@Copyright 1991 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/91/3003-0291 $03.40
Toxic Effects of Ingested Lead Shots in Domestic Fowls HIRAI, T O S H I H I R O KAWAMOTO, AND YASUSH! KODAMA*
MANABU
Department o f Environmental Health, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka Yahata Nishi-ku, Kitakyusyu, 807, Japan Received November 4, 1990; Accepted December 17, 1990
ABSTRACT Lead poisoning from ingested shots is thought to be a major cause of high mortality in waterfowls throughout the world, and some millions of fowls die each year. However, there have been no other Japanese studies regarding lead toxicity in birds from ingested lead shots. We used domestic fowls instead of waterfowls as the experimental birds, in order to make clear the distribution and the toxic effects of lead shot in the birds. In a 1-wk follow-up study, two, four, and eight # 4 lead shots were administered orally. A dosedependent increase of the lead concentrations in blood, brain, liver, kidney, lung, spleen, bone, and epidermis of the gizzard was observed. In the 12-wk follow-up study, twenty domestic fowls were used and eight # 4 lead shots were administered to the experimental birds. Lead concentrations in brain, liver, kidney, bone, ovary, fat tissue, and breast muscle increased more than in the 1-wk follow-up study. The observed lead concentrations of organs in the domestic fowls were lower than those of the other species used in past studies. The blood lead concentrations increased up to the third week and a remarkable suppression of 3-aminolevulinic acid dehydratase activity in red blood cells and elevation of free erythropoietic protoporphyrin were observed in the exposed group. Body weight loss, loss of hair, and neurological symptoms were also observed. However, there were no mortalities during the 1- and 12-wk studies. *Author to whom all correspondence and reprint requests should be addressed.
BiologicalTraceElementResearch
291
vol. 30, 1991
HiraL Kawamoto, and Kodama
292
Index Entries: Lead toxicity; lead shot; oral ingestion; domestic fowl; FEP; ALA-D; distribution of lead.
INTRODUCTION Lead poisoning from ingested shots is t h o u g h t to be a major cause of high mortality in waterfowls t h r o u g h o u t the world, and some millions of fowls die each. year. In Japan, the first episode of lead toxicity in swans was reported in April 1989, but there have been no other studies regarding lead toxicity in birds from ingested lead shots. Experimental ingestion of five or more # 4 lead shots by Canada geese resulted in the death of all birds (1). A total of 200 shots were ingested by geese over a 12-wk period; however, there were no cases of mortality and the birds exhibited no outward signs of illness (2). These were the two extremes. Between these two extremes, reports have s h o w e d various results. Therefore, we have not been able to reach a clear conclusion regarding the cause of the death of waterfowls, even if some reports have suggested lead poisoning. In order to evaluate the extent of lead exposure, ~-aminolevulinic acid dehydratase (ALA-D) activity and free erythrocyte p r o t o p o r p h y r i n concentrations (FEP) are m e a s u r e d a n d s h o w a good correlation to lead concentration of blood (3). Techniques are available for monitoring the presence of ingested lead shots in gizzards of birds, such as direct examination of dead birds and X-ray photographs of live or d e a d birds. Even w h e n no lead shot is found in the gizzard of a waterfowl, it is impossible to d e n y recent lead ingestion (4). A n analysis of hepatic lead concentration reveals that a substantial n u m b e r of waterfowls have high concentrations, indicative of recent acute dosing, without the presence of ingested lead shot in gizzards (5). In such circumstances, from the point of view of environmental toxicology, it seems very important to k n o w the fate of lead and the early toxic effects on birds after the ingestion of lead shots. H o w e v e r , the waterfowls and other birds used in the past studies s h o w e d rather high concentrations of lead before the experiments, so there was only a relative increase of lead in organs after the administration of lead shots. Domestic fowls generally have little lead in the b o d y and, since we m a d e sure that the lead concentrations in the organs of domestic fowls were lower than the detectable level in our preliminary experiment, we decided to use domestic fowls for our studies. We force-fed a sublethal dose of lead shots to domestic fowls and followed up for I w k and for 12 wk. After the follow-up periods, we m e a s u r e d the lead concentrations of the organs and tried to clarify the lead distribution after oral ingestion of lead shots a n d the appearance of toxicological effects.
Biological Trace Element Research
Vol. 30, 1991
Toxic Effects of Lead in Fowls
293
MATERIALS AND METHODS Lead Shots The lead shots used in this experiment were #4 lead shots, which were obtained from two gun shops in Kitakyusyu, Japan.
Birds Female domestic fowls (Gallus domesticus Brisson; 100 d after birth) were purchased from Kyudo Company, Japan. They were allowed free access to water and commercial basal diet (Clea lot 'No. 0-6240) for domestic fowls, which contained all nutrient requirements. The birds were housed in four partially shaded dog cages (70 x 80 x 70 cm) located on concrete blocks. Metal mesh was laid on the floor of cage and plastic trays were placed under the cages to collect excrement.
1-wk Follow-tip Study Twenty birds were randomly divided into four groups and were acclimated for 1 wk prior to the ingestion of lead shots. On the first day of the experiment, the birds of groups 1, 2, and 3 were administered two, four, and eight lead shots, respectively. The lead shots were concealed in small pieces of apples and force-fed. The other five birds, which received only pieces of apples, served as the control group. Prior to the dosage, all birds were weighed and blood samples were drawn from the wing veins. Both exposed and control birds were weighed, and blood (6 mL) was collected on the third and the seventh day after ingestion. Blood was drawn directly into heparinized syringes to prevent coagulation, and then immediately chilled on ice. On the seventh day, all experimental birds were sacrificed by decapitation and their organs (brain, lung, heart, liver, kidney, spleen, ovary, gall bladder, gizzard, intestine, and so on) were dissected and weighed. Sternal bone, breast muscle, and fat tissue adhering to the intestines were partially dissected. The collected samples were then frozen until analysis.
12-wk Follow-tip Study Twenty birds were divided into two groups and each group, composed of 10 birds, was housed in the same dog cages that were used in the 1-wk study. On the first day of the study the exposure group was administered eight #4 lead shots in the same manner as during the 1-wk study. Ten birds, serving as the control group, received no lead shots. Prior to the experiment, all birds were weighed and blood was drawn from the wing veins. The blood samples were collected from both the exposure and the control groups at 3 and 7 d, and 3, 4, 6, 8, and 12 wk after ingestion.
Biological Trace Element Research
Vol. 30, 1991
294
HiraL Kawamoto, and Kodama
Eggs laid during the experimental period were weighed and preserved in a cold room at 4~ At the end of the experiment, all experimental birds were treated in the same manner as in the 1-wk experiment.
Analytical Procedures Red blood cell (RBC) count and hematocrit (Hct) value were determined in fresh blood and hemoglobin (Hb) concentrations were analyzed using the cyanomethemoglobin method after centrifugation (2000 rpm for 5 min) of hemolysate in order to remove nuclear material (6). ALA-D was assayed by the European Standardized Method (7,8) and FEP was determined by spectrofluorometric determination using rhodamine B as the standard substance (9). The concentrations of lead in the blood samples and the organ tissues were determined by flameless atomic-absorption spectrophotometry using the Zeeman effect correction (Hitachi polarized Zeeman AAS Model Z 8000). The eggs laid between 8 and 10 wk were divided into three groups, that is, egg shells, egg whites, and egg yolks. The lead concentrations of each group were determined.
Statistical Analysis Student's unpaired t-test and analysis of variance were used to compare the mean values.
RESULTS Table 1 shows the organ weights at 12 wk after ingestion. The weights of the kidneys and brains of experimental animals were significantly greater than those of the control group. In the 1-wk study, there was no significant difference between the organ weights of the control group and those of the exposed groups. Table 2 shows the lead concentrations in the organs. The lead concentration of brain, liver, kidney, bone, lung, and spleen increased dosedependently in the 1-wk follow-up study. The lead concentrations in brain, liver, kidney, bone, ovary, fat tissue, and muscle after 12 wk increased more than those after 1 wk. In particular, the lead concentration in fat tissue was more than 30 times higher than that after 1 week.
1-wk Follow-Up Study The kinetics of lead shots that were dosed to domestic fowls were investigated for 1 wk. As shown in Fig. 1, the lead concentration in the blood of each exposure group continued to increase throughout the observation period. It is suspected that the amount of absorption was larger than that of excretion for the initial week. The lead concentration Biological Trace Element Research
Vol. 30, 1991
Toxic Effects of Lead in Fowls
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Biological Trace Element Research
VoL 30, 1991
Hirai, Kawamoto, and Kodama
296
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Toxic Effects of Ingested Lead Shots in Domestic Fowls HIRAI, T O S H I H I R O KAWAMOTO, AND YASUSH! KODAMA*
MANABU
Department o f Environmental Health, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka Yahata Nishi-ku, Kitakyusyu, 807, Japan Received November 4, 1990; Accepted December 17, 1990
ABSTRACT Lead poisoning from ingested shots is thought to be a major cause of high mortality in waterfowls throughout the world, and some millions of fowls die each year. However, there have been no other Japanese studies regarding lead toxicity in birds from ingested lead shots. We used domestic fowls instead of waterfowls as the experimental birds, in order to make clear the distribution and the toxic effects of lead shot in the birds. In a 1-wk follow-up study, two, four, and eight # 4 lead shots were administered orally. A dosedependent increase of the lead concentrations in blood, brain, liver, kidney, lung, spleen, bone, and epidermis of the gizzard was observed. In the 12-wk follow-up study, twenty domestic fowls were used and eight # 4 lead shots were administered to the experimental birds. Lead concentrations in brain, liver, kidney, bone, ovary, fat tissue, and breast muscle increased more than in the 1-wk follow-up study. The observed lead concentrations of organs in the domestic fowls were lower than those of the other species used in past studies. The blood lead concentrations increased up to the third week and a remarkable suppression of 3-aminolevulinic acid dehydratase activity in red blood cells and elevation of free erythropoietic protoporphyrin were observed in the exposed group. Body weight loss, loss of hair, and neurological symptoms were also observed. However, there were no mortalities during the 1- and 12-wk studies. *Author to whom all correspondence and reprint requests should be addressed.
BiologicalTraceElementResearch
291
vol. 30, 1991
HiraL Kawamoto, and Kodama
292
Index Entries: Lead toxicity; lead shot; oral ingestion; domestic fowl; FEP; ALA-D; distribution of lead.
INTRODUCTION Lead poisoning from ingested shots is t h o u g h t to be a major cause of high mortality in waterfowls t h r o u g h o u t the world, and some millions of fowls die each. year. In Japan, the first episode of lead toxicity in swans was reported in April 1989, but there have been no other studies regarding lead toxicity in birds from ingested lead shots. Experimental ingestion of five or more # 4 lead shots by Canada geese resulted in the death of all birds (1). A total of 200 shots were ingested by geese over a 12-wk period; however, there were no cases of mortality and the birds exhibited no outward signs of illness (2). These were the two extremes. Between these two extremes, reports have s h o w e d various results. Therefore, we have not been able to reach a clear conclusion regarding the cause of the death of waterfowls, even if some reports have suggested lead poisoning. In order to evaluate the extent of lead exposure, ~-aminolevulinic acid dehydratase (ALA-D) activity and free erythrocyte p r o t o p o r p h y r i n concentrations (FEP) are m e a s u r e d a n d s h o w a good correlation to lead concentration of blood (3). Techniques are available for monitoring the presence of ingested lead shots in gizzards of birds, such as direct examination of dead birds and X-ray photographs of live or d e a d birds. Even w h e n no lead shot is found in the gizzard of a waterfowl, it is impossible to d e n y recent lead ingestion (4). A n analysis of hepatic lead concentration reveals that a substantial n u m b e r of waterfowls have high concentrations, indicative of recent acute dosing, without the presence of ingested lead shot in gizzards (5). In such circumstances, from the point of view of environmental toxicology, it seems very important to k n o w the fate of lead and the early toxic effects on birds after the ingestion of lead shots. H o w e v e r , the waterfowls and other birds used in the past studies s h o w e d rather high concentrations of lead before the experiments, so there was only a relative increase of lead in organs after the administration of lead shots. Domestic fowls generally have little lead in the b o d y and, since we m a d e sure that the lead concentrations in the organs of domestic fowls were lower than the detectable level in our preliminary experiment, we decided to use domestic fowls for our studies. We force-fed a sublethal dose of lead shots to domestic fowls and followed up for I w k and for 12 wk. After the follow-up periods, we m e a s u r e d the lead concentrations of the organs and tried to clarify the lead distribution after oral ingestion of lead shots a n d the appearance of toxicological effects.
Biological Trace Element Research
Vol. 30, 1991
Toxic Effects of Lead in Fowls
293
MATERIALS AND METHODS Lead Shots The lead shots used in this experiment were #4 lead shots, which were obtained from two gun shops in Kitakyusyu, Japan.
Birds Female domestic fowls (Gallus domesticus Brisson; 100 d after birth) were purchased from Kyudo Company, Japan. They were allowed free access to water and commercial basal diet (Clea lot 'No. 0-6240) for domestic fowls, which contained all nutrient requirements. The birds were housed in four partially shaded dog cages (70 x 80 x 70 cm) located on concrete blocks. Metal mesh was laid on the floor of cage and plastic trays were placed under the cages to collect excrement.
1-wk Follow-tip Study Twenty birds were randomly divided into four groups and were acclimated for 1 wk prior to the ingestion of lead shots. On the first day of the experiment, the birds of groups 1, 2, and 3 were administered two, four, and eight lead shots, respectively. The lead shots were concealed in small pieces of apples and force-fed. The other five birds, which received only pieces of apples, served as the control group. Prior to the dosage, all birds were weighed and blood samples were drawn from the wing veins. Both exposed and control birds were weighed, and blood (6 mL) was collected on the third and the seventh day after ingestion. Blood was drawn directly into heparinized syringes to prevent coagulation, and then immediately chilled on ice. On the seventh day, all experimental birds were sacrificed by decapitation and their organs (brain, lung, heart, liver, kidney, spleen, ovary, gall bladder, gizzard, intestine, and so on) were dissected and weighed. Sternal bone, breast muscle, and fat tissue adhering to the intestines were partially dissected. The collected samples were then frozen until analysis.
12-wk Follow-tip Study Twenty birds were divided into two groups and each group, composed of 10 birds, was housed in the same dog cages that were used in the 1-wk study. On the first day of the study the exposure group was administered eight #4 lead shots in the same manner as during the 1-wk study. Ten birds, serving as the control group, received no lead shots. Prior to the experiment, all birds were weighed and blood was drawn from the wing veins. The blood samples were collected from both the exposure and the control groups at 3 and 7 d, and 3, 4, 6, 8, and 12 wk after ingestion.
Biological Trace Element Research
Vol. 30, 1991
294
HiraL Kawamoto, and Kodama
Eggs laid during the experimental period were weighed and preserved in a cold room at 4~ At the end of the experiment, all experimental birds were treated in the same manner as in the 1-wk experiment.
Analytical Procedures Red blood cell (RBC) count and hematocrit (Hct) value were determined in fresh blood and hemoglobin (Hb) concentrations were analyzed using the cyanomethemoglobin method after centrifugation (2000 rpm for 5 min) of hemolysate in order to remove nuclear material (6). ALA-D was assayed by the European Standardized Method (7,8) and FEP was determined by spectrofluorometric determination using rhodamine B as the standard substance (9). The concentrations of lead in the blood samples and the organ tissues were determined by flameless atomic-absorption spectrophotometry using the Zeeman effect correction (Hitachi polarized Zeeman AAS Model Z 8000). The eggs laid between 8 and 10 wk were divided into three groups, that is, egg shells, egg whites, and egg yolks. The lead concentrations of each group were determined.
Statistical Analysis Student's unpaired t-test and analysis of variance were used to compare the mean values.
RESULTS Table 1 shows the organ weights at 12 wk after ingestion. The weights of the kidneys and brains of experimental animals were significantly greater than those of the control group. In the 1-wk study, there was no significant difference between the organ weights of the control group and those of the exposed groups. Table 2 shows the lead concentrations in the organs. The lead concentration of brain, liver, kidney, bone, lung, and spleen increased dosedependently in the 1-wk follow-up study. The lead concentrations in brain, liver, kidney, bone, ovary, fat tissue, and muscle after 12 wk increased more than those after 1 wk. In particular, the lead concentration in fat tissue was more than 30 times higher than that after 1 week.
1-wk Follow-Up Study The kinetics of lead shots that were dosed to domestic fowls were investigated for 1 wk. As shown in Fig. 1, the lead concentration in the blood of each exposure group continued to increase throughout the observation period. It is suspected that the amount of absorption was larger than that of excretion for the initial week. The lead concentration Biological Trace Element Research
Vol. 30, 1991
Toxic Effects of Lead in Fowls
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Biological Trace Element Research
VoL 30, 1991
Hirai, Kawamoto, and Kodama
296
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