South Dakota State College

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1 THE EFFECT OF ARSANILIC ACID AND 3-NITRO-4- HYDROXYPHENYLARSONIC ACID ON SELENIUM POISONING IN THE PIG 1, 2 RICHARD C. WAHLSTROM, LESLIE D. KAMSTRA, AND OscAR E. OLSON~ S South Dakota State College ELENIUM poisoning in pigs has been discussed by Moxon (1937) and Miller and Schoening (1938). The toxic effects of selenium in certain animals can be counteracted by arsenic, as sodium arsenite (Moxon, 1938, 1941; Rhian and Moxon, 1943; Moxon et al., 1944). However, the toxicity of sodium arsenite makes its general use on farms and ranches in seleniferous areas somewhat impractical. Recently Hendrick et al. (1953) have shown that both arsanilic acid and 3-nitro- 4-hydroxyphenylarsonic acid give partial protection against the toxicity of selenum in the rat, the degree of protection depending upon how much of the arsenical was used. The investigations reported here were made to determine the effectiveness of arsanilic acid and 3-nitro-4-hydroxyphenylarsonic acid as selenium poisoning preventives in pigs. Experiment 1 Experimental Procedure Thirty purebred Hampshire, Duroc and Spotted Poland China pigs were allotted to six experimental treatments on the basis of initial weight, breed, litter and sex. All lots were fed and watered ad libitum in concrete pens. Weights of pigs were determined at 14-day intervals throughout the experiment. Each pig was removed from the experiment when it weighed 200 lb. or after 112 days on the experiment. The basal ration was composed of ground yellow corn, solvent soybean oil meal, tankage, steamed bonemeal, trace mineralized salt and a vitamin premix contributing the following per lb. of ration: 6 mg. niacin, 5 rag. pantothenic acid, 1 rag. riboflavin, 125 mg. choline chloride, 4 mcg. vitamin B12, 1000 USP units of vitamin A and 125 USP units of vitamin D. The calculated protein content of the basal a Contribution from the Departments of Animal Husbandry and Station Biochemistry and approved ior publication by the Director of the Agricultural Experiment Station. Publication No. 328 of the Journal Series. Grateful acknowledgment is made to Abbott Laboratories, North Chicago, Illinois for supplying the arsanilic acid, to Dr. Salsbury's Laboratories for the 3-nitro-4-hydroxyphenylarsonic acid, to Merck and Company, Inc., Rahway, New Jersey for the B-vitamins and to Nopco Chemical Company, Harrison, New Jersey for vitamins A and D. 105

2 106 WAHLSTRO.~Y[~ KAMSTRA AND OLSON ration was 15.5 percent. Arsanilic acid was used at a level of 0.01 percent of the total ration and 3-nitro-4-hydroxyphenylarsonic acid at a percent level. Selenium was added as sodium selenite to furnish 7 p.p.m, of selenium in the total ration. The arsenical compounds and the selenium were premixed with soybean oil meal and steamed bonemeal before being mixed with the remainder of the ground ration to insure optimum mixing of these compounds in the basal ration. Experiment 2 The animals used in this trial were purebred Duroc and Spotted Poland China pigs weighing approximately 29 lb. initially. Twenty pigs of each breed were allotted into five outcome groups. Where outcome groups could not be made up of littermates, other comparable pigs were selected. Within the outcome groups, the pigs were assigned to the experimental treatments at random. In order to obtain as little variation as possible in initial weights half of the pigs in each lot were started on the experiment one week prior to the other pigs. The experiment was terminated 14 weeks after the first pigs were started on test. The basal ration differed from that used in Experiment 1 only in that it contained sodium selenite furnishing 10 p.p.m, of selenium to the total ration. The levels of arsenicals used in this trial were 0.02 and 0.01 percent of the total ration as arsanilic acid and 0.(305 and percent where 3-nitro-4-hydroxyphenylarsonic acid was used. The arsenicals used in these experiments have been reported to prolong blood clotting time in chicks fed rations containing no alfalfa meal or supplemental vitamin K (Griminger et al., 1953; Sweet et al., 1954). Therefore, it was decided 'to determine if the higher levels of arsenicals used in Experiment 2 had any effect on blood clotting time. Blood was obtained by puncturing the ear vein of each pig in Lots 1, 2 and 4 and the clotting time determined by the capillary tube method. Experiment 1 Results and Discussion The results of the first experiment together with the various experimental treatments are shown in table 1. The effectiveness of the two arsenicals used in this study in protecting against selenium toxicity is shown in the growth response of Lot 4 compared to Lots 5 and 6. The average daily gain of Lot 4 is somewhat out of proportion with that of the other groups since the death of the weaker pig raises the final weight

3 SELENIUlV~ POISONING 107 of this group. Three of the five pigs allotted to Lot 4 developed typical selenium toxicity symptoms during the test. Two Duroc pigs showed loss of hair, cracked hoofs and emaciation five weeks after the experiment was started. One of these pigs eventually recovered while the other died at the end of ten weeks. On autopsy this pig exhibited ex- TABLE 1. RESULTS OF ARSENICAL SUPPLEMENTS IN RATIONS WITH AND WITHOUT SELENIUM FED TO WEANLING PIGS Comparison Lot Number and Treatment 1 2 ~ Basal + Basal + Selenium + 3-nitro-4 3-nitro-4 Hydroxy- Basal + Hydroxy- Basal + phenyl- Selenium + phenyl- Arsanilic arsonic Basal + Arsanilic arsonic Basal Acid Acid Selenium Acid Acid Number of pigs 5 41 Av. initial wt., lb Av. final wt., lb Av. number of days on test Av. daily gain, lb Av. daily feed per pig, lb Av. feed per ]b. gain, lb Does not include one unthrifty pig removed during the test. 2 Does not include one pig that died due to selenium poisoning. Does not include one pig that died due to gastric hemorrhage tensive atrophy and cirrhosis of the liver which is typical of selenium poisoning. The other pig that showed the effects of selenium poisoning was a Hampshire. Its hoofs cracked and it became quite lame. The failure of any of the pigs in Lots 5 and 6 to show any of the above symptoms indicates that the arsenicals were giving some protection. The arsanilic acid at the 0.01 percent level used in this trial did not give complete protection as these pigs gained only 1.36 lb. per

4 108 WAHLSTRO1VL~ KAMSTRA AND OLSON day while those receiving the non-seleniferous diet supplemented with arsanilic acid gained 1~59 lb. per day. Complete protection from selenium poisoning appeared to be given by 3-nitro-4-hydroxyphenylarsonic acid since Lots 3 and 6 had identical rates of gain. The slightly faster rate of gain of the pigs in these two lots is in agreement with the work of Carpenter (1951) who also found 3-nitro-4-hydroxyphenylarsonic acid to stimulate growth in weanling pigs. TABLE 2. COMPARISON OF DIFFERENT LEVELS OF ARSENICALS IN COUNTERACTING SELENIUM TOXICITY IN WEANLING PIGS Lot Number and Treatment Comparison Basal Basal Basal+.O05% Basal+.0025% +.02% -[-.01% 3-nitro-4 Hy- 3-nitro-4 Hy- Arsanilic Arsanilic droxyphenyl- droxyphenyl- Basal Acid Acid arsonic Acid arsonic Acid Number of pigs Av. initial wt., lb Av. 10 week wt., lb "* " 96 Av. final wt., lb Av. daily gain, lb * * 1.09 Av. daily feed per pig, lb Av. feed per lb. gain, lb Does not include two pigs removed and autopsied on 84th day. Significant at 0.05 level. ~ Significant at 0.01 level. Experiment 2 The second experiment was planned to confirm the results of Experiment 1 and to obtain information on other levels of arsenical supplementation. The weight data for this experiment at 10 weeks, before any of 'the pigs had died, is included with the summary of the final results presented in table 2. A significantly greater rate of gain was shown by the pigs receiving either of the arsenicals at the higher levels than by the control lot. This difference was much more pronounced at the end of ten weeks when all of the pigs were still on the experiment. However, even after the two most severely affected pigs had been removed from Lot 1 this lot showed a slower rate of gain than all lots except Lot 5. Arsanilic acid at the 0.02 percent level and 3-nitro-4-hydroxyphenylarsonic acid at the percent level appeared to be equally effective

5 SELENIU1V~ POISONING 109 in counteracting selenium toxicity in this trial. Both of these arsenicals were well tolerated at these levels. Arsanilic acid at the 0.01 percent level was partially effective, the slower rate of gain being due mainly to one pig in that lot that gained poorly during the first few weeks of the experiment. Although several pigs in Lot 5 gained very slowly, 3-nitro-4-hydroxyphenytarsonic acid at the percent level apparently gave some protection since the pigs did not show the visible signs of selenium toxicity as exhibited by two of the pigs in Lot 1. That a breed difference in susceptibility to selenium poisoning may exist seems likely since in both experiments the Duroc pigs were most TABLE 3. INFLUENCE OF ARSENICAL SUPPLEMENTS ON BLOOD CLOTTING TIME IN PIGS Av. Blood Clotting Time, Min. and Standard Deviation Lot No. Treatment 53rd Day 95th Day 1 None ~ arsanilic acid 1.6* "* ~ 3-nitro-4-hydroxyphenylarsonic acid 1.9" Significant at 0.05 level. "~ Significant at 0.01 level. severely affected and showed the toxic symptoms at an earlier date. In neither experiment did the Spotted Poland China pigs exhibit any of the visible symptoms of selenium toxicity, A great deal of variation also was found in the growth response of individual pigs in the lots receiving selenium or selenium plus the lower levels of arsenicals. Some pigs are apparently more susceptible to selenium toxicity than others. In both trials efficiency of the gains made by the pigs on the seleniferous rations were affected only slightly by the arsenicals. The main cause of the slower growth seemed to be due to a reduced feed intake. This might be expected in view of the dislike for toxic diets reported by Miller and Schoening (1938). The effect of the two arsenical supplements on blood clotting time is presented in table 3. The addition of either arsanilic acid or 3-nitro-4-hydroxyphenylarsonic acid to the ration increased the blood clotting time. However, even the longest blood clotting time (4.1 minutes) does not appear to be long enough to be of any practical significance.

6 110 WAHLSTROM~ KAMSTRA AND OLSON In both trials chemical analyses indicated that the arsenicals may prevent at least a part of the selenium from being deposited in the hair as reported by Moxon (1941). However, the results were variable and more data are needed before any definite conclusions can be made. Summary Two experiments involving a total of 70 pigs were conducted to determine the effectiveness of arsanilic acid and 3-nitro-4-hydroxyphenylarsonic acid in counteracting the effects of chronic selenium poisoning in pigs. Excellent protection against up to I0 p.p.m, of selenite selenium was given by these two arsenicals at levels of 0.02 percent and percent respectively. Partial protection, as evidenced by reduced mortality or increased growth, was shown at the lower levels used (arsanilic acid 0.01 percent and 3-nitro-4-hydroxyphenylarsonic acid percent). No toxic effects due to the arsenicals were observed at any time during either experiment. In Experiment 1 a slight growth response was shown by the pigs fed the arsenical supplements in a nonseleniferous ration. A slight increase in blood clotting time was observed in Experiment 2 by the pigs fed the high levels of arsenicals. Literature Cited Carpenter, Lawrence E The effect of 3-nitro-4-hydroxyphenylarsonic acid on the growth of swine. Arch. Biochem. and Biophys. 32:181. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. IV[. Scott Factors influencing blood clotting time in the chick. Science 118:379. Hendrick, Catherine, Harlan L. Klug and Oscar E. Olson Effect of 3-nitro- 4-hydroxyphenylarsonic acid on selenium poisoning in the rat. Jour. Nutr. 51:131. Miller, W. T. and H. W. Schoening Toxicity of selenium fed to swine in the form of sodium selenite. Jour. Agric. Res. 56:831. Moxon, A. L Alkali disease or selenium poisoning. So. Dak. Agri. Exp. Sta. Bul. No Moxon, A. L The effect of arsenic on the toxicity of se]eniferous grains. Science 88:81. Moxon, A. L The influence of arsenic on selenium poisoning in hogs. Proc. So. Dak. Acad. Sci. 21:34. Moxon, A. L., M. A. Rhian, H. D. Anderson and O. E. Olson Growth of steers on seleniferous range. JOURNAL OF ANIMAL SCIENCE 3:299. Rhian, M., and A. L. Moxon Chronic selenium poisoning in dogs and its prevention by arsenic. Jour. Pharmacol. and Exptl. Therap. 78:249. Sweet, G. B., G. L. Romoser and G. F. Combs Further observations on the effect of sulfaquinoxaline, p-aminophenylarsenic acid, and oxytetracyeline on blood clotting time of chicks. Poul. Sci. 33:430.