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Intestinal absorption of 5 chromium compounds in young black ducks (Anas rubripes)
Toxicology Letters, 6 (1980) 193-197 o Elsevier/North-Holland Biomedical Press
193
INTESTINAL ABSORPTION OF 5 CHROMIUM COMPOUNDS IN YOUNG BLACK DUCKS (AiVAS RUBRIPES) W.C. EASTIN Jr., S.D. HASELTINE and H.C. MURRAY Environmental Physiology and Toxicology Project, U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, MD 20811 (U.S.A.) (Received January 16th, 1980) (Accepted April 4th, 1980)
SUMMARY
An in vivo intestinal perfusion technique was used to measure the absorption rates of five Cr compounds in black ducks. Cr was absorbed from saline solutions of KCr(S04 )2 and Cr03 at a rate about 1.5 to 2.0 times greater than from solutions of Cr, Cr(N03 )3, and Cr(CSH,02)3. These results suggest the ionic form of Cr in solution may be an important factor in determining absorption of Cr compounds from the small intestine.
INTRODUCTION
Industrial and urban complexes which release Cr in liquid effluent wastes are concentrated along the eastern seaboard of the United States. Thus Cr concentrations are higher in coastal freshwater streams along the Atlantic as compared with the Gulf and Pacific coasts. In a Maine river receiving tannery effluents, sediment Cr levels reached 25 000 ppm Cr [l] . In Rhode Island, bivalves and polychaetes in an area receiving the outfall from an electroplating plant contained 20-25 ppm Cr [ 21. Similar facilities in operation throughout the northeast may be releasing enough Cr to reach equally high levels in indigenous invertebrates. Reviews of the occurrence, function, and toxicity of Cr in biological systems have recently been published [3-51. Although Cr is considered an essential metal for maintenance of normal glucose metabolism [3, 61, high concentrations of Cr in the diet can result in toxicity [3-51. Black ducks breed along the Atlantic coast from the Maritime Provinces to Virginia. During the reproductive season potentially contaminated invertebrates make up more than 80% of the diets of adult and juvenile black ducks [7] It has been reported that the absorption of Cr in mammals depends on the valence state of this metal [5]. Although not conclusive, it would appear that if high concentrations of Cr, regardless of valence state, get into the circulation there is a toxic affect [3, 81. There is little information on the absorption of
194
Cr in non-mammalian intestinal absorption
vertebrates. The present study examined the relative of five Cr compounds in juvenile black ducks.
METHODS
Pairs of black ducks were housed in 9.1 m X 4.6 m outdoor pens, fed a commercial breeder mash and water ad lib., and allowed to hatch and rear their own young. Two ducklings from each of 20 broods were randomly selected when brood mean weight reached 175 g (mean weight it S.D. for selected birds was 188.5 f 54.6 g). Ducklings ranged in age from 11 to 17 days. Ducklings were anesthetized with ketamine HCl (200 mg/kg i.m.). Laparotomy (3 cm) was performed on the left side and the upper half of the small intestine delivered into a warm saline-moistened gauze sponge. The intestine was ligated about 1 cm from the pylorus. An entrance canula was inserted and secured % cm below the ligature. A second ligation was made at Meckel’s diverticulum; an intestinal incision was made anterior to this point. The intestine was flushed with warm physiological saline (0.9% NaCl) followed by air and the exit canula was established. The bile ducts were ligated and the exposed intestine was replaced in the abdominal cavity. The incision was closed with a hemostat and a heating pad was used to help maintain body temperature. Perfusion fluid was recirculated through the intestine with a Buchler polystaltic pump at a rate of 2 ml/min. After 20 min equilibration, 20 ml of fresh solution was perfused for an additional 20 min and frozen for subsequent analyses. Preperfusion samples were saved from each trial for comparison to the recirculated perfusion solutions. Stock solutions of 1 mg Cr/ml for each of the chromium compounds were prepared as follows. Chromium potassium sulfate [CrK(S04 )Z -12 Hz 01; chromium trioxide, (CrOB ); and chromium nitrate, [Cr(N03 )3 09 H2 0] were dissolved in 0.9% NaCl. 100 mg pelleted chromium (Cr) was dissolved in 10 ml 1 N HCl overnight and then diluted 1 to 10 with 0.9% NaCl. The organic compound, 2,+pentanedione chromium [Cr(C, H, O2 )3 ] , was dissolved in 70% ethyl alcohol. Perfusion solutions containing (10 ppm Cr) were made up fresh on the day of each trial; 1 ml of the appropriate stock solution was added to 99 ml of 0.9% NaCl containing 50 mg/l phenol red as a nonabsorbable volume marker. The pH was adjusted to approx. 6.5 with dropwise addition of 1 M NaOH. After perfusion, birds were killed by an overdose of Equi-Thesin@ . The perfused intestine was dried overnight at 100” C and then weighed. In test solutions, Phenol red concentrations were measured spectrophotometrically and chromium concentrations were determined by atomic absorption spectrometry (Perkin-Elmer Model 303). Net chromium flux was determined by the formula:
195
Net Cr flux =
Vl[Cr,(PR)
- Cri]
W
where Vi = initial volume (ml); Cr, and Cri = the final and initial Cr concentrations in pmol/ml; PR = phenol ratio = orginal cont./final cont.; and W = dry weight (g) of the perfused intestinal segment. Net flux is expressed as number of pmol Cr/20 min, g dry weight. Chromium absorption rates were compared by one-way analysis of variance and subsequently by Duncan’s multiple range test. RESULTS
AND DISCUSSION
All but one of the ducklings completed the perfusion trial. Cr was absorbed from solutions of KCr (SO4 )* and Cr03 at equivalent rates but at a significantly greater rate (1.5-2.0 X ) than from solutions of Cr, Cr(N03 )3, and Cr(C, H, O2 )3 (Table I). The equal absorption rates of trivalent Cr as KCr(S04 )* and hexavalent Cr as CrG3 in young black ducks are of particular interest. In mammals, trivalent Cr is poorly absorbed from the gastrointestinal tract [9--111 when compared with hexavalent Cr [3,4,9, lo]. This would initially suggest that there is a difference in intestinal absorption of Cr between ducks and mammals. However, Gray and Sterling, in discussing the marked affinity of the red cell for hexavalent Cr in their tracer experiments, suggested that the ionic form of Cr (i.e. 51Cr042- and %r3+ ) determines the membrane permeability of this metal and thus, the differential binding by the cell [12]. The present findings with immature black ducks are in general agreement with an ionic charge concept; Cr was best absorbed from solutions containing the anionic Cr complex compared with solutions containing cationic Cr (Table I). In reviewing those mammalian studies indicating a Cr absorption difference based on valence, it was found that the results can also be explained on an anionic vs cationic Cr basis [3,4, 9, lo]. Based on ionic charge, the results from perfusion studies with young black ducks do not conflict with previous absorption data and suggest the ionic form of Cr in solution may be an important factor in determining relative absorption of Cr compounds across the small intestine. Studies on the toxic effects of high levels of Cr have dealt mostly with mammalian species. Although the results are not easy to generalize, they have shown Cr accumulates in liver, kidney, bone, and spleen [&lo, 111. Cr has also been linked to disorders in glucose metabolism [3,13, 141, to changes in serum cholesterol levels [15], and to impaired kidney [16] and liver [8] function. Few studies have been done with other vertebrates. The only avian study dealing with Cr ingestion included 30 and 100 ppm Cr as Na2 Cr04 in the feed of growing male chicks for up to 32 days [17]. No mortality, depression in growth rate, or efficiency of food utilization was noted. The effects of Cr on reproduction are also not resolved. For example, in
196 TABLE I CHROMIUM ABSORPTION IN BLACK DUCKLINGS Chromium compound
n
Chromium absorptiona’b
Valence
Charge on Cr complex
KCr(S0, ).12 H, 0 CrG, Cr (pellets) Cr(C, H, 0, )3 Cr(N0, )3 a9 H, 0
9 7 7 8 8
1.516 1.482 0.979 1.021 0.772
3 6 3 3 3
+ + +
f 0.421* ?: 0.272* * 0.552** i: 0.439** * 0.289**
aMean f S.D.; rmol/20 min, g dry weight intestine. bValues with different asterisks are significantly different from each other (P < 0.05). Analysis by Duncan’s multiple range test.
guinea pigs, reproduction appeared normal when adult females were given up to 50 ppm CrC13 in their diet [ 181. However, Cr03 injected into pregnant hamsters was embryotoxic [19, 201. Also, the LD5,, of Naz Crz 0, injected into chicken eggs was about 10 times lower than Cr(N0, )3 [21] and Cr03 injected into the air sacs of embryonating chicken eggs at different days of development showed embryolethal and teratogenic effects [ 221. The present study suggests that the anionic form of Cr is better absorbed than the cationic form from the black duck small intestine. These findings also stress the importance of considering the ionic state of Cr when planning dietary toxicity studies. Because there is an increased probability for waterfowl to come into contact with effluents containing high levels of Cr more information about the toxic effects of this metal on these species is needed. ACKNOWLEDGEMENTS
We thank Doug Louk and Tom Heazel for technical aid and Gary Heinz for editorial assistance. Donna Vaurio and Patty McDonald typed the manuscript. REFERENCES 1 R.D. Duval, Benthic macro-invertebrate and heavy metal survey of the Sebasticook River, Maine, Senior thesis, University of Maine, Orono, ME, 1977, 83 pp. 2 D.K. Phelps, G. Telek and R. Lapan, Assessment of heavy metal distribution within the food web, in Pearson and Frangipani (Eds.), Marine Pollution and Marine Waste Disposal, Pergamon, New York, 1975. 3 W. Mertz, Chromium occurrence and function in biological systems, Physiol. Rev., 49 (1969) 2,163-239. 4 National Research Council, Committee on biological effects of atmospheric pollutants, medical and biological effects of environmental pollutants, Chromium, National Acad. Sci., Washington, DC, 1974. 5 E.J. Underwood, Chromium, Trace Elements in Human and Animal Nutrition, 3rd ed., Academic Press, New York, 1971.
197 6 W. Mertz, The newer essential trace elements, chromium, tin, vanadium, nickel, and silicon, Proc. Nutr. Sot., 33 (1974) 307-313. ‘7 K.J. Beinecke, Invertebrate feeding by black ducks in Maine wetlands, Proc. N.E. Fish Wildl. Conf., New Haven, CT, 1975, pp. 170-182. 8 SK. Tandon, D.K. Saxena, J.S. Gaur and S.V. Chandra, Comparative toxicity of trivalent and hexavalent chromium, alterations in blood and liver, Environ. Res., 15 (1978) 90-99. 9 R.M. Donaldson Jr. and R.F. Barreras, Intestinal absorption of trace quantities of chromium, J. Lab. Clin. Med., 68 (1966) 484-493. 10 RD. MacKenzie, R.A. Anwar, R.U. Byerrum and C.A. Hoppert, Absorption and distribution of Cr5’ in the albino rat, Arch. Biochem. Biophys., 79 (1959) 200-205. 11 W.J. Visek, I.B. Whitney, U.S.G. Kuhn III, and C.L. Comar, Metabolism of Crsl by animals as influenced by chemical state, Proc. Sot. Exp. Biol. Med., 84 (1953) 610-615. 12 S.J. Gray and K. Sterling, The tagging of red cells and plasma proteins with radioactive chromium, J. Clin. Invest., 29 (1950) 1604-1613. 13 I. Dinu and L. Boghianu, Influence of chromium on the activity of some hepatic enzymes, Rev. Roum. Biochim., 10 (1973) 2,105-111. 14 T. Ghafghazi, A. Maghbareh and R. Barnett, Chromium-induced hyperglycemia in the rat, Toxicology, 12 (1979) 47-52. 15 H.W. Staub, G. Reussner and R. Thiessen Jr., Serum cholesterol reduction by chromium in hypercholesterolemic rats, Science, 166 ( 1969) 746-747. 16 P.S. Simavoryan, Effect of sexivalent chromium on concentration and dilution capacity of dog kidney, Tr. Erevan. Med. Inst., Min. Zdravookhr. Arm. SSR No. 14, (1965) 207-214. 17 G.L. Rosomer, W.A. Dudley, L.J. Machlin and L. Loveless,‘Toxieity of vanadium and chromium for the growing chick, Poul. Sci., 40 (1961) 5, 1171-1173. 18 A.M. Preston, R.P. Dowdy, M.A. Preston and J.N. Freeman, Effect of dietary chromium on glucose tolerance and serum cholesterol in guinea pigs, J. Nutr., 106 (1976) 1391-1397. 19 T.F. Gale, Embryotoxic effects of chromium trioxide in hamsters, Environ. Res. 16 (1978) 101-109. 20 T.F. Gale and J.D. Brunch III, The effect of the time of administration of chromium trioxide on the embryotoxic response in hamsters, Teratology, 19 (3.979) 1, 81-86. 21 L.P. Ridgway and D.A. Karnofsky, The effects of metals on the chick embryo: Toxicity and production of abnormalities in development, Ann. N.Y. Acad. Sci., 55 (1952) 203-215. 22 S.H. Gilani and M. Marano, Chromium poisoning and chick embryogenesis, Environ. Res., 19 (1979) 427-431.
193
INTESTINAL ABSORPTION OF 5 CHROMIUM COMPOUNDS IN YOUNG BLACK DUCKS (AiVAS RUBRIPES) W.C. EASTIN Jr., S.D. HASELTINE and H.C. MURRAY Environmental Physiology and Toxicology Project, U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, MD 20811 (U.S.A.) (Received January 16th, 1980) (Accepted April 4th, 1980)
SUMMARY
An in vivo intestinal perfusion technique was used to measure the absorption rates of five Cr compounds in black ducks. Cr was absorbed from saline solutions of KCr(S04 )2 and Cr03 at a rate about 1.5 to 2.0 times greater than from solutions of Cr, Cr(N03 )3, and Cr(CSH,02)3. These results suggest the ionic form of Cr in solution may be an important factor in determining absorption of Cr compounds from the small intestine.
INTRODUCTION
Industrial and urban complexes which release Cr in liquid effluent wastes are concentrated along the eastern seaboard of the United States. Thus Cr concentrations are higher in coastal freshwater streams along the Atlantic as compared with the Gulf and Pacific coasts. In a Maine river receiving tannery effluents, sediment Cr levels reached 25 000 ppm Cr [l] . In Rhode Island, bivalves and polychaetes in an area receiving the outfall from an electroplating plant contained 20-25 ppm Cr [ 21. Similar facilities in operation throughout the northeast may be releasing enough Cr to reach equally high levels in indigenous invertebrates. Reviews of the occurrence, function, and toxicity of Cr in biological systems have recently been published [3-51. Although Cr is considered an essential metal for maintenance of normal glucose metabolism [3, 61, high concentrations of Cr in the diet can result in toxicity [3-51. Black ducks breed along the Atlantic coast from the Maritime Provinces to Virginia. During the reproductive season potentially contaminated invertebrates make up more than 80% of the diets of adult and juvenile black ducks [7] It has been reported that the absorption of Cr in mammals depends on the valence state of this metal [5]. Although not conclusive, it would appear that if high concentrations of Cr, regardless of valence state, get into the circulation there is a toxic affect [3, 81. There is little information on the absorption of
194
Cr in non-mammalian intestinal absorption
vertebrates. The present study examined the relative of five Cr compounds in juvenile black ducks.
METHODS
Pairs of black ducks were housed in 9.1 m X 4.6 m outdoor pens, fed a commercial breeder mash and water ad lib., and allowed to hatch and rear their own young. Two ducklings from each of 20 broods were randomly selected when brood mean weight reached 175 g (mean weight it S.D. for selected birds was 188.5 f 54.6 g). Ducklings ranged in age from 11 to 17 days. Ducklings were anesthetized with ketamine HCl (200 mg/kg i.m.). Laparotomy (3 cm) was performed on the left side and the upper half of the small intestine delivered into a warm saline-moistened gauze sponge. The intestine was ligated about 1 cm from the pylorus. An entrance canula was inserted and secured % cm below the ligature. A second ligation was made at Meckel’s diverticulum; an intestinal incision was made anterior to this point. The intestine was flushed with warm physiological saline (0.9% NaCl) followed by air and the exit canula was established. The bile ducts were ligated and the exposed intestine was replaced in the abdominal cavity. The incision was closed with a hemostat and a heating pad was used to help maintain body temperature. Perfusion fluid was recirculated through the intestine with a Buchler polystaltic pump at a rate of 2 ml/min. After 20 min equilibration, 20 ml of fresh solution was perfused for an additional 20 min and frozen for subsequent analyses. Preperfusion samples were saved from each trial for comparison to the recirculated perfusion solutions. Stock solutions of 1 mg Cr/ml for each of the chromium compounds were prepared as follows. Chromium potassium sulfate [CrK(S04 )Z -12 Hz 01; chromium trioxide, (CrOB ); and chromium nitrate, [Cr(N03 )3 09 H2 0] were dissolved in 0.9% NaCl. 100 mg pelleted chromium (Cr) was dissolved in 10 ml 1 N HCl overnight and then diluted 1 to 10 with 0.9% NaCl. The organic compound, 2,+pentanedione chromium [Cr(C, H, O2 )3 ] , was dissolved in 70% ethyl alcohol. Perfusion solutions containing (10 ppm Cr) were made up fresh on the day of each trial; 1 ml of the appropriate stock solution was added to 99 ml of 0.9% NaCl containing 50 mg/l phenol red as a nonabsorbable volume marker. The pH was adjusted to approx. 6.5 with dropwise addition of 1 M NaOH. After perfusion, birds were killed by an overdose of Equi-Thesin@ . The perfused intestine was dried overnight at 100” C and then weighed. In test solutions, Phenol red concentrations were measured spectrophotometrically and chromium concentrations were determined by atomic absorption spectrometry (Perkin-Elmer Model 303). Net chromium flux was determined by the formula:
195
Net Cr flux =
Vl[Cr,(PR)
- Cri]
W
where Vi = initial volume (ml); Cr, and Cri = the final and initial Cr concentrations in pmol/ml; PR = phenol ratio = orginal cont./final cont.; and W = dry weight (g) of the perfused intestinal segment. Net flux is expressed as number of pmol Cr/20 min, g dry weight. Chromium absorption rates were compared by one-way analysis of variance and subsequently by Duncan’s multiple range test. RESULTS
AND DISCUSSION
All but one of the ducklings completed the perfusion trial. Cr was absorbed from solutions of KCr (SO4 )* and Cr03 at equivalent rates but at a significantly greater rate (1.5-2.0 X ) than from solutions of Cr, Cr(N03 )3, and Cr(C, H, O2 )3 (Table I). The equal absorption rates of trivalent Cr as KCr(S04 )* and hexavalent Cr as CrG3 in young black ducks are of particular interest. In mammals, trivalent Cr is poorly absorbed from the gastrointestinal tract [9--111 when compared with hexavalent Cr [3,4,9, lo]. This would initially suggest that there is a difference in intestinal absorption of Cr between ducks and mammals. However, Gray and Sterling, in discussing the marked affinity of the red cell for hexavalent Cr in their tracer experiments, suggested that the ionic form of Cr (i.e. 51Cr042- and %r3+ ) determines the membrane permeability of this metal and thus, the differential binding by the cell [12]. The present findings with immature black ducks are in general agreement with an ionic charge concept; Cr was best absorbed from solutions containing the anionic Cr complex compared with solutions containing cationic Cr (Table I). In reviewing those mammalian studies indicating a Cr absorption difference based on valence, it was found that the results can also be explained on an anionic vs cationic Cr basis [3,4, 9, lo]. Based on ionic charge, the results from perfusion studies with young black ducks do not conflict with previous absorption data and suggest the ionic form of Cr in solution may be an important factor in determining relative absorption of Cr compounds across the small intestine. Studies on the toxic effects of high levels of Cr have dealt mostly with mammalian species. Although the results are not easy to generalize, they have shown Cr accumulates in liver, kidney, bone, and spleen [&lo, 111. Cr has also been linked to disorders in glucose metabolism [3,13, 141, to changes in serum cholesterol levels [15], and to impaired kidney [16] and liver [8] function. Few studies have been done with other vertebrates. The only avian study dealing with Cr ingestion included 30 and 100 ppm Cr as Na2 Cr04 in the feed of growing male chicks for up to 32 days [17]. No mortality, depression in growth rate, or efficiency of food utilization was noted. The effects of Cr on reproduction are also not resolved. For example, in
196 TABLE I CHROMIUM ABSORPTION IN BLACK DUCKLINGS Chromium compound
n
Chromium absorptiona’b
Valence
Charge on Cr complex
KCr(S0, ).12 H, 0 CrG, Cr (pellets) Cr(C, H, 0, )3 Cr(N0, )3 a9 H, 0
9 7 7 8 8
1.516 1.482 0.979 1.021 0.772
3 6 3 3 3
+ + +
f 0.421* ?: 0.272* * 0.552** i: 0.439** * 0.289**
aMean f S.D.; rmol/20 min, g dry weight intestine. bValues with different asterisks are significantly different from each other (P < 0.05). Analysis by Duncan’s multiple range test.
guinea pigs, reproduction appeared normal when adult females were given up to 50 ppm CrC13 in their diet [ 181. However, Cr03 injected into pregnant hamsters was embryotoxic [19, 201. Also, the LD5,, of Naz Crz 0, injected into chicken eggs was about 10 times lower than Cr(N0, )3 [21] and Cr03 injected into the air sacs of embryonating chicken eggs at different days of development showed embryolethal and teratogenic effects [ 221. The present study suggests that the anionic form of Cr is better absorbed than the cationic form from the black duck small intestine. These findings also stress the importance of considering the ionic state of Cr when planning dietary toxicity studies. Because there is an increased probability for waterfowl to come into contact with effluents containing high levels of Cr more information about the toxic effects of this metal on these species is needed. ACKNOWLEDGEMENTS
We thank Doug Louk and Tom Heazel for technical aid and Gary Heinz for editorial assistance. Donna Vaurio and Patty McDonald typed the manuscript. REFERENCES 1 R.D. Duval, Benthic macro-invertebrate and heavy metal survey of the Sebasticook River, Maine, Senior thesis, University of Maine, Orono, ME, 1977, 83 pp. 2 D.K. Phelps, G. Telek and R. Lapan, Assessment of heavy metal distribution within the food web, in Pearson and Frangipani (Eds.), Marine Pollution and Marine Waste Disposal, Pergamon, New York, 1975. 3 W. Mertz, Chromium occurrence and function in biological systems, Physiol. Rev., 49 (1969) 2,163-239. 4 National Research Council, Committee on biological effects of atmospheric pollutants, medical and biological effects of environmental pollutants, Chromium, National Acad. Sci., Washington, DC, 1974. 5 E.J. Underwood, Chromium, Trace Elements in Human and Animal Nutrition, 3rd ed., Academic Press, New York, 1971.
197 6 W. Mertz, The newer essential trace elements, chromium, tin, vanadium, nickel, and silicon, Proc. Nutr. Sot., 33 (1974) 307-313. ‘7 K.J. Beinecke, Invertebrate feeding by black ducks in Maine wetlands, Proc. N.E. Fish Wildl. Conf., New Haven, CT, 1975, pp. 170-182. 8 SK. Tandon, D.K. Saxena, J.S. Gaur and S.V. Chandra, Comparative toxicity of trivalent and hexavalent chromium, alterations in blood and liver, Environ. Res., 15 (1978) 90-99. 9 R.M. Donaldson Jr. and R.F. Barreras, Intestinal absorption of trace quantities of chromium, J. Lab. Clin. Med., 68 (1966) 484-493. 10 RD. MacKenzie, R.A. Anwar, R.U. Byerrum and C.A. Hoppert, Absorption and distribution of Cr5’ in the albino rat, Arch. Biochem. Biophys., 79 (1959) 200-205. 11 W.J. Visek, I.B. Whitney, U.S.G. Kuhn III, and C.L. Comar, Metabolism of Crsl by animals as influenced by chemical state, Proc. Sot. Exp. Biol. Med., 84 (1953) 610-615. 12 S.J. Gray and K. Sterling, The tagging of red cells and plasma proteins with radioactive chromium, J. Clin. Invest., 29 (1950) 1604-1613. 13 I. Dinu and L. Boghianu, Influence of chromium on the activity of some hepatic enzymes, Rev. Roum. Biochim., 10 (1973) 2,105-111. 14 T. Ghafghazi, A. Maghbareh and R. Barnett, Chromium-induced hyperglycemia in the rat, Toxicology, 12 (1979) 47-52. 15 H.W. Staub, G. Reussner and R. Thiessen Jr., Serum cholesterol reduction by chromium in hypercholesterolemic rats, Science, 166 ( 1969) 746-747. 16 P.S. Simavoryan, Effect of sexivalent chromium on concentration and dilution capacity of dog kidney, Tr. Erevan. Med. Inst., Min. Zdravookhr. Arm. SSR No. 14, (1965) 207-214. 17 G.L. Rosomer, W.A. Dudley, L.J. Machlin and L. Loveless,‘Toxieity of vanadium and chromium for the growing chick, Poul. Sci., 40 (1961) 5, 1171-1173. 18 A.M. Preston, R.P. Dowdy, M.A. Preston and J.N. Freeman, Effect of dietary chromium on glucose tolerance and serum cholesterol in guinea pigs, J. Nutr., 106 (1976) 1391-1397. 19 T.F. Gale, Embryotoxic effects of chromium trioxide in hamsters, Environ. Res. 16 (1978) 101-109. 20 T.F. Gale and J.D. Brunch III, The effect of the time of administration of chromium trioxide on the embryotoxic response in hamsters, Teratology, 19 (3.979) 1, 81-86. 21 L.P. Ridgway and D.A. Karnofsky, The effects of metals on the chick embryo: Toxicity and production of abnormalities in development, Ann. N.Y. Acad. Sci., 55 (1952) 203-215. 22 S.H. Gilani and M. Marano, Chromium poisoning and chick embryogenesis, Environ. Res., 19 (1979) 427-431.