- Email: [email protected]
Chronic Toxicity to Laying Hens and Degradation of Bayer 187791,2
648
J. J. MULKERN AND R. E. CLEGG
and indications that more than one component might be present. There was no evidence of the fast, high-phosphorus component present in all the other ages
and 14th days the nitrogen was removed more rapidly than the phosphorus, but between the 14th and 19th days the reverse was the case.
studied.
REFERENCES SUMMARY
Chronic Toxicity to Laying Hens and Degradation of Bayer 1877912 MARTIN SHERMAN,3
G. H.
TAKEI, 3
R. B.
HERRICK 4 AND
E. Ross 4
College of Tropical Agriculture, University of Hawaii, Honolulu, Hawaii 96822 (Received for publication August 24, 1967)
I
NSECTICIDES administered via treated feed or water over a prolonged period have been found to have little detrimental effect on hens yet prevented fly
1 Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 901. 2 This investigation was supported in part byPublic Health Service Research Grant UI-00111 from the National Center for Urban and Industrial Health.
breeding in the droppings (Ross and Sherman, 1960; Sherman and Ross, 1960a; Sherman et al., 1963). It was reported earlier that droppings from chicks fed Bayer 18779 (0-ethyl-0-isopropyl-0-phthaloximido phosphorothioate) at 100 p.p.m. over a short period of time caused greater than 90% mortality in the larvae of 3 4
Department of Entomology. Department of Animal Sciences.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
Employing electrophoretic-radiochemical techniques the changes in embryo yolk phosphoproteins were investigated. The yolk proteins from 0-, 7-, 14- and 19-day embryos were analyzed by free electrophoresis and the percentage of nitrogen and phosphorus in the various components determined. The patterns and phosphorus distribution in the 7-day yolk samples were similar to those of fresh yolk. However, the 14- and 19-day electrophoretic patterns and phosphorus distribution showed considerable variation from the fresh yolk. In general, the fast moving peaks disappeared and the remaining components demonstrated very low mobilities and very low total phosphorus. The P / N ratios indicated that between the 7th
Clegg, R. E., R. E. Hein, C. H. Suelter and R. H. McFarland, 1955. The distribution of radioactive phosphorus in the electrophoretic components of yolk proteins. Poultry Sci. 34: 210-214. Hawk, P. B., B. L. Oser and W. H. Summerson, 1947. Practical Physiological Chemistry, 12th Edition. Blakiston, Philadelphia, Pa. Needham, J., 1942. Biochemistry and Morphogenesis, Cambridge University Press. Needham, J., 1954. Thoughts on the biochemistry of the developing hen's egg. Ann. Nutr. et aliment, 8: 195-205. Romanoff, A. L., and A. Romanoff, 1949. The Avian Egg. John Wiley & Sons, New York. Schenck, E. G., 1932. Untersuchungen iiber das Verhalten der Eriversstaffe. Z. Physiol. Chem. 211: 111-153. Tiselius, A., and E. A. Kabat, 1939. An electrophoretic study of immune sera and purified preparations. J. Exptl. Med. 69: 119-121. Williams, M. A., W. A. DaCosta, L. H. Newman and L. M. Marshall, 1954. Free amino acids in the yolk during the development of the chick. Nature, 173: 4901.
TOXICITY OF BAYER
several species of flies and had no detrimental effect on the chicks (Sherman and Komatsu, 1963; Sherman et ah, 1964). Blood plasma cholinesterase activity of the chicks was 27% inhibited. The present paper reports the results of an extensive study of the effects of this insecticide administered in the feed of laying hens over a 36-week period.
The Bayer 18779 used at the start of the experiment was the same technical grade material that was used in the shortterm studies mentioned above (Sherman, et at., 1964). This material, a yellow amorphous substance, was dissolved in acetone and added to the basal ration at the rate of 100 p.p.m. The ration containing this insecticide was fed for approximately 13 weeks. A later shipment of this insecticide from the manufacturer6 was 5
Bayer Farbenfabrik, via Chemagro Corp., Kansas City, Mo.
649
a white crystalline material with a melting range of 38.5-41.0°C. Because of the obvious differences in physical appearance of the new material, preliminary chick studies were carried out to determine the relative toxicity to fly maggots. The results of these studies showed that the resultant droppings from chicks fed the refined insecticide were less toxic to the maggots than droppings from chicks fed comparable levels of the original technical material. Therefore, in subsequent mixes the refined Bayer 18779 was added to the feed at 200 p.p.m. The experimental feed was fed for a total of 36 weeks. The hens were also observed for a 2 week post-treatment period during which time they received the untreated control ration. Egg production was recorded daily while egg weights, albumen height and shell thickness were determined on all eggs laid for 3 consecutive days in each 4 week period. Feed consumption and individual body weights were obtained every 4 weeks during the course of the experiment and at the end of the posttreatment period. Manure was collected approximately biweekly, from dropping pans set beneath the cages and the toxicity to fiist-instar larvae of Musca domestica (L.), Fannia pusio (Wiedemann), Chrysomya megacephala (F.), and Parasarcophaga argyrostotna (Robineau-Desvoidy) was determined by methods described by Sherman and Ross (1960a, b) and Sherman et ah, (1962). Two series of organoleptic tests were held during the experimental period. The first series was conducted after 25 weeks on experiment while the second series was conducted during the last week. Each series consisted of tests conducted on 3 consecutive days using the same 9-member panel for all tests. The panel was
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
MATERIALS AND METHODS
Eighty pullets of a commercial strain of White Leghorns were reared in battery brooders until 7 weeks of age when they were moved to wirefloor developer pens until 18 weeks of age. At this time, they were moved to individual cages. At 22 weeks of age, the pullets were leg banded, weighed, and distributed at random into 10" laying cages, 2 birds per cage, and started on experiment. One-half of the birds selected at random were continued on the control ration while the other half received the insecticide-treated feed. The basal diet consisted of the following percentage composition: ground corn, 69.75; soybean meal, 12.0; tuna meal, 5.0; meat and bone meal, 2.5; dehydrated alfalfa meal, 3.0; derluorinated phosphate, 3.0; oyster shell flour, 4.0; iodized salt, 0.5; microingredient premix, 0.25; and DLmethionine, 0.06.
18779
650
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
was first observed with a sample at a concentration of approximately 1 /ig./ml. in distilled water. The u.v. spectra of this sample was measured, scanning a range of from 200 myu. to 350 m/z. The reference cell contained the reference solvent of methanol in distilled water. Timed scans were made over a period of a little over ten days. Degradation was allowed to take place in 100 ml. volumetric flasks from which aliquots were taken, measured, and then replaced. Three additional experiments using the same procedures were conducted to study the degradation under both acid and basic conditions. Bayer 18779 at a concentration of 5 /ig./ml. was dissolved in tap water, deionized distilled water, and basic deionized distilled water. Measurements of pH changes in the solutions were also taken. The degradation studies were carried out in a room maintained at a temperature of 21° to 23° C. and a relative humidity of 40 to 50 percent. Statistical analyses were made where applicable using Abbott's formula (Abbott, 1925) and the one-tailed t test (Cochran and Cox, 1950). RESULTS AND DISCUSSION
No mortality that could be attributable to the insecticidal treatment occurred in the hens. During the experimental period, there was 11% mortality in both the untreated controls and animals receiving the Bayer 18779 treatment. Autopsy showed that death was attributable to visceral avian leucosis in 72% of the cases. Body Weight.—There was no detrimental effect on body weight due to the ingestion of Bayer 18779 (Fig. 1). The mean gain in body weight of hens receiving the Bayer 18779 was 446 g. while the control hens gained 336 g. during the course of the experiment. Feed Consumption.—Figure 2 expresses
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
asked to evaluate the eggs with respect to flavor and odor using a scale of 1 (best) to 6 (poorest). For each test, 5 eggs were chosen at random from hens receiving Bayer 18779 and 5 from hens on the untreated diet. These eggs were hard cooked, allowed to stand overnight, and sliced the following morning just prior to testing. Five hens from each of the treatment groups were used to determine the effect of treatment on blood plasma cholinesterase activity. Blood was obtained by venipuncture just prior to treatment, 2, 4, and 7 days after start of treatment, then weekly for a total of 8 weeks and biweekly for the duration of the experiment. Blood was also sampled 1, 2, 4, 7, and 14 days after the hens were withdrawn from treated feed and placed on the untreated control ration. The blood plasma was analyzed colorimetrically as described by Cook (1954) and Fallscheer and Cook (1956). In vitro cholinesterase studies using outdated human plasma as the enzyme source showed Bayer 18779 to be unstable in aqueous media. In order to determine this instability, Bayer 18779 in diethyl ether was added to distilled water to produce a stock solution containing 0.08 iig. of Bayer 18779 per ml. The inhibitory effect of 0.020, 0.008, and 0.004 fig. from this stock solution was analyzed over a period of 2 days. A more intensive study of the degradation of Bayer 18779 was made by means of UV spectrophotometry. The measurements were made with a Bausch and Lomb Spectronic 505 recording spectrophotometer, using 10 mm. silica cells, and methanol as the solvent. An accurately weighed sample of Bayer 18779 was dissolved in analytical grade methanol and was used as a stock solution for subsequent degradation studies. A trial run
TOXICITY OF BAYER
651
O lU
120110100 90^z 80CONTROL 7 0 /BAYER18779(EGG) z o 60 UJ.O 5 0 _'BAYER18779(FEED)-
z < w 2
-120 110 100 90 80 70 60 50
i—n—i—i—i—i—rr 4 8 12 16 20 242832 3638 WEEKS OF EXPERIMENT
2 m > z ?rm Qm §c O
z
FIG. 2. Mean feed comsumption and egg production of White Leghorn hens receiving Bayer 18779treated feed as compared to the untreated control. Two-week post-treatment period is also shown.
appears that at least partial acclimatization took place. Overall, the control hens laid at an average rate of 72.22% compared to 67.38% for the hens fed Bayer 18779. Economic Factors.—Table 1 summarizes the effects of the Bayer 18779 treatment on various factors of economic importance. The continuous administration of Bayer 18779-treated feed had no detrimental effect on shell thickness, egg weight or interior egg quality as measured by albumen height. Feed efficiency was greater in the control group, requiring only 1.78 kg. per dozen eggs compared to 1.97 kg. for the group receiving Bayer 18779 in the feed. This difference in feed efficiency was statistically significant at the 5% level of probability. TABLE 1.—Mean shell thickness, weight, and interior quality of eggs produced by White Leghorn hens receiving Bayer 18779-treated feed for a 36-week period*
0 4 8 12 16 202428323638 WEEKS OF EXPERIMENT FIG. 1. Body weights of White Leghorn hens receiving untreated and Bayer 18779-treated feed over a 36-week period. Two-week post-treatment period is also shown.
Treatment (p.p.m.) 100-200 0
Shell Egg thickness weight (0.001 inch) (gms.) 13.5 13.1
56.6 56.8
• No significant differences.
Albumin height (Haugh units) 83.1 82.3
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
feed consumption of the treated hens as a percentage of the feed consumed by the control hens. The birds on the treated feed consumed between 92 and 102% as much feed as the control group, so that the total feed consumption per bird for the hens receiving Bayer 18779 was only slightly and not significantly lower than that consumed by the control hens, 26.16 kg. compared to 27.12 kg. The mean feed consumption per hen/day was 112.95 g. of untreated feed and 109.28 g. of Bayer 18779-treated feed. Egg Production.—Figure 2 also expresses the egg production of the treated hens compared with that of the control hens. Egg production of the insecticidetreated birds, during the initial 4 week period, was only 50% of that of the control birds. In as much as the treated birds consumed 92% as much feed as the control birds during this same period, this depression was probably due to a physiological effect of the insecticide. Since, after this initial depression, production increased to 91-109% of the controls, it
18779
652
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
TABLE 2.—Organoleptic evaluation* of hard-cooked eggs from hens receiving Bayer 18779-ireated feed Treatment (p.p.m.)
Flavor
Odor
200 0
1st Series 1.14 1.14
1.19 1.26
200 0
2nd Series 1.33 1.25
1.19 1.11
Organoleptic Evaluation.—Table 2 summarizes the taste panel's results. There were no deleterious effects of feeding Bayer 18779 on egg flavor or odor. In Vivo Cholinesterase Activity.—The effect on blood plasma cholinesterase activity of Bayer 18779 treatment and withdrawal from treatment is given in Figure 3. During the initial 13 week period when 100 p.p.m. of the technical Bayer 18779 was included in the rations, cholinesterase inhibition increased to a high of 89% after 6 weeks of treatment and declined to 50% by the 13th week. A similar increase and decline in inhibition occurred after each subsequent feeding of freshly prepared insecticide-treated feed at 13 and 27 weeks (marked with arrows). This loss of activity when the crystalline material was used was greater than with the more technical Bayer
10 20 30 WEEKS OF EXPERIMENT
FIG. 3. Inhibition and recovery of plasma cholinesterase in Bayer 18779-treated White Leghorn hens. Hens removed from treated feed at 36 weeks. Arrows depict feed change.
18779 despite the higher concentration. Inhibitory action was increased to a high of 95% when fresh Bayer 18779-mixed feed was given the hens at 27 weeks, and with time the activity decreased to an average of 40% cholinesterase inhibition. The loss in cholinesterase inhibition activity appears related to the time of storage of the insecticide-mixed feed and indicates that the Bayer 18779 is undergoing some sort of degradatory change. Full recovery of enzyme activity occurred within 2 weeks after removal of the hens from the treated diet. Larval Mortality.—Table 3 summarizes the toxicity of droppings from the hens to larvae of Musca domestica, Fannia pusio, Chysomya megacephala, and Parasarco-
TABLE 3.—Toxicity to fly larvae of droppings from hens receiving Bayer 18779-treated feed Corrected percent larval mortality
Treatment (p.p.m.)
Time on experiment (wks.)
Musca domestica
Fannia pusio
100* 0» 200b 0b
1-12 1-12 13-36 13-36
94.8 12.6 53.3 8.1
12.0 8.6 3.6 5.7
a b
Mean values based on 15 replications of 10 larvae each. Mean values based on 36 replications of 10 larvae each.
Chrysomya megacephala 100 3.4 2.8 8.1
Parasarcophaga argyrosloma 18.8 12.0 1.8
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a Mean values based on a 9-member taste panel scoring from 1 for the highest quality to 6 for the lowest. No significant differences were found due to treatment.
TOXICITY
or
70-
50O i
30-
10-
1—i—r
r
•004 -008 020 BAYER 18779 (gamma) FIG. 4. In vitro inhibition of cholinesterase activity by Bayer 18779 in water over a 2-day period. Human blood plasma was used as the enzyme source.
18779
653 TAP HjO, pH 7.90 DBONIZED DISTILLED Hp, pH 6.40
,.»„^._
DBONIZED DISTILLED HJO, ADJUSTED INITIALLY TO pH a 2 0
100 TIME (HOURS)
150
200
FIG. 5. Degradation of Bayer 18779 in various water solutions as measured by absorption at 220-5 ra/i. pH values are those of the water media prior to adding Bayer 18779.
pound on human blood plasma cholinesterase was rapidly reduced with time. In the UV spectrophotometric study, two absorption bands were observed, at 220.5 (±0.5) m/i. and 297.7 (±0.5) m> The absorption peak at 220.5 mp. was more pronounced than the one at 297.7 imt., and followed Beer's law in the concentration range of 10 -5 to 10 -6 moles/ liter. This linearity was further confirmed by comparing samples in a Beckman DU spectrophotometer. The peak at 297.7 mjtt. was also studied for its linearity, according to Beer's law, but proved to be too feeble to be feasible for analytical work. Changes in absorption began within a few hours. Finally when the sample was measured after 10 days, the spectra showed a complete breakdown of Bayer 18779. This sample was checked again at a later date verifying the degradation which proved to be irreversible. Degradation was also followed in deionized distilled water, basic deionized distilled water, and tap water (Fig. 5). Each trial indicated degradation of Bayer 18779. Though essentially the same techniques used in the initial trial were followed, these latter experiments did not reach a point of complete degradation. Instead, they approached, in each case, an
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phaga argyrostoma. During the first 12 weeks of experiment, the use of the more technical Bayer 18779 resulted in excellent control of larvae of the house fly and the blow fly (C. megacephala) but in poor control of F. pusio and the flesh fly (P. argyrostoma). At 13 weeks the crystalline Bayer 18779 was incorporated into the experimental diet with a subsequent loss of larvicidal activity despite the use of the higher concentration. Subsequent manure samples caused less than 10% mortality in larvae of F. pusio, P. argyrostoma, and C. megacephala and mortality of house fly larvae gradually dropped from 100% to 0% between 13 and 27 weeks of treatment. Stability Studies.—Figure 4 summarizes the instability of Bayer 18779 in distilled water over a 2-day period in terms of in vitro cholinesterase inhibition. As can be seen the inhibitory effect of this com-
BAYER
654
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
SUMMARY
Two grades of technical Bayer 18779 were administered continuously for 36 weeks in the feed to White Leghorn laying hens initially at 100 p.p.m. and after 13 weeks at 200 p.p.m. The toxicity of droppings from these hens to larvae of Musca domestica (L.), Fannia pusio (Wiedemann), Chrysomya megacephala (F.), and Parasarcophaga argyrostoma (Robineau-Desvoidy) was determined. Treatment had no significant effect on hen mortality, body weight, feed consumption, egg production, shell thickness, egg weight, internal egg quality, flavor, or odor. Feed efficiency, however, was significantly depressed. Blood plasma cholinesterase activity was inhibited by treatment but this inhibition decreased with age of feed mixture suggesting instability of the Bayer 18779. Instability was substantiated by in vitro degradation studies using enzymatic and spectrophotometric means. Bayer 18779 applied in the feed of laying hens was too un-
stable to effect a high level protection against the breeding of maggots in the droppings from these hens. ACKNOWLEDGEMENT
The authors are indebted to M. T. Y. Chang for technical assistance in carrying out this study. REFERENCES Abbott, W. S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Cochran, W. G., and G. M. Cox, 1950. Experimental Designs. John Wiley and Sons, New York. Cook, J. W., 1954. Report on determination of insecticides by enzymatic methods. J. Assoc. Offic. Agric. Chem. 37: 561-564. Fallscheer, H. O., and J. W. Cook, 1956. Report on enzymatic methods for insecticides. Studies on the conversion of some thionophosphates and a dithiophosphate to in vitro cholinesterase inhibitors. J. Assoc. Offic. Agric. Chem. 39: 691-697. Ross, E., and M. Sherman, 1960. The effect of selected insecticides on growth and egg production. when administered continuously in the feed, Poultry Sci. 39:1203-1211. Sherman, M., and G. H. Komatsu, 1963. Maggot development in droppings from chicks fed organophosphorus insecticide-treated rations. J. Econ. Entomol. 56: 847-850. Sherman, M., and E. Ross, 1960a. Toxicity to house fly larvae of droppings from chickens fed insecticide-treated rations. J. Econ. Entomol. 53: 429-432. Sherman, M., and E. Ross, 1960b. Toxicity to house fly larvae of droppings from chicks given dipterex-treated water. J. Econ. Entomol. 53: 1066-1070. Sherman, M., E. Ross and M. T. Y. Chang, 1964. Acute and subacute toxicity of several organophosphorus insecticides to chicks. Toxicol. Appl. Pharmacol. 6: 147-153. Sherman, M., E. Ross and G. H. Komatsu, 1962. Differential susceptibility in maggots of several species to droppings from chickens fed insecticidetreated rations. J. Econ. Entomol. 55: 990-993. Sherman, M., E. Ross, F. F. Sanchez and M. T. Y. Chang, 1963. Chronic toxicity of dimethoate to hens. J. Econ. Entomol. 56: 10-15.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
equilibrium condition of their own. Initial degradation was correlated with hydrogen ion concentration. The more basic solutions degraded more rapidly initially. The pH values shown in Figure 5 are those of the water media prior to adding the Bayer 18779. Within 15 minutes of adding Bayer 18779, the pH values of the solutions were remeasured and it was found that the tap water solution had decreased from 7.90 to 7.73, the basic deionized water solution had dropped from 8.20 to 6.51 while the acidic deionized water had further decreased from 6.40 to pH 5.85. The loss in absorbance was probably due to a decomposition of the phthaloximido moiety.
J. J. MULKERN AND R. E. CLEGG
and indications that more than one component might be present. There was no evidence of the fast, high-phosphorus component present in all the other ages
and 14th days the nitrogen was removed more rapidly than the phosphorus, but between the 14th and 19th days the reverse was the case.
studied.
REFERENCES SUMMARY
Chronic Toxicity to Laying Hens and Degradation of Bayer 1877912 MARTIN SHERMAN,3
G. H.
TAKEI, 3
R. B.
HERRICK 4 AND
E. Ross 4
College of Tropical Agriculture, University of Hawaii, Honolulu, Hawaii 96822 (Received for publication August 24, 1967)
I
NSECTICIDES administered via treated feed or water over a prolonged period have been found to have little detrimental effect on hens yet prevented fly
1 Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 901. 2 This investigation was supported in part byPublic Health Service Research Grant UI-00111 from the National Center for Urban and Industrial Health.
breeding in the droppings (Ross and Sherman, 1960; Sherman and Ross, 1960a; Sherman et al., 1963). It was reported earlier that droppings from chicks fed Bayer 18779 (0-ethyl-0-isopropyl-0-phthaloximido phosphorothioate) at 100 p.p.m. over a short period of time caused greater than 90% mortality in the larvae of 3 4
Department of Entomology. Department of Animal Sciences.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
Employing electrophoretic-radiochemical techniques the changes in embryo yolk phosphoproteins were investigated. The yolk proteins from 0-, 7-, 14- and 19-day embryos were analyzed by free electrophoresis and the percentage of nitrogen and phosphorus in the various components determined. The patterns and phosphorus distribution in the 7-day yolk samples were similar to those of fresh yolk. However, the 14- and 19-day electrophoretic patterns and phosphorus distribution showed considerable variation from the fresh yolk. In general, the fast moving peaks disappeared and the remaining components demonstrated very low mobilities and very low total phosphorus. The P / N ratios indicated that between the 7th
Clegg, R. E., R. E. Hein, C. H. Suelter and R. H. McFarland, 1955. The distribution of radioactive phosphorus in the electrophoretic components of yolk proteins. Poultry Sci. 34: 210-214. Hawk, P. B., B. L. Oser and W. H. Summerson, 1947. Practical Physiological Chemistry, 12th Edition. Blakiston, Philadelphia, Pa. Needham, J., 1942. Biochemistry and Morphogenesis, Cambridge University Press. Needham, J., 1954. Thoughts on the biochemistry of the developing hen's egg. Ann. Nutr. et aliment, 8: 195-205. Romanoff, A. L., and A. Romanoff, 1949. The Avian Egg. John Wiley & Sons, New York. Schenck, E. G., 1932. Untersuchungen iiber das Verhalten der Eriversstaffe. Z. Physiol. Chem. 211: 111-153. Tiselius, A., and E. A. Kabat, 1939. An electrophoretic study of immune sera and purified preparations. J. Exptl. Med. 69: 119-121. Williams, M. A., W. A. DaCosta, L. H. Newman and L. M. Marshall, 1954. Free amino acids in the yolk during the development of the chick. Nature, 173: 4901.
TOXICITY OF BAYER
several species of flies and had no detrimental effect on the chicks (Sherman and Komatsu, 1963; Sherman et ah, 1964). Blood plasma cholinesterase activity of the chicks was 27% inhibited. The present paper reports the results of an extensive study of the effects of this insecticide administered in the feed of laying hens over a 36-week period.
The Bayer 18779 used at the start of the experiment was the same technical grade material that was used in the shortterm studies mentioned above (Sherman, et at., 1964). This material, a yellow amorphous substance, was dissolved in acetone and added to the basal ration at the rate of 100 p.p.m. The ration containing this insecticide was fed for approximately 13 weeks. A later shipment of this insecticide from the manufacturer6 was 5
Bayer Farbenfabrik, via Chemagro Corp., Kansas City, Mo.
649
a white crystalline material with a melting range of 38.5-41.0°C. Because of the obvious differences in physical appearance of the new material, preliminary chick studies were carried out to determine the relative toxicity to fly maggots. The results of these studies showed that the resultant droppings from chicks fed the refined insecticide were less toxic to the maggots than droppings from chicks fed comparable levels of the original technical material. Therefore, in subsequent mixes the refined Bayer 18779 was added to the feed at 200 p.p.m. The experimental feed was fed for a total of 36 weeks. The hens were also observed for a 2 week post-treatment period during which time they received the untreated control ration. Egg production was recorded daily while egg weights, albumen height and shell thickness were determined on all eggs laid for 3 consecutive days in each 4 week period. Feed consumption and individual body weights were obtained every 4 weeks during the course of the experiment and at the end of the posttreatment period. Manure was collected approximately biweekly, from dropping pans set beneath the cages and the toxicity to fiist-instar larvae of Musca domestica (L.), Fannia pusio (Wiedemann), Chrysomya megacephala (F.), and Parasarcophaga argyrostotna (Robineau-Desvoidy) was determined by methods described by Sherman and Ross (1960a, b) and Sherman et ah, (1962). Two series of organoleptic tests were held during the experimental period. The first series was conducted after 25 weeks on experiment while the second series was conducted during the last week. Each series consisted of tests conducted on 3 consecutive days using the same 9-member panel for all tests. The panel was
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
MATERIALS AND METHODS
Eighty pullets of a commercial strain of White Leghorns were reared in battery brooders until 7 weeks of age when they were moved to wirefloor developer pens until 18 weeks of age. At this time, they were moved to individual cages. At 22 weeks of age, the pullets were leg banded, weighed, and distributed at random into 10" laying cages, 2 birds per cage, and started on experiment. One-half of the birds selected at random were continued on the control ration while the other half received the insecticide-treated feed. The basal diet consisted of the following percentage composition: ground corn, 69.75; soybean meal, 12.0; tuna meal, 5.0; meat and bone meal, 2.5; dehydrated alfalfa meal, 3.0; derluorinated phosphate, 3.0; oyster shell flour, 4.0; iodized salt, 0.5; microingredient premix, 0.25; and DLmethionine, 0.06.
18779
650
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
was first observed with a sample at a concentration of approximately 1 /ig./ml. in distilled water. The u.v. spectra of this sample was measured, scanning a range of from 200 myu. to 350 m/z. The reference cell contained the reference solvent of methanol in distilled water. Timed scans were made over a period of a little over ten days. Degradation was allowed to take place in 100 ml. volumetric flasks from which aliquots were taken, measured, and then replaced. Three additional experiments using the same procedures were conducted to study the degradation under both acid and basic conditions. Bayer 18779 at a concentration of 5 /ig./ml. was dissolved in tap water, deionized distilled water, and basic deionized distilled water. Measurements of pH changes in the solutions were also taken. The degradation studies were carried out in a room maintained at a temperature of 21° to 23° C. and a relative humidity of 40 to 50 percent. Statistical analyses were made where applicable using Abbott's formula (Abbott, 1925) and the one-tailed t test (Cochran and Cox, 1950). RESULTS AND DISCUSSION
No mortality that could be attributable to the insecticidal treatment occurred in the hens. During the experimental period, there was 11% mortality in both the untreated controls and animals receiving the Bayer 18779 treatment. Autopsy showed that death was attributable to visceral avian leucosis in 72% of the cases. Body Weight.—There was no detrimental effect on body weight due to the ingestion of Bayer 18779 (Fig. 1). The mean gain in body weight of hens receiving the Bayer 18779 was 446 g. while the control hens gained 336 g. during the course of the experiment. Feed Consumption.—Figure 2 expresses
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 23, 2015
asked to evaluate the eggs with respect to flavor and odor using a scale of 1 (best) to 6 (poorest). For each test, 5 eggs were chosen at random from hens receiving Bayer 18779 and 5 from hens on the untreated diet. These eggs were hard cooked, allowed to stand overnight, and sliced the following morning just prior to testing. Five hens from each of the treatment groups were used to determine the effect of treatment on blood plasma cholinesterase activity. Blood was obtained by venipuncture just prior to treatment, 2, 4, and 7 days after start of treatment, then weekly for a total of 8 weeks and biweekly for the duration of the experiment. Blood was also sampled 1, 2, 4, 7, and 14 days after the hens were withdrawn from treated feed and placed on the untreated control ration. The blood plasma was analyzed colorimetrically as described by Cook (1954) and Fallscheer and Cook (1956). In vitro cholinesterase studies using outdated human plasma as the enzyme source showed Bayer 18779 to be unstable in aqueous media. In order to determine this instability, Bayer 18779 in diethyl ether was added to distilled water to produce a stock solution containing 0.08 iig. of Bayer 18779 per ml. The inhibitory effect of 0.020, 0.008, and 0.004 fig. from this stock solution was analyzed over a period of 2 days. A more intensive study of the degradation of Bayer 18779 was made by means of UV spectrophotometry. The measurements were made with a Bausch and Lomb Spectronic 505 recording spectrophotometer, using 10 mm. silica cells, and methanol as the solvent. An accurately weighed sample of Bayer 18779 was dissolved in analytical grade methanol and was used as a stock solution for subsequent degradation studies. A trial run
TOXICITY OF BAYER
651
O lU
120110100 90^z 80CONTROL 7 0 /BAYER18779(EGG) z o 60 UJ.O 5 0 _'BAYER18779(FEED)-
z < w 2
-120 110 100 90 80 70 60 50
i—n—i—i—i—i—rr 4 8 12 16 20 242832 3638 WEEKS OF EXPERIMENT
2 m > z ?rm Qm §c O
z
FIG. 2. Mean feed comsumption and egg production of White Leghorn hens receiving Bayer 18779treated feed as compared to the untreated control. Two-week post-treatment period is also shown.
appears that at least partial acclimatization took place. Overall, the control hens laid at an average rate of 72.22% compared to 67.38% for the hens fed Bayer 18779. Economic Factors.—Table 1 summarizes the effects of the Bayer 18779 treatment on various factors of economic importance. The continuous administration of Bayer 18779-treated feed had no detrimental effect on shell thickness, egg weight or interior egg quality as measured by albumen height. Feed efficiency was greater in the control group, requiring only 1.78 kg. per dozen eggs compared to 1.97 kg. for the group receiving Bayer 18779 in the feed. This difference in feed efficiency was statistically significant at the 5% level of probability. TABLE 1.—Mean shell thickness, weight, and interior quality of eggs produced by White Leghorn hens receiving Bayer 18779-treated feed for a 36-week period*
0 4 8 12 16 202428323638 WEEKS OF EXPERIMENT FIG. 1. Body weights of White Leghorn hens receiving untreated and Bayer 18779-treated feed over a 36-week period. Two-week post-treatment period is also shown.
Treatment (p.p.m.) 100-200 0
Shell Egg thickness weight (0.001 inch) (gms.) 13.5 13.1
56.6 56.8
• No significant differences.
Albumin height (Haugh units) 83.1 82.3
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feed consumption of the treated hens as a percentage of the feed consumed by the control hens. The birds on the treated feed consumed between 92 and 102% as much feed as the control group, so that the total feed consumption per bird for the hens receiving Bayer 18779 was only slightly and not significantly lower than that consumed by the control hens, 26.16 kg. compared to 27.12 kg. The mean feed consumption per hen/day was 112.95 g. of untreated feed and 109.28 g. of Bayer 18779-treated feed. Egg Production.—Figure 2 also expresses the egg production of the treated hens compared with that of the control hens. Egg production of the insecticidetreated birds, during the initial 4 week period, was only 50% of that of the control birds. In as much as the treated birds consumed 92% as much feed as the control birds during this same period, this depression was probably due to a physiological effect of the insecticide. Since, after this initial depression, production increased to 91-109% of the controls, it
18779
652
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
TABLE 2.—Organoleptic evaluation* of hard-cooked eggs from hens receiving Bayer 18779-ireated feed Treatment (p.p.m.)
Flavor
Odor
200 0
1st Series 1.14 1.14
1.19 1.26
200 0
2nd Series 1.33 1.25
1.19 1.11
Organoleptic Evaluation.—Table 2 summarizes the taste panel's results. There were no deleterious effects of feeding Bayer 18779 on egg flavor or odor. In Vivo Cholinesterase Activity.—The effect on blood plasma cholinesterase activity of Bayer 18779 treatment and withdrawal from treatment is given in Figure 3. During the initial 13 week period when 100 p.p.m. of the technical Bayer 18779 was included in the rations, cholinesterase inhibition increased to a high of 89% after 6 weeks of treatment and declined to 50% by the 13th week. A similar increase and decline in inhibition occurred after each subsequent feeding of freshly prepared insecticide-treated feed at 13 and 27 weeks (marked with arrows). This loss of activity when the crystalline material was used was greater than with the more technical Bayer
10 20 30 WEEKS OF EXPERIMENT
FIG. 3. Inhibition and recovery of plasma cholinesterase in Bayer 18779-treated White Leghorn hens. Hens removed from treated feed at 36 weeks. Arrows depict feed change.
18779 despite the higher concentration. Inhibitory action was increased to a high of 95% when fresh Bayer 18779-mixed feed was given the hens at 27 weeks, and with time the activity decreased to an average of 40% cholinesterase inhibition. The loss in cholinesterase inhibition activity appears related to the time of storage of the insecticide-mixed feed and indicates that the Bayer 18779 is undergoing some sort of degradatory change. Full recovery of enzyme activity occurred within 2 weeks after removal of the hens from the treated diet. Larval Mortality.—Table 3 summarizes the toxicity of droppings from the hens to larvae of Musca domestica, Fannia pusio, Chysomya megacephala, and Parasarco-
TABLE 3.—Toxicity to fly larvae of droppings from hens receiving Bayer 18779-treated feed Corrected percent larval mortality
Treatment (p.p.m.)
Time on experiment (wks.)
Musca domestica
Fannia pusio
100* 0» 200b 0b
1-12 1-12 13-36 13-36
94.8 12.6 53.3 8.1
12.0 8.6 3.6 5.7
a b
Mean values based on 15 replications of 10 larvae each. Mean values based on 36 replications of 10 larvae each.
Chrysomya megacephala 100 3.4 2.8 8.1
Parasarcophaga argyrosloma 18.8 12.0 1.8
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a Mean values based on a 9-member taste panel scoring from 1 for the highest quality to 6 for the lowest. No significant differences were found due to treatment.
TOXICITY
or
70-
50O i
30-
10-
1—i—r
r
•004 -008 020 BAYER 18779 (gamma) FIG. 4. In vitro inhibition of cholinesterase activity by Bayer 18779 in water over a 2-day period. Human blood plasma was used as the enzyme source.
18779
653 TAP HjO, pH 7.90 DBONIZED DISTILLED Hp, pH 6.40
,.»„^._
DBONIZED DISTILLED HJO, ADJUSTED INITIALLY TO pH a 2 0
100 TIME (HOURS)
150
200
FIG. 5. Degradation of Bayer 18779 in various water solutions as measured by absorption at 220-5 ra/i. pH values are those of the water media prior to adding Bayer 18779.
pound on human blood plasma cholinesterase was rapidly reduced with time. In the UV spectrophotometric study, two absorption bands were observed, at 220.5 (±0.5) m/i. and 297.7 (±0.5) m> The absorption peak at 220.5 mp. was more pronounced than the one at 297.7 imt., and followed Beer's law in the concentration range of 10 -5 to 10 -6 moles/ liter. This linearity was further confirmed by comparing samples in a Beckman DU spectrophotometer. The peak at 297.7 mjtt. was also studied for its linearity, according to Beer's law, but proved to be too feeble to be feasible for analytical work. Changes in absorption began within a few hours. Finally when the sample was measured after 10 days, the spectra showed a complete breakdown of Bayer 18779. This sample was checked again at a later date verifying the degradation which proved to be irreversible. Degradation was also followed in deionized distilled water, basic deionized distilled water, and tap water (Fig. 5). Each trial indicated degradation of Bayer 18779. Though essentially the same techniques used in the initial trial were followed, these latter experiments did not reach a point of complete degradation. Instead, they approached, in each case, an
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phaga argyrostoma. During the first 12 weeks of experiment, the use of the more technical Bayer 18779 resulted in excellent control of larvae of the house fly and the blow fly (C. megacephala) but in poor control of F. pusio and the flesh fly (P. argyrostoma). At 13 weeks the crystalline Bayer 18779 was incorporated into the experimental diet with a subsequent loss of larvicidal activity despite the use of the higher concentration. Subsequent manure samples caused less than 10% mortality in larvae of F. pusio, P. argyrostoma, and C. megacephala and mortality of house fly larvae gradually dropped from 100% to 0% between 13 and 27 weeks of treatment. Stability Studies.—Figure 4 summarizes the instability of Bayer 18779 in distilled water over a 2-day period in terms of in vitro cholinesterase inhibition. As can be seen the inhibitory effect of this com-
BAYER
654
M. SHERMAN, G. H. TAKEI, R. B. HERRICK AND E. ROSS
SUMMARY
Two grades of technical Bayer 18779 were administered continuously for 36 weeks in the feed to White Leghorn laying hens initially at 100 p.p.m. and after 13 weeks at 200 p.p.m. The toxicity of droppings from these hens to larvae of Musca domestica (L.), Fannia pusio (Wiedemann), Chrysomya megacephala (F.), and Parasarcophaga argyrostoma (Robineau-Desvoidy) was determined. Treatment had no significant effect on hen mortality, body weight, feed consumption, egg production, shell thickness, egg weight, internal egg quality, flavor, or odor. Feed efficiency, however, was significantly depressed. Blood plasma cholinesterase activity was inhibited by treatment but this inhibition decreased with age of feed mixture suggesting instability of the Bayer 18779. Instability was substantiated by in vitro degradation studies using enzymatic and spectrophotometric means. Bayer 18779 applied in the feed of laying hens was too un-
stable to effect a high level protection against the breeding of maggots in the droppings from these hens. ACKNOWLEDGEMENT
The authors are indebted to M. T. Y. Chang for technical assistance in carrying out this study. REFERENCES Abbott, W. S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Cochran, W. G., and G. M. Cox, 1950. Experimental Designs. John Wiley and Sons, New York. Cook, J. W., 1954. Report on determination of insecticides by enzymatic methods. J. Assoc. Offic. Agric. Chem. 37: 561-564. Fallscheer, H. O., and J. W. Cook, 1956. Report on enzymatic methods for insecticides. Studies on the conversion of some thionophosphates and a dithiophosphate to in vitro cholinesterase inhibitors. J. Assoc. Offic. Agric. Chem. 39: 691-697. Ross, E., and M. Sherman, 1960. The effect of selected insecticides on growth and egg production. when administered continuously in the feed, Poultry Sci. 39:1203-1211. Sherman, M., and G. H. Komatsu, 1963. Maggot development in droppings from chicks fed organophosphorus insecticide-treated rations. J. Econ. Entomol. 56: 847-850. Sherman, M., and E. Ross, 1960a. Toxicity to house fly larvae of droppings from chickens fed insecticide-treated rations. J. Econ. Entomol. 53: 429-432. Sherman, M., and E. Ross, 1960b. Toxicity to house fly larvae of droppings from chicks given dipterex-treated water. J. Econ. Entomol. 53: 1066-1070. Sherman, M., E. Ross and M. T. Y. Chang, 1964. Acute and subacute toxicity of several organophosphorus insecticides to chicks. Toxicol. Appl. Pharmacol. 6: 147-153. Sherman, M., E. Ross and G. H. Komatsu, 1962. Differential susceptibility in maggots of several species to droppings from chickens fed insecticidetreated rations. J. Econ. Entomol. 55: 990-993. Sherman, M., E. Ross, F. F. Sanchez and M. T. Y. Chang, 1963. Chronic toxicity of dimethoate to hens. J. Econ. Entomol. 56: 10-15.
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equilibrium condition of their own. Initial degradation was correlated with hydrogen ion concentration. The more basic solutions degraded more rapidly initially. The pH values shown in Figure 5 are those of the water media prior to adding the Bayer 18779. Within 15 minutes of adding Bayer 18779, the pH values of the solutions were remeasured and it was found that the tap water solution had decreased from 7.90 to 7.73, the basic deionized water solution had dropped from 8.20 to 6.51 while the acidic deionized water had further decreased from 6.40 to pH 5.85. The loss in absorbance was probably due to a decomposition of the phthaloximido moiety.