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Importance of Feed as an Unavoidable Source of Pesticide Contamination in Poultry Meat and Eggs
Importance of Feed as an Unavoidable Source of Pesticide Contamination in Poultry Meat and Eggs 1. R E S I D U E S I N F E E D S T U F F S 1 A . C. WALDRON AND E . C. NABER
Pesticide Analytical Laboratory, Cooperative Extension Service, Department of Entomology and Department of Poultry Science. The Ohio State University and Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (Received for publication October 9, 1973)
POULTRY SCIENCE 53: 1359-1371, 1974
INTRODUCTION ESTICIDE contamination of poultry meat and eggs resulting from feeding of low level concentrations of pesticides or from pesticide contamination of the housing environment has been studied by many workers. Hixen and Muma (1947) reported that the flavor of poultry meat was adversely affected by the feeding or environmental application of benzene hexachloride. Furman and Bankowski (1949) showed that benzene hexachloride is readily absorbed by poultry and subsequently it has been shown that most chlorinated organic insecticides are absorbed through the skin or the digestive tract. Contamination of poultry meat and eggs by lindane (Ivey et al., 1961; Ware and Naber,
P
1. Ohio Agricultural Research and Development Center Journal Article No. 73-73. Supported by U.S.D.A., A.R.S. Research and Service Contract No. 12-14-100-8952(44).
1961, 1962); DDT (Draper et al, 1950; Liska etal, 1964); methoxychlor (Olney etal., 1962; Thompson et al., 1967) kepone and mirex (Naber and Ware, 1965); and chlordanepyrethrum (Mountney and Quisenberry, 1957) has been reported from feeding and /or environmental application of these compounds. Cummings et al. (1966,1967) fed diets containing combinations of lindane, DDT, heptachlor epoxide, dieldrin, and endrin to hens for 14 weeks and showed transfer of residues to eggs and tissues. In 1965 the United States Department of Agriculture reported that minute pesticide residues were present in all 2600 samples of poultry meat tested in a nationwide survey. The residue levels in almost all cases were well below the concentration considered as a safe daily intake level in the human diet. Thus no attempts were made to define the sources of contamination as a means to correct the situation and/or protect the poultry producer from contamination that could re-
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ABSTRACT General and broad occurrences of minute organochlorine pesticide residues were indicated in feedstuffs used in poultry feeds in the United States. Corn, soybean meal, alfalfa meal, fish meal, and fat samples were collected and submitted bimonthly over a 24 month period from Maine, Georgia, Texas, Ohio, and California and analyzed for aldrin, dieldrin, heptachlor, heptachlor epoxide, lindane, DDE, DDD, o,p-DDT, p,p'-DDT, and methoxychlor. Concentration of individual pesticides when occurring were generally in the range of 10 to 50 p.p.b. although higher concentrations occasionally occurred. DDT and its metabolites were the most frequently occurring organochlorine pesticides detected. The highest concentration and frequency of residues detected were associated with fish meal and fat samples. Occurrence of residues in corn and soybean meal was generally infrequent and at very low concentrations. The presence of pesticide residues was very closely correlated with the areas of high crop production and hence greater pesticide use. In general it appears that poultry feeds formulated from feedstuff sources as analyzed in this study (corn and soybean meal constituting by far the major part of the poultry ration) would not contain organochlorine pesticide residues large enough to cause any major contamination problems with poultry products.
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A. C. WALDRON AND E. C. NABER
It thus becomes important to establish the level of pesticide residues naturally occurring in poultry feed ingredients over which the poultry producer has no control, to establish the transfer of these residues to poultry meat and eggs when raised on such feeds relative to the tissue and egg contamination, and to establish a realistic tolerance level for certain pesticides in poultry products and feedstuffs that will protect the poultry producer and at the same time not cause concern in the human diet. The purpose of this research is to provide data required to establish the extent of contamination in the feed supply. EXPERIMENTAL PROCEDURES Collection of Samples. Four samples each of soybean meal, corn or corn meal, alfalfa meal, fish meal, and fat were collected bimonthly over a period of 24 months from Ohio, Maine, Georgia, Texas, and California. Samples were collected by State Departments
of Agriculture personnel and submitted to The Ohio State University Poultry Science Department where they were stored (generally at -20° C. in a freezer) until analyzed. A total of 1162 samples were collected during the two year period. Grain samples were ground into meal prior to analysis. Extraction and Cleaning Procedures. Extraction procedures for the feed ingredient samples followed in general the principles published in the literature for extraction of organochlorine insecticides (Mills etal., 1963; Minyard and Jackson, 1963; Cummings et al., 1967; Waldron et al, 1968; and Waldron and Goleman, 1969) but with modifications adaptable to the techniques of this laboratory. Additional modifications in procedures, solvent systems, elution techniques, gas chromatographic operation, etc. that increased laboratory efficiency were developed during the course of the study (Waldron, 1971). 1. Corn meal and soybean meal. A 50-gram subsample of each submitted sample was extracted with 300 ml. of glass distilled petroleum ether by blending for 8 minutes in a Waring Blendor set at a moderate speed that would not cause heating of the solvent during blending. The extract, after permitting the solids to settle, was filtered through Whatman # 1 filter paper and collected in a 16-oz. narrow neck bottle. The blendor and filtration equipment. were covered with the lid and aluminium foil, respectively, to prevent evaporation of the petroleum ether. An aliquot of extract equal to one half of the sample (150 ml.) was transferred to a 500-ml. evaporation flask, 20 ml. of propanediol cyclic carbonate added and the petroleum ether removed by evaporation with a flash evaporator. The carbonate-oil solution was transferred to a 60-ml. separatory funnel and chilled for about one hour in the freezer to separate residual oil or fat. The chilled carbonate solution was filtered through glass
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move his produce from the market. In most cases the poultry producer must rely on external sources for his feeds and consequently could become a victim of a condition over which he has no control. There has been concern for several years about the potential that exists in adulterating poultry products with pesticide residues. The problem may have international implications because considerable quantities of poultry products are exported and considerable quantities of poultry feed ingredients (such as fish meal) are imported. The increased use of pesticides in crop protection during recent years increases the possibility of feedstuff contamination and the consequent exposure of poultry to these products. Because some pesticides, such as some chlorinated hydrocarbons, are retained and stored in body fat tissues the potential for contamination of poultry tissues and, consequently, eggs becomes significant. Such contamination could result in significant economic losses.
PESTICIDE RESIDUES IN FEEDSTUFFS
glass-distilled acetone for 3-4 minutes. Two hundred ml. of pesticide quality acetonitrile was added causing the fat to coagulate during an additional 3-4 minutes of blending, and leaving the pesticide residue in solution. The rest of the procedure followed that for corn meal except that greater patience was required in permitting the oil phase to separate. 4. Alfalfa meal. A 25-gram subsample of each submitted alfalfa meal sample was extracted by blending with 300 ml. of acetonitrile for 5-6 minutes (later samples were extracted with 400 ml. of acetone). The extract was filtered and an aliquot representing 1/2 of the sample was combined with 700 ml. of approximately 0.18 N H 2 S 0 4 and 150 ml. of petroleum ether for extraction in a separatory funnel. The aqueous phase was extracted a second time with petroleum ether and the combined organic phases concentrated and placed on the florisil column and the procedure for corn meal followed. In later extractions with acetone, 500 ml. of 2% NajSC^ was used instead of sulfuric acid. Gas Chromatographic Analysis. Aliquots of the sample extracts were injected on a 6-ft. glass column containing a 1:1 mixture of 10.45% DC-200 and 15.14% QF-1 on Gas Chrom Q. A Barber Coleman Series 5000 gas chromatograph equipped with a Tritium foil electron capture detector was operated at a column temperature of 190° C , detector temperature of 210° C , and injector temperature of 215° C. The voltage was set at 4 volts with sensitivity at 1000 and attenuation at 1 which have by our laboratory experimentation proved to be the most sensitive settings possible with the least amount of instrument noise and background in operation of the chromatograph and analysis of organochlorine pesticide residues. Concentration levels of organochlorine pesticides ranging from approximately 0.02 ng. lindane, heptachlor, or aldrin; 0.04 ng. DDE; 0.08 ng. dieldrin;0.2ng.DDT;to l.Ong. methoxychlor
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wool and collected in a 1-liter separatory funnel. The residual oil in the 60-ml. funnel and that collected on the glass wool was extracted with an additional 20 ml. of propanediol cyclic carbonate and the separation procedure repeated. Two hundred-fifty ml. of 2% Na 2 S0 4 solution was added to the sample solution in the 1-liter separatory funnel and the pesticide residue extracted by shaking vigorously for one minute with 200 ml. of petroleum ether. The aqueous solution was extracted a second time with 100 ml. of petroleum ether, the organic phases combined in a 500-ml. evaporation flask, and the volume reduced to 10-20 ml. by evaporation. The extract was transferred to a 20 x 250-mm. reservoir type chromatography column (Kontes, Teflon stopcock) containing a 4-inch bed of activated Florisil (Kensington Corp.—activated at 650° C. and stored after opening the bottle at 140° C.) over a glass wool plug and capped with about 1 inch of anhydrous NajSC),. The material on the column was eluted in sequence with 200 ml. of petroleum ether and then 120 ml. of 25% diethyl ether in petroleum ether. Each eluate was collected separately and reduced to 2-10 ml. volume in an all glass flash evaporator prior to analysis by gas chromatography. 2. Fish meal. A 50-gram subsample of each submitted fish meal sample was extracted in a similar manner to the corn meal. The extract was centrifuged in 250 ml. bottles in a Universal floor model centrifuge at 1025 x g for 15 minutes to separate the solids. Later the carbonate-oil solution was centrifuged in 15 ml. stoppered test tubes in a clinical centrifuge at about half maximum speed for 10 minutes to separate residual oil prior to the chilling. Four hundred ml. of 2% N a ^ C ^ solution was used during the partitioning of the pesticide residues into petroleum ether. 3. Fat and oils. A 50-gram subsample of each submitted fat sample was extracted by blending (and thus dissolving) in 200 ml. of
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A. C. WALDRON AND E. C. NABER
TABLE 1.—Percent recovery from samples Insecticide Lindane Heptachlor Aldrin Hept. epoxide DDE Dieldrin o,p-DDT DDD p,p'-DDT Methoxychlor Average percentage
Corn
Soybean
Alfalfa
Fish meal
Fat
86 87 99 84 104 67 98 85 102 99
106 87 81 117 93 105 90 98 97 110
92 65 75 90 80 87 78 83 84 96
90 101 102 94 109 115 111 107 104 89
97 73 63 96 66 83 61 89 78 77
91
~9%~
~83~
102
78
RESULTS AND DISCUSSION The efficiency of extraction and the average recovery of the insecticide through the methods used are outlined in Table 1. The recovery of insecticides was high in almost every case indicating satisfactory procedures. Variation between similar repetitive recoveries was at a narrow range well within the accepted standards for residue analysis. During the 24 month collection period approximately equal numbers of samples were submitted by each of the states cooperating in the survey. However it was interesting to note that with the exception of those samples submitted from Texas and California the majority of the corn, soybean and alfalfa meal samples originated in the Midwest despite the fact that they were submitted from Maine and Georgia. A large percentage of
the fish meal samples submitted by all states were of foreign origin, notably South America, Canada and Europe. A serious fire that occurred in our laboratory near the completion of the project terminated analytical work before all the samples could be analyzed. The percentage completion of samples was: soybean 95%, corn 96%, alfalfa meal 95%, fat 95%, and fish meal 56%. Based upon statistical analysis of data from the samples that were analyzed during the first 2 years versus the last 2 years of the analytical period, it was determined that the lack of data from the unanalyzed samples would not significantly alter the over-all conclusions. A summary of the feedstuff s contaminated with pesticide residue in relation to the origin of the samples is given in Tables 2 through 6. The contamination of feedstuffs with the pesticide residues investigated was quite common. For example, up to 68% of the soybean samples, 67% of the corn, 94% of the alfalfa, 98% of the fish meal, and 98% of the fat samples contained minute residues of at least one of the insecticides under investigation. In some samples only one insecticide residue and its metabolite were detected; but in the majority of samples multiple insecticide residues were present. DDT and its metabolites were the most commonly found contaminants in the five feedstuff commodities. Aldrin-dieldrin and heptachlor-heptachlor epoxide had somewhat
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(order of increasing retention time) gave approximately half scale recorder response under the conditions specified above. Confirmation of the identity of the pesticide residue was accomplished by injecting samples of extract on an identical column in the same gas chromatographic oven, but attached to a microcoulometric detector. All samples were analyzed for ten compounds. These were aldrin, dieldrin, heptachlor, heptachlor epoxide, p,p'-DDT, o,p'DDT, DDE, DDD, lindane and methoxychlor.
All
Northeast
Pacific coast
Southwest
Southeast
Plains states
Area of sample origin Midwest
Number of samples Description colof Heptalected information chlor 98 % of samples with resi- 19 due 7 % of samples with resi0 due 31 % of samples with resi- 13 due 47 % of samples with resi- 23 due % of samples with resi47 4 due % of samples with resi2 0 due No. of samples with 37 234 residue % of samples with resi- 16 due 1-24 Range of residue p.p.b. Av. residue for + sam7.3 ples (p.p.b.) Av. res. for all samples 1.2 (p.p.b.) % of samples > 10 3.4 p.p.b. % of samples > 50 0 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b. 83 35 1-278 16.9 6.0 6.4 3.0 1.7 0
8 1-7 2.3 0.2 0 0 0 0
0
38
30
23
29
Aldrin 42
18
0
6
6
0
0
Heptachlor epoxide 11
0
0
0
5.1
1.3
1-28 5.1
26
61
0
28
19
6
14
Dieldrin 38
0
0
0
10.7
3.4
1-47 7.9
43
101
50
43
30
48
57
P.P'DDT 50
TABLE 2.—Summary of feedstuffs contaminated with pesticide residues relative
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24
35
46
Southeast
Southwest
Pacific coast
203
2
Northeast
All
91
Area of sample origin Midwest
Number of samples collected
Heptachlor % of samples with resi- 20 due % of samples with resi0 due % of samples with resi8 due % of samples with resi- 34 due % of samples with resi7 due No. of samples with 35 residue % of samples with resi- 17 due Range of residue p.p.b. 1-29 Av. residue for + sam7.9 pies (p.p.b.) Av. residue for all sam1.4 pies p.p.b. % of samples > 10 5.4 p.p.b. % of samples > 50 0 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b.
Description of information
65 32 1-1909 48.7 15.6 4.9 1.0 1.0 1.0
12 1-36 5.0 0.6 1.5 0 0 0
26
49
21
0
Aldrin 34
24
9
26
0
0
Heptachlor epoxide 13
0
1.0
1.0
3.0
2.5
1-203 13.3
19
38
2
37
13
0
Dieldrin 25
0
0.5
1.0
8.4
3.9
1-131 8.1
48
97
50
60
54
0
P»P'DDT 40
TABLE 3.—Summary of feedstuffs contaminated with pesticide residues relati
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All
Pacific coast
Southwest
Southeast
Plains states
Northeast
Area of sample origin Midwest
Number of samples Description Heptacolof chlor lected information 73 % of samples with resi- 44 due 15 % of samples with resi- 13 due 16 % of samples with resi- 25 due 24 % of samples with resi- 25 due % of samples with resi- 28 46 due % of samples with resi- 16 45 due 226 No. of samples with 68 residue % of samples with resi- 30 due Range of residue p.p.b. 2-130 Av. residue for + sam- 18.2 ples (p.p.b.) Av. residue for all sam5.5 ples (p.p.b.) % of samples > 10 13.7 p.p.b. % of samples > 50 2.2 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b. P.P'DDT 77 80 81 75 65 93 176 78 1-532 45.4 35.3 55.8 14.2 8.0 0.4
Dieldrin 58 60 31 58 33 42 105 46 1-183 15.4 7.2 17.3 3.0 0.8 0
Aldrin 51 33 25 29 30 20 77 34 1-322 23.4 8.0 16.8 3.0 1.7 0
Heptachlor epoxide 33 13 25 17 24 33 62 27 1-138 15.3 4.2 10.2 1.3 0.4 0
TABLE 4.—Summary of feedstuffs contaminated with pesticide residues relative
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34
31
18
16
13
South America
Northeast
Midwest
Southwest
Pacific coast
127
2
Europe
All
9
Canada
Area of sample origin
Number of samples collected
% of samples > 500 p.p.b.
Description of information % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue No. of samples with residue % of samples with residue Range of residue p.p.b. Av. residue for + sampies (p.p.b.) Av. res. for ail samples (p.p.b.) % of samples > 10 p.p.b. % of samples > 50 p.p.b. % of samples > 100 1-116 11.2 2.0 4.7 0.8 0.8
1-64 10.4 2.5 6.3 0.8 0 0
18
24
0
23
31
6
22
26
12
0
31
8
6
61
35
12
0
11
Heptachlor
Heptachlor epoxide 22
0
0
2.4
11.8
4.4
1-78 10.1
43
55
15
38
67
32
53
0
56
Aldrin
0.8
0.8
1.6
11.8
23.0
1-2450 53.1
43
55
23
56
67
42
24
50
78
Dieldrin
0
7.0
18.9
50.4
30.8
1-266 39.5
78
99
62
88
94
87
82
100
100
P,P'DDT
TABLE 5.—Summary of feedstuffs contaminated with pesticide residue relati
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232
48
Pacific coast
All
36
Southwest
51
Southeast
8
41
Northeast
Southcentral
45
Midwest
Area of sample origin
Number of samples collected
Description of Heptainformation chlor % of samples with resi4 due % of samples with resi- 12 due % of samples with resi8 due % of samples with resi- 13 due % of samples with resi- 58 due % of samples with resi- 13 due No. of samples with 38 residue % of samples with resi- 16 due Range of residue p.p.b. 1-734 Av. residue for + sam- 69.1 ples (p.p.b.) Av. res. for all samples 11.3 (p.p.b.) % of samples > 10 8.2 p.p.b. % of samples > 50 3.9 p.p.b. % of samples > 100 2.6 p.p.b. % of samples > 500 0.9 p.p.b. 35 15 1-8395 260 39.3 6.5 2.2 1.3 0.4
25 1-62 9.8 2.4 6.9 0.4 0 0
19
11
0
24
12
13
Aldrin
58
19
36
50
16
17
38
Heptachlor epoxide
46
71
0
3.0
6.0
41.8
19.2
27.2
0
0.9
3.9
22.0
9.7
1-448 21.3
106
164
1-389
48
47
38
24
66
47
P,P'DDT
77
81
63
51
71
76
Dieldrin
TABLE 6.—Summary of feedstuffs contaminated with pesticide residue rela
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A. C. WALDRON AND E. C. NABER
dieldrin, DDE, and DDD respectively. As indicated earlier, DDT and its metabolites were the most frequently occurring residues in all feedstuffs and exhibited no particular relationship to any one area of collection. It was noted that the percentage incidence and the concentration level of DDE and DDD was generally much greater than the parent DDT compound in fat samples indicating a high degree of metabolism in the animal prior to processing for feedstuff purposes. Considerable metabolism also had occurred in fish indicated by the very similar incidence and concentration of the metabolites to the parent DDT. The frequency of contamination of feedstuffs by individual pesticides was evaluated by the chi square test of independence and association while a least squares analysis of variance was used to evaluate quantitative differences in pesticide levels among the feedstuffs (Steel and Torrie, 1960). Statistical evaluation of the residue data required the pooling of individual pesticide residue data of all similar samples submitted from the collection agencies. The data presented in Table 7 shows that the incidence (percentage of samples with residue) of heptachlor residues was greater (p < 0.05) in alfalfa meal than it was in soybean meal, corn meal or fat. Incidence of heptachlor in fish meal was intermediate between alfalfa meal and other feedstuffs, but not significantly different from any of the feedstuffs. The level (av. residue for + samples) of heptachlor in fat was significantly greater (p < 0.05) than that found in other feedstuffs. Heptachlor epoxide incidence was greater in alfalfa meal and fat than it was in soybean meal and corn meal. Incidence in fish meal was again intermediate and not statistically different from the others. Feedstuff levels of heptachlor epoxide found were not significantly different, but the higher level in alfalfa meal approached significance. The incidence of aldrin in fat was lower than the incidence of this compound in other
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less occurrence than the DDT compounds but the percent of samples exhibiting contamination was still appreciable. Lindane residues at very low concentrations occurred in 36 and 40% of the fat and fish meal samples, respectively, and in 15% or less of the other feedstuffs. With the exception of the alfalfa samples where 16% had residue the occurrence of methoxychlor in the feedstuffs was of little significance ranging from 6% in soybeans to 1% in fat and at very low concentration levels. The occurrence of pesticide residues in feedstuffs appeared to be closely associated with those areas of high crop production and hence supposedly high pesticide use. However, the concentration level of pesticide residue in those individual samples giving positive detection was not significantly different between the areas of collection. The concentration levels of pesticide residues found were generally very low as can be observed in Tables 2-6. Where residues occurred they were generally at average concentrations of less than 50 p.p.b., however, with occasional samples at higher concentrations of certain pesticides and only 0.1% exceeding 500 p.p.b. of any one of the 10 pesticides. The only pesticide residues that occurred in concentrations greater than 50 p.p.b. in soybean meal samples were 3% showing aldrin residue and 0.4% showing DDE and methoxychlor residues. One percent of the corn meal samples had aldrin, dieldrin, and p,p'-DDT residues; 0.5% had o,p-DDT and DDD; and 2% had methoxychlor residues in excess of 50 p.p.b. Other feedstuffs showed slightly higher concentrations of contamination from some pesticides in that 14.2, 9.3, and 8.0% of the alfalfa meal samples exceeded 50 p.p.b. of p,p'-DDT, DDE, and methoxychlor respectively; 18.9, 20.5 and 11.8% of the fish meal samples exceeded 50 p.p.b. of p,p'-DDT, DDE, and DDD respectively; and 6, 46.6 and 26.3% of the fat samples exceeded 50 p.p.b. of
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PESTICIDE RESIDUES IN FEEDSTUFFS
TABLE 7.—Comparison of the incidence and amount of pesticide residues found in feedstuffs Feedstuff Pesticide Heptachlor
%' Av 2
Heptachlor epoxide
% Av
Aldrin
% Av
Dieldrin
% Av
p,p'-DDT
% Av
o,p-DDT
%
DDE
% Av
DDD
% Av
Lindane
% Av
Methoxychlor
% Av
Corn meal
Alfalfa meal
Fish meal
Fats
16* 7.3* 8* 2.3* 35* 16.9* 26*" 5.1* 43* 7.9* 23* 5.6* 18* 2.9* 12* 5.3* 15* 2.7* 6* 17.7*
17* 7.9* 12* 5.0* 32* 48.7* 19* 13.3*" 48* 8.1* 25* 4.3* 21* 0.8* T 61.5" 15* 4.7* 6* 74.2*
30" 18.2* 27" 15.3* 34* 23.4* 46" 15.4*" 78" 45.4" 46" 20.0" 63" 23.4" 32" 51.9" 12* 14.5" 16" 361"
24*" 10.4* 18*" 11.2* 43* 10.1* 43" 53.1" 78" 39.5" 39" 13.4*" 89" 150c 68 c 29.7* 40" 14.2" 3* 83.0*
16* 69.1" 25" 9.8* 15" 260" 71 c 27.2" 46* 21.3*" 20* 21.6" 90" 93. l c 86 c 62.9" 36" 11.7" 1* 121*
'Percentage of samples found to contain residue (Incidence). Values on the same horizontal line bearing different superscript letters are significantly different from each other (p < 0.05) using the chi2 square method in pair comparisons. Average residue in p.p.b. for positive samples. Values on the same horizontal line bearing different superscript letters are significantly different from each other (p < 0.05) using the least squares analysis of variance. feedstuffs although those samples of fat containing aldrin had significantly more of the compound than the other feedstuffs. Dieldrin incidence was lowest in corn meal, significantly increased in alfalfa meal and fish meal, and was of highest incidence in fat. Dieldrin levels in fish meal and fat were significantly greater than those found in soybean meal. The low incidence of aldrin and high incidence of dieldrin in fat indicates metabolic conversion of aldrin to dieldrin while the fat was associated with a living plant or animal. Incidence of p,p'-DDT and o,p-DDT followed the same pattern in alfalfa meal and fish meal by showing significantly higher incidences of the two isomers than that in soybean meal, corn meal or fat. This would be expected because technical DDT contains both isomers. However, more samples of all
feedstuffs were found to contain p,p'-DDT than o,p-DDT. In part this difference is due to the ratios of the two compounds found in commercial products and the limits of detection in analysis. Soybean meal and corn meal contained the lowest levels of both DDT isomers while alfalfa meal contained significantly higher levels of the isomers. Fish meal and fat were intermediate in concentration except that fish meal contained more of the p,p'-DDT and fat more of the o,p-DDT than did soybean meal or corn meal. In addition, the percentage of samples containing > 50 p.p.b. of p,p'-DDT was significantly greater for alfalfa meal and fish meal than for the other feedstuffs. The DDE metabolite occurred in a larger percentage of the samples of alfalfa meal, fish meal and fat than it did in soybean meal and corn meal. DDE levels
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Av
Soybean meal
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A. C. WALDRON AND E. C. NABER
even low concentrations of pesticide residue in feed to the poultry and hence any potential accumulation in tissues and eggs. The data produced by this survey of feedstuffs indicates that contamination of poultry via their normal food supply may not be grounds for excessive concern relative to pesticide residues in poultry products. Evaluation of the significance of pesticide residue accumulation in poultry products after prolonged consumption of feed contaminated with the pesticide concentration levels indicated in the survey reported herein will be the subject of a subsequent publication. ACKNOWLEDGEMENTS The authors wish to acknowledge the contribution to this study made by the technicians, Carol Day in the Poultry Department in receiving, logging, and storing all feedstuff samples, and Kay MacAllister, Carl Robb, and Melvin Aden, in the Pesticide Analytical Laboratory in conducting routine laboratory analysis. Also, acknowledgement is extended to Drs. George Mountney and Robert Cook, formerly professor and Chairman respectively of the O.S.U. Poultry Department, under whose direction the Project was originally initiated and to Dr. D. Lyle Goleman, Chairman of the Entomology Department O.S.U. and O.A.R.D.C. for coordination activities. REFERENCES Cummings, J. G., K. T. Zee, V. Turner, F. Quinn and R. E. Cook, 1966. Residues in eggs from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Ass. Off. Agr. Chetn. 49: 354-364. Cummings, J. G., M. Eidelman, V. Turner, D. Reed, K. T. Zee and R. E. Cook, 1967. Residues in poultry tissues from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Ass. Off. Agr. Chem. 50: 418-425. Draper, C. I., C. Biddulph, D. A. Greenwood, J. R. Harris, W. Binns and M. L. Miner, 1950. Concentration of DDT in tissues of chickens fed varying levels of DDT in the diet. Poultry Sci. 29: 756.
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were found to be highest in fish meal and fat, intermediate in alfalfa meal and lowest in soybean meal and corn meal. The data indicate that fish meal and fat tend to accumulate more of the DDE since the ratio of DDE to DDT is larger in these two feedstuffs. The pattern of DDD incidence resembles that of DDE but overall incidence is somewhat reduced. When compared to DDE the amounts of DDD found in positive samples of soybean meal, corn meal and alfalfa meal were greater and the amounts of DDD found in fish meal and fat were smaller. However, concentration of DDD in corn meal, alfalfa meal, and fat was greater than the DDD concentration in soybean meal and fish meal. Both fish meal and fat samples exceeded 50 p.p.b. more often than corn meal and soybean meal for both DDD and DDE. Alfalfa meal was intermediate and like fish meal in the case of DDE but like corn meal in the case of DDD. Lindane residues were found more frequently in fish meal and fat than in the three plant feedstuffs. Levels of lindane were greater in alfalfa meal, fish meal and fat than in soybean meal and corn meal. Incidence of methoxychlor was significantly greater in alfalfa meal than in the other feedstuffs. In addition, the amount of methoxychlor and the percentage of samples exceeding 50 p.p.b. was also greater for alfalfa meal. Limited numbers of positive samples made other comparisons meaningless. The results of the research reported herein confirms the suspicion that low levels of pesticide contamination (< 50 p.p.b.) are a common occurrence in the feedstuffs that are used in formulating poultry rations throughout the United States. However, only 0.1% of the feedstuffs examined exceeded 500 p.p.b. of any of the 10 pesticides. Due to the concept of zero tolerance of pesticide residues in poultry products it is necessary to determine the significance of transfer of
PESTICIDE RESIDUES IN FEEDSTUFFS
J. Econ. Ent. 55: 477-479. Steel, R. G. D. and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Thompson, E. M., G. J. Mountney and G. W. Ware, 1967. Methoxychlor residues in chicken eggs. J. Econ. Ent. 60: 235-237. United States Department of Agriculture, 1965. USDA study shows pesticide residues in poultry samples. Feedstuffs, 37(33): 1, 70. Waldron, A. C , H. E. Kaeser, D. L. Goleman, J. R. Staubus and H. D. Niemczyk, 1968. Heptachlor and heptachlor epoxide on fall-treated alfalfa and in milk and cow tissues. Agric. Food Chem. 16: 627-631. Waldron, A. C , and D. L. Goleman, 1969. Ethyl and methyl parathion in green and cured alfalfa. Agric. Food Chem. 17: 1066-1069. Waldron, A. C , 1971. Analysis for organochlorine pesticide residues in agricultural commodities, soil and water. Unpublished report by Ohio Cooperative Extension Service, Ohio State University, Columbus. Ware, G. W., and E. C. Naber, 1961. Lindane in eggs and chicken tissues. J. Econ. Ent. 54: 675-677. Ware, G. W., and E. C. Naber, 1962. Lindane and BHC in egg yolks following recommended uses for louse and mite control. J. Econ. Ent. 55: 568-570.
NEWS AND NOTES (Continued from page 1358) nity had their bursas intact. ' "Glick was so intrigued with this discovery that he wrote a paper which he submitted to the magazine, Science. The editors felt it was not of sufficient interest to their subscribers and turned it down. Glick subsequently saw it published in The Journal of Poultry Science. ' "There it might have languished and become of interest only to chicken scholars if it had not been accidentally picked up by Dr. Harold Wolfe of the University of Wisconsin, who was then working in the broad field of immunity. Wolfe, a friend of mine, told me during a meeting of immunologists that he had taken the bursas out of newly hatched chicks and found an absence of antibodies, the same effect as noted by Chang. The bursa was evidently essential to antibody production and to immunity." 'Goodpaused. "Thestrange thingwasthatachicken
also has a thymus, which on the basis of my own observations I would have expected to regulate the immune system. This led me, and independently, Dr. J. F. A. P. Miller in England, to remove thymuses from newborn animals to see what happens. It dawned on me suddenly that the chickens might have two immune systems. This idea was strengthened when a student stood up at the meeting to make just that same provocative statement," Dr. Good smiled. ' "I was quite excited about all this, and I brought in a team of scientists to work out the role of the two organs. Other labs started in, and there was intense competition. Max Cooper of our lab made one of the definitive experiments when he rounded up some newly hatched chicks and divided them into two groups. In one group he removed the thymus; in the other he took out the bursa, and in both he destroyed with radiation any vestiges of embryonic immunity.
(Continued on page 1386)
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Furman, D. P., and R. A. Bankowski, 1949. Absorption of benzene hexachloride in poultry. J. Econ. Ent. 42: 980-982. Hixen, E., and M. H. Muma, 1947. Effect of benzene hexachloride on the flavor of poultry meat. Science, 106: 422-423. Ivey, M. C , R. H. Roberts, H. D. Mann and H. V. Claborn, 1961. Lindane residues in chickens and eggs following poultry house sprays. J. Econ. Ent. 54: 487-488. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman, 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Mills, P. A., J. H. Onley and R. A. Gaither, 1963. Rapid method for chlorinated pesticide residues in nonfatty foods. J. Ass. Off. Agr. Chem. 46: 186-191. Minyard, J. P., and E. R. Jackson, 1963. Pesticide residues in commercial animal feeds. J. Ass. Off. Agr. Chem. 46: 843-859. Mountney, G. J., and J. H. Quisenberry, 1957. Contamination of broilers with chlordane and pyrethrum. Poultry Sci. 36: 923-924. Naber, E. C , and G. W. Ware, 1965. Effect of kepone and mirex on reproductive performance in the laying hen. Poultry Sci. 44: 875-880. Olney, C. E., W. E. Donaldson and T. W. Kerr, 1962. Methoxychlor in eggs and chicken tissues.
1371
Pesticide Analytical Laboratory, Cooperative Extension Service, Department of Entomology and Department of Poultry Science. The Ohio State University and Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (Received for publication October 9, 1973)
POULTRY SCIENCE 53: 1359-1371, 1974
INTRODUCTION ESTICIDE contamination of poultry meat and eggs resulting from feeding of low level concentrations of pesticides or from pesticide contamination of the housing environment has been studied by many workers. Hixen and Muma (1947) reported that the flavor of poultry meat was adversely affected by the feeding or environmental application of benzene hexachloride. Furman and Bankowski (1949) showed that benzene hexachloride is readily absorbed by poultry and subsequently it has been shown that most chlorinated organic insecticides are absorbed through the skin or the digestive tract. Contamination of poultry meat and eggs by lindane (Ivey et al., 1961; Ware and Naber,
P
1. Ohio Agricultural Research and Development Center Journal Article No. 73-73. Supported by U.S.D.A., A.R.S. Research and Service Contract No. 12-14-100-8952(44).
1961, 1962); DDT (Draper et al, 1950; Liska etal, 1964); methoxychlor (Olney etal., 1962; Thompson et al., 1967) kepone and mirex (Naber and Ware, 1965); and chlordanepyrethrum (Mountney and Quisenberry, 1957) has been reported from feeding and /or environmental application of these compounds. Cummings et al. (1966,1967) fed diets containing combinations of lindane, DDT, heptachlor epoxide, dieldrin, and endrin to hens for 14 weeks and showed transfer of residues to eggs and tissues. In 1965 the United States Department of Agriculture reported that minute pesticide residues were present in all 2600 samples of poultry meat tested in a nationwide survey. The residue levels in almost all cases were well below the concentration considered as a safe daily intake level in the human diet. Thus no attempts were made to define the sources of contamination as a means to correct the situation and/or protect the poultry producer from contamination that could re-
1359
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ABSTRACT General and broad occurrences of minute organochlorine pesticide residues were indicated in feedstuffs used in poultry feeds in the United States. Corn, soybean meal, alfalfa meal, fish meal, and fat samples were collected and submitted bimonthly over a 24 month period from Maine, Georgia, Texas, Ohio, and California and analyzed for aldrin, dieldrin, heptachlor, heptachlor epoxide, lindane, DDE, DDD, o,p-DDT, p,p'-DDT, and methoxychlor. Concentration of individual pesticides when occurring were generally in the range of 10 to 50 p.p.b. although higher concentrations occasionally occurred. DDT and its metabolites were the most frequently occurring organochlorine pesticides detected. The highest concentration and frequency of residues detected were associated with fish meal and fat samples. Occurrence of residues in corn and soybean meal was generally infrequent and at very low concentrations. The presence of pesticide residues was very closely correlated with the areas of high crop production and hence greater pesticide use. In general it appears that poultry feeds formulated from feedstuff sources as analyzed in this study (corn and soybean meal constituting by far the major part of the poultry ration) would not contain organochlorine pesticide residues large enough to cause any major contamination problems with poultry products.
1360
A. C. WALDRON AND E. C. NABER
It thus becomes important to establish the level of pesticide residues naturally occurring in poultry feed ingredients over which the poultry producer has no control, to establish the transfer of these residues to poultry meat and eggs when raised on such feeds relative to the tissue and egg contamination, and to establish a realistic tolerance level for certain pesticides in poultry products and feedstuffs that will protect the poultry producer and at the same time not cause concern in the human diet. The purpose of this research is to provide data required to establish the extent of contamination in the feed supply. EXPERIMENTAL PROCEDURES Collection of Samples. Four samples each of soybean meal, corn or corn meal, alfalfa meal, fish meal, and fat were collected bimonthly over a period of 24 months from Ohio, Maine, Georgia, Texas, and California. Samples were collected by State Departments
of Agriculture personnel and submitted to The Ohio State University Poultry Science Department where they were stored (generally at -20° C. in a freezer) until analyzed. A total of 1162 samples were collected during the two year period. Grain samples were ground into meal prior to analysis. Extraction and Cleaning Procedures. Extraction procedures for the feed ingredient samples followed in general the principles published in the literature for extraction of organochlorine insecticides (Mills etal., 1963; Minyard and Jackson, 1963; Cummings et al., 1967; Waldron et al, 1968; and Waldron and Goleman, 1969) but with modifications adaptable to the techniques of this laboratory. Additional modifications in procedures, solvent systems, elution techniques, gas chromatographic operation, etc. that increased laboratory efficiency were developed during the course of the study (Waldron, 1971). 1. Corn meal and soybean meal. A 50-gram subsample of each submitted sample was extracted with 300 ml. of glass distilled petroleum ether by blending for 8 minutes in a Waring Blendor set at a moderate speed that would not cause heating of the solvent during blending. The extract, after permitting the solids to settle, was filtered through Whatman # 1 filter paper and collected in a 16-oz. narrow neck bottle. The blendor and filtration equipment. were covered with the lid and aluminium foil, respectively, to prevent evaporation of the petroleum ether. An aliquot of extract equal to one half of the sample (150 ml.) was transferred to a 500-ml. evaporation flask, 20 ml. of propanediol cyclic carbonate added and the petroleum ether removed by evaporation with a flash evaporator. The carbonate-oil solution was transferred to a 60-ml. separatory funnel and chilled for about one hour in the freezer to separate residual oil or fat. The chilled carbonate solution was filtered through glass
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move his produce from the market. In most cases the poultry producer must rely on external sources for his feeds and consequently could become a victim of a condition over which he has no control. There has been concern for several years about the potential that exists in adulterating poultry products with pesticide residues. The problem may have international implications because considerable quantities of poultry products are exported and considerable quantities of poultry feed ingredients (such as fish meal) are imported. The increased use of pesticides in crop protection during recent years increases the possibility of feedstuff contamination and the consequent exposure of poultry to these products. Because some pesticides, such as some chlorinated hydrocarbons, are retained and stored in body fat tissues the potential for contamination of poultry tissues and, consequently, eggs becomes significant. Such contamination could result in significant economic losses.
PESTICIDE RESIDUES IN FEEDSTUFFS
glass-distilled acetone for 3-4 minutes. Two hundred ml. of pesticide quality acetonitrile was added causing the fat to coagulate during an additional 3-4 minutes of blending, and leaving the pesticide residue in solution. The rest of the procedure followed that for corn meal except that greater patience was required in permitting the oil phase to separate. 4. Alfalfa meal. A 25-gram subsample of each submitted alfalfa meal sample was extracted by blending with 300 ml. of acetonitrile for 5-6 minutes (later samples were extracted with 400 ml. of acetone). The extract was filtered and an aliquot representing 1/2 of the sample was combined with 700 ml. of approximately 0.18 N H 2 S 0 4 and 150 ml. of petroleum ether for extraction in a separatory funnel. The aqueous phase was extracted a second time with petroleum ether and the combined organic phases concentrated and placed on the florisil column and the procedure for corn meal followed. In later extractions with acetone, 500 ml. of 2% NajSC^ was used instead of sulfuric acid. Gas Chromatographic Analysis. Aliquots of the sample extracts were injected on a 6-ft. glass column containing a 1:1 mixture of 10.45% DC-200 and 15.14% QF-1 on Gas Chrom Q. A Barber Coleman Series 5000 gas chromatograph equipped with a Tritium foil electron capture detector was operated at a column temperature of 190° C , detector temperature of 210° C , and injector temperature of 215° C. The voltage was set at 4 volts with sensitivity at 1000 and attenuation at 1 which have by our laboratory experimentation proved to be the most sensitive settings possible with the least amount of instrument noise and background in operation of the chromatograph and analysis of organochlorine pesticide residues. Concentration levels of organochlorine pesticides ranging from approximately 0.02 ng. lindane, heptachlor, or aldrin; 0.04 ng. DDE; 0.08 ng. dieldrin;0.2ng.DDT;to l.Ong. methoxychlor
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wool and collected in a 1-liter separatory funnel. The residual oil in the 60-ml. funnel and that collected on the glass wool was extracted with an additional 20 ml. of propanediol cyclic carbonate and the separation procedure repeated. Two hundred-fifty ml. of 2% Na 2 S0 4 solution was added to the sample solution in the 1-liter separatory funnel and the pesticide residue extracted by shaking vigorously for one minute with 200 ml. of petroleum ether. The aqueous solution was extracted a second time with 100 ml. of petroleum ether, the organic phases combined in a 500-ml. evaporation flask, and the volume reduced to 10-20 ml. by evaporation. The extract was transferred to a 20 x 250-mm. reservoir type chromatography column (Kontes, Teflon stopcock) containing a 4-inch bed of activated Florisil (Kensington Corp.—activated at 650° C. and stored after opening the bottle at 140° C.) over a glass wool plug and capped with about 1 inch of anhydrous NajSC),. The material on the column was eluted in sequence with 200 ml. of petroleum ether and then 120 ml. of 25% diethyl ether in petroleum ether. Each eluate was collected separately and reduced to 2-10 ml. volume in an all glass flash evaporator prior to analysis by gas chromatography. 2. Fish meal. A 50-gram subsample of each submitted fish meal sample was extracted in a similar manner to the corn meal. The extract was centrifuged in 250 ml. bottles in a Universal floor model centrifuge at 1025 x g for 15 minutes to separate the solids. Later the carbonate-oil solution was centrifuged in 15 ml. stoppered test tubes in a clinical centrifuge at about half maximum speed for 10 minutes to separate residual oil prior to the chilling. Four hundred ml. of 2% N a ^ C ^ solution was used during the partitioning of the pesticide residues into petroleum ether. 3. Fat and oils. A 50-gram subsample of each submitted fat sample was extracted by blending (and thus dissolving) in 200 ml. of
1361
1362
A. C. WALDRON AND E. C. NABER
TABLE 1.—Percent recovery from samples Insecticide Lindane Heptachlor Aldrin Hept. epoxide DDE Dieldrin o,p-DDT DDD p,p'-DDT Methoxychlor Average percentage
Corn
Soybean
Alfalfa
Fish meal
Fat
86 87 99 84 104 67 98 85 102 99
106 87 81 117 93 105 90 98 97 110
92 65 75 90 80 87 78 83 84 96
90 101 102 94 109 115 111 107 104 89
97 73 63 96 66 83 61 89 78 77
91
~9%~
~83~
102
78
RESULTS AND DISCUSSION The efficiency of extraction and the average recovery of the insecticide through the methods used are outlined in Table 1. The recovery of insecticides was high in almost every case indicating satisfactory procedures. Variation between similar repetitive recoveries was at a narrow range well within the accepted standards for residue analysis. During the 24 month collection period approximately equal numbers of samples were submitted by each of the states cooperating in the survey. However it was interesting to note that with the exception of those samples submitted from Texas and California the majority of the corn, soybean and alfalfa meal samples originated in the Midwest despite the fact that they were submitted from Maine and Georgia. A large percentage of
the fish meal samples submitted by all states were of foreign origin, notably South America, Canada and Europe. A serious fire that occurred in our laboratory near the completion of the project terminated analytical work before all the samples could be analyzed. The percentage completion of samples was: soybean 95%, corn 96%, alfalfa meal 95%, fat 95%, and fish meal 56%. Based upon statistical analysis of data from the samples that were analyzed during the first 2 years versus the last 2 years of the analytical period, it was determined that the lack of data from the unanalyzed samples would not significantly alter the over-all conclusions. A summary of the feedstuff s contaminated with pesticide residue in relation to the origin of the samples is given in Tables 2 through 6. The contamination of feedstuffs with the pesticide residues investigated was quite common. For example, up to 68% of the soybean samples, 67% of the corn, 94% of the alfalfa, 98% of the fish meal, and 98% of the fat samples contained minute residues of at least one of the insecticides under investigation. In some samples only one insecticide residue and its metabolite were detected; but in the majority of samples multiple insecticide residues were present. DDT and its metabolites were the most commonly found contaminants in the five feedstuff commodities. Aldrin-dieldrin and heptachlor-heptachlor epoxide had somewhat
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(order of increasing retention time) gave approximately half scale recorder response under the conditions specified above. Confirmation of the identity of the pesticide residue was accomplished by injecting samples of extract on an identical column in the same gas chromatographic oven, but attached to a microcoulometric detector. All samples were analyzed for ten compounds. These were aldrin, dieldrin, heptachlor, heptachlor epoxide, p,p'-DDT, o,p'DDT, DDE, DDD, lindane and methoxychlor.
All
Northeast
Pacific coast
Southwest
Southeast
Plains states
Area of sample origin Midwest
Number of samples Description colof Heptalected information chlor 98 % of samples with resi- 19 due 7 % of samples with resi0 due 31 % of samples with resi- 13 due 47 % of samples with resi- 23 due % of samples with resi47 4 due % of samples with resi2 0 due No. of samples with 37 234 residue % of samples with resi- 16 due 1-24 Range of residue p.p.b. Av. residue for + sam7.3 ples (p.p.b.) Av. res. for all samples 1.2 (p.p.b.) % of samples > 10 3.4 p.p.b. % of samples > 50 0 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b. 83 35 1-278 16.9 6.0 6.4 3.0 1.7 0
8 1-7 2.3 0.2 0 0 0 0
0
38
30
23
29
Aldrin 42
18
0
6
6
0
0
Heptachlor epoxide 11
0
0
0
5.1
1.3
1-28 5.1
26
61
0
28
19
6
14
Dieldrin 38
0
0
0
10.7
3.4
1-47 7.9
43
101
50
43
30
48
57
P.P'DDT 50
TABLE 2.—Summary of feedstuffs contaminated with pesticide residues relative
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24
35
46
Southeast
Southwest
Pacific coast
203
2
Northeast
All
91
Area of sample origin Midwest
Number of samples collected
Heptachlor % of samples with resi- 20 due % of samples with resi0 due % of samples with resi8 due % of samples with resi- 34 due % of samples with resi7 due No. of samples with 35 residue % of samples with resi- 17 due Range of residue p.p.b. 1-29 Av. residue for + sam7.9 pies (p.p.b.) Av. residue for all sam1.4 pies p.p.b. % of samples > 10 5.4 p.p.b. % of samples > 50 0 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b.
Description of information
65 32 1-1909 48.7 15.6 4.9 1.0 1.0 1.0
12 1-36 5.0 0.6 1.5 0 0 0
26
49
21
0
Aldrin 34
24
9
26
0
0
Heptachlor epoxide 13
0
1.0
1.0
3.0
2.5
1-203 13.3
19
38
2
37
13
0
Dieldrin 25
0
0.5
1.0
8.4
3.9
1-131 8.1
48
97
50
60
54
0
P»P'DDT 40
TABLE 3.—Summary of feedstuffs contaminated with pesticide residues relati
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All
Pacific coast
Southwest
Southeast
Plains states
Northeast
Area of sample origin Midwest
Number of samples Description Heptacolof chlor lected information 73 % of samples with resi- 44 due 15 % of samples with resi- 13 due 16 % of samples with resi- 25 due 24 % of samples with resi- 25 due % of samples with resi- 28 46 due % of samples with resi- 16 45 due 226 No. of samples with 68 residue % of samples with resi- 30 due Range of residue p.p.b. 2-130 Av. residue for + sam- 18.2 ples (p.p.b.) Av. residue for all sam5.5 ples (p.p.b.) % of samples > 10 13.7 p.p.b. % of samples > 50 2.2 p.p.b. % of samples > 100 0 p.p.b. % of samples > 500 0 p.p.b. P.P'DDT 77 80 81 75 65 93 176 78 1-532 45.4 35.3 55.8 14.2 8.0 0.4
Dieldrin 58 60 31 58 33 42 105 46 1-183 15.4 7.2 17.3 3.0 0.8 0
Aldrin 51 33 25 29 30 20 77 34 1-322 23.4 8.0 16.8 3.0 1.7 0
Heptachlor epoxide 33 13 25 17 24 33 62 27 1-138 15.3 4.2 10.2 1.3 0.4 0
TABLE 4.—Summary of feedstuffs contaminated with pesticide residues relative
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34
31
18
16
13
South America
Northeast
Midwest
Southwest
Pacific coast
127
2
Europe
All
9
Canada
Area of sample origin
Number of samples collected
% of samples > 500 p.p.b.
Description of information % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue % of samples with residue No. of samples with residue % of samples with residue Range of residue p.p.b. Av. residue for + sampies (p.p.b.) Av. res. for ail samples (p.p.b.) % of samples > 10 p.p.b. % of samples > 50 p.p.b. % of samples > 100 1-116 11.2 2.0 4.7 0.8 0.8
1-64 10.4 2.5 6.3 0.8 0 0
18
24
0
23
31
6
22
26
12
0
31
8
6
61
35
12
0
11
Heptachlor
Heptachlor epoxide 22
0
0
2.4
11.8
4.4
1-78 10.1
43
55
15
38
67
32
53
0
56
Aldrin
0.8
0.8
1.6
11.8
23.0
1-2450 53.1
43
55
23
56
67
42
24
50
78
Dieldrin
0
7.0
18.9
50.4
30.8
1-266 39.5
78
99
62
88
94
87
82
100
100
P,P'DDT
TABLE 5.—Summary of feedstuffs contaminated with pesticide residue relati
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232
48
Pacific coast
All
36
Southwest
51
Southeast
8
41
Northeast
Southcentral
45
Midwest
Area of sample origin
Number of samples collected
Description of Heptainformation chlor % of samples with resi4 due % of samples with resi- 12 due % of samples with resi8 due % of samples with resi- 13 due % of samples with resi- 58 due % of samples with resi- 13 due No. of samples with 38 residue % of samples with resi- 16 due Range of residue p.p.b. 1-734 Av. residue for + sam- 69.1 ples (p.p.b.) Av. res. for all samples 11.3 (p.p.b.) % of samples > 10 8.2 p.p.b. % of samples > 50 3.9 p.p.b. % of samples > 100 2.6 p.p.b. % of samples > 500 0.9 p.p.b. 35 15 1-8395 260 39.3 6.5 2.2 1.3 0.4
25 1-62 9.8 2.4 6.9 0.4 0 0
19
11
0
24
12
13
Aldrin
58
19
36
50
16
17
38
Heptachlor epoxide
46
71
0
3.0
6.0
41.8
19.2
27.2
0
0.9
3.9
22.0
9.7
1-448 21.3
106
164
1-389
48
47
38
24
66
47
P,P'DDT
77
81
63
51
71
76
Dieldrin
TABLE 6.—Summary of feedstuffs contaminated with pesticide residue rela
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1368
A. C. WALDRON AND E. C. NABER
dieldrin, DDE, and DDD respectively. As indicated earlier, DDT and its metabolites were the most frequently occurring residues in all feedstuffs and exhibited no particular relationship to any one area of collection. It was noted that the percentage incidence and the concentration level of DDE and DDD was generally much greater than the parent DDT compound in fat samples indicating a high degree of metabolism in the animal prior to processing for feedstuff purposes. Considerable metabolism also had occurred in fish indicated by the very similar incidence and concentration of the metabolites to the parent DDT. The frequency of contamination of feedstuffs by individual pesticides was evaluated by the chi square test of independence and association while a least squares analysis of variance was used to evaluate quantitative differences in pesticide levels among the feedstuffs (Steel and Torrie, 1960). Statistical evaluation of the residue data required the pooling of individual pesticide residue data of all similar samples submitted from the collection agencies. The data presented in Table 7 shows that the incidence (percentage of samples with residue) of heptachlor residues was greater (p < 0.05) in alfalfa meal than it was in soybean meal, corn meal or fat. Incidence of heptachlor in fish meal was intermediate between alfalfa meal and other feedstuffs, but not significantly different from any of the feedstuffs. The level (av. residue for + samples) of heptachlor in fat was significantly greater (p < 0.05) than that found in other feedstuffs. Heptachlor epoxide incidence was greater in alfalfa meal and fat than it was in soybean meal and corn meal. Incidence in fish meal was again intermediate and not statistically different from the others. Feedstuff levels of heptachlor epoxide found were not significantly different, but the higher level in alfalfa meal approached significance. The incidence of aldrin in fat was lower than the incidence of this compound in other
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less occurrence than the DDT compounds but the percent of samples exhibiting contamination was still appreciable. Lindane residues at very low concentrations occurred in 36 and 40% of the fat and fish meal samples, respectively, and in 15% or less of the other feedstuffs. With the exception of the alfalfa samples where 16% had residue the occurrence of methoxychlor in the feedstuffs was of little significance ranging from 6% in soybeans to 1% in fat and at very low concentration levels. The occurrence of pesticide residues in feedstuffs appeared to be closely associated with those areas of high crop production and hence supposedly high pesticide use. However, the concentration level of pesticide residue in those individual samples giving positive detection was not significantly different between the areas of collection. The concentration levels of pesticide residues found were generally very low as can be observed in Tables 2-6. Where residues occurred they were generally at average concentrations of less than 50 p.p.b., however, with occasional samples at higher concentrations of certain pesticides and only 0.1% exceeding 500 p.p.b. of any one of the 10 pesticides. The only pesticide residues that occurred in concentrations greater than 50 p.p.b. in soybean meal samples were 3% showing aldrin residue and 0.4% showing DDE and methoxychlor residues. One percent of the corn meal samples had aldrin, dieldrin, and p,p'-DDT residues; 0.5% had o,p-DDT and DDD; and 2% had methoxychlor residues in excess of 50 p.p.b. Other feedstuffs showed slightly higher concentrations of contamination from some pesticides in that 14.2, 9.3, and 8.0% of the alfalfa meal samples exceeded 50 p.p.b. of p,p'-DDT, DDE, and methoxychlor respectively; 18.9, 20.5 and 11.8% of the fish meal samples exceeded 50 p.p.b. of p,p'-DDT, DDE, and DDD respectively; and 6, 46.6 and 26.3% of the fat samples exceeded 50 p.p.b. of
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PESTICIDE RESIDUES IN FEEDSTUFFS
TABLE 7.—Comparison of the incidence and amount of pesticide residues found in feedstuffs Feedstuff Pesticide Heptachlor
%' Av 2
Heptachlor epoxide
% Av
Aldrin
% Av
Dieldrin
% Av
p,p'-DDT
% Av
o,p-DDT
%
DDE
% Av
DDD
% Av
Lindane
% Av
Methoxychlor
% Av
Corn meal
Alfalfa meal
Fish meal
Fats
16* 7.3* 8* 2.3* 35* 16.9* 26*" 5.1* 43* 7.9* 23* 5.6* 18* 2.9* 12* 5.3* 15* 2.7* 6* 17.7*
17* 7.9* 12* 5.0* 32* 48.7* 19* 13.3*" 48* 8.1* 25* 4.3* 21* 0.8* T 61.5" 15* 4.7* 6* 74.2*
30" 18.2* 27" 15.3* 34* 23.4* 46" 15.4*" 78" 45.4" 46" 20.0" 63" 23.4" 32" 51.9" 12* 14.5" 16" 361"
24*" 10.4* 18*" 11.2* 43* 10.1* 43" 53.1" 78" 39.5" 39" 13.4*" 89" 150c 68 c 29.7* 40" 14.2" 3* 83.0*
16* 69.1" 25" 9.8* 15" 260" 71 c 27.2" 46* 21.3*" 20* 21.6" 90" 93. l c 86 c 62.9" 36" 11.7" 1* 121*
'Percentage of samples found to contain residue (Incidence). Values on the same horizontal line bearing different superscript letters are significantly different from each other (p < 0.05) using the chi2 square method in pair comparisons. Average residue in p.p.b. for positive samples. Values on the same horizontal line bearing different superscript letters are significantly different from each other (p < 0.05) using the least squares analysis of variance. feedstuffs although those samples of fat containing aldrin had significantly more of the compound than the other feedstuffs. Dieldrin incidence was lowest in corn meal, significantly increased in alfalfa meal and fish meal, and was of highest incidence in fat. Dieldrin levels in fish meal and fat were significantly greater than those found in soybean meal. The low incidence of aldrin and high incidence of dieldrin in fat indicates metabolic conversion of aldrin to dieldrin while the fat was associated with a living plant or animal. Incidence of p,p'-DDT and o,p-DDT followed the same pattern in alfalfa meal and fish meal by showing significantly higher incidences of the two isomers than that in soybean meal, corn meal or fat. This would be expected because technical DDT contains both isomers. However, more samples of all
feedstuffs were found to contain p,p'-DDT than o,p-DDT. In part this difference is due to the ratios of the two compounds found in commercial products and the limits of detection in analysis. Soybean meal and corn meal contained the lowest levels of both DDT isomers while alfalfa meal contained significantly higher levels of the isomers. Fish meal and fat were intermediate in concentration except that fish meal contained more of the p,p'-DDT and fat more of the o,p-DDT than did soybean meal or corn meal. In addition, the percentage of samples containing > 50 p.p.b. of p,p'-DDT was significantly greater for alfalfa meal and fish meal than for the other feedstuffs. The DDE metabolite occurred in a larger percentage of the samples of alfalfa meal, fish meal and fat than it did in soybean meal and corn meal. DDE levels
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Av
Soybean meal
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A. C. WALDRON AND E. C. NABER
even low concentrations of pesticide residue in feed to the poultry and hence any potential accumulation in tissues and eggs. The data produced by this survey of feedstuffs indicates that contamination of poultry via their normal food supply may not be grounds for excessive concern relative to pesticide residues in poultry products. Evaluation of the significance of pesticide residue accumulation in poultry products after prolonged consumption of feed contaminated with the pesticide concentration levels indicated in the survey reported herein will be the subject of a subsequent publication. ACKNOWLEDGEMENTS The authors wish to acknowledge the contribution to this study made by the technicians, Carol Day in the Poultry Department in receiving, logging, and storing all feedstuff samples, and Kay MacAllister, Carl Robb, and Melvin Aden, in the Pesticide Analytical Laboratory in conducting routine laboratory analysis. Also, acknowledgement is extended to Drs. George Mountney and Robert Cook, formerly professor and Chairman respectively of the O.S.U. Poultry Department, under whose direction the Project was originally initiated and to Dr. D. Lyle Goleman, Chairman of the Entomology Department O.S.U. and O.A.R.D.C. for coordination activities. REFERENCES Cummings, J. G., K. T. Zee, V. Turner, F. Quinn and R. E. Cook, 1966. Residues in eggs from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Ass. Off. Agr. Chetn. 49: 354-364. Cummings, J. G., M. Eidelman, V. Turner, D. Reed, K. T. Zee and R. E. Cook, 1967. Residues in poultry tissues from low level feeding of five chlorinated hydrocarbon insecticides to hens. J. Ass. Off. Agr. Chem. 50: 418-425. Draper, C. I., C. Biddulph, D. A. Greenwood, J. R. Harris, W. Binns and M. L. Miner, 1950. Concentration of DDT in tissues of chickens fed varying levels of DDT in the diet. Poultry Sci. 29: 756.
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were found to be highest in fish meal and fat, intermediate in alfalfa meal and lowest in soybean meal and corn meal. The data indicate that fish meal and fat tend to accumulate more of the DDE since the ratio of DDE to DDT is larger in these two feedstuffs. The pattern of DDD incidence resembles that of DDE but overall incidence is somewhat reduced. When compared to DDE the amounts of DDD found in positive samples of soybean meal, corn meal and alfalfa meal were greater and the amounts of DDD found in fish meal and fat were smaller. However, concentration of DDD in corn meal, alfalfa meal, and fat was greater than the DDD concentration in soybean meal and fish meal. Both fish meal and fat samples exceeded 50 p.p.b. more often than corn meal and soybean meal for both DDD and DDE. Alfalfa meal was intermediate and like fish meal in the case of DDE but like corn meal in the case of DDD. Lindane residues were found more frequently in fish meal and fat than in the three plant feedstuffs. Levels of lindane were greater in alfalfa meal, fish meal and fat than in soybean meal and corn meal. Incidence of methoxychlor was significantly greater in alfalfa meal than in the other feedstuffs. In addition, the amount of methoxychlor and the percentage of samples exceeding 50 p.p.b. was also greater for alfalfa meal. Limited numbers of positive samples made other comparisons meaningless. The results of the research reported herein confirms the suspicion that low levels of pesticide contamination (< 50 p.p.b.) are a common occurrence in the feedstuffs that are used in formulating poultry rations throughout the United States. However, only 0.1% of the feedstuffs examined exceeded 500 p.p.b. of any of the 10 pesticides. Due to the concept of zero tolerance of pesticide residues in poultry products it is necessary to determine the significance of transfer of
PESTICIDE RESIDUES IN FEEDSTUFFS
J. Econ. Ent. 55: 477-479. Steel, R. G. D. and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Thompson, E. M., G. J. Mountney and G. W. Ware, 1967. Methoxychlor residues in chicken eggs. J. Econ. Ent. 60: 235-237. United States Department of Agriculture, 1965. USDA study shows pesticide residues in poultry samples. Feedstuffs, 37(33): 1, 70. Waldron, A. C , H. E. Kaeser, D. L. Goleman, J. R. Staubus and H. D. Niemczyk, 1968. Heptachlor and heptachlor epoxide on fall-treated alfalfa and in milk and cow tissues. Agric. Food Chem. 16: 627-631. Waldron, A. C , and D. L. Goleman, 1969. Ethyl and methyl parathion in green and cured alfalfa. Agric. Food Chem. 17: 1066-1069. Waldron, A. C , 1971. Analysis for organochlorine pesticide residues in agricultural commodities, soil and water. Unpublished report by Ohio Cooperative Extension Service, Ohio State University, Columbus. Ware, G. W., and E. C. Naber, 1961. Lindane in eggs and chicken tissues. J. Econ. Ent. 54: 675-677. Ware, G. W., and E. C. Naber, 1962. Lindane and BHC in egg yolks following recommended uses for louse and mite control. J. Econ. Ent. 55: 568-570.
NEWS AND NOTES (Continued from page 1358) nity had their bursas intact. ' "Glick was so intrigued with this discovery that he wrote a paper which he submitted to the magazine, Science. The editors felt it was not of sufficient interest to their subscribers and turned it down. Glick subsequently saw it published in The Journal of Poultry Science. ' "There it might have languished and become of interest only to chicken scholars if it had not been accidentally picked up by Dr. Harold Wolfe of the University of Wisconsin, who was then working in the broad field of immunity. Wolfe, a friend of mine, told me during a meeting of immunologists that he had taken the bursas out of newly hatched chicks and found an absence of antibodies, the same effect as noted by Chang. The bursa was evidently essential to antibody production and to immunity." 'Goodpaused. "Thestrange thingwasthatachicken
also has a thymus, which on the basis of my own observations I would have expected to regulate the immune system. This led me, and independently, Dr. J. F. A. P. Miller in England, to remove thymuses from newborn animals to see what happens. It dawned on me suddenly that the chickens might have two immune systems. This idea was strengthened when a student stood up at the meeting to make just that same provocative statement," Dr. Good smiled. ' "I was quite excited about all this, and I brought in a team of scientists to work out the role of the two organs. Other labs started in, and there was intense competition. Max Cooper of our lab made one of the definitive experiments when he rounded up some newly hatched chicks and divided them into two groups. In one group he removed the thymus; in the other he took out the bursa, and in both he destroyed with radiation any vestiges of embryonic immunity.
(Continued on page 1386)
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Furman, D. P., and R. A. Bankowski, 1949. Absorption of benzene hexachloride in poultry. J. Econ. Ent. 42: 980-982. Hixen, E., and M. H. Muma, 1947. Effect of benzene hexachloride on the flavor of poultry meat. Science, 106: 422-423. Ivey, M. C , R. H. Roberts, H. D. Mann and H. V. Claborn, 1961. Lindane residues in chickens and eggs following poultry house sprays. J. Econ. Ent. 54: 487-488. Liska, B. J., B. E. Langlois, G. C. Mostert and W. J. Stadelman, 1964. Residues in eggs and tissues of chickens on rations containing low levels of DDT. Poultry Sci. 43: 982-984. Mills, P. A., J. H. Onley and R. A. Gaither, 1963. Rapid method for chlorinated pesticide residues in nonfatty foods. J. Ass. Off. Agr. Chem. 46: 186-191. Minyard, J. P., and E. R. Jackson, 1963. Pesticide residues in commercial animal feeds. J. Ass. Off. Agr. Chem. 46: 843-859. Mountney, G. J., and J. H. Quisenberry, 1957. Contamination of broilers with chlordane and pyrethrum. Poultry Sci. 36: 923-924. Naber, E. C , and G. W. Ware, 1965. Effect of kepone and mirex on reproductive performance in the laying hen. Poultry Sci. 44: 875-880. Olney, C. E., W. E. Donaldson and T. W. Kerr, 1962. Methoxychlor in eggs and chicken tissues.
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