4-D are generally more toxic to fish than are the
    various salts or the acid. In flow-through systems, the 96-h LC50
    values for rainbow trout ranged from 0.69 mg/L for the isopropyl ester
    (Drottar & Swigert, 1996a) to 7.2 mg/L for the ethylhexyl ester (Mayes
    et al., 1990a). The 96-h LC50 values for the various salts ranged
    from > 120 mg/L for the diethanolamine salt (Graves & Peters, 1991f)
    to 2840 mg/L for the isopropanolamine salt (Alexander et al., 1983d).
    The values for the dimethylamine salt (Alexander et al., 1983c),
    triisopropanolamine salt (Mayes et al., 1989b), and acid (Alexander et
    al., 1983b) also fall within this range. The 96-h LC50 values for
    rainbow trout fry and smolts were very low, irrespective of the


        Table 4. Toxicity of 2,4-D to estuarine and marine organisms

                                                                                                                                              
    Oxganism              Flow/    Temperature   Salinity    pH         Active ingredient     Exposure       2,4-D conc.   Reference
                          Static   (°C)                                                       parameter      (mg ai/L)
                                                                                                                                              

    Bay mussel (Mytilus            17.2-18.6     22.9-24.5   6.4-7.8    Free acid             96-h LC50      259           Liu & Lee (1975)
    edulis)                                                                                   96-h EC50      262
                                                                                              (attachment)

    (trocophore larva)             17.2-18.6     22.9-24.5   6.4-7.8    Free acid             48-h EC50      211.7         Liu & Lee (1975)
                                                                                              (normal
                                                                                              development)

    Tidewater silverside  Flow     21.2-22.8                 6.9-8.0    Ethylhexyl estera     96-h LC50      > 1.1b        Ward & Boeri 
    (Menidia beryllina)                                                                       96-h NOEC      1.1           (1991c)c
                          Flow     21.7-22.8                 7.8-8.4    Isooctyl ester        96-h LC50      > 0.24b       Ward & Boeri 
                                                                                              96-h NOEC      0.24          (1991d)c
                          Flow     21.5-22.7                 8.2-8.5    Dimethylamine salta   96-h LC50      469b          Ward (1991b)c
                          Flow     21.3-22.7                 5.98.3     Acid                  96-h LC50      175b          Vaishnav et al.
                                                                                                                           (1990a)c
                          Flow                                          Triisopropanolamine   96-h LC50      376b          Sousa (1990a)
                                                                                              96-h NOEC      60.7

    Atlantic silverside   Flow                                          Diethanolamine salt   96-h LC50      > 118b        Graves & Peters 
    (Menidia menidia)                                                                                                      (1991b)
                          Flow                                          Isopropylamine salt   96-h LC50      298b          Sousa (1990b)
                                                                                              96-h NOEC      < 26.9

    Eastern oyster        Static   19-21                     7.5-8.5    Dimethylamine salta   48-h EC50      > 210-< 320   Heitmuller (1975)c
    (Crassostrea 
    virginica) (larva)    Flow     21.2-23.2                 7.7-8.0    Dimethylamine salta   96-h EC50      136b          Ward (1991c)c
    (shell deposition)                                                                        96-h NOEC      40.6
                          Flow     18.1-19.6                 7.3-7.8    Ethylhexyl estera     96-h EC50      >0.71b        Ward & Boeri 
                                                                                              96-h NOEC      0.71          (1991e)c
                          Flow     15.3-17.7                 7.3-7.4    Ethylhexyl ester      96-h EC50      > 0.21b       Ward & Boeri 
                                                                                              96-h NOEC      0.21          (1991f)c

    Table 4. (continued)

                                                                                                                                              
    Oxganism              Flow/    Temperature   Salinity    pH         Active ingredient     Exposure       2,4-D conc.   Reference
                          Static   (°C)                                                       parameter      (mg ai/L)
                                                                                                                                              

                          Flow     20-21                     3.7 8.1    Acid                  96-h EC50      146b          Wade & Overman 
                                                                                                                           (1991)c
                          Flow     21.1-22.1                 7.0-8.0    Acid                  96-h EC50      57b           Ward et al. 
                                                                                              96-h NOEC      30            (1993)c
                          Flow                                          Diethanolamine salt   96-h EC50      > 112b        Graves & Peters 
                                                                                              96-h NOEC      < 6.9         (1991c)
                          Flow                                          Isopropylamine salt   96-h EC50      63.9b         Dionne (1990a)
                                                                                              96-h NOEC      31.7
                          Flow                                          Triisopropanolamine   96-h EC50      179b          Dionne (1990b)
                                                                                              96-h NOEC      77.2
                          Flow     29            25                     Isooctyl ester        96-h EC50      1.0           Mayer (1987)
                                                                                              (shell growth)
                          Flow     28            25                     Propylene glycol      96-h EC50      0.055         Mayer (1987)
                                                                        butyl ethyl ester     (shell growth)

    Copepod (Nitocra               21            7           7.8        Butoxyethanol         96-h LC50      3.1           Linden et al.
    spinipes)                                                                                                              (1979)

    Grass shrimp          Flow     21.2-22.8                 7.6-7.8    Ethylhexyl estera     96-h LC50      > 1.4b        Ward & Boeri 
    (Palaemonetes pugio)                                                                      96-h NOEC      > 1.4         (1991g)c
                          Flow     21.2-22.9                 7.3-8.1    Ethylhexyl ester      96-h LC50      > 0.14b       Ward & Boeri 
                                                                                              96-h NOEC      0.14          (1991h)c

    Pink shrimp           Flow     3.6-8.3                   21.1-22.6  Acid                  96-h LC50      554b          Vaishnav et al.
    (Panaeus duorarum)                                                                                                     (1990b)c
                          Static   19-21                     7.5-8.5    Dimethylamine salta   96-h NOEC      > 1000        Heitmuller
                                                                                                                           (1975)c
                          Flow     20.7-22.6                 8.2-8.5    Dimethylamine salta   96-h LC50      181b          Ward (1991d)c
                                                                                              96-h NOEC      65
                          Flow                                          Diethanolamine salt   96 h LC50      > 99.6b       Graves & Peters 
                                                                                              96-h NOEC                    (1991d)

    Table 4. (continued)

                                                                                                                                              
    Oxganism              Flow/    Temperature   Salinity    pH         Active ingredient     Exposure       2,4-D conc.   Reference
                          Static   (°C)                                                       parameter      (mg ai/L)
                                                                                                                                              

                          Flow                                          Isopropylamine salt   96-h LC50      623b          Sousa (1990c)
                                                                                              96-h NOEC      140
                          Flow                                          Triisopropanolamine   96-h LC50      744b          Sousa (1990d)
                                                                                              96-h NOEC      410

    Brown shrimp 
    (Panaeus aztecus)
    (juvenile)            Static   26            30                     Butoxyethanol         48-h LC50      5.6           Mayer (1987)
    (adult)               Flow     29            26                     Isooctyl              48-h LC50      0.48

    Dungeness crab 
    (Cancer magister)
    (first zoel)          Static   13            25                     Acid (technical)      96-h LC50      > 10          Caldwell (1977)
    (first instar 
    juvenile)             Static   13            25                     Acid (technical)      96-h LC50      > 100

    Fiddler crab          Static   19-21                     7.5-8.5    Dimethylamine salta   96-h NOEC      >1000         Heitmuller (1975)c
    (Uca pugilator)

    Blue crab (juvenile)
    (Callinectes 
    sapidus)              Static   24            29                     Propylene glycol      48-h LC50      2.8           Mayer (1987)
                                                                        butyl ethyl ester

    Estuarine crab 
    (Chasmagnathus
    granulata) 
    (first zoeI)          Static                             6.5-7.5    Isobutoxyethanol      24-h LC50      4.5-13.5      Rodriguez & Amin
                                                                        ester                                              (1991)
                                                                                              48-h LC50      1.06
                                                                                              72-h LC50      0.43
                                                                                              96-h LC50      0.30

    Table 4. (continued)

                                                                                                                                              
    Oxganism              Flow/    Temperature   Salinity    pH         Active ingredient     Exposure       2,4-D conc.   Reference
                          Static   (°C)                                                       parameter      (mg ai/L)
                                                                                                                                              

    (juvenile)                                                                                24-h LC50      > 6.4
                                                                                              48-h LC50      > 6.4
                                                                                              72-h LC50      5.55
                                                                                              96 h LC50      2.89
                                                 12          6.5-7.5    Isobutoxyethanol      24-h LC50      > 10          Rodriguez & 
                                                                        ester                 48 h LC50      > 10          Lombardo (1991)
                                                                                              72-h LC50      6.73
                                                                                              96-h LC50      3.37
    (adult)                        22-24         12          6.5-7.5    Isobutoxyethanol      4-week LC50    > 50          Rodriguez et al. 
    (juvenile)                                                          ester                                30.36         (1992)

    Estuarine crab                               12          6.5-7.5    Isobutoxyethanol      24-h LC50      > 400         Rodriguez & 
    (Uca uruguayensis)                                                  ester                 48-h LC50      > 400         Lombardo (1991)
                                                                                              72-h LC50      213
                                                                                              96-h LC50      130

                                   22-24         12          6.5-7.5    Isobutoxyethanol      4-week LC50    > 30          Rodriguez et al. 
                                                                        ester                                              (1992)
                                                                                                                                              

    a Formulated product
    b Reliable data based on measured concentrations
    c Study conducted according to international guidelines and good laboratory practice

    Table 5. Toxicity of 2,4-D to fish in early life stages

                                                                                                                                              

    Organism               Flow/   Temp        Alkalinity  Hardness  pH        Active ingredient     Exposure      2,4-D conc.  Reference
                           Static  (°C)                                                              parameter     (mg ai/L)
                                                                                                                                              

    Fathead minnow         Flow    24.9-25.7   41-50       70-75     7.1-7.6   2-Ethylhexyl ester    32-d NOEC     0.16         Mayes et al. 
    (Pimephales promelas)                                                                            32-d MATC     0.12m        (1990a)a
                           Flow    25.0-25.6   23-49       68-74     6.5-7.7   Acid                  32-d NOEC     63.4m        Mayes et al. 
                                                                                                     32-d MATC     80.4         (1990b)a
                           Flow    25.3-25.8   47-52       70-76     7.1-7.8   Dimethylamine salt    28-d NOEC     17.1b        Dill et al. 
                                                                                                     28-d MATC     22           (1990)a
                           Flow                                                Diethanolamine salt   33-d NOEC     29.1b        Graves & Peters
                                                                                                                                (1991e)
                           Flow                                                Butoxyethyl ester     96-h LC50     0.95b        Mayes et al. 
                                                                                                                                (1989a)
    Bleak (Alburnus                                                            Sodium salt           12-h LC50     159.4        Biro (1979)
    alburnus) (embryo)                                                                               24-h LC50     129.0
                                                                                                     36-h LC50     63.9
                                                                                                     48-h LC50     12.9
    (larvae)                                                                                         12-h LC50     111.2
                                                                                                     24-h LC50     70.6
                                                                                                     36-h LC50     62.1
                                                                                                     48-h LC50     51.6

    Goldfish (Carassius    Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        4-day LC50    > 187        Birge et al. 
    auratus) (embryo)      Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        4-day LC50    > 201        (1979)
    (4-day post-hatch)     Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        8-day LC50    133.1
                           Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        8-day LC50    119.1

    Largemouth bass 
    (Micropterus salmoides)
    (embryo)               Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        3.5-day LC50  165.4        Birge et al. 
                           Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        3.5-day LC50  160.7        (1979)
    (4-day post-hatch)     Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        7.5-day LC50  108.6
                           Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        7.5-day LC50  81.6

    Table 5. Toxicity of 2,4-D to fish in early life stages

                                                                                                                                              

    Organism               Flow/   Temp        Alkalinity  Hardness  pH        Active ingredient     Exposure      2,4-D conc.  Reference
                           Static  (°C)                                                              parameter     (mg ai/L)
                                                                                                                                              

    Rainbow trout          Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        23-day LC50   11.0         Birge et al. 
    (Oncorhynchus mykiss)                                                                                                       (1979)
    (embryo)               Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        23-day LC50   4.2
    (4-day post-hatch)     Flow    18.2-25.8   66.7        53.3      7.84      Potassium salt        27-day LC50   11.0
                           Flow    18.2-25.8   65.3        197.5     7.78      Potassium salt        27-day LC50   4.2
                                                                                                                                              

    a Results generated in accordance with international guidelines and good laboratory practice
    b Reliable data based on measured concentrations

    Table 6. Toxicity of 2,4-D to fish

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

    Rainbow trout          Static   19-21       51          78         8.4      Isooctyl ester        96-h LC50   > 5         Alexander 
    (Oncorhynchus mykiss)                                                                                                     et al. (1983a)a
                           Flow     11.7-12.7   46          70         7.4-7.7  Ethylhexyl esterb     96-h LC50   7.2c        Mayes et al. 
                                                                                                      192-h LC50  4.6         (1990c)a
                           Static   12-20                              7.0-8.2  Acid                  96-h LC50   358         Alexander et 
                                                                                                                              al. (1983b)a
                           Static   19-21                   47         7.1      Dimethylamine salta   96-h TL50   377         Bentley (1974)a
                                                                                                      96-h NOEL   210
                           Static   12-20       77-84       90-108     7.8-8.0  Dimethylamine salt    96-h LC50   250         Alexander et al.
                                                                                                                              (1983e)a
                           Flow                                                 Dimethylamine salt    48-h LC50   240         Bogers & Enninger
                                                                                                                              (1990a)
                                                                                                      72-h LC59   240
                                                                                                      96-h LC50   240
                           Static   14                      Soft       6.3      Diethanolamine        96-h LC50   409         Wan et al. (1991)
                                                                       6.3      Isooctyl ester        96-h LC50   167
                                                            Inter-     7.5      Diethanolamine        96-h LC50   511
                                                            mediate    7.5      Isooctyl ester        96-h LC50   164
                                                            Hard       8        Diethanolamine        96-h LC50   744
                                                                       8        Isooctyl ester        96-h LC50   79
                           Flow     18.2-25.8   66.7        53.5       7.84     Potassium salt        27-d LC50   0.032c      Birge et al.(1979)
                           Flow     18.2-25.8   65.3        197.5      7.78     Potassium salt        27-d LC50   0.022c      Birge et al.(1979)
                           Flow                                                 Diethanolamine salt   96-h LC50   > 120c      Graves & Peters
                                                                                                                              (1991f)
                           Flow                                                 Isopropylamine salt   96-h LC50   2840        Alexander et al.
                                                                                                                              (1983d)
                           Flow                                                 Triisopropanolamine   96-h LC50   317         Mayes et al.
                                                                                                                              (1989c)
                           Flow                                                 Butoxyethyl ester     96-h LC50   2.0         Alexander et al.
                                                                                                                              (1983d)
                           Flow                                                 Isopropyl ester       96-h LC50   0.69c       Drottar & Swigert
                                                                                                                              (1996a)

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

                           Flow                                                 Isopropyl esterb      96-h LC50   0.78c       Drottar & Swigert
                                                                                                                              (1996e)
    (fingerlings)          Flow     15          7.6         21.6       4.54     Acid                  96-h LC50   < 100c      Doe et al. (1988)
                                                                       5.6                            96-h LC50   < 400
                                                                       6.8                            96-h LC50   < 1000
                                                                       8.48                           96-h LC50   > 1000
    (fry)                  Flow     15          18          17         7.1      Butoxyethanol esterb  96-h LC50   0.518c      Finlayson & Verrue
                                                                                Total 2,4-D           96-h LC50   0.642c      (1985)
                                                                                Propylene glycol      96-h LC50   0.329c
                                                                                butyl ethyl esterb
                                                                                Total 2,4-D           96-h LC50   0.514c
    (smolts)               Flow     15          18          17         7.1      Butoxyethanol esterb  96-h LC50   0.468c      Finlayson & Verrue
                                                                                                                              (1985)
                                                                                Total 2,4-D           96-h LC50   1.338c
                           Flow     15          18          17         7.1      Propylene glycol      96-h LC50   0.342c      Finlayson & Verrue
                                                                                butyl ethyl esterb                            (1985)
                                                                                Total 2,4-D           96-h LC50   1.555c
    Loading factor         Static   14          18          17         7.1      Butoxyethanol esterb  96-h LC50   1.206c      Finlayson & Verrue
    4.2 g fish/L                                                                Total 2,4-D           96-h LC50   1.422c      (1985) 
    Loading factor         Static   15          18          17         7.1      Butoxyethanol esterb  96-h LC50   3.689c      Flnlayson & Verrue
    9.8 g fish/L                                                                Total 2,4-D           96-h LC50   4.487c      (1985) 

    Bluegill (Lepomis      Static   19-21       51          78         8.4      Isooctyl ester        96-h LC50   > 5         Alexander et al.
    machrochirus)                                                                                                             (1983a)a 
                           Static   12-20                              7.0-8.2  Acid                  96-h LC50   263         Alexander et al.
                                                                                                                              (1983b)a
                           Static   19-21                   47         7.1      Dimethylamine saltb   96-h TL50   387         Bentley (1974)a
                                                                                                      96-h NOEL   280 
                           Static   12-20       77-84       91-108     7.8-8.0  Dimethylamine salt    96-h LC50   524         Alexander et al.
                                                                                                                              (1983e)a 
                           Flow                                                 Diethanolamine salt   96-h LC50   > 121c      Graves & Peters
                                                                                                                              (1991g) 

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

                           Flow                                                 Isopropylamine salt   96-h LC50   1700        Alexander et al.
                                                                                                                              (1983d) 
                           Flow                                                 Triisopropanolamine   96-h LC50   432         Mayes et al. 
                                                                                                                              (1989a) 
                           Flow                                                 Butoxyethyl ester     96-h LC50   0.61        Alexander et al.
                                                                                                                              (1983d) 
                           Flow                                                 Isopropyl ester       96-h LC50   0.31c       Drottar & Swigert
                                                                                                                              (1996c) 
                           Flow                                                 Isopropyl esterb      96-h LC50   0.31c       Drottar & Swigert
                                                                                                                              (1996d)
    Fathead minnow         Static   19-21       51          78         8.4      Isooctyl ester        96-h LC50   >5          Alexander et al.
    (Pimephales promelas)                                                                                                     (1983a)a
                           Static   12-20                              7.0-8.2  Acid                  96-h LC50   320         Alexander et al.
                                                                                                                              (1983b)a
                           Static   12-20       77-84       90-108     7.8-8.0  Dimethylamine salt    96-h LC50   344         Alexander et al.
                                                                                                                              (1983e)a
                                                                                Isopropylamine salt   96-h LC50   2180        Alexander et al.
                                                                                                                              (1983d) 
                                                                                Butoxyethyl ester     96-h LC50   2.5         Alexander et al.
                                                                                                                              (1983d)

    Striped bass           Static   20                      50         7.2      Free acid             24-h LC50   85.6        Rehwoldt et al. 
    (Morone saxatillis)                                                                               96-h LC50   70.1        (1977)

    Banded killifish       Static   20                      50         7.2      Free acid             24-h LC50   306.2       Rehwoldt et al. 
    (Fundulus diaphanus)                                                                              96-h LC50   26.7        (1977)

    Pumpkinseed sunfish    Static   20                      50         7.2      Free acid             24-h LC50   120         Rehwoldt et al. 
    (Lepomis gibbosus)                                                                                96-h LC50   94.6        (1977)

    White perch            Static   20                      50         7.2      Free acid             24-h LC50   55.5        Rehwoldt et al.
    (Roccus americanus)                                                                               96-h LC50   40          (1977)

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

    American eel           Static   20                      50         7.2      Free acid             24-h LC50   427.2       Rehwoldt et al. 
    (Anguilla rostrata)                                                                               96-h LC50   300.6       (1977)

    Carp (Cyprinus carpio) Static   20                      50         7.2      Free acid             24-h LC50   175.2       Rehwoldt et al. 
                                                                                                                              (1977)
                                    22                      141-223    7.0-7.5  Acid                  24-h LC50   310         Neskovic et al. 
                                                                                                      48-h LC50   295         (1994)
                                                                                                      96-h LC50   270
                           Static                                               Dimethylamine salt    48-h LC50   >560-<1000  Bogen & Enninger
                                                                                                      72-h LC50   >560-<1000  (1990b)
                                                                                                      96-h LC50   >560-<1000
                           Static   20                      50         7.2      Free acid             96-h LC50   96.5        Rehwoldt et al. 
                                                                                                                              (1977)

    Guppy (Lebistes        Static   20                      50         7.2      Free acid             24-h LC50   76.7        Rehwoldt et al. 
    reticulata)                                                                                       96-h LC50   70.7        (1977)

    Carp (Cirrhina                                                              Dimethylamine salt    96-h LD50   > 100       Singh & Yadav 
    mrigla hamilton)                                                                                                          (1978)
    fingerlings

    Grass carp             Flow     13                      270        8.1      Amine salt            24-h LC50   3080        Tooby et al. 
    (Ctenopharyngodon                                                                                 48-h LC50   2540        (1980)
    idella)                                                                                           96-h LC50   1313

    Bleak (Alburnus        Static   10          15                     7.8      Butoxyethanol ester   96-h LC50   3.2-3.7     Linden et al. 
    alburnus)                                                                                                                 (1979)

    Mosquito fish          Static   21-22                                       Amine salt            24-h LC50   500         Johnson (1978)
    (Gambusia affinis)                                                                                48-h LC50   445
                                                                                                      96-h LC50   405

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

    Mullet (Mugil          Static                                               Sodium salt           24-h LC50   68.0        Tag El-Din et al.
    cephalus)                                                                                         96-h LC50   32.0        (1981)

    Punti (Puntius ticto)  Static   23.5                                        Ethyl ester           24-h LC50   1.6         Verma et al. 
                                                                                                                              (1984)

    Medaka (Oryzias                                                             Sodium salt           48-h LC50   > 40        Hashimoto & 
    latipes)                                                                                                                  Nishiuchi (1978)
                                                                                                                              in Hidaka et al.
                                                                                                                              (1984)

    Pink salmon            Static   14                      Soft       6.3      Diethanolamine        96-h LC50   291         Wan et al. (1991)
    (Oncorhynchus                                                               Isooctyl ester        96-h LC50   30
    gorbuscha) (fry)                                        Inter-     7.5      Diethanolamine        96-h LC50   363
                                                            mediate             Isooctyl ester        96-h LC50   70
                                                            Hard       8.0      Diethanolamine        96-h LC50   438
                                                                                Isooctyl ester        96-h LC50   21

    Alaska coho salmon     Static   14                      Soft       6.3      Diethanolamine        96-h LC50   472         Wan et al. (1991)
    (Oncorhynchus kisutch)                                                      Isooctyl ester        96-h LC50   156
    (fingerling)                                            Inter-     7.5      Diethanolamine        96-h LC50   493
                                                            mediate             Isooctyl ester        96-h LC50   158
                                                            Hard       8.0      Diethanolamine        96-h LC50   662
                                                                                Isooctyl ester        96-h LC50   63

    Chinook salmon         Flow     9           18          17         7.1      Butoxyethanol esterb  96-h LC50   0.315c      Finlayson & Verrue
    (Oncorhynchus                                                               Total 2,4-D           96-h LC50   0.373c      (1985)
    tshawytscha) (fry)
    (smolts)               Flow     15          18          17         7.1      Butoxyethanol esterb  96-h LC50   0.375c
                                                                                Total 2,4-D           96-h LC50   1.250c
                                                                                Propylene glycol      96-h LC50   0.246c
                                                                                butyl ethyl esterb
                                                                                Total 2,4-D           96-h LC50   1.117c

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

    Goldfish (Carassius    Flow     18.2-25.8   66.7        53.3       7.84     Potassium salt        8-d LC      18.2c       Birge et al.(1979)
    auratus)               Flow     18.2-25.8   65.3        197.5      7.78     Potassium salt        8-d LC1     8.9c        Birge et al.(1979)

    Largemouth bass        Flow     18.2-25.8   66.7        53.3       7.84     Potassium salt        7.5-d LC1   13.1c       Birge et al.(1979)
    (Micropterus           Flow     18.2-25.8   65.3        197.5      7.78     Potassium salt        7.5-d LC1   3.2c        Birge et al.(1979)
    salmoides)

    Cutthroat trout (Salmo                                                      Butyl ester           96-h LC50   0.78        Woodward (1982)
    clarki) (juvenile)                                                          Propylene glycol      96-h LC50   0.77        Woodward (1982)
                                                                                butyl ethyl ester
                                                                                Isooctyl ester        96-h LC59   > 50        Woodward (1982)
                                    5                       40         7.2      Butyl ester           96-h LC50   490         Woodward & Mayer
                                    10                      40         7.2                            96-h LC50   540         (1978)
                                    15                      40         7.2                            96-h LC50   770
                                    5                       40         7.2      Propylene glycol      96-h LC50   490
                                                                                butyl ethyl ester
                                    10                      40         7.2                            96-h LC50   1030
                                    15                      40         7.2                            96-h LC50   780
                                    10                      40         6.5      Butyl ester           96-h LC50   750         Woodward & Mayer
                                    10                      40         7.5                            96-h LC50   740         (1978)
                                    10                      40         8.5                            96-h LC50   835
                                    10                      40         6.5      Propylene glycol      96-h LC50   930
                                                                                butyl ethyl ester
                                    10                      40         7.5                            96-h LC50   1220
                                    10                      40         8.5                            96-h LC50   930
                                    10                      40         8.5                            96-h LC50   1075
                                    10                      40         7.8      Butyl ester           96-h LC50   860         Woodward & Mayer
                                    10                      170        7.8                            96-h LC50   860         (1978)
                                    10                      300        7.8                            96-h LC50   860
                                    10                      40         7.8      Propylene glycol      96-h LC50   1000
                                                                                butyl ethyl ester

    Table 6. (continued)

                                                                                                                                               

    Organism               Flow/    Temp        Alkalinity  Hardness   pH       Active ingredient     Exposure    2,4-D conc. Reference
                           Static   (°C)                                                              pmameter    (mg ai/L)
                                                                                                                                               

                                    10                      170        7.8                            96-h LC50   860
                                    10                      300        7.8                            96-h LC50   1000

    Lake trout (Salvelinus          5                       40         7.2      Butyl ester           96-h LC50   600         Woodward & Mayer
    namaycush)                      10                      40         7.2                            96-h LC50   640         (1978)
                                    15                      40         7.2                            96-h LC50   820
                                    5                       40         7.2      Propylene glycol      96-h LC50   700
                                                                                butyl ethyl ester
                                    10                      40         7.2                            96-h LC50   630
                                    15                      40         7.2                            96-h LC50   1000
                                    10                      40         6.5      Butyl ester           96-h LC50   820         Woodward & Mayer
                                    10                      40         7.5                            96-h LC50   840         (1978)
                                    10                      40         8.5                            96-h LC50   1170
                                    10                      40         6.5      Propylene glycol      96-h LC50   840
                                                                                butyl ethyl ester
                                    10                      40         7.5                            96-h LC50   1125
                                    10                      40         7.8      Butyl ester           96-h LC50   880         Woodward & Mayer
                                    10                      170        7.8                            96-h LC50   930         (1978)
                                    10                      300        7.8                            96-h LC50   1075
                                    10                      40         7.8      PGBEE ester           96-h LC50   1050
                                    10                      170        7.8                            96-h LC50   1200
                                    10          300         7.8                 96 h LC50             1150
                                                                                                                                               

    a Results generated in accordance with international guidelines and good laboratory practice
    b Formulated product
    c Reliable data based on measured concentrations
    

    formulation. As the pH increased, the toxicity of 2,4-D acid to
    fingerling  Salmo gairdneri decreased drastically. At pH 8.5, the
    96-h LC50 value was more than an order of magnitude higher than at pH
    4.5 (> 1000 mg/L and < 90 mg/L, respectively) (Doe et al., 1988).

    Similar 96-h LC50 values were seen for bluegills, the values for
    esters ranging from 0.31 mg/L for the isopropyl ester and formulated
    isopropyl ester (Drottar & Swigert, 1996c,d) to 0.61 mg/L for the
    butoxyethyl ester (Mayes et al., 1989c). The 96-h values for the salts
    of 2,4-D ranged from > 121 mg/L for the diethanolamine salt (Graves &
    Peters, 1991g) to 1700 mg/L for the isopropylamine salt (Alexander et
    al., 1983d). The 96-h LCd, values for the esters in fathead minnow
    ranged from 2.5 mg/L for the butoxyethyl ester (Alexander et al.,
    1983e) to > 5 mg/L for the isooctyl ester (Alexander et al., 1983a),
    whereas the values for the salts ranged from 344 mg/L for the
    dimethylamine salt (Alexander et al., 1983c) to 2180 mg/L for the
    isopropylamine salt (Alexander et al., 1983d). The LC50, values for
    seven species of fish in static systems were 55.5-427.2 mg/L for 24 h
    and 26.7-300.6 mg/L for 96 h (Rehwoldt et al., 1977). The 96-h LC50
    values for the free acid, the butyl ester, and the isooctyl ester in
    various species of salmon ranged from < 1 to 50 mg/L, most values
    being < 1 mg/L (Meehan et al., 1974).

    6.2.3.2  Other effects on vertebrates

    Embryos and larvae of the fathead minnow,  Pimephales promelas, were
    exposed to the butoxyethyl ester of 2,4-D in a flow-through system at
    concentrations as high as 416.1 µg/L for 32 days. The NOEC was 80.5
    µg/L, and the maximum acceptable toxicant concentration was 96.0 µg/L
    (Mayes et al., 1989a).

    In a study of the inhibitory effect of 2,4-D sodium salt (80%) on the
    hatching of carp eggs, the average hatch was 92% with none deformed at
    25 mg/dm, whereas none hatched at 100 mg/dm (Kapur & Yaduv, 1982).
    Carp embryos and larvae were exposed to the sodium salt of 2,4-D (both
    pure and as an 85% formulation) and maintained at 23°C for 34 days.
    The formulated product at 50 mg ai/L was not harmful to embryos, but
    it induced behavioural changes, disturbances in feeding, some
    morphopathological changes, and ultimately death in larvae (Kamler et
    al., 1974).

    3.3  Terrestrial organisms

    3.3.1  Plants

    As 2,4-D is a herbicide and plant growth regulator, it is well
    established that it can affect a number of species of terrestrial
    plants, particularly non-grass species.

    In field trials with paraquat in combination with other herbicides,
    including 2,4-D, for the control of barley, wheat, and oats conducted
    in 1982 and 1983, the phytotoxicity of paraquat was slightly reduced
    when applied in combination with commercially formulated mixtures that

    included 2,4-D as the dimethylamine salt and acid (O'Donovon &
    O'Sullivan, 1986). Repeated applications of 2,4-D (as the 50% EC
    formulation, 0.5 kg/ha) had no significant effect on either alfalfa
    yields or weed suppression between treatments applied in the spring
    and fall (Waddington, 1987). Applications of the iso-octyl ester of
    2,4-D at 0.56 kg/ha post-emergence in combination with atrazine
    resulted in the most effective control of Russian thistle and kochia
    in dryland corn during the 1987, 1988, and 1989 growing seasons. The
    corn yields were highest with these treatments in 1987 and 1989
    (Blackshaw, 1990). In a review of management systems for conservation
    fallow on southern Canadian prairies, repeated use of 2,4-D was
    reported to effectively control most weeds (Blackshaw & Lindwall,
    1995). As a component of a study on the effect of soil heat treatment
    and microflora on the efficacy of glyphosate in bean seedlings, the
    diethanolamine salt of 2,4-D was also evaluated. The quantity of
    sprayed 2,4-D required to kill 50% of the bean seedlings was similar
    in autoclaved and raw soil (Levesque et al., 1992).

    Application of 6 µg/µl of 2,4-D every second day for a total of five
    applications to the furled fourth leaf of the main shoot apex of wild
    oats  (Avena fatua L.) in greenhouses reduced imazamethabenz-induced
    tillering but did not increase the efficacy of imazamethabenz (Chao et
    al., 1994). In a study of the effect of 2,4-D on spore germination and
    appressorial formation on  Colletotrichum gloeosporioides f. sp.
     malvae, a bioherbicide, various concentrations of the isooctyl ester
    and amine salt had no significant effect (Grant et al., 1990).

    An investigation was carried out on the efficacy of and residues from
    the use of the butoxyethanol ester (granule) or dimethylamine salt of
    2,4-D in plants, water, and sediment in Buckhorn Lake, Ontario,
    Canada, over a three-year period. More than 20 indigenous species of
    plants were examined (Carpentier et al., 1988). The results confirmed
    the finding that the persistence of 2,4-D is controlled in part by the
    organic content of the sediments and that it moves laterally through
    water. The two forms of 2,4-D had essentially the same efficacy
    (Bothwell & Daley, 1981). 2,4-D was among the least toxic herbicides
    with regard to the rates of phosphate and ammonium assimilation in
    lake phytoplankton when compared with inhibition of photosynthesis by
    the 14C-bicarbonate assimilation technique. (Brown & Lean, 1995).
    Eurasian watermilfoil was exposed to 2,4-D at 0.5 mg acid equivalent/L
    for 12, 24, 36, 48, 60, or 72 h or to 1.0 and 2.0 mg acid equivalent/L
    for 12, 24, 36, and 48 h; two untreated controls were available. The
    plants were treated when the shoot apices reached to within 5-10 cm of
    the water surface, after two weeks. As expected, plant damage
    increased with increasing concentrations and exposure times. Control
    should be achieved by exposure to a minimum 2,4-D concentration of 0.5
    mg acid equivalent/L for more than 72 h and 1.0 mg acid equivalent/L
    for more than 24-36 h (Green & Westerdahl, 1990).

    3.3.2  Invertebrates

    3.3.2.1  Toxicity to arthropods

    (a)  Bees

    In studies conducted according to the guidelines of the Council of
    Europe (CoE)/European Plant Protection Organization (EPPO) and US
    guidelines, the 72-h LD50, values of the dimethylamine salt and the
    ethylhexyl ester of 2,4-D to for honeybees exposed orally and by
    contact were > 100 mg/bee (Palmer & Krueger, 1997a,b,c,d).

    Four nuclei of bees  (Apis mellifera L.) were moved into four areas
    of a cage containing a heavy stand of London rocket  (Sisymbrium 
     irio) for seven days of acclimatization before spraying. Dead bee
    traps were placed for each hive. Immediately before spraying, five
    wire cages each containing about 75 adult bees were placed in each
    area, and dead bees were counted for 24 and 48 h after spraying at 2.2
    kg ai/ha. No significant difference was observed between test and
    control values (Moffet, 1972).

    Eight hectares of flowering clover pasture, which was the sole source
    of nectar for field bees from an apiary of 35 hives, received an
    application of 3.5 kg/ha of the sodium salt of 2,4-D. Half of the area
    was dusted under ideal conditions at 8.00 h and half the following day
    at 6.00 h. Sufficient drift occurred to affect clover over an area of
    42 ha. No bees were dusteci, as application took place before they
    began to fly. There were 3500 bees/ha before dusting, 3000 on the day
    after application, and fewer as the plants withered. No evidence of
    repellence was observed. Nectar bees were picked off clover flowers
    while they were collecting and placed in observation cages with
    feeders of sugar syrup. The mortality within two days of application
    among 243 field bees collected was 22%, and behavioural changes were
    seen, including agitation, quivering, and attempts to sting each
    other. No deaths occurred among control bees collected from a site two
    miles away. No adverse effect was observed on brood or hive activity.
    In a further test, while the nectar and pollen were being brought into
    the hive and the queen was laying heavily, 200 foragers were heavily
    dusted with 2,4-D mixture as they entered the hive. No deaths among
    adult bees and no disorganization or effect on broods were observed
    (Palmer-Jones, 1966).

    The sodium salt of 2,4-D was considered to be non-toxic to bees 
     (Apis mellifera) exposed for up to 96 h at 11 µg/bee, the highest
    dose tested (Atkins et al., 1975).

    The 24-h oral LD50 value for the sodium salt in bees  (Apis 
     mellifera carnica) was 62.1-92.0 µg/bee, depending on the location
    of the colony and diet (Wahl & Ulm, 1983). When 2,4-D was given in 60%
    sucrose syrup at concentrations of 100-1000 mg/kg to newly emerged
    worker bees, no deaths were observed (Morton & Moffet, 1972).

    As reported by WHO (1989), feeding of worker honey bees  (Apis 
     mellifera) with 2,4-D salts in sucrose syrup gave 24-h LC50 values
    of 104 and 115 µg/bee (Jones & Connell, 1954; Beran & Neururer, 1955).
    2,4-D acid was fed to honey bees in 60% sucrose syrup at 10, 100, or
    1000 mg/L and the half-time, i.e. the time for 50% of the bees in a
    cage to die, was monitored. The half-time at the two lowest doses was
    significantly longer than that of controls (37.2 days at 10 mg/L and
    40.4 days at 00 mg/L, with a control value of 33.4 days) but was
    significantly reduced at 1000 mg/L (18.6 days). The butoxyethanol and
    isooctyl (commercial formulation) esters of 2,4-D had no effect on
    survival times when fed at the same doses. The dimethylamine salt of
    2,4-D (commercial formulation) had no effect at 10 or 100 mg/L but
    shortened the half-time at 1000 mg/L (Morton et al., 1972).

     (b)  Other non-target arthropods

    The herbicide U46 Combi fluid, containing 350 g/L 2,4-D acid and 300
    g/L (4-chloro-2-methylphenoxy) acetic acid (MCPA), was tested in the
    laboratory by applying 1.8 mg of a 1.5% formulation in water per cm2
    on the microhymenopterous egg parasite  Trichogramma cacoeciae. The
    effect was determined by counting the reduction in parasitism, as
    compared with the control, after eight days. Three replicates of ± 350
    females each and three control replicates with a similar number of
    animals were used. The adults were exposed for seven days, and the
    parasitic capacity was evaluated after 12 days. The average numbers of
    parasitized host eggs per adult wasp at the end of the study were
    13.47, 9.42, and 15.6 (average, 12.83) in the three replicates treated
    with U46 Combi fluid and 19.33, 21.37, and 22.29 (average, 20.99) in
    the control cages. The average reduction in parasitic capacity was
    calculated to be 38.9%. As the reduction was < 50%, U46 Combi fluid
    was classified as harmless to  T. cacoeciae on the basis of the
    classification of the International Organization for Biological
    Control of Noxious Animals and Plants (Konig, 1989).

    BAS 009011H, containing 350 g/L 2,4-D dimethylamine salt and 300 g/L
    MCPA dimethylamine salt, was tested in  Poecilus cupreus aged five to
    six weeks. Thirty insects in five replicates of six at an equal sex
    ratio were used to test BAS 009011H and for the control and reference
    (Afugan) groups. The test concentrations were equivalent to the
    maximum recommended rate of 8 L formulation/ha in 400 L of water. No
    deaths occurred among the beetles, and no behavioural effects were
    seen; 73% of the insects in the reference group died. In the first
    week, the feeding rate of treated beetles was similar to the controls,
    but in the second week feeding fell to 55% of control values. Exposure
    was therefore extended to 28 days, in order to observe any delayed
    effect. Feeding increased in the third week, so that the total
    reduction in feeding during the study was < 30%. The number of pupae
    eaten by beetles was 3.95 for controls and 2.78 for beetles treated
    with BAS 009011H (Dohmen, 1990).

    The LC50 of the formulated amine salt of 2,4-D at 4 lb/gal (0.4 kg/L)
    to two mite species was 0.22 kg/ha for  Neoseiulus fallacis and 0.98
    kg ai/ha for  Tetranychus urticae (Rock & Yeargan, 1973).

    Application of 2,4-D at a rate of 2.2 kg ai/ha to leafy spurge
     (Euphorbia esula) and cyprus spurge  (E. cyparissias) had no effect
    on unsprayed larvae of the leafy spurge hawk moth  (Hyles 
     euphoribiae) which fed on treated leaves or on the number of adults
    that emerged from pupae obtained from the mating of treated or
    untreated adults (Rees & Fay, 1989).

    As reported by WHO (1989), beneficial coccinellid larvae were sprayed
    with a preparation of mixed amine salts of 2,4-D at a rate equivalent
    to 0.56 kg acid equivalent/ha 1, 3, 6, 9, or 12 days after hatching.
    The development period was lengthened when the larvae were treated on
    days 3, 6, 9, or 12, but there was no effect when they were sprayed on
    the first day after hatching. The rate of mortality before pupation
    was more than doubled in all treated groups, but that during pupation
    was no different from that of controls (Adams, 1960).

    Adult thistle-rosette weevils  (Ceuthorhynchidius horridus), which
    are used for the biological control of musk thistle, were dosed with
    2,4-D amine salt at five concentrations of 0.17-147.8 kg/ha. No
    significant change in mortality rate was observed up to 175 days after
    treatment at doses <- 1.68 kg/ha, but the rate was significantly
    increased at 16.8 and 84 kg/ha three days after treatment, and at
    147.8 kg/ha mortality was increased both on day 3 and subsequently.
    The five-day LC50 values were 70.2 kg/ha for males and 61.4 kg/ha for
    females, which are 41.8 times the recommended application rate of 2,4-
    D for males and 36.6 times that for females (Trumble & Kok, 1980).
    European cockroaches  (Blatella germanica) reared on food containing
    1000 mg/kg showed negligible effects on reproduction (Riviere, 1976).

    Wheat plants were wetted with a 0.3% solution of mixed isopropyl and
    butyl esters of 2,4-D and exposed to females of the wheat stem sawfly
     (Cephus cinctus). The plants were sprayed seven days before
    oviposition, at the time of oviposition, or 7, 14, or 21 days after
    oviposition. The eggs took about seven days to hatch. The highest rate
    of mortality among larvae (96.4%) occurred after spraying at the time
    of egg laying, and the effectiveness of 2,4-D in killing larvae
    decreased with time of exposure, larval mortality being 68.1% 7 days,
    60.8% 14 days, and 37% 21 days after oviposition, with mortality in
    controls of 30%. When plants were sprayed seven days before egg
    laying, larval mortality was 46.9%. Adult flies were not affected
    (Gall & Dogger, 1967).

    Two species of beetle (Carabidae),  Bembidion femoratum and 
     B. ustulatum, exposed to sand dosed with 2,4-D at 1 g/m2 had
    mortality rates > 50% within four days of exposure.  B. ustulatum 
    showed 100% mortality within 10 days of exposure to 1 g/m2 and within
    four days of exposure to 2 g/m2. About 20% of the individuals of 
     B. femoratum survived 14-day exposures to 1 or 2 g/m2 (Muller
    (1971).

    3.3.2.2  Toxicity to earthworms

    The toxicity of the dimethylamine salt of 2,4-D was tested in the
    earthworm  (Eisenia foetida) at concentrations of 10-1000 mg ai/kg of
    soil for up to 14 days. The 14-day LC50 was 350 mg/kg. No deaths or
    sublethal effects were seen at doses < 100 mg ai/kg, equal to 200
    mg formulation/kg, after 14 days. All earthworms at the highest
    concentration died (Adema & Roza, 1989). When earthworms were exposed
    to 2,4-D acid sprayed onto filter paper in glass vials, the calculated
    48-h LC50 value was 61.6 (95% confidence interval, 41-92.4) µg/cm2
    (Roberts & Dorough, 1984).

    3.3.2.3  Other effects on invertebrates

    Above-ground application of 2,4-D on field crops at 1250 g ai/ha for
    over 20 months did not reduce the epigeal predator fauna
    (staphylinids, carabids, and spiders). Field processing for planting
    was reported to be more harmful (Everts et al., 1989). 'Relatively
    insoluble' 2,4-D applied to a forest floor at a rate of 33.6 kg/ha
    tended to remain in the litter layer when decomposition was initiated.
    Fully treated substrate resulted in 50% mortality of adult millipeds
     (Scytonotus simplex) by day 7 (Hoy, 1985).

    No effect on soil microarthropods was reported when turf was treated
    with a mixture of MCPA at the recommended rate and the butyl ester of
    2,4-D at 10 times the recommended rate (Rapoport & Cangioli, 1963).

    Adult millipeds  (Scytonotus simplex) were exposed to 2,4-D at a rate
    equivalent to 33.6 kg/ha, either uniformly to filter paper substrate,
    to half the substrate, or added to half the food consisting of
    air-dried red alder leaves. The mortality rate was highest for
    millipeds on fully treated substrate, lower when they were exposed
    only to contaminated food, and lowest when they were exposed to
    substrate half of which was treated with twice the dose of the fully
    treated substrate. Those on fully treated substrate began to die on
    the first day and more than 50% were dead by day 7 (Hoy, 1985).

    After a single application of 2,4-D (Rapoport & Cangioli, 1963) or
    repeated applications for 10 and 11 years (Davies, 1965; Bieringer,
    1969), none or very little effect was seen on collembolan and mite
    populations. Population increases seen after use of 2,4-D were
    ascribed to increased microbiological and bacteriological activity in
    the soil, and a population decrease 11 months after 2,4-D application
    may have been related to lower plant residues in the soil (Prasse,
    1975).

    3.3.3  Vertebrates

    3.3.3.1  Toxicity to birds

    The US Environmental Protection Agency requires data on the toxicity
    of pesticides to mallard ducks  (Anas platyrhynchos) and bobwhite
    quail  (Colinus virginianus). Those data are presented in Table 7. In


        Table 7. Toxicity of 2,4-D to birds

                                                                                                                                         

    Species                Sex   Age          Route    Active ingredient         Exposure       2,4-D conc.a     Reference
                                                                                 parameter
                                                                                                                                         

    Mallard duck                 Mature       Oral     Isooctyl ester            Acute LD50     663              Beavers (1984a)
    Anas platyrhynchos           9 days       Diet     Isooctyl ester            Acute LC50     > 5620           Beavers (1984b)
                                 10 days      Diet     Acid                      Acute LC50     > 5620           Culotta et al. (1990a)
                                 14 days      Diet     Dimethylamine salt        8-day LC50     > 4640           Fink (1974a)
                                 10 days      Diet     Dimethylamine salt        Acute LC50     > 5620           Long et al. (1990a)
                                                                                 NOEC           562
                           M     4 months     Oral     Acid                      Acute LD50     > 2000           Hudson et al. (1984)
                           M     3-5 months   Oral     Sodium salt               Acute LD50     > 2025           Hudson et al. (1984)
                           M     7 months     Oral     Amine salt                Acute LD50     < 2000           Hudson et al. (1984)
                           F     3-5 months   Oral     Acid (technical grade)    Acute LD50     > 1000           Hudson et al. (1984)
                                              Oral     Isopropylamine salt       Acute LD50     > 398            Beavers (1983a)
                                 10 days      Diet     Isopropyl ester           Acute LC50     > 5930           Palmer &Beavers (1996a)
                                 10 days      Diet     Butoxyethyl ester         Acute LC50     > 5620           Grimes et al. (1990a)
                                 23 days      Diet     Butoxyethanol ester       5-day LC50     > 5000           Hill et al. (1975)
                                 17 days      Diet     Dimethylamine salt        5-day LC50     > 5000           Hill et al. (1975)
                                 10 days      Diet     Diethanolamine salt       Acute LC50     > 5620           Hoxter et al. (1991)
                                 9 days       Diet     Isopropylamine salt       Acute LC50     > 5620           Beavers (1983b)
                                 10 days      Diet     Triisopropanolamine salt  Acute LC50     > 5620           Driscoll et al. (1990a)

    Bobwhite quail               21 weeks     Oral     Dimethylamine salt        Acute LD50     500              Hoxter et al. (1990a)
    (Colinus virginianus)                                                        No mortality   250
                                 11 days      Diet     Isooctylester             Acute LC50     > 5620           Beavers (1984c)
                                 10 days      Diet     Acid                      Acute LC50     > 5620           Culotta et al. (1990b)
                                                                                 No mortality   3160
                                 14 days      Diet     Dimethylamine salt        8-day LC50     > 4640           Fink (1974b)
                                 10 days      Diet     Dimethylamine salt        Acute LC50     > 5620           Long et al. (1990b)
                                                                                 No mortality   3160
                                 23 weeks     Oral     Isopropyl ester           Acute LD50     1879             Palmer & Beavers (1996b)
                                                                                 No mortality   308
                                 19 weeks     Oral     Butoxyethyl ester         Acute LD50     > 2000           Lloyd et al. (1990)
                                 18 weeks     Oral     Diethanolamine salt       Acute LD50     595              Campbell et al. (1991)
                                 27 weeks     Oral     Triisopropanolamine       Acute LD50     405              Culotta et al. (1990c)
                                 10 days      Diet     Isopropyl ester           Acute LC50     > 5930           Palmer & Beavers (1996c)

    Table 7. (continued)

                                                                                                                                         

    Species                Sex   Age          Route    Active ingredient         Exposure       2,4-D conc.a     Reference
                                                                                 parameter
                                                                                                                                         

                                 10 days      Diet     Butoxyethyl ester         Acute LC50     > 5620           Grimes et al. (1990b)
                                 23 days      Diet     Butexyethanol ester       5-day LC50     > 5000           Hill et al. (1975)
                                 23 days      Diet     Dimethylamine salt        5-day LC50     > 5000           Hill et al. (1975)
                                 12 days      Diet     Isopropylamine salt       Acute LC50     > 5620           Beavers (1983c)
                                 10 days      Diet     Diethanolamine salt       Acute LC50     > 5620           Hoxter et al. (1991)
                                 10 days      Diet     Triisopropanolamine salt  Acute LC50     > 5620           Driscoll et al. (1990b)

    Japanese quail         M     2 months     Oral     Acid (technical grade)    Acute LD50     668 (530-842)    Hudson et al. (1984)
    (Coturnix japonica)          14 days      Diet     Acetamide salt            5-day LC50     > 5000           Hill et al. (1975)
                                 12 days      Diet     Butoxyethanol ester       5-day LC50     > 5000           Hill et al. (1975)
                                 20 days      Diet     Dimethylamine salt        5-day LC50     > 5000           Hill et al. (1975)

    Pheasant               F     3-4 months   Oral     Acid (technical grade)    Acute LD50     472 (340-654)    Hudson et al. (1984)
    (Phesianus colchicus)        10 days      Diet     Butoxyethanol ester       5-day LC50     > 5000           Hill et al. (1975)
                                 10 days      Diet     Dimethylamine salt        5-day LC50     > 5000           Hill et al. (1975)

    Chukar partridge       M,F   4 months     Oral     Acid (technical grade)    Acute LD50     200-400          Hudson et al. (1984)
    (Alectoris chukar)

    Rock dove (Columba     M,F                Oral     Acid (technical grade)    Acute LD50     668 (530-842)    Hudson et al. (1984)
    livia)
                                                                                                                                         

    a Oral doses, mg/kg body weight; dietary doses, ppm
    

    studies conducted according to the guidelines of the US Environmental
    Protection Agency, the oral LD50 values for mallard ducks are
    generally high, ranging from > 398 mg/kg for the isopropylamine salt
    (Beavers, 1983a) to > 2000 mg/kg for the sodium salt and acid (Hudson
    et al., 1984). In similar studies, the oral LD50 values for northern
    bobwhite quail ranged from 405 mg/kg for the triisopropanolamine salt
    (Culotta et al., 1990c) to > 2000 mg/kg for the butoxyethyl ester
    (Lloyd et al., 1990). The dietary LC50 values in mallard ducks and
    northern bobwhite quail were all > 5000 ppm, for the isooctyl ester,
    isopropyl ester, butoxyethyl ester, dimethylamine salt, diethanolamine
    salt, isopropylamine salt, triisopropanolamine salt, and acid
    (Beavers, 1983b, 1984b; Driscoll et al., 1990a; Grimes et al., 1990b;
    Long et al., 1990a; Hoxter et al., 1991; Palmer & Beavers, 1996a), and
    the dietary LC50 values for the isopropylamine salt, diethanolamine
    salt, and triisopropanolamine salt in northern bobwhite quail all
    exceeded 5620 ppm, even in a five-day study in 23-day-old birds.
    Similar toxicity was seen for Japanese quail  (Coturnix japonica) and
    pheasants  (Phesianu colchicus) with the acetamide, dimethylamine
    salt, and butoxyethanol ester (Hill et al., 1975).

    2,4-D is less toxic to vertebrates treated in the diet than dosed
    orally. Furthermore, the toxicity of all of the forms tested was low.
    Data submitted by the Industry Task Force II on 2,4-D Research Data
    was consistent with values in the literature. Owing to its good
    solubility in water (23 180 ppm at pH 7), its low log P value (-0.95
    to-0.75 at pH 7), and its rapid excretion after uptake, 2,4-D does not
    tend to accumulate to any significant extent in the environment; owing
    to its low toxicity to birds when given by the dietary route,
    secondary poisoning is unlikely to occur.

    3.3.3.2  Toxicity to birds' eggs

    As reported by WHO (1989,), several studies have been conducted on the
    toxicity of various 2,4-D formulations to birds eggs. No adverse
    effects were found on the hatchability of eggs or on the incidence of
    deformities or mortality of hatched chicks after the eggs of pheasants
    or chickens were sprayed with an isooctyl ester formulation of 2,4-D
    on day 13 of incubation at a dose equivalent to 0.28 kg/ha (Kopischke,
    1972). Similarly, spraying of chicken's eggs before incubation with an
    amine salt of 2,4-D at concentrations of up to 15 times the
    recommended field application rate of 3 kg/ha had no effect on
    hatching success or on the survival of chicks three to four weeks
    after hatching (Somers et al., 1974). Spraying chicken's eggs on day
    0, 4, or 18 of incubation with 2,4-D propylene glycol butyl ethyl
    ester formulation at up to 10 times the field application rate also
    had no effect on the hatchability or on survival or growth of chicks
    after hatching (Somers et al., 1978a). Birds hatched from eggs
    similarly treated by spraying showed no significant adverse effects in
    reproductive performance, i.e. egg laying performance of females and
    testicular weight and sperm count in males (Somers et al., 1978b). No
    effect on egg hatching rate or on body weight or malformation rate was
    seen in chicks after the eggs of Japanese quail, pheasants, and
    chickens were sprayed at 20 kg/ha before incubation or three days

    after the start of incubation (Hilbig et al., 1976a). In a follow-up
    study on the reproductive performance of birds hatched from the
    treated eggs, no effects were seen on laying capacity, fertility, or
    hatchability of the eggs (Hilbig et al., 1976b).

    The effects of 2,4-D dimethylamine salt on the eggs of Japanese quail,
    grey partridge, and red-legged partridge were studied by spraying eggs
    at doses equivalent to the recommended application rate of 1.2 kg/ha
    and at two higher doses equivalent to 2.4 and 6 kg/ha. No effects were
    seen on hatching rate or embryonic or chick mortality during the first
    month after hatching or on embryonic or chick malformations.
    Histopathological examination of partridge thyroids revealed no
    effects. When the residues of 2,4-D were measured in the partridge
    eggs that had received the highest dose, very little 2,4-D was found
    to have penetrated the eggshell; the highest level of residue measured
    was a total egg content of 19.3 µg in an 11-g egg, 15 days after
    treatment. The lack of effect of 2,4-D on sprayed eggs was attributed
    to its poor penetration (Grolleau et al., 1974). No adverse effect of
    2,4-D was seen on hatchability and no increase was seen in
    abnormalities in pheasant or quail chicks from eggs sprayed 24 h
    before hatching with a dose 12 times higher than that recommended for
    application. Only at a dose 30 times higher than the recommended rate
    did hatchability fall by 10-15%, relative to controls. No increased
    incidence of abnormalities was reported at this dose (Spittler, 1976).

    When mallard eggs were immersed for 30 s in aqueous emulsions of
    2,4-D, the calculated LC50 was equivalent to a field application rate
    of 216 (155-300) kg/ha, which is 32 times the recommended field
    application rate (Hoffman & Albers, 1984). Chicken eggs injected with
    10, 100, or 200 mg/kg 2,4-D, equivalent to 0.5, 5, or 10 mg/egg, had
    hatching rates of 80-90%, 70%, and 50% of the solvent control hatch
    rate at the three doses, respectively (Dunachie & Fletcher, 1967). In
    a similar study, an injection of < 1 mg/egg of the dimethylamine
    salt of 2,4-D had no effect, whereas injections of 2 mg/egg reduced
    both hatchability and survival of hatched chicks; 5 mg/egg reduced the
    hatching rate to 15% of control levels, and there were no surviving
    chicks after one week. No hatching occurred after an injection of 10
    mg/egg. When eggs were treated by immersion in solutions of 2,4-D for
    10 s, no effect was seen in a solution of 10 g/L and only a slight
    effect in 50 g/L. Hatching success and survival of chicks up to four
    weeks post-hatch was more than 80% of control values after immersion
    in 50 g/L (Gyrd-Hansen & Dalgaard-Mikkelsen, 1974)œ

    3.3.3.3  Effects on mammals

    The acute toxicity of 2,4-D, its diethanolamine, dimethylamine,
    isopropylamine, and  triisopropanolamine salts, and the 2-butoxyethyl
    and 2-ethylhexyl) esters was determined in rats and rabbits. After
    oral administration, the isopropylamine salt was the least toxic
    (LD50 = 2322 mg ai/kg for male rats and 1646 for female rats; Carreon
    et al., 1983), and the LD50 for the acid was 699 mg ai/kg (Myer,
    1981a). The LD50 values for all of the other salts and esters fell
    between these ranges; those for the dimethylamine salt and ethyhexyl

    esters were 863 and 896 mg/kg bw, respectively (Myer, 1981 b; Jeffrey,
    1987a; Berdasco et al., 1989a; Schults et al., 1990a). After dermal
    administration to rabbits, none of the salts or esters was toxic, with
    LD50 values > 2000 mg ai/kg (Myer, 1981c,d,e; Carreon et al., 1983;
    Jeffrey, 1987b; Berdasco et al., 1989b; Schults et al., 1990b). After
    administration by inhalation, the triisopropanolamine salt was the
    least toxic, with an LC50 > 10.7 mg ai/L (Nitschke & Stebbins,
    1991); all of the other salts and esters had LC50 values of
    > 1.79-5.39 mg ai/L (Streeter & Young, 1983; Auletta & Daly, 1986;
    Streeter et al., 1987; Jackson & Hardy, 1990; Cieszlak, 1992).

    3.3.3.4  Effects on amphibia

    The results of studies conducted according to the CoE/EPPO and US
    guidelines on the toxicity of 2,4-D to amphibia are shown in Table 8
    (Palmer & Krueger, 1997e,f). LC50 values for 2,4-D ethythexyl ester
    could not be determined owing to its rapid hydrolysis and very low
    solubility in water. The 96-h acute LC50 values based on measured
    concentrations were 359 mg/L for the 2,4-D acid and 337 mg/L for the
    dimethylamine salt. The 96-h LC50 for the acid in Indian toads 
     (Bufo melanostictus) was 8.05 mg/L, and the 24-h LC50 was 13.77
    mg/L (Vardia et al., 1984); the 48-h value in common frogs  (Rana 
     temporaria) was 50 mg/L (Cooke, 1972), while the amine salt was
    relatively nontoxic, with 96-h LC50 values of 200-288 mg ai/L
    (Johnson, 1976). Although the studies on amphibia are limited to only
    a few species, 2,4-D acid appears to be more toxic in these species
    than the dimethylamine salt.

    4.  Risk assessment based on agricultural use

    The information on use and application rates used in this risk
    assessment refers to the agricultural use of 2,4-D within the European
    Union and the United States. It should be possible to extrapolate the
    assessment to other agricultural uses at similar application rates
    elsewhere in the world. 2,4-D can be formulated in a variety of
    different salts (e.g. dimethylamine, sodium, diethanolamine,
    triisopropanolamine, and isopropylamine salts) and esters (e.g. 2-
    ethylhexyl and butoxyethyl), but the dimethylamine salt and ethylhexyl
    ester account for about 95% of the global use of 2,4-D (personal
    communicaton from the Industry Task Force II on 2,4-D Research Data).
    The following risk assessment is therefore restricted to these forms
    of 2,4-D. As all forms are rapidly transformed to the acid, data for
    the dimethylamine salt and ethylhexyl ester can be converted to the
    acid equivalent by multiplying the value by the following molecular
    mass-based correction factor:

                           molecular mass of 2,4-D acid (221)
    Correction factor =                                          
                         molecular mass of dimethylamine (265) or
                              ethylhexylester (333)


        Table 8. Toxicity of 2,4-D to amphibia (tadpoles)

                                                                                                                                              

    Organism             Flow/   Temperature   Alkalinity   Hardness   pH     Active ingredient   Exposure    2,4-D conc.  Reference
                         static  (°C)                                                             parameter   (mg ai/L)
                                                                                                                                              

    Leopard frog                 22                                           Acid                48-h LC50   462a         Palmer & Krueger 
    (Ranapipiens)                                                                                 72-h LC50   445a         (1997e)
                                                                                                  96-h LC50   359a
                                 22                                           Dimethylamine salt  48-h LC50   480a         Palmer & Krueger 
                                                                                                  72-h LC50   376a         (1997f)
                                                                                                  96-h LC50   337a

    Indian toad (Bufo            25            210          220        8.3    Free acid           24-h LC50   13.77        Vardia et al. 
    melanostictus)                                                                                48-h LC50   9.03         (1984)
                                                                                                  96-h LC50   8.05

    Frog                 Static  21-22                                        Amine salt          24-h LC50   321          Johnson (1976)
    (Limnodynastes                                                                                48-h LC50   300
    peroni)                                                                                       96-h LC50   287

    Toad (Bufo           Static  21-22                                        Amine salt          24-h LC50   346          Johnson (1976)
    marinus)                                                                                      48-h LC50   333
                                                                                                  96-h LC50   288

    Common frog (Rana            17-29                                        Free acid           48-h LC50   > 50         Cooke (1972)
    temporaria)
                                                                                                                                              

    a Reliable data based on measured concentrations
    

    In order to convert data for the acid to the salt or ester equivalent,
    the acid value should be multiplied by the inverse of the above
    correction factor.

    The main uses of 2,4-D are shown in Table 9. It can be applied by
    either conventional tractor-mounted or -drawn hydraulic sprayers or by
    air, for instance in forestry use. This risk assessment is based on
    the following applications (converted to the dimethylamine salt or
    ethylhexyl ester equivalent), which are representative of real maximum
    application rates:

    Single ground-based           2.69 kg/ha  dimethylamine salt
    hydraulic application
    (maximum rate = 2.24 kg       3.37/ha kg ethylexyl ester
    2,4-D acid/ha)

    Single aerial application     5.37 kg/ha dimethylamine salt
    (maximum rate = 4.48 kg       6.75 kg/ha ethylhexyl ester
    2,4-D acid/ha)

    Aquatic weed control          water concentration, 1.13-4 mg/L
                                  dimethylamine salt

    The risk assessment is based on the principle of calculating
    toxicity:exposure ratios (TERs; see Figure 1) from the CoE/EPPO
    Environmental Risk Assessment models and trigger values.

    4.1  Microorganisms

    The most important sources of exposure of soil microorganisms to 2,4-D
    are likely to be ground or aerial applications at maximal individual
    or seasonal rates of up to 4.28 kg/ha dimethylamine salt or 5.4 kg/ha
    ethylhexyl ester. In studies in the laboratory, concentrations of
    2,4-D (form unstated) of < 10 µg/g soil had no effect on soil
    respiration or nitrification and were not toxic to various
    denitrifying microorganisms, and concentrations up to 25 µg/g soil had
    no effect on soil bacteria, fungi, or actinomyces. These
    concentrations are equal to application rates of 7.5 and 18.75 kg
    2,4-D/ha, respectively, based on a soil depth of 5 cm and a soil
    density of 1.5 g/cm3 and correspond to about 1.4 and 4.7 times the
    maximum recommended application rate (both single and seasonal). The
    risk to soil microorganisms from use of 2,4-D should thus be low. In
    another study, applications of the dimethylamine salt and isooctyl
    ester at rates corresponding to 0.95 kg 2,4-D/ha, resulted in 10-15%
    and 27-29% reductions in populations of soil bacteria, fungi, and
    actinomyces, respectively. As the trigger for concern in the CoE/EPPO
    microorganism risk assessment scheme is > 30%, the risk to soil
    microorganisms from use of 2,4-D is low. This is reinforced by the
    reports in section 5.3 that little or no residue of 2,4-D is found in
    soils in the field.

    FIGURE 1

        Table 9. Main uses of 2,4-D, in kg/ha of acid

                                                                                        

    Crop             Labelled             Typical                     Maximum seasonal
                     application rate     application rate            application rate
                                                                   
                                          USAa     European Unionb
                                                                                        

    Cereals          0.28-1.4             0.49     0.41-0.9           2.0
    Corn             0.28-1.4             0.50     1.2                3.4
    Sorghum          0.28 1.1             0.50                        1.1
    Soya beans       0.56-1.1             0.53                        1.1
    Sugar cane       1.1-2.2              0.90                        4.5
    Rice             0.56-1.7             1.1                         1.7
    Pasture          0.56-2.2             0.63     0.9-1.5            4.5
    Top fruit        0.56-2.2             1.2                         4.5
    Turf             1.1-2.2              -        0.9-1.5            4.5
    Non crop land    0.56-4.5             1.2                         4.5
    Fallow/stubble   0.56-3.4             -        0.9-2.9            2.2
    Forests          0.56-4.5             2.5                         4.5
    Aquatic weeds    2-4 mg/L in          1.13 mg/L
                     treated water
                                                                                        

    a typical average rate
    b European usage
    

    4.2  Aquatic organisms

    The main potential sources of risk to aquatic organisms from the use
    of 2,4-D are overspray during aerial use at 5.37 kg dimethylamine
    salt/ha or 6.75 kg ethylhexyl ester/ha (4.48 kg 2,4-D acid
    equivalents/ha), spray drift from ground-based hydraulic applications
    at 2.69 kg dimethylamine salt/ha or 3.37 kg ethylhexyl ester/ha (2.24
    kg 2,4-D acid equivalents/ha), or use to control aquatic weeds at UP
    to 5 mg dimethylamine salt/L treated water. For each of these
    situations, the predicted environmental concentration (PEC) in a 30-cm
    static surface-water body arising from ground-based spray drift 1 m
    from the edge of a spray boom (from Ganzelmeier et al., 1995) or
    aerial overspray, was calculated from the European Union model, as
    follows:

                    maximum application rate (kg 2,4-D/ha) × A 
                                  (% spray drift)
    PEC (mg/L) =                                                
                                        300

    where A = 5 for ground-based hydraulic spray applications 1 m from
    edge of boom and 100 for overspray from aerial applications.

    4.2.1  Acute risk to freshwater pelagic organisms

    The results of toxicity tests reported in section 6.2 shows that the
    ethylhexyl ester is significantly more toxic to aquatic organisms than
    the acid. Section 4.2.3 indicates that the ethylhexyl ester degrades
    rapidly to 2,4-D acid in water, with a reported DT50 of 6.2 h
    (section 4.2.1). Therefore, the risk to aquatic organisms from the
    ethylhexyl ester is somewhere between that posed by the ester and that
    of the less toxic acid. In order to take into consideration both of
    these risks, the inital assessment is based on the worst-case scenario
    for the ethylhexyl ester; if a risk is identified, a further
    assessment is carried out for the acid. Similarly for algae, 2,4-D
    acid is about an order of magnitude less toxic than either the
    dimethylamine salt or the ethylhexyl ester. Therefore, when the data
    on the ester or salt indicate a risk to algae, it is re-assessed on
    the basis of data for the acid. In contrast, 2,4-D acid is an order of
    magnitude more toxic to fish than the dimethylamine salt, which,
    however, degrades rapidly to the acid in water. Therefore, the risk to
    fish from the dimethylamine salt is re-assessed with data for the acid
    when the TER for the dimethylamine is less than an order of magnitude
    higher than the EPPO trigger value.

    The acute LC50 and EC50 values for the most sensitive fish (Tables 5
    and 6) were 250 mg/L dimethylamine salt and 7.2 mg/L ethylhexyl ester,
    with a 96-h LC50 as low as 1 mg/L for the ester. The values for the
    most sensitive aquatic invertebrate (Tables 3 and 4) were 184 mg/L for
    the dimethylamine salt and 5.2 mg/L for the ester. Although an LC50
    of 4.0 mg/L for the dimethylamine salt to  Daphnia has been reported,
    the more recent results of studies conducted according to guidelines
    and good laboratory practice were used. The values for the most
    sensitive algal species (Table 2) were 5.28 mg/L dimethylamine salt
    and 4.1 mg/L ethylhexyl ester, and those for the most sensitive
    aquatic plant species were 0.58 mg/L dimethylamine salt and 0.5 mg/L
    ethylhexyl ester.

    The acute LC50 and EC50 values for the most sensitive aquatic
    organisms to 2 4-D acid (Tables 3-6) were 26.7 mg/L for fish, 79 mg/L
    for aquatic invertebrates, and 29 mg/L for algae.

    4.2.1.1  Spray drift from ground-based applications

    The acute PEC for spray drift (1 m from the edge of a spray boom,
    based on the assumptions listed in section 7.2) into a 30-cm static
    water body at the maximum application rate (see above) is 0.045 mg/L
    for the dimethylamine salt and 0.056 mg/L for the ethylhexyl ester.
    Therefore, the TERs based on such PECs and the LC50 and EC50 values
    for the dimethylamine and ethylhexyl forms of 2,4-D, respectively are:
    fish, 5555 and 128 (22.2 from published data); aquatic invertebrates,
    4088 and 93; algae, 117 and 73; and higher aquatic plants, 12.9 and
    8.9. On the basis of the CoE/EPPO risk assessment scheme for aquatic
    organisms, these TERs (> 10) indicate a low acute risk to fish,
    aquatic invertebrates, and algae; however, for higher aquatic plants,
    the TER for the dimethylamine salt indicates a low risk but that for

    the ethylhexyl ester indicates a potential risk. The ethylhexyl ester
    is, however, rapidly degraded to the acid in water. The acid was less
    toxic to  Lemna, with a 14-day EC50 of 3.3 mg/L, which gives a TER
    of 88.4 on the basis of a PEC of 0.037 mg/L, and indicates a low risk
    to higher aquatic plants.

    4.2.1.2  Overspray from aerial spray applications

    The acute PEC for aerial overspray in a 30-cm static water body at the
    maximum application rate on the basis of the assumptions listed in
    section 7.2, is 1.79 mg/L dimethylamine salt or 2.25 mg/L ethylhexyl
    ester. The TERs for the dimethylamine and ethylhexyl forms of 2,4-D,
    respectively, based on these PECs and the LC50 and EC50 values given
    above are: fish, 140 and 3.2 (1.8 in published data); aquatic
    invertebrates, 103 and 2.3; algae, 3.0 and 1.8; and higher aquatic
    plants, 0.32 and 0.22. On the basis of the CoE/EPPO risk assessment
    scheme for aquatic organisms, the TERs for the dimethylamine indicate
    a low acute risk to both fish and aquatic invertebrates (i.e. TERs
    >10); however, the TERs for the dimethylamine to algae and higher
    aquatic plants and those for the ethylhexyl ester to all aquatic
    organisms indicate a high acute risk. As both the esters and salts of
    2,4-D are rapidly degraded to the acid, the risk was re-assessed on
    the basis of data for 2,4-D acid, giving a PEC of 1.49 mg/L and TERs
    of 17.9 for fish, 53 for aquatic invertebrates, 19.4 for algae, and
    2.2 for higher aquatic plants  (Lemna), indicating low acute risks to
    fish, aquatic invertebrates, and algae but a potential risk to higher
    aquatic plants from aerial use of 2,4-D.

    4.2.1.3  Aquatic weed control

    For the control of aquatic weeds, water can be treated with up to 4
    mg/L 2,4-D. Use of the ethylhexyl ester is not recommended for aquatic
    weed control. On the basis of the PEC and the LC50 and EC50 values
    for the dimethylamine, the TERs are: fish, 50; aquatic invertebrates,
    36.8; algae, 1.06; and higher aquatic plants, 0.12. On the basis of
    the CoE/EPPO risk assessment scheme for aquatic organisms, these TERs
    indicate a low acute risk to fish and aquatic invertebrates; however,
    the TERs for the dimethylamine salt to algae and aquatic plants and
    the TERs for the ethylhexyl ester to all aquatic organisms indicate a
    high acute risk. As both the esters and salts of 2,4-D are rapidly
    degraded to the acid, the risk was re-assessed on the basis of data
    for 2,4-D acid, giving a PEC of 4 mg/L and TERs of 5.8 for algae and
    0.8 for higher aquatic plants. These TERs still indicate a potential
    risk; however, since the application is for the control of aquatic
    weeds, the risk can be ignored. The low TER for fish indicates that
    the use may pose an acute risk; however, that risk should be balanced
    against the risks of other alternatives, such as not controlling weeds
    (e.g. algal bloom leading to water deoxygenation) and the potential
    damage caused by manual weed control, which may both pose higher risks
    to fish and other aquatic organisms.

    4.2.2  Long-term risk to freshwater pelagic organisms

    The long-term NOEC values reported in Table 5 for the most sensitive
    aquatic species tested were 17.1 mg/L for dimethylamine salt and 0.12
    mg/L for the ethylhexyl ester for fish at early life stages and 27.5
    mg/L dimethylamine salt and 0.015 mg/L ethylhexyl ester for aquatic
    invertebrates. As both the ester and the salt degrade rapidly in water
    to 2,4-D acid (e.g. the DT50 for the ester is 6.2 h), the assessment
    of long-term risk was based on data on the long-term NOECs of the
    acid, i.e. 63.4 mg/L for the most sensitive fish (Table 5) and 79 mg/L
    for aquatic invertebrates (Table 3).

    4.2.2.1  Levels of 2,4-D acid in surface water

    Instead of using PECs in this risk assessment, actual field levels of
    2,4-D (assumed to be the acid) in various surface waters (as reported
    in section 5.2) were used. The levels of the acid in surface waters
    generally ranged from 0.00008 mg/L (in a small watershed in
    Saskatchewan, Canada) to 0.0021 mg/L (in ground- and surface waters in
    the United Kingdom). Although a higher level, 0.029 mg/L, was reported
    in Saskatchewan, it was attributed to back-siphoning of spray
    solution. As this source of contamination is considered to be
    accidental, it was not used in this risk assessment, which addresses
    the risk from normal or good agricultural use. The TERs based on the
    measured levels of 2,4-D in surface water and the NOEC values are all
    well in excess of the CoE/EPPO trigger of concern (i.e. TERs > 10 for
    fish and aquatic invertebrates) and indicate a low risk to these
    organisms.

    4.2.2.2  Aquatic weed control

    Water can be treated with up to 4 mg/L of 2,4-D for the control of
    aquatic weeds. On the basis of this PEC, the TERs based on the NOEC
    for 2,4-D acid are 15.4 for fish and 19.7 for aquatic invertebrates,
    indicating low risks.

    4.2.3  Risk to sediment-dwelling invertebrates

    2,4-D partitions and can persist in aquatic sediments, particularly
    under anaerobic conditions. Levels up to 0.17 mg/kg sediment (assumed
    to be 2,4-D acid) were found in a pond three days after application of
    8.86 kg/ha of the dimethylamine salt (section 4.2.4). As the highest
    seasonal application rate is 4.48 kg 2,4-D acid equivalents/ha, which
    is about half the rate used in the above study, the level of 2,4-D
    would be 0.085 mg/kg sediment. On the basis of this PEC and the LC50
    of 86.7 mg 2,4-D acid/L for the most sensitive sediment-dwelling
    invertebrate species tested, the oligochaete worm  Lumbriculus 
     variegatus, the TER for sediment-dwelling invertebrates would be
    1020, indicating a low risk.

     Risk to amphibia

    The LC50 values shown in Table 8 for tadpoles of a variety of frog
    species range from 8.05 to 359 mg 2,4-D acid equivalents/L. On the
    basis of the lowest LC50 value of 8.05 mg/L and PEC values of 0.037,
    1.49, and 5 mg/L 2,4-D acid for spray drift from ground-based
    applications, aerial applications, and aquatic weed control,
    respectively, the TERs are 214, 5.36, and 1.1, indicating a low risk
    to amphibia; however, contamination of surface waters arising from
    overspray during aerial application and aquatic weed control might
    pose a risk to amphibia, as indicated by TERs < 10. The risk from
    aquatic weed control should be balanced against the risks from other
    alternatives, however, such as not controlling weeds (e.g. algal bloom
    leading to water deoxygenation) and potential damage caused by manual
    weed control.

    4.2.5  Bioaccumulation

    No data were submitted on bioaccumulation in fish. Residues of < 5-
    102 µg/kg of 2,4-D have been monitored, indicating a mimimal risk of
    bioaccumulation.

     Terrestrial organisms

     Plants

    2,4-D is a translocated, selective herbicide used to control a variety
    of broad-leaved weeds (section 6.3.1). Consequently, any risk to
    broad-leaved non-target plants is to be expected from its mode of
    action and consequent area of use. 2,4-D has been reported to cause
    phytotoxic damage to neighbouring crops, attributed to vapour drift;
    however, such drift was associated mainly with use of the volatile
    ester forms of 2,4-D. Use of the butyl ester is now restricted and
    continues to decline globally, in part due to the campaigning by the
    registrants (personal communication from Industry Task Force II on
    2,4-D Research Data).

    4.3.2  Invertebrates

    4.3.2.1  Bees

    Bees may be exposed to 2,4-D while they forage on flowering weeds in
    treated crops; the main uses of 2,4-D are not on flowering crops. The
    LD50 values for dimethylamine salt and the ethylhexyl ester in
    honeybees were > 100 µg/bee for both oral and contact exposure. On
    the basis of the maximum individual application rate of 4.48 kg 2,4-D
    acid equivalents/ha, the hazard quotient would be < 45. As the
    CoE/EPPO trigger for concern is a hazard quotient > 50, the acute
    risk to honeybees from use of 2,4-D at high application rates should
    be low. Studies in which these forms of 2,4-D were fed to bees in
    syrup showed no effect on survival at 100 mg/L. In field studies,
    application of 2,4-D at 2.24 kg/ha had no significant effect on caged
    foraging bees. In view of the restricted exposure of bees, the risk

    should be low. Furthermore, 2,4-D has not been implicated in any
    incidents of poisoning of honeybees in the Wildlife Incident
    Investigation scheme in the United Kingdom (personal communication
    from the Industry Task Force II on 2,4-D Research Data).

    4.3.2.2  Other non-target arthropods

    Few of the studies used in the risk assessment were conducted
    according to internationally recognized guidelines or good laboratory
    practices, and most predate the use of such standards by a
    considerable time. Additionally, the test substances were usually not
    adequately characterized or identified and in some cases were
    mixtures. Non-target arthropods may be exposed to 2,4-D during any of
    its various agricultural and non-agricultural uses. Standard
    laboratory tests conducted with a mixture of 2,4-D acid and
    dimethylamine salt (2.8 kg 2,4-D/ha) and MCPA reduced the fecundity of
    the parasitic wasp  Trichogramma cacoeciae by 38.9%, caused a
    transient 55% drop (not significant over the whole test period) in the
    feeding rate of the carabid beetle  Poecilus cupreus, and had no
    significant effect on the staphylinnid beetle  Aleochara bilineata. 
    Doubled pre-pupal mortality and increased development time were seen
    in larval coccinellids sprayed directly with 2,4-D acid at an
    application rate of 0.56 kg/ha. Similarly, mortality rates of 36-96.4%
    were seen among wheat-stem sawflies at a concentration of 0.3% of the
    mixed isopropyl and butyl esters of 2,4-D; a mortality rate of 50% was
    reported for  Bembidion femoratum and  B. ustulatum at a rate equal
    to 10 kg/ha, i.e. 2.5 times the maximum recommended rate, for the
    predatory mites  Neoseiulus fallacis and  Tetranychus utricae after
    applications of 0.22 and 0.98 kg/ha of the amine salt, respectively,
    and for adult millipedes exposed to 33.6 kg/ha (8.4 times the maximum
    recommended application rate). No significant effect was reported on
    thistle-rosette weevils treated with various amine salts of 2,4-D at a
    rate of 1.68 kg/ha or on leafy spurge hawk moth larvae treated at 2.2
    kg/ha; and in field studies, no significant effects on epigeal
    predator fauna (staphylinids, carabids, and spiders) were seen with
    application of 2,4-D at 1.25 kg/ha. Similarly, single and repeated
    field applications of 2,4-D (rate unstated) resulted in little or no
    effect on soil collembolans or mites.

    The risk assessment is limited by the standard of the available data.
    The CoE/EPPO and International Organization for Biological Control of
    Noxious Animals and Plants trigger for concern with regard to
    non-target arthropods, is an effect > 30%. The limited laboratory
    data indicate that 2,4-D may have insecticidal activity, particularly
    at high application rates, and hence may pose a risk to this
    compartment of the terrestrial environment. Limited field data at
    lower, more typical rates of application (< 1.25 kg/ha) indicate,
    however, that this risk may not be realized in the field.

    4.3.2.3  Earthworms

    Earthworms may be exposed after single or multiple applications of
    2,4-D to a wide variety of crops but particularly after its use on
    grass, fallow land, and stubble. On the basis of a maximum application
    rate of 5.37 kg 2,4-D dimethylamine salt/ha, a soil depth of 5 cm, and
    a soil density of 1.5 g/cm3, the PEC for the dimethylamine salt is
    7.2 mg/kg soil. A study in  Eisenia foetida with the dimethylamine
    salt gave a 14-day LC50 of 350 mg/kg soil. The TER for earthworms
    would thus be 49, which is above the CoE/EPPO trigger value of 10. The
    acute risk to earthworms of the use of the dimethylamine should
    therefore be low. This conclusion is reinforced by the reported levels
    of 2,4-D in soil (section 5.3), which are well below the PEC used in
    the above calculation.

    4.3.3  Vertebrates

    Vertebrates are likely to be exposed to 2,4-D while grazing on treated
    or contaminated vegetation or eating contaminated insects. The
    estimated residues on food items represent the maximum values for
    pesticides immediately after application and do not take into
    consideration the degradation of 2,4-D in the environment (which is
    rapid). Furthermore, it is assumed that all food consumed contains the
    maximum residue levels. For the purpose of this risk assessment, the
    worst-case maximum application rates of 5.37 kg dimethylamine salt/ha
    and 6.75 kg ethylhexyl ester/ha (4.48 kg 2,4-D acid equivalents/ha)
    from aerial application and 2.69 kg dimethylamine salt/ha and 3.37 kg
    ethylhexyl ester/ha (2.24 kg 2,4-D acid equivalents/ha) for
    ground-based and aerial spraying of pastures were used.

    4.3.3.1  Birds

    Table 7 gives LD50 values of 500 mg/kg bw for the dimethylamine salt
    and 663 mg/kg bw for the isooctyl ester for bobwhite quail and mallard
    ducks, respectively, the most sensitive species tested. The dietary
    LC50 values for the mallard duck, the most sensitive species tested,
    were > 4640 ppm dimethylamine salt and > 5620 ppm isooctyl ester.
    The indicator birds used in the risk assessment were a 3-kg greylag
    goose  (Anser anser), with a total daily food consumption of 900 g
    vegetation, as the grazing bird (Owen, 1975) and an 11-g blue tit
     (Parus caeruleus), with a total daily food consumption of 8.23 g, as
    the small insectivorous bird (Kenaga, 1973).

     (a) Aerial applications (4.48 kg 2,4-D acid equivalents/ha for 
     insectivorous birds and 2.24 kg 2,4-D acid equivalents/ha for 
     grazing birds)

     Grazing birds: The residues on short grass measured initially were
    either 290 mg/kg of grass for 2,4-D dimethylamine salt or 459 mg/kg
    for the ethylhexyl ester. If a 3 -kg greylag goose fed exclusively on
    contaminated grass, it would ingest 261 mg dimethylamine salt or 413
    mg ethylhexyl ester, equal to 87 mg/kg bw of the salt and 138 mg/kg bw
    of the ester. Consequently, the acute oral TERs would be 5.7 for the

    salt and 4.8 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating a high risk to grazing birds from aerial
    applications of both 2,4-D dimethylamine salt and ethylhexyl ester.
    The short-term results of feeding studies, which represent more
    realistic exposure, would give TERs, based on the LC50 values, of
    > 16 and > 12 for the salt and ester forms, respectively. These TERs
    are above the CoE/EPPO trigger for high risk but may be below the
    trigger for medium risk (< 100). As the residues in grass on day 7
    had declined to between one-half and one-third of those on day 0 and
    as 2,4-D has not been implicated in incidents of poisoning in birds
    when used normally (UK Wildlife Incident Investigation Scheme), the
    risk to grazing birds from 2,4-D is unlikely to be high (personal
    communication from the Industry Task Force II on 2,4-D Research Data).

     Insectivorous birds: The initial residues on small insects after
    aerial overspray (based on 29 times the application rate in kg/ha as
    mg/kg; CoE/EPPO vertebrate risk assessment scheme) would be 156 mg/kg
    insects for the dimethylamine salt or 196 mg/kg for the ethylhexyl
    ester. On the basis of the values for acute oral toxicity, if an 11-g
    blue tit fed exclusively on contaminated insects, it would ingest 1.3
    mg of the salt or 1.6 mg of the ester, equal to 118 and 145 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 4.2 for the
    salt and 4.6 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating a high risk. The short-term results of feeding
    studies, which represent more realistic exposure, would give TERs,
    based on the LC50 values, of > 30 and > 29 for the salt and the
    ester, respectively. These TERs are just below the CoE/EPPO trigger
    for medium risk, but it should be noted that they are given as
    'greater than'. As 2,4-D is more likely to be used in early growth
    stages, it is likely that large insects dominate the diet of these
    birds. As the initial residues on large insects are an order of
    magnitude lower than on small ones, the acute oral TERs would rise to
    46 for the salt and 42 for the ester, and the short-term dietary TERs
    would rise to > 300 and > 290, respectively. Therefore, the acute
    risk to small insectivorous birds from aerial applications of 2,4-D
    dimethylamine salt or ethylhexyl ester is considered to be low. It
    should be noted that 2,4-D has not been implicated in incidents of
    poisoning in birds when used normally (UK Wildlife Incident
    Investigation Scheme), which confirms that the risk to insectivorous
    birds 2,4-D is low (personal communication from the Industry Task
    Force II on 2,4-D Research Data).

     (b) Ground-based application of 2.24 kg 2,4-D acid equivalents/ha

     Grazing birds: The residues measured initially on short grass after
    ground-based applications were 290 mg/kg grass of 2,4-D dimethylamine
    salt and 459 mg/kg ethylhexyl ester. If a 3-kg greylag goose fed
    exclusively on such contaminated grass, it would ingest 261 mg of the
    salt or 413 mg of the ester, equal to 87 and 138 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 5.7 for the
    salt and 4.8 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating a high risk to grazing birds from aerial
    applications of both 2,4-D dimethylamine salt and ethylhexyl ester.

    The short-term results of feeding studies, which represent more
    realistic exposure, would give TERs, based on the LC50 values, of
    > 16 and > 12 for the salt and ester forms, respectively. These TERs
    are above the CoE/EPPO trigger for high risk but may be below the
    trigger for medium risk (< 100). As the residues in grass on day 7
    had declined to between one-half and one-third of those on day 0 and
    as 2,4-D has not been implicated in incidents of poisoning in birds
    when used normally (UK Wildlife Incident Investigation Scheme), the
    risk to grazing birds from 2,4-D is unlikely to be high (personal
    communication from the Industry Task Force II on 2,4-D Research Data).

     Insectivorous birds: The initial residues on small insects after
    aerial overspray (based on 29 times the application rate in kg/ha as
    mg/kg; CoE/EPPO vertebrate risk assessment scheme) would be 78 mg/kg
    insects of the dimethylamine salt or 97.7 mg/kg ethylhexyl ester. On
    the basis of the values for acute oral toxicity, if an 11-g blue tit
    fed exclusively on contaminated insects, it would ingest 0.64 mg of
    the salt or 0.8 mg of the ester, equal to 58.2 and 72.7 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 9.1 for the
    salt and 8.6 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating a potential risk to insect-eating birds. The
    short-term results of feeding studies, which represent more realistic
    exposure, would give TERs, based on the LC50 values, of > 59.5 and
    > 57.5 for the salt and the ester, respectively. These TERs may be
    below the CoE/EPPO trigger for medium risk (< 100), but it should be
    noted that they are given as 'greater than'. As 2,4-D is more likely
    to be used in early growth stages, it is likely that large insects
    dominate the diet of these birds. As the initial residues on large
    insects are an order of magnitude lower than on small ones, the acute
    oral TERs would rise to 86 for the salt and 91 for the ester, and the
    short-term dietary TERs would rise to > 300 and > 290, respectively.
    Similarly, the short-term TERs would rise to > 595 for the salt and
    > 755 for the ester. As these revised dietary TERs are > 100, the
    acute risk to small insectivorous birds from ground-based application
    of both 2,4-D dimethylamine salt or ethylhexyl ester is considered to
    be low (personal communication from the Industry Task Force II on
    2,4-D Research Data).

    4.3.3.2  Mammals

    LD50 values of 863 mg/kg bw for the dimethylamine salt and 896 mg/kg
    bw for the ethylhexyl ester were reported for the rat, the most
    sensitive species tested. The indicator mammals used in the risk
    assessment were a 1200-g rabbit  (Oryctolagus cuniculus), with a
    total dally food consumption of 500 g vegetation (Ross, personal
    communication), as the grazing mammal and an 18-g shrew  (Sorex 
     araneus), with a total daily food consumption of 18 g (Churchfield,
    1986), as the small insectivorous mammal.

     Aerial applications (4.48 kg 2,4-D acid equivalents/ha for 
     assessing the risk to insectivorous mammals and 2.24 kg/ha for 
     assessing the risk to grazing mammals)

     Grazing mammals: The residues measured initially on short grass
    after aerial applications were 290 mg/kg grass for 2,4-D dimethylamine
    salt and 459 mg/kg for the ethylhexyl ester. If a 1200-g rabbit fed
    exclusively on such contaminated grass, it would ingest 145 mg of the
    salt or 230 mg of the ester, equal to 121 and 192 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 7.1 for the
    salt and 4.7 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating a high risk to grazing mammals from aerial
    applications of both 2,4-D dimethylamine salt and ethylhexyl ester. As
    the residues in grass on day 7 had declined to between one-half and
    one-third of those on day 0 and as 2,4-D has not been implicated in
    incidents of poisoning in birds when used normally (UK Wildlife
    Incident Investigation Scheme), the risk to grazing mammals from 2,4-D
    is unlikely to be high (personal communication from the Industry Task
    Force II on 2,4-D Research Data).

     Insectivorous mammals: The initial residues on large insects after
    aerial overspray (based on 2.7 times the application rate in kg/ha as
    mg/kg; CoE/EPPO vertebrate risk assessment scheme) would be 14.5 mg/kg
    insects for the dimethylamine salt or 18.2 mg/kg for the ethylhexyl
    ester. On the basis of the values for acute oral toxicity, if an 18-g
    shrew fed exclusively on contaminated insects, it would ingest 0.26 mg
    of the salt or 0.33 mg of the ester, equal to 14.4 and 18.3 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 60 for the
    salt and 49 for the ester. These TERs are below the CoE/EPPO trigger
    value of 100, indicating a medium risk to insect-eating mammals from
    aerial application of 2,4-D, but it should be noted that 2,4-D has not
    been implicated in incidents of poisoning in mammals when used
    normally (UK Wildlife Incident Investigation Scheme), suggesting that
    the risk to insectivorous mammals from 2,4-D is unlikely to be high.
    (personal communication from the Industry Task Force II on 2,4-D
    Research Data)

     (b) Ground-based application of 2.24 kg 2,4-D acid equivalents/ha 

     Grazing mammals: The residues measured initially on short grass
    after ground-based applications were 290 mg/kg grass for 2,4-D
    dimethylamine salt and 459 mg/kg for the ethylhexyl ester. If a 1200-g
    rabbit fed exclusively on such contaminated grass, it would ingest 145
    mg of the salt or 230 mg of the ester, equal to 121 and 192 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 7.1 for the
    salt and 4.7 for the ester. These TERs are below the CoE/EPPO trigger
    value of 10, indicating, a high risk to grazing mammals from
    ground-based applications of both 2,4-D dimethylamine salt and
    ethylhexyl ester. As the residues in grass on day 7 had declined to
    between one-half and one-third of those on day 0 and as 2,4-D has not
    been implicated in incidents of poisoning in birds when used normally
    (UK Wildlife Incident Investigation Scheme), the risk to grazing

    mammals from 2,4-D is unlikely to be high (personal communication from
    the Industry Task Force II on 2,4-D Research Data).

     Insectivorous mammals: The initial residues on large insects after
    aerial overspray (based on 2.7 times the application rate in kg/ha as
    mg/kg; CoE/EPPO vertebrate risk assessment scheme) would be 7.26 mg/kg
    insects for the dimethylamine salt or 9.1 mg/kg for the ethylhexyl
    ester. On the basis of the values for acute oral toxicity, if an 18-g
    shrew fed exclusively on contaminated insects, it would ingest 0.13 mg
    of the salt or 0.16 mg of the ester, equal to 7.2 and 8.9 mg/kg bw,
    respectively. Consequently, the acute oral TERs would be 120 for the
    salt and 101 for the ester. These TERs are above the CoE/EPPO trigger
    value of 100, indicating a low risk to small insect-eating mammals
    from ground-based application of 2,4-D (personal communication from
    the Industry Task Force II on 2,4-D Research Data).

    5.  Evaluation of effects on the environment

    2,4-Dichlorophenoxyacetic acid (2,4-D) is a selective herbicide, which
    is available as the free acid, salts, and esters. It has low
    volatility and should not partition to air after application. Amine
    salt formulations of 2,4-D are less volatile than butyl, ethyl, or
    isopropyl ester formulations.

    The 2-ethylhexyl ester is hydrolysed under alkaline conditions
    (half-life, 48 days at pH 7 and 2.2 days at pH 9). 2,4-D may be
    degraded slowly by photolysis. Its half-life in aqueous solution was
    4.5 days under aerobic conditions and 312 days under anaerobic
    conditions. The major breakdown product was carbon dioxide;
    2,4-dichlorophenol, 2,4-dichloroanisole, and 4-chlorophenoxyacetic
    acid were formed as intermediates. The half-lives of 2,4-D in natural
    waters after aerial application of its dimethylamine salt ranged from
    1.1 to 20 days. 2,4-D formulations were found to be rapidly hydrolysed
    or biodegraded in ponds and lakes. There was no evidence that 2,4-D
    bioaccumulates in aquatic organisms.

    The behaviour of 2,4-D salts and esters in soils is greatly influenced
    by the organic matter content and pH: 2,4-D is more strongly adsorbed
    in soils with a higher organic matter content and/or lower pH values.
    The rapid biodegradation of 2,4-D in soil prevents significant
    downward movement under normal field conditions. Run-off from treated
    soil has been estimated at 0.01 - 1% of the applied 2,4-D; the maximum
    recorded concentration after run-off was about 0.2 µg/L. In
    non-sterile soils, various esters of 2,4-D are hydrolysed very rapidly
    (> 72% within 72 h). A number of microbial organisms rapidly degrade
    2,4-D, with half-lives of 1.25 h to 40 days, usually between 3 and 10
    days. The dimethylamine salt dissociates rapidly, leaving 2,4-D that
    then undergoes further degradation.

    Field trials in the United States with the dimethylamine salt or the
    ethylhexyl ester at 2.24 kg acid equivalents/ha on grass resulted in
    maximum initial residues at day 0 of 120 or 153 mg acid
    equivalents/kg, respectively. These initial residues had decreased by
    one-half to one-third by day 7.

    In general, populations of aerobic bacteria, actinomycetes, and fungi
    in soils were not affected by 2,4-D at 25 ppm. Application of the
    isooctyl ester at a rate of 0.95 kg/ha, however, reduced the
    populations of bacteria by 26.3%, those of fungi by 19.5%, and those
    of actinomycetes by 30%; the dimethylamine salt reduced these
    populations by about half as much.

    2,4-D applied at the maximum recommended rate stimulated (by 10%) the
    growth of  Skeletonema costatum, whereas it inhibited that of
     Navicula pelliculosa (24%) and  Lemna gibba (75%). The five-day
    EC50 values for the toxicity of 2,4-D and its salts and esters differ
    widely with respect to both algal species and compounds, ranging from
    0.23 mg ai/L for the ethylhexyl ester in  Skeletonema costatum to 153
    mg ai/L for the dimethylamine salt in  Anabaena flosaquae. The acute
    toxicity of 2,4-D to the aquatic higher plant  Lemna gibba also
    depended upon the salt or ester used, with 14-day EC50 values of 3.3
    mg/L for 2,4-D, 0.58 mg/L for the dimethylamine salt, and 0.5 mg/L for
    the ethylhexyl ester. At concentrations of 0.001-100 mg/L, 2,4-D had
    no effect on chlorophyll production in several algal species.
     Anabaenopsis raciborskii tolerated up to 800 µg/ml of 2,4-D in
    liquid culture. 

    Many studies have been performed on invertebrate freshwater,
    estuarine, and marine species, including  Daphnia, Gammarus, 
     Macrobranchium, Crassostrea, Palaernonetes, Panaeus, and  Uca, and
    many forms of 2,4-D have been evaluated, including 2,4-D itself, the
    dimethylamine, diethanolamine, isopropylamine, tri-isopropanolamine,
    and sodium salts, and the ethylhexyl, butoxyethyl, and isopropyl
    esters. 2,4-D and its salts are generally less toxic to these
    organisms than the ester forms. The 48-h toxicity (LC50) values to
     Daphnia magna ranged from 5.2 mg ai/L for the isooctyl ester to 184
    mg ai/L for the dimethylamine salt, and the 21-day NOEC values ranged
    from 0.0015 mg ai/L for the ethylhexyl ester to 27.5 mg ai/L for the
    dimethylamine salt and 79 mg ai/L for the acid.

    Grass shrimp avoided water containing concentrations of 1 or 10 mg/L
    of the butoxyethyl ester. In a study covering the life cycle of
     Daphnia magna, the NOEC was 23.6 mg/L diethanolamine salt. In a
    study of the long-term toxicity study of the butoxyethyl ester in
     Daphnia magna, the maximum acceptable toxicant concentration was
     0.70-0.29 mg/L.

    The 96-h LC50 values for frog and toad tadpoles ranged from 8 mg/L
    for the free acid to 477 mg/L for the dimethylamine saltœ

    The effects of 2,4-D and its salts and esters have been studied on
    various growth stages of fish species such as  Oncorhynchus, 
     Lepomis, Pimephales, Gambusia, Micropterus, and  Salmo. Generally,
    2,4-D and its salts are less toxic to fish than are the esters Typical
    96-h LC50 values for adult fish were 5-10 mg ai/L for the isooctyl
    ester, 200-400 mg ai/L for 2,4-D, and 250-500 mg ai/L for the
    diethanolamine salt, although lower figures have been reported. Fish
    in early life stages appear to be more sensitive, with 32-day NOEC
    values ranging from 0.12 mg ai/L for the ethylhexyl ester to 17.1 mg
    ai/L for the diethanolamine salt and 63.4 mg ai/L for the acid.

    The NOEC for embryos and larvae of the fathead minnow,  Pimephales 
     promelas, exposed to < 416.1 µg/L of the butoxyethyl ester for 32
    days was 80.5 µg/L, and the maximum acceptable toxicant concentration
    was estimated to be 96 µg/L. The sodium salt of 2,4-D did not inhibit
    the hatching of carp eggs at 25 mg/L, but at a concentration of 100
    mg/L none hatched. At 50 mg ai/L, the sodium salt was not harmful to
    carp embryos but induced behavioural changes, some morphopathological
    alterations, and, ultimately, death in carp larvae.

    The LD50 values for the dimethylamine salt and ethylhexyl ester for
    the honeybee after oral exposure and contact were > 100 µg/bee. No
    toxic effects have been seen in bees in the field.

    2,4-D (in combination with MCPA) did not harm.  Trichogramma 
     cacoeciae at 1.5% in water or  Aleochara bilineata at the
    recommended rate. Mixed amine salts and mixed isopropyl esters of
    2,4-D were toxic to coccinellid larvae and to sawflies. No
    reproductive effects were observed in European cockroaches reared on
    food containing 1000 mg/kg 2,4-D (unspecified).

    Application of 2,4-D at 1250 g ai/L in field crops did not affect
    staphylinids, carabids, or spiders during a 20-month observation
    period. Some adult millipedes exposed to 2,4-D at a rate of 33.6 kg/ha
    died on the first day, and the rate was 50% higher than that of
    controls by day 7.

    The 14-day LC50 for earthworms exposed to the dimethylamine salt was
    350 mg/kg soil, but no deaths occurred at concentrations < 100 mg
    ai/kg. A 48-h LC50 of 61.6 µg/cm2 was reported for earthworms
    exposed on filter paper.

    The LD50 values for birds are 200 to > 2000 mg/kg bw for mallards,
    bobwhite quail, Japanese quail, pheasants, chukar partridges, and rock
    doves The dietary LC50 values for the acid, salt, and ester exceeded
    4640 mg/kg diet for mallards, bobwhite quail, Japanese quaff, and
    pheasants. Application of formulations at doses greater than the
    recommended rate did not adversely affect the reproductive performance
    of pheasants, quail, partridges, or chickens.

    The oral LD50 values for the acid and its salts and esters in rats
    and rabbits were 699-2322 mg/kg bw. The dermal LD50 value for rabbits
    was > 2000 mg/kg bw, and the LC50 value after inhalation was
    1.8-10.7 mg/L.

    5.1  Risk assessment

    The information on use and application rates used in this risk
    assessment is based on the agricultural use of 2,4-D within the
    European Union and the United States. 2,4-D can be formulated as a
    variety of salts (e.g. dimethylamine, sodium, diethanolamine,
    tri-isopropanolamine, and isopropylamine) and esters (ethylhexyl,
    isooctyl, and butoxyethyl); however, the dimethylamine salt and
    ethylhexyl ester account for about 95% of the global use of 2,4-D.
    This risk assessment is therefore restricted to the use of those
    compounds. Both are rapidly hydrolysed to 2,4-D. The main uses of
    2,4-D include application to cereals, corn, sorghum, soya beans, sugar
    cane, rice, pasture, top fruit, turf, non-cropland, fallow land,
    forests, and aquatic weeds. Applications can be made by either
    conventional tractor-mounted or -drawn hydraulic sprayers or by aerial
    application (e.g. in forests) at rates of 0.25-4.48 kg acid
    equivalent/ha.

    This risk assessment is based on the principle of calculating
    toxicity:exposure ratios (TERs) and on the CoE/EPPO Environmental Risk
    Assessment models and trigger values.

    5.1.1  Aquatic environment

    The main risk to aquatic organisms from the use of 2,4-D is due to
    overspray during aerial use, spray drift from ground-based hydraulic
    applications, or use to control aquatic weeds. The CoE/EPPO risk
    assessment scheme for aquatic organisms showed low acute risk (TERs
    > 10) to fish, aquatic invertebrates, and algae due to spray drift
    arising from ground-based hydraulic applications and to overspray
    during aerial applications. A potential acute risk (TER < 10) to
    higher aquatic plants and amphibia due to overspray during aerial
    application was identified. The use of 2,4-D to control aquatic weeds
    also presents a potential acute risk (TER < 10) to algae and higher
    aquatic plants, but this risk can be ignored as these organisms are
    the targets of 2,4-D used in this way. A potential acute risk to
    amphibia remains from use to control aquatic weeds, but this risk must
    be balanced against those associated with alternative means of aquatic
    weed control, such as no control (e.g. algal bloom leading to water
    deoxygenation) and manual weed control, both of which may pose a
    higher risk to fish and other aquatic organisms. The ethylhexyl ester
    is not recommended for this use.

    Owing to the very rapid degradation of the salts and esters of 2,4-D
    in water, the long-term risk to aquatic organisms from these compounds
    was considered to be low. The primary breakdown product, 2,4-D acid,
    is, however, more persistent in water; therefore, the long-term risk
    assessment was based on its use. The levels of 2,4-D measured in

    surface waters after approved uses range from 0.00008 mg/L in small
    watersheds in Saskatchewan, Canada, to 0.0021 mg/L in ground- and
    surface waters in the United Kingdom. These values indicate that the
    long-term risk to fish and invertebrates living in water columns and
    sediments is low.

     Terrestrial environment

     (a) Microorganisms

    The most important sources of exposure of soil microorganisms to 2,4-D
    are likely to be ground and aerial applications. The results of
    laboratory studies indicate that application of 2,4-D at rates of 7.5
    kg/ha aerially and 18.75 kg/ha on the ground should pose a low risk to
    soil microorganisms; these rates correspond to 1.4 and 4.7 times the
    maximum recommended single or seasonal application rate. Application
    of the dimethylamine salt and the isooctyl ester at rates
    corresponding to 0.95 kg 2,4-D/ha resulted in 10-30% reductions in
    populations of soil bacteria, fungi, and actinomyces; the ester caused
    greater reductions than the salt. As the trigger for concern in the
    CoE/EPPO microorganism risk assessment scheme is an effect > 30%, the
    risk to soil microorganisms from the use of 2,4-D should be low.

     (b) Plants

    2,4-D is a translocated, selective herbicide used to control a variety
    of broad-leaved weeds. Consequently, although it may pose a risk to
    broad-leaved non-target plants, this is to be expected from its mode
    of action and consequent use.

     (c) Invertebrates

    Bees may be exposed to 2,4-D while foraging flowering weeds present in
    treated crops. At the maximum individual application rate of 4.48 kg
    acid equivalent/ha, the hazard quotients for toxicity after oral
    exposure and contact were > 45 for both the dimethylamine salt and
    the ethylhexyl ester. As the CoE/EPPO trigger for concern is a hazard
    quotient > 50, the acute risk to honeybees from the use of 2,4-D at
    this rate of application should be low. This conclusion is supported
    by the fact that 2,4-D has not been implicated in any incident of
    poisoning of honeybees in the UK Wildlife Incident Investigation
    Scheme.

    Arthropods may be exposed to 2,4-D during its many agricultural and
    non-agricultural uses. On the basis of the CoE/EPPO triggers for
    concern with regard to effects on non-target arthropods in laboratory
    studies (i.e. effects > 30%), 2,4-D may pose a risk to arthropods at
    high rates of application; however, the data on which this conclusion
    is based either related to a joint formulation with MCPA or were old
    and perhaps unreliable. Limited data from studies in the field with
    the lower, more typical rates of application (< 1.25 kg/ha)
    indicate that this risk may not be present in the field.

    Earthworms may be exposed after single or multiple applications of
    2,4-D to a wide variety, of crops but in particular during its use on
    grass, fallow land, and stubble. The TER at a maximum rate of
    application of the dimethylamine salt at 5.37 kg/ha is greater than
    the CoE/EPPO trigger value of 10, which indicates that the acute risk
    to earthworms from the use of 2,4-D should be low.

     (d) Vertebrates

    Vertebrates are likely to be exposed to 2,4-D while grazing on treated
    or contaminated vegetation or consuming contaminated insects.
    Estimates of residues on food items represent the maximum values
    determined immediately after application and do not take into account
    the rapid degradation of 2,4-D in the environment. The risk assessment
    further assumes that all food consumed contains 2,4-D at the level of
    the MRL.

    The short-term dietary TERs, based on measured initial residues on
    short grass after application of 2.24 kg acid equivalent/ha, indicate
    a potential medium risk (10 < TER < 100) to grazing birds from both
    aerial and ground-based applications. The initial residues declined to
    one-half or one-third within seven days of application. Furthermore,
    2,4-D has not been implicated in any incident of poisoning in birds as
    a result of normal use, suggesting that the risk to grazing birds is
    unlikely to be high. The short-term dietary TERs based on initial
    residues on large insects predicted in the CoE/EPPO vertebrate risk
    assessment scheme indicate a low acute risk (TER > 100) to small
    insectivorous birds from both aerial and ground applications (4.48 kg
    acid equivalent/ha and 2.24 kg acid equivalent/ha, respectively).
    Large insects are likely to constitute a higher proportion of both
    avian and mammalian diets than small insects during early growth stage
    or pm-emergence use.

    The acute oral TERs based on measured initial residues on short grass
    arising from application at 2.24 kg acid equivalent/ha indicate a
    potentially high risk (TER < 10) to grazing mammals from both aerial
    and ground-based applications, but the initial residues had declined
    to one-half or one-third within seven days of application. The acute
    oral TERs based on predicted initial residues on large insects
    indicate, however, a medium acute risk (10 < TER < 100) from aerial
    applications and a low acute risk (TER > 100) from ground-based
    applications, to small insectivorous mammals. It should be noted that
    2,4-D has not been implicated in any incidents of poisoning in mammals
    as a result of normal use. This suggests that the risk to mammals from
    2,4-D is unlikely to be high.

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    emergence (tier II) (Document No. 5097-91-0389-BE-001). Unpublished
    study from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Bakus, P. (1992c) Effect of 2,4-D DEAS on vegetative vigor of plants
    (tier II) (Document No. 5283-92-0156-BE-001). Unpublished study from
    Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Industry
    Task Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Bakus, P. (1992d) Effect of 2,4-D DEAS on seed germination/seedling
    emergence (tier II) (Document No. 5282-92-0155-BE-001). Unpublished
    study from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Bakus, P. (1993a) Supplemental dose testing of 2,4-D acid: Vegetative
    vigor of plants (Document No. 5464-92-0380-BE-001). Unpublished study
    from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Bakus, P. (1993b) Supplemental dose testing of 2,4-D acid: Seed
    germination/seedling emergence (tier II) (Document No. 5464 92 0379-
    B]E-001). Unpublished study from Ricerca, Inc., Painesville, Ohio,
    USA. Submitted to WHO by Industry Task Force II on 2,4-D Research
    Data, Indianapolis, Indiana, USA.

    Bakus, P. (1993c) Supplemental dose testing of 2,4-D DMAS: Vegetative
    vigor of plants (tier II) (Document No. 5464-92-0380-BE-003).
    Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. (1993d) Supplemental dose testing of 2,4-D DMAS: Seed
    germination/seedling emergence (tier II) (Document No. 5464-92-0379-
    BE-003). Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. (1993e) Supplemental dose testing of 2,4-D 2-EHE: Vegetative
    vigor of plants (tier II) (Document No. 5464-92-0380-BE-002).
    Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. (1993f) Supplemental dose testing of 2,4-D 2-EHE: Seed
    gemination/seedling emergence (tier II) (Document No. 5464-0379-BE-
    002). Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. (1995) Effect of 2,4-D 2-ethylhexyl ester on seedling
    emergence (tier II) (Document No. 6128-94-0140-BE-001). Unpublished
    study from Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Bakus, P. & Crosby, K. (1992a) Effect of 2,4-D DMAS on vegetative
    vigor of plants (tier II) (Document No. 3722-90-0408-BE-001).
    Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. & Crosby, K. (1992b) Effect of 2,4-D DMAS on seed
    germination/seedling emergence (tier II) (Document No. 3722-90-0407-
    BE-001). Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. & Crosby, K. (1992c) Effect of 2,4-D 2-EHE on vegetative
    vigor of plants (tier II) (Document No. 3722-90-0410-BE-001).
    Unpublished study from Ricerca, Inc., Painesville, Ohio, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Bakus, P. & Crosby, K. (1992d) Effect of 2,4-D 2-EHE on seed
    germination/seedling emergence (tier II) (Document No. 3722-90-0409-
    BE-001 ). Unpublished study from Ricerca, Inc., Painesville, Ohio,
    USA. Submitted to WHO by Industry Task Force II on 2,4-D Research
    Data, Indianapolis, Indiana, USA.

    Barney, W.P. (1994) Aquatic field dissipation study of 2,4-D DMAS in
    Louisiana (Study No. 2001RI-Louisiana). Unpublished study from
    Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995a) Terrestrial field dissipation study of 2,4-D DMAS
    on wheat in North Carolina (Study No. 2000WH06). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995b) Terrestrial field dissipation study of 2,4-D 2-EHE
    on wheat in North Carolina (Study No. 2000WH08). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney, W.P. (1995c) Terrestrial field dissipation study of 2,4-D DMAS
    on turf in North Carolina (Study No. 2000TF02). Unpublished study from
    Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995d) Terrestrial field dissipation study of 2,4-D DMAS
    on bare soil in North Carolina (Study No. 2000BS02). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney, W.P. (1995e) Terrestrial field dissipation study of 2,4-D 2-
    EHE on turf in North Carolina (Study No. 2000TF04). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995f) Terrestrial field dissipation study of 2,4-D DMAS
    on bare soil in North Carolina (Study No. 2000WH02). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995g) Terrestrial field dissipation study of 2,4-D 2-EHE
    on bare soil in North Carolina (Study No. 2000WH04). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney W.P. (1995h) Terrestrial field dissipation study of 2,4-D 2-EHE
    on bare soil in North Carolina (Study No. 2000BS04). Unpublished study
    from Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney, W.P. (1995i) Terrestrial field dissipation study of 2,4-D DMAS
    on pasture in Texas (Study No. 2000PA02). Unpublished study from
    Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney, W.P. (1995j) Terrestrial field dissipation study of 2,4-D
    2-EHE on pasture in Texas (Study No. 2000PA04). Unpublished study from
    Environmental Technologies Institute, Inc., North Carolina, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Barney, W.P. (1995k) Forest field dissipation study of
    2,4-dichlorophenoxyacetic acid, dimethylamine salt in Oregon (Study
    No. 2002FO01). Unpublished study from Environmental Technologies
    Institute, Inc., North Carolina, USA. Submitted to WHO by Industry
    Task Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Barney, W.P. (1996) Forest field dissipation study of
    2,4-dichlorophenoxyacetic acid, isooctyl (2-ethylhexyl) ester in
    Georgia (Study No. 2002FO02). Unpublished study from Environmental
    Technologies Institute, Inc., North Carolina, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

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    Beavers, J.B. (1983a) An acute oral toxicity study in the mallard with
    2,4-D isopropylamine salt (Project No. 103-226). Unpublished study
    from Wildlife International, Inc., Maryland, USA. Submitted to WHO by
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    USA.

    Beavers, J.B. (1983b) A dietary LC50 in the mallard with 2,4-D
    isopropylamine salt (Project No. 103-225). Unpublished study from
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    Beavers, J.B. (1983c) A dietary LC50 in the bobwhite with 2,4-D
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    Beavers, J.B. (1984a) (2,4-Dichlorophenoxy acetic acid isooctyl
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    Maryland, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Beavers, J.B. (1984b) (2,4-Dichlorophenoxy) acetic acid isooctyl
    ester: A dietary LC50 study with the mallard (Study ID: 103-228).
    Unpublished study from Wildlife International, Ltd, Maryland, USA.
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    Indianapolis, Indiana, USA.

    Beavers, J.B. (1984c) (2,4-Dichlorophenoxy) acetic acid isooctyl
    ester: A dietary LC50 study with the northern bobwhite (Study ID:
    103-227). Unpublished study from Wildlife International, Ltd,
    Maryland, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Behrens, R. & Elakkad, M.A. (1981) Influence of rainfall on the
    phytotoxicity of foliarly applied 2,4-D.  Weed Sci., 29, 349.

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     macrochirus) and rainbow trout  (Salmo gairdneri) (Study ID:
    256940). Unpublished study from Bionomics, E.G. & G., Massachusetts,
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    Indiana, USA.

    Berdasco, N.M., Schuetz, D.J., Yano, B.L. & Mizell, M.J. (1989b)
    2,4-Dichlorophenoxyacetic acid triisopropanolamine salt: Acute dermal
    toxicity study in Fischer 344 rats (Report No. K-008866-002D).
    Unpublished study from Dow Chemical Co., Midland, Michigan, USA.
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    of herbicide chemicals in silt loam soil. In:  Proceedings of the 
     46th Purdue Industrial Waste Conference, p. 591. Submitted to WHO by
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    USA.

    Bhanamurthy, V.B., Subramanian, S. & Rajukkannu, K. (1989) Degradation
    of 2,4-D ethyl ester under varying soil moisture conditions. 
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    2,4-dichlorophenoxyacetates.  Chemosphere, 1, 3.

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     Chemicals to Embryo-larval Stages of Fish (EPA 560-1179-007).
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    Birmingham, B.C. & Colman, B. (1985) Persistence and fate of 2,4-D
    butoxyethanol ester in artificial ponds.  J. Environ. Qual., 14, 100.

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    persistence of the herbicide Aquakleen in small artificial ponds and
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     Tech. Rep. Fish. Aquat. Sci., 1151, 12-23.

    Biro, P. (1979) Acute effects of the sodium salt of 2,4-D on the early
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    Bogers, M. & Enninger, I.C. (1990a) 2,4-D acid (as DMA salt). 96-Hour
    acute toxicity study (LCs0) in the rainbow trout (Project 019620).
    Unpublished study from RCC Notox, Netherlands. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Bogers, M. & Enninger, I.C. (1990b) 2,4-D acid (as DMA salt). 96-Hour
    acute toxicity study (LC50) in the carp (static) (Project 018977).
    Unpublished study from RCC Notox, Netherlands. Submitted to WHO by
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    Bollag, J.M., Lui, S.Y. & Minard, R.D. (1980) Cross-coupling of
    phenolic humus constituents and 2,4-dichlorophenol  Soil Sci. Soc. 
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    Bothwell, M.L. & Daley, R.J. (1981)  Selected Observations on the 
     Persistence and Transport of Residues from Aqua-Kleen(R) 20% 
     (2,4-D) Treatments in Wood and Kalamalka Lakes, BC, West Vancouver,
    British Columbia, National Water Research Institute.

    Boval, B. & Smith, J.M. (1973) Photodecomposition of
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    uptake of the herbicide 2,4-D-butyl applied to barley leaves. 
     Pestic. Sci., 36, 101.

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    Physico-chemical factors affecting uptake by roots and translocation
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    lake phytoplankton measured using photosynthetic inhibition compared
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    Bruns, G.W., Nelson, S. & Erickson, D.G. (1991) Determination of MCPA,
    Bromoxynil, 2,4-D, Trifluralin, Triallate, Picloram, and
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    salt): Mobility and degradation in soil in outdoor lysimeters (RCC
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    Switzerland. Submitted to WHO by Industry Task Force II on 2,4-D
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    Burke, B.A. (1994) Rate of de-esterification of [ring-14C]
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     Dungeness Crab (EPA-600/3-77-131), Gulf Breeze, Florida, US
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    Campbell, S., Grimes, J. & Smith, G.J. (1991) Diethanolamine salt of
    2,4-D: An acute oral toxicity study with the northern bobwhite
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    Inc., Maryland, USA. Submitted to WHO by Industry Task Force II on
    2,4-D Research Data, Indianapolis, Indiana, USA.

    Carpentier, A.G., MacKenzie, D.L. & Frank, R. (1988) Residues and
    efficacy of two formulations of 2,4-D on aquatic macrophytes in
    Buckhorn Lake, Ontario. J.  Aquat. Plant Manage., 26, 29.

    Carreon, R.E., et al. (1983) 2,4-Dichlorophenoxyacetic acid
    isopropylamine salt: Acute toxicological properties. Unpublished study
    from Dow Chemical Company, Michigan, USA. Submitted to WHO by Industry
    Task Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Cavalier, T.C., Lavy, T.L. & Mattice, J.D. (1991) Persistence of
    selected pesticides in ground-water samples.  Ground Water, 29, 225.

    Cessna, A.J. (1990) The determination of residues of 2,4-D in
    post-emergence-treated  Triticale. Pestic. Sci., 30, 141.

    Cessna, A.J. (1993) Relative foliar uptake of a tank mixture of 2,4-D
    and Dicamba by wheat.  Weed Sci., 41, 682.

    Cessna, A.J. & Hunter, J.H. (1993) Residues of 2,4-D and Dicamba in
    wheat following postemergence field application as a tank mixture.
     Can. J. Plant Sci., 73, 345.

    Cessna, A.J., Waddington, J. & Bittman, S. (1989) Residues of 2,4-D
    and Picloram in aspen poplar and soil after application with a roller.
     Can. J. Plant Sci., 69, 205.

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    2,4-dichlorophenoxyacetic acid (Project ID: ML-AL 87-40047).
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    to WHO by Industry Task Force II on 2,4-D Research Data, Indianapolis,
    Indiana, USA.

    Chamarro, E. & Esplugas, S. (1993) Photodecomposition of
    2,4-dichlorophenoxyacetic acid: Influence of pH.  Chem. Tech. 
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    biodegradation in lake water.  J. Environ. Qual., 18, 153.

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    2,4-D Research Data, Indianapolis, Indiana, USA.

    Cloutier, J.N. (1983) Removal of the herbicide 2,4-D by adsorption on
    peat. MSc Thesis, Laval University, Quebec (in French).

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    using batch equilibrium technique (Project No. 012/011/006/89).
    Unpublished study from Center For Hazardous Materials Research,
    Pittsburgh, Pennsylvania, USA. Submitted to WHO by Industry Task Force
    II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Comeau, Y., Greer, C.W. & Samson, R. (1993) Role of inoculum
    preparation and density on the bioremediation of 2,4-D contaminated
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    2-ethylhexyl ester in a buffered aqueous solution at pH 5 by natural
    sunlight (Project No. 390W). Unpublished study from PTRL-West,
    Richmond, California, USA. Submitted to WHO by Industry Task Force II
    on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Concha, M. & Shepler, B.S. (1993b) Aerobic aquatic metabolism of
    [14C] 2,4-D acid (Project No. 393W). Unpublished study from
    PTRL-West, Inc., Richmond, California, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Concha, M. & Shepler, B.S. (1994a) Anaerobic aquatic metabolism of
    [14C]2,4-D acid (Project No. 394W). Unpublished study from PTRL-West,
    Inc., Richmond, California, USA. Submitted to WHO by Industry Task
    Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Concha, M. & Shepler, B.S. (1994b) Aerobic soil metabolism of
    [14C]2,4-dichlorophenoxyacetic acid (Project No. 391W). Unpublished
    study from PTRL-West, Inc., Richmond, California, USA. Submitted to
    WHO by Industry Task Force II on 2,4-D Research Data, Indianapolis,
    Indiana, USA.

    Concha, M., Shepler, K. & Zabik, S.E. (1993a) Hydrolysis of
    [14C]2,4-D ethylhexyl ester in soil slurries (Project No. 403W).
    Unpublished study from PTRL-West, Inc., Richmond, California, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Concha, M., Shepler, K. & Zabik, S.E. (1993b) Hydrolysis of
    [14C]2,4-D ethylhexyl ester in natural water (Project No. 395W).
    Unpublished study from PTRL-West, Inc., Richmond, California, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

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    spawn and tadpoles.  Environ. Pollut., 3, 51.

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    at pH 5, 7, and 9 (Project No. 002/001/ 001/88, C8-208). Unpublished
    study from Center for Hazardous Materials Research, Pittsburgh,
    Pennsylvania, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Creeger, S. (1989b) Aqueous photodegradation of
    2,4-dichlorophenoxyacetic acid in p14 7 buffered solution (Project No.
    002/001/002/88, C28-208). Unpublished study from Center for Hazardous
    Materials Research, Pittsburgh, Pennsylvania, USA. Submitted to WHO by
    Industry Task Force II on 2,4-D Research Data, Indianapolis, Indiana,
    USA.

    Creeger, S. (1989c) Photodegradation of 2,4-dichlorophenoxyacetic acid
    on soil (Project No. C28-208). Unpublished study from Center for
    Hazardous Materials Research, Pittsburgh, Pennsylvania, USA. Submitted
    to WHO by Industry Task Force II on 2,4-D Research Data, Indianapolis,
    Indiana, USA.

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     J. Inst. Water Environ. Manage., 5, 389-395.

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    II) (Document No. 6819-96-0078-BE-001). Unpublished study from
    Ricerca, Inc., Painesville, Ohio, USA. Submitted to WHO by Industry
    Task Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Cullimore, D.R. (1981) The enumeration of 2,4-D degraders in
    Saskatchewan soils.  Weed Sci., 29, 440.

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    Graves, W.C. & Peters, G.T. (1991b) Diethanolamine salt of 2,4-D: A
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    Graves, W.C. & Peters, G.T. (1991f) Diethanolamine salt of 2,4-D: A
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    96-hour flow-through acute toxicity test with the bluegill  (Lepomis 
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    Hatfield, M.W. (1995c) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995e) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995f) Field soil dissipation of the 2-ethylhexyl
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    study from American Agricultural Services, Inc., North Carolina, USA.
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    Hatfield, M.W. (1995g) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995h) Field soil dissipation of the 2-ethylhexyl
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    Hatfield, M.W. (1995i) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995j) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995k) Field soil dissipation of the dimethylamine
    salt of 2,4-D on a bare soil in a wheat use pattern in North Dakota
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    Force II on 2,4-D Research Data, Indianapolis, Indiana, USA.

    Hatfield, M.W. (19951) Field soil dissipation of the 2-ethylhexyl
    ester of 2,4-D on bare soil in a wheat use pattern in North Dakota
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    Hatfield, M.W. (1995o) Field soil dissipation of the dimethylamine
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    Hatfield, M.W. (1995p) Field soil dissipation of the 2-ethylhexyl
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    Hatfield, M.W. (1995q) Field soil dissipation of the 2-ethlyhexyl
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    Hatfield, M.W. (1995r) Aquatic dissipation of the dimethylamine salt
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    Hilbig, V., Lucas, S.K. & Sebek, V. (1976b) Studies on the
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    Hinshalwood, A.M. & Kirkwood, R.C. (1988) The effect of simultaneous
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    Holmes, C.M. & Peters, G.T. (1991) Diethanolamine salt of 2,4-D: A
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    Holst, R.W., Yopp, J.H. & Kapusta, G. (1982) Effect of several
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    Hoxter, K.A., Grimes, Y., Smith, G.J. & Lynn, S.P. (1990b)
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    Hoxter, K.A., Grimes, J. Smith, G.J. & Lynn, S.P. (1991)
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    bobwhite (Project No. 281-107). Unpublished study from Wildlife
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    Hughes, J.S. (1990a) The toxicity of 2,4-D, 2-ethylhexyl ester to
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    Hughes, J.S. (1990b) The toxicity of 2,4-D, 2-ethylhexyl ester to
     Anabaena flos-aquae (Study ID: B460-07-1). Unpublished study from
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    Hughes, J.S. (1990c) The toxicity of 2,4-D, butoxyethyl ester to
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    Hughes, J.S. (1990d) The toxicity of 2,4-D, dimethylamine salt to
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    Hughes, J.S. (1990e) The toxicity of 2,4-D, 2-ethylhexyl ester to
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    Hughes, J.S. (1990f) The toxicity of 2,4-D, dimethylamine salt to
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    Hughes, J.S. (1990g) The toxicity of 2,4-D, 2-ethylhexyl ester to
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    Hughes, J.S. (1990h) The toxicity of 2,4-D, butoxyethyl ester to
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    Hughes, J.S. (1990i) The toxicity of 2,4-D, butoxyethyl ester to
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    Malcolm Pirnie, Inc., New York, USA. Submitted to WHO by Industry Task
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    Hughes, J.S. (1990j) The toxicity of 2,4-D, dimethylamine salt to
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    Hughes, J.S. (1990k) The toxicity of 2,4-D, butoxyethyl ester to
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    Hughes, J.S. (1990l) The toxicity of 2,4-D, dimethylamine salt to
     Lemna gibba G3 (Study ID: 0460-05-11007). Unpublished study from
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    Hughes, J.S. (1990m) The toxicity of 2,4-D to  Selenastrum 
     capricornutum (Study ID: 0460-05-1100-1). Unpublished study from
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    Hughes, J.S. (1990n) The toxicity of 2,4-D, 2-ethylhexyl ester to
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    Hughes, J.S. (19900) The toxicity of 2,4-D, dimethylamine salt to
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    Hughes, J.S. (1994) The toxicity of 2,4-D triisopropanolamine salt to
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994a) The toxicity of
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994b) The toxicity of
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994c) The toxicity of
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994d) The toxicity of
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994e) The toxicity of
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    Hughes, J.S., Williams, T.L. & Conder, L.A. (1994f) The toxicity of
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    Hughes J.S., Williams, T.L. & Conder, L.A. (1994g) The toxicity of
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    2-ethylhexyl ester to the daphnid,  Daphnia magna (Study ID: 9041-D).
    Unpublished study from Resource Analysts, Inc., New Hampshire, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Ward, T.J. & Boeri, R.L. (1991c) Acute flow-through toxicity of
    Esteron 99 herbicide to the tidewater silverside,  Menidia beryllina 
    (Study ID: 9038-D). Unpublished study from Resource Analysts, New
    Hampshire, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Ward. T.J. & Boeri, R.L. (1991d) Acute flow-through toxicity of 2,4-D,
    2-ethylhexyl ester to the tidewater silverside,  Menidia beryllina 
    (Study ID: 9035-D). Unpublished study from Resource Analysts, Inc.,
    New Hampshire, USA. Submitted to WHO by Industry Task Force II on
    2,4-D Research Data, Indianapolis, Indiana, USA.

    Ward, T.J. & Boeri, R.L. (1991e) Acute flow-through mollusk shell
    deposition test with Esteron 99 herbicide (Study ID: 9037-D).
    Unpublished study from Resource Analysts, Inc., New Hampshire, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Ward, T.J. & Boeri, R.L. (1991f) Acute flow-through mollusk shell
    deposition test with 2,4-D, 2-ethylhexyl ester (Study ID: 9034-D).
    Unpublished study from Resource Analysts, Inc., New Hampshire, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Ward, T.J. & Boeri, R.L. (1991g) Acute flow-through toxicity of 2,4-D,
    2-ethylhexyl ester to the grass shrimp,  Palaemonetes pugio (Study
    ID: 9036-D). Unpublished study from Resource Analysts, Inc., New
    Hampshire, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Ward, T.J. & Boeri, R.L. (1991h) Acute flow-through toxicity of
    Esteron 99 herbicide m grass shrimp,  Palaemonetes pugio (Study ID:
    9039-D). Unpublished study from Resource Analysts, Inc., New
    Hampshire, USA. Submitted to WHO by Industry Task Force II on 2,4-D
    Research Data, Indianapolis, Indiana, USA.

    Ward, T.I., Maguzu, J.P. & Boeri, R.L. (1993) 2,4-D: Acute
    flow-through mollusc shell deposition test (Study No. 286-DE).
    Unpublished study from Wilbury Laboratories, Inc., Massachusetts, USA.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Watson, J.R. (1977) Seasonal variation in the biodegradation of 2,4-D
    in river water.  Water Res., 11, 153.

    Wauchope, R.G. (1978) The pesticide content of surface water draining
    from agricultural fields--A review.  J. Environ. Qual., 7, 459.

    WHO (1989)  2,4-Dichlorophenoxyacetic Acid (2,4-D)--Environmental 
     Aspects (Environmental Health Criteria 84), Geneva.

    Wilson, R.G., Jr & Cheng, H.H. (1976) Breakdown and movement of 2,4-D
    in the soil under field conditions.  Weed Sci., 24, 461.

    Wilson, R.G., Jr & Cheng, H.H. (1978) Fate of 2,4-D in a naff silt
    loam soil.  J. Environ. Qual., 7, 281.

    Wolf, D.C. & Martin, J.P. (1976) Decomposition of fungal mycelia and
    humic-type polymers containing carbon 14 from ring and side-chain
    labeled 2,4-D and Chlorpropham.  Soil Sci. Soc. Am. J., 40, 700.

    Woodward, D.F. (1982) Acute toxicity of mixtures of range management
    herbicides to cutthroat trout.  J. Range Manage., 35, 539-540.

    Woodward, D.F. & Mayer, F. (1978) Toxicity of three herbicides (butyl,
    isooctyl, and propylene glycol butyl ether esters of 2,4-D) to
    cutthroat trout and lake trout.  Tech Pap. US Fish Wildlife Serv., 
    97, 6.

    Zepp, R.G., Wolfe, N.L., Gordon, J.A. & Baughman, G.L. (1975) Dynamics
    of 2,4-D esters in surface waters.  Environ. Sci. Technol., 9, 1144.

    Zepp, R.G., Wolfe, N.L., Baughman, G.L. & Gordon, J.A. (1976) Chemical
    and photochemical alteration of 2,4-D esters in the aquatic
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    Zohner, A. (1990a) Determination of the mobility of soil-aged residues
    by soil column leaching test for 14C-2,4-D acid (Report No. 989).
    Unpublished study from Agrolinz Agrarchemicalien GmbH, Linz, Austria.
    Submitted to WHO by Industry Task Force II on 2,4-D Research Data,
    Indianapolis, Indiana, USA.

    Zohner, A. (1990b) Determination of the mobility of soil-aged residues
    by soil column leaching test for 14C-2,4-D acid (Report No. 1013).
    Unpublished study from Agrolinz Agrarchemicalien GmbH. Submitted to
    WHO by Industry Task Force II on 2,4-D Research Data, Indianapolis,
    Indiana, USA.

    Zsoldas, F. & Haunold, E. (1979) Effects of pH changes on ion and
    2,4-D uptake of wheat roots.  Physiol. Plant., 47, 77.
    


    See Also:
       Toxicological Abbreviations