PESTICIDE RESIDUES IN FOOD - 1979
Sponsored jointly by FAO and WHO
EVALUATIONS 1979
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Expert Group on Pesticide Residues
Geneva, 3-12 December 1979
CHLORPYRIFOS-METHYL
Explanation
In 1975 the Meeting evaluated information on chlorpyrifos-methyl. An
ADI was recommended, as well as MRLs for a number of commodities.
Further information on residues in animal tissues, fat and eggs
following feeding of residues in animal feeds was required before
additional maximum residue limits could be recommended.
The 11th Session of the CCPR noted that Germany (F.R.) possessed data
supporting changes in the proposed MRLs and the addition of MRLs for
other commodities (Alinorm 79/24-A, para. 133).
New information was received on residues in animal tissues, fat and
eggs from the feeding of rations fortified with chlorpyrifos-methyl at
several levels; on residues in milk resulting from the feeding of
feeds fortified at several levels, and on validated analytical methods
for feeds and animal tissues, fat and eggs. This information is
assessed in the following monograph addendum.
RESIDUES IN FOOD AND THEIR EVALUATION
RESIDUES FROM SUPERVISED TRIALS
These studies were designed to indicate residues of chlorpyrifos-
methyl and its predominant metabolite, 3, 5, 6-trichloro-2-pyridinol,
in animal tissues, fat, milk and eggs from the feeding of rations
fortified at various levels. Although the studies were not designed
to indicate residues that actually result from good agricultural
practices, they can be used to make reasonable estimates thereof.
Cow's Milk and Cream
Three cows were fed 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-methyl
in the diet starting at the lowest level and increasing the dosage
every 2 weeks (Kuper, 1978a). Milk samples for each cow were obtained
by combining equal portions of milk from two successive milkings.
Residues were monitored throughout the feeding period and up to two
weeks after removal of treated feed. Equal portions of milk from each
of the three cows were composited for cream samples. Average residues
of chlorpyrifosmethyl and 3,5,6-trichloro-2-pyridinol in milk and in
cream from composited milk are summarized in Table 1. Gross controls
were <0.002 mg/kg chlorpyrifos or pyridinol metabolite equivalent.
TABLE 1. Average Residues of Chlorpyrifos-Methyl and 3,5,6-trichloro-2-pyridinol
Found in Milk and in Composited Cream samples from Cows Fed Chlorpyrifos-Methyl
Average Residues Found, mg/kg1,4
Chlorlpyrifos-Methyl Chlorpyrifos-Methyl 3,5,6-Trichloro-2-Pyridinol
Mg/kg Milk Cream Milk Cream
3 - - ND4
10 <O.01 <0.01
30 <0.01 0.09 0.02 0.02
(0.07-0.11) (0.01-0.02)
100 0.03 0.43 0.05 0.06
(0.02-0.04) (0.31-0.50) (0.03-0.07) (0.06-0.07)
1002 <0.01 <0.01
(ND-<0.01)
1003 ND ND
1 Range is in parentheses if different than average value
2 After 2 day withdrawal from feed
3 After 4 day withdrawl from feed
4 ND = less than twice the average of controls. < indicates a response greater than
twice the average of controls but less than the 0.01 mg/kg verified sensitivity of the
method. Values are adjusted for methodology recovery and controls.
The data indicate that chlorpyrifos-methyl preferentially concentrates
in the fat of milk whereas the pyridinol metabolite distributes
equally in the aqueous and fat phases. It also indicates that
residues of 3,5,6-trichloro-2-pyridinol can equal or exceed residues
of chlorpyrifos-methyl in milk. Relative to milk, the concentration
of chlorpyrifos-methyl in cream is proportional to the increase in the
fat content. At the 100 mg/kg feeding level, residues of
chlorpyrifos-methyl and its pyridinol metabolite decrease to <0.01
mg/kg after a 2-day withdrawal period.
At the 10 mg/kg feeding level, the level of the highest proposed MRL
on feed items that would contribute significantly to residues in
animal products, residues in milk were 0.01 mg/kg (method sensitivity)
for both chlorpyrifos-methyl and the pyridinol metabolite. This
confirms the 0.01 ppm MRL previously recommended for milk (FAO/WHO,
1976).
Calves
Cattle feed fortified at 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-
methyl was fed ad libitum to three calves at each level for a period
of 28 days with residue determinations for chlorpyrifos-methyl and
3,5,6-trichloro-2-pyridinol up to 28 days after withdrawal (Kuper,
1978b). Residues in the fortified feed ranged from 70% of theoretical
at the 30 mg/kg level to 130% at the 1 mg/kg level, averaging 92% of
theoretical over all levels. Data were not provided to show whether
initial residue levels in the feed were maintained over the feeding
period. Assuming a half-life similar to that of stored grains (1-6
months) possible 50% but probably no more than 10 - 30% of the
chlorpyrifos-methyl would have dissipated. Results of this feeding
study are summarized in Table 2. Controls were <0.001 mg/kg in all
tissues for chlorpyrifos-methyl and <0.011 mg/kg (liver) for the
pyridinol metabolite.
At the 100 mg/kg feeding level maximum residues of chlorpyrifos-methyl
at the end of 28 days were 0.05 mg/kg in kidney, 0.91 mg/kg in fat,
and less than the validated method sensitivity for muscle and liver.
Maximum residues of the pyridinol metabolite at this feeding level
were 0.12, 2.17, 1.4, and 0.13 mg/kg respectively in muscle, liver,
kidney and fat. After 7 days withdrawal from treated feed, residues
of chlorpyrifos-methyl and its pyridinol metabolite were less than the
validated method sensitivities of 0.01 mg/kg and 0.05 mg/kg
respectively.
The chlorpyrifos-methyl therefore concentrates primarily in the fat
and to a lesser extent in the kidney. The pyridinol on the other
hand, concentrates primarily in the liver and kidney.
The highest MRL recommended for animal feed items which would be
expected to contribute the most to potential residues of
chlorpyrifos-methyl and 3,4,6-trichloro-2-pyridinol is 10 mg/kg in raw
grains. Maximum residues from feeding at the 10 mg/kg level were 0.03
mg/kg chlorpyrifos-methyl in fat and 0.49 mg/kg pyridinol metabolite
in liver. Although the cattle diet would probably not be 100% raw
grains, up to 80% could be. Considering that initial feed
fortification levels could have dissipated during the 28 day period,
that some initial residues were less than 100% theoretical, that no
data were available to demonstrate whether a 28 day feeding period was
sufficient for tissue residues to peak (milk data gave some
circumstantial indication that it would have), and control values up
to 0.011 mg/kg for pyridinol, the residue data would indicate a need
for a 0.05 mg/kg MRL for residues of chlorpyrifos-methyl in the meat,
fat and meat by-products of cattle.
Chickens and Eggs
Mature White Leghorn laying hens were fed, ad libitum, feed
fortified at 0, 1, 3, 10, 30 and 100 mg/kg chlorpyrifos-methyl for a
period of 28 days (Kuper 1978c). Residues in the fortified feed
ranged from 70% of theoretical at the 30 mg/kg feeding level to 110%
at the 1 mg/kg level (85% average over all levels). No data were
available to indicate whether initial residues in feed were maintained
throughout the 28 day feeding period, or whether tissue residues
peaked within that period.
Muscle, fat, liver and egg tissues were analyzed for
chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol with methods
validated on 0.01 and 0.05 mg/kg respectively. Analyses were
continued up to 14 days after withdrawal of treated feed. Eggs were
sampled on alternate days and composited per group. Some individual
eggs were analysed to demonstrate that the range of residues for
individual eggs was essentially the same as for composited samples.
Maximum apparent residues in chicken control were 0.004 mg/kg
chlorpyrifos-methyl in muscle and 0.001 mg/kg pyridinol metabolite in
liver and 0.011 mg/kg of the metabolite in eggs.
Average residues, corrected for controls and method recovery are
summarized in Table 3 which shows increasing residues with increasing
ingestion levels. At the 100 mg/kg feeding level, maximum
chlorpyrifos-methyl residues at 0.1 mg/kg in chicken fat and 0.03
mg/kg in eggs, again demonstrating its affinity for fat tissues
relative to other tissues. Maximum residues of the pyridinol
metabolite at this feeding level are 0.12 mg/kg in liver and 0.08
mg/kg in eggs, which is relatively low compared to similar feeding
levels in calves. Even so, residues of the metabolite in chicken
tissues may equal, or exceed, those of chlorpyrifos-methyl. Residues
of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in chicken and
egg tissues are generally less than validated method sensitivities
after a 7 day withdrawal from treated feed. Although not shown in
Table 3, both chlorpyrifos-methyl and its pyridinol metabolite showed
a greater affinity for egg yolk than the white.
TABLE 2. Average Residues of Chlorpyrifos-Methyl and 3,5,6-Trichloro-2-Pyridinol found in tissues of Calves fed Chlorpyrifos-Methyl
for 28 days
Feed Rate
Mg/Kg Average Residues in Tissues, Mg/Kg1
in Feed Chlorpyrifos-Methyl 3,5,6-Trichloro-2-Pyridinol
Muscle Liver Kidney Fat Muscle Liver Kidney Fat
1 - - - ND2 - 0.07 <0.05 -
(ND-<0.01) (0.06-0.07)
3 - - - 0.02 - 0.15 0.07 -
(<0.01-0.02) (0.13-0.19) (0.07-0.08)
10 - - - 0.03 - 0.44 0.21 <0.05
(0.02-0.03) (0.39-0.49) (0.13-0.35)
30 ND ND <0.01 0.09 <0.05 1.17 0.63 0.06
(ND-<0.01) (ND-<0.01) (0.04-0.16) (ND-<0.05) (0.96-1.54) (0.55-0.78) (0.05-0.06)
100 <0.01 ND 0.03 0.77 0.08 2.16 1.22 0.88
(ND-<0.01) (0.02-0.05) (0.65-0.91) (0.05-0.12) (2.15-2.17) (0.93-1.4) (<0.05-0.13)
1003 ND ND ND ND ND <0.05 <0.05 <0.05
(ND-<0.01)
1004 - - - ND - ND ND -
(0.00-<0.01)
1 Range for three calves in parentheses if different than average. All valves adjusted for controls and recovery of methods.
2 ND = less than twice the average of controls.
3 After 7 day withdrawal from treated feed.
4 After 14 day withdrawal from treated feed.
TABLE 3. Average Residues of Chlorpyrifos-Methyl and 3,5,6-Trichloro-2-Pyridinol found in Tissues of Chickens fed Chlorpyrifos-Methyl
for 28 Days
Level
In Feed Average Residues in Tissues, Mg/Kg1
Mg/Kg Chlorpyrifos-Methyl 3,5,6-Trichloro-2-Pyridinol
Muscle Liver Kidney Fat Muscle Liver Kidney Fat
10 - <0.01 - ND - - <0.05 <0.05
(ND-<0.01) (ND-<0.01) (ND-<0.05)
30 ND 0.01 ND <0.01 <0.05 <0.05 <0.05 <0.05
(<0.01-0.02) (<0.01-0.02) (<0.05-0.06)
100 0.01 0.08 ND 0.02 <0.05 <0.05 0.06 0.06
(0.05-0.1) (0.01-0.03) (ND-<0.05) (0.05-0.12) (<0.05-0.08)
100 ND6 ND6 ND6 0.023 <0.056 <0.056 <0.056
<0.053
(<0.05-0.06)
<0.014 <0.057
(ND-0.05)
1 Range in parentheses if different than average.
2 ND = less than twice average for controls.
3 After two day withdrawal from treated feed.
4 After four day withdrawal from treated feed.
5 After six day withdrawal from treated feed.
6 After seven day withdrawal from treated feed.
7 After 14 day withdrawal from treated feed.
At the 10 mg/kg feeding level, the highest recommended MRL for a feed
item which would contribute significantly to chicken feed, maximum
residues were less than validated method sensitivity for both
chlorpyrifos-methyl and its pyridinol metabolite in all chicken
tissues and eggs. This is true even at the 30 mg/kg feeding level for
average tissues.
The available information would support an MRL of 0.05 mg/kg combined
residues of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in the
meat, fat and meat by-products of chickens and in eggs.
FATE OF RESIDUES
No additional data were submitted for review.
METHODS OF RESIDUE ANALYSIS
Validated methods were received for the determination of
chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol in animal tissues
and eggs. These appear similar to methods previously submitted with
some variations for specific tissues. These methods are also similar
to methods for chlorpyrifos and its pyridinol hydrolytic product as
available in the U.S. Food and Drug Administration's Pesticide
Analytical Manual, Volume II.
Method ACR 77.6 is used for the determination of chlorpyrifos-methyl
in bovine and chicken tissues and in eggs (Kuper, 1977). Residues are
extracted with acetone, except in the case of fat, in which case
hexane is used. The acetone from the filtrate is evaporated.
Residues are taken up in hexane. All hexane extracts are partitioned
with acetonitrile, followed-by cleanup on a de-activated silica gel
column. Quantitation is by gas chromatography using a flame
photometric detector. Average recoveries range from 78 ± 6 to 95 ± 6
percent over the 0.01-1 mg/kg fortification levels. The method is
validated at 0.01 mg/kg for the specified tissues.
A modification of ACR 77.6 (ACR 77.6.S1) is used for the determination
of chlorpyrifos-methyl in milk and cream (Kuper, 1978d). Methanol and
sodium chloride is added to warmed milk or cream before shaking with
hexane. After centrifugation and repeat extractions, the procedure is
continued as in ACR 77.6 with the acetonitrile partitioning of the
hexane extract. Over the 0.01 to 1.0 mg/kg fortification level
average recoveries in milk and cream are 87 ± 2 and 84 ± 4 percent
respectively. The method is validated for milk and cream at 0.01
mg/kg.
Method ACR 78.9 is used for the determination of
3,5,6-trichloro-2-pyridinol in bovine muscle, liver, kidney and fat
(Kuper, 1978e). Residues are extracted with methanol and
chromatographed on acidic alumina, eluting with hydrochloric acid.
The eluate is partitioned with benzene. In the case of liver and
kidney, the benzene phase is partitioned with sodium bicarbonate
solution. The aqueous phase is acidified and partitioned with
benzene. Residues of all tissues are derivatized with N,O-bis
(trimethylsilyl) acetamide for determination by electron capture gas
chromatography. Average recoveries at the 0.05 - 2.0 mg/kg
fortification levels range from 83.5 to 92 ± 4 percent. The method is
validated to 0.05 mg/kg for bovine tissues.
A modified version of ACR 78.9 (ACR 78.9.S1) is used for the
determination of 3,5,6-trichloro-2-pyridinol in the muscle, fat, liver
and eggs of chickens (Kuper, 1978f). The procedure is basically the
same as for bovine liver and kidney except a 3% OV-17 gas
chromatography column is used in lieu of 6% DC 200. Average
recoveries in chicken tissues and eggs over the 0.05 - 1.0 mg/kg
fortification levels range from 88 ± 3 to 93 ± 4 percent. The method
is validated at 0.05 mg/kg for chicken tissues and eggs.
A modified version of 78.9 (78.9 S2) is used for the determination of
3,5,6-trichloro-2-pyridinol in milk and cream (see Kuper, 1978d, p.
5). The procedure is basically the same as used for bovine liver and
kidney except the initial extraction is with benzene lieu of
methanol. Recoveries over the concentration range of 0.01-1.0 mg/kg
average 93 ± 3 percent for milk and 88 ± 4 percent for cream. The
method is validated at 0.01 mg/kg for milk and cream.
APPRAISAL
Following the evaluation in 1975, the Meeting required information on
residues in animal tissues, fat and eggs following the feeding of
chlorpyrifos-methyl residues in animal feeds before additional maximum
residue limits could be recommended. Other studies were listed as
desirable. Data on residues in animal tissues, fat and eggs,
additional data for milk, and validated analytical methodologies have
been provided and are evaluated.
Calves, dairy cows and hens were fed diets containing
chlorpyrifos-methyl at concentrations ranging from 0 - 100 ppm.
Tissues, milk, fat and eggs were analyzed for chlorpyrifos-methyl and
3,5,6-trichloro-2-pyridinol. Analytical methods similar to those
previously evaluated were validated in all tissues down to 0.01 mg/kg
chlorpyrifos-methyl and 0.05 mg/kg 3,5,6-trichloro-2-pyridinol. In
the case of milk both chlorpyrifos-methyl and its pyridinol hydrolytic
product were validated at 0.01 mg/kg.
In calves, residues of chlorpyrifos-methyl were maximal in the fat at
the end of 28 days feeding. Except for trace residues in the kidney
at exaggerated feeding levels, residues were less than method
sensitivity in muscle and liver. Maximum residues of the pyridinol
were in muscle and liver. 7 days after withdrawal of the diet
containing 100 mg/kg chlorpyrifos-methyl, residues of
chlorpyrifos-methyl and the pyridinol were below validated method
sensitivity.
In hens, maximum residues of chlorpyrifos-methyl were found in fat and
maximum of 3,5,6-trichloro-2-pyridinol in the liver. While the
pyridinol concentrates in the liver of hens as it does in the liver of
calves, the concentration of residues in hens is significantly lower
than in the liver of calves receiving similar concentrations in feed.
In eggs, residues of the 3,5,6-trichloro-2-pyridinol are 2-3 times
those of chlorpyrifos-methyl. Egg yolk showed a greater affinity than
the white for both chlorpyrifos-methyl and the pyridinol. Except for
one sample, residues of chlorpyrifos-methyl and the pyridinol in hen
tissues and eggs were less than method sensitivities 7 days after
withdrawal from the diet containing 100 mg/kg chlorpyrifos-methyl.
Chlorpyrifos-methyl was found to concentrate in the fat of milk
whereas the 3,5,6-trichloro-2-pyridinol distributed equally in the
aqueous and fat phases. Residues of chlorpyrifos-methyl in cream
increased over that of milk proportionally to the increased fat
content. Residues of the pyridinol on the other hand remain
relatively constant. The study confirms conclusions on milk residues
reached by the 1975 Meeting.
None of the animal tissue, milk or egg samples were analyzed for the
oxygen analogue of chlorpyrifos-methyl or possible conjugated
residues. As the 1975 Meeting concluded, data indicate little
likelihood of the oxygen analogue occurring. However there appears to
be little experimental evidence to demonstrate the absence of
conjugated residues in animal tissues. It has been demonstrated that
the pyridinol is conjugated in the urine as the glucuronide. More,
definitive experimental evidence to demonstrate the presence or
absence of conjugated moieties in animal tissues is desirable.
No analyses were conducted for animal tissue, milk or egg residues of
O-methyl-O-hydrogen-O(3,5,6-trichloro-2-pyridyl) phosphorothionate
which has been found in appreciable quantities in the urine of ewes
and rats (1975 Evaluations). This information is desirable.
The data indicate that in animal tissues, milk and eggs maximum
residues of 3,5,6-trichloro-2-pyridinol usually exceed maximum
residues of chlorpyrifos-methyl although not necessarily in the same
tissues.
Even the limited data on 3,5,6-trichloro-2-pyridinol residues in
plants (1975 Evaluation, p. 126) indicate that residues of the
pyridinol can exceed those of chlorpyrifos-methyl after 1-2 weeks from
application.
The data on residues in animal tissues, fat, and eggs are adequate to
fulfill data requirements from the 1975 Evaluations and to support
recommendations for additional animal products.
RECOMMENDATIONS
On the basis of additional information before the Meeting, the
previously recommended MRL for milk is confirmed and the following
MRLs are recommended. The recommended figures refer to residues of
chlorpyrifos-methyl.
Commodity Limit, mg/kg
Carcase meat of cattle, cattle fat, 0.05
cattle by-products
Meat of chickens, chicken fat, 0.05
chicken by-products
FURTHER WORK OR INFORMATION
Desirable
1. Analysis of milk, tissues, fat and eggs from animals fed feed with
chlorpyrifos-methyl fortified rations for possible residues of
O-methyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate.
2. Residue data for the carcase meat, fat and meat by-products of
pigs from feeding of chlorpyrifos-methyl fortified rations.
3. Additional information or work listed as desirable by the 1975
Meeting.
REFERENCES
Kuper, A.W. Residues of Chlorpyrifos-Methyl and
3,5,6-Trichloro-2-Pyridinol in Milk and Cream from Cows fed
Chlorpyrifos-Methyl. (1978a) Unpublished Report GH-C 1161, Dow
Chemical U.S.A., Midland, Michigan. December 27, 1978.
Kuper, A.W. Residues of Chlorpyrifos-Methyl and
3,5,6-Trichloro-2-Pyridinol in Tissues from Calves fed with
Chlorpyrifos-Methyl. (1978b) Unpublished Report GH-C 1118, Dow
Chemical U.S.A., Midland, Michigan. July 5 1978.
Kuper, A.W. Residues of Chlorpyrifos-Methyl and
3,5,6-Trichloro-2-Pyridinol in Tissues and Eggs from Chickens fed
Chlorpyrifos-Methyl. (1978c) Unpublished Report GH-C 1155, Dow
Chemical U.S.A., Midland, Michigan. November 15, 1978.
Kuper, A.W. Determination of Residues of Chlorpyrifos-Methyl in
Bovine Tissues. (1977) Unpublished Method ACR 77.6, Dow Chemical
U.S.A., Midland, Michigan, April 11, 1977.
Kuper, A.W. Determination of Residues of Chlorpyrifos-Methyl in Milk
and Cream. Modification ACR 77.6.S1 of Method ACR 77.6, Dow Chemical
U.S.A., Midland, Michigan. November 9, 1978. (1978d).
Kuper, A.W. Determination of Residues of 3,5,6-Trichloro-2-Pyridinol
in Bovine Tissues. (1978e) Unpublished Method ACR 78.9, Dow Chemical
U.S.A., Midland, Michigan. May 12, 1978.
Kuper, A.W. Determination of Residues of 3,5,6-Trichloro-2-Pyridinol
in Chicken Tissues. Modification ACR 78.9.S1 of Method ACR 78.9, Dow
Chemical, U.S.A., Midland, Michigan. July 13, 1978. (1978f).
See Also:
Toxicological Abbreviations
Chlorpyrifos-methyl (WHO Pesticide Residues Series 5)
Chlorpyrifos-methyl (Pesticide residues in food: 1991 evaluations Part II Toxicology)
Chlorpyrifos-methyl (Pesticide residues in food: 1992 evaluations Part II Toxicology)