- Email: [email protected]
Behavior and toxicity of antimony in the short-tailed field vole (Microtus agrestis)
ECOTOXICOLOGY
AND
ENVIRONMENTAL
Behavior
of Biology,
21,16.5-
170( 199I)
and Toxicity of Antimony in the Short-Tailed Field Vole (Microtus agrestis)
N.AINSWORTH,’ School
SAFETY
J.A. COOKE,AND M.S. JOHNSON*
Sunderland Polytechnic, and Evolutionary Biology, Received
Sunderland, England: Liverpool University, November
and *Department qf Environmental Liverpool, England
23. I989
Laboratory experiments were undertaken to study uptake and retention of antimony and to investigate whether the elevated organ antimony concentrations found previously in a population of Microfus agrestis at a contaminated site could cause harmful effects.Antimony trioxide in the diet produced elevated organ concentrations, but even in a 60-day experiment no harmful effects were evident. An equilibrium between uptake and excretion of antimony seemed to be rapidly established and progressive increases in organ concentrations did not occur. When dietary intake was terminated antimony was rapidly cleared. Comparison of findings from the laboratory and field suggested that inhalation was an additional route of intake in the field. It seems that present levels of antimony are unlikely to cause toxic effects in the wild population. o 1991 Academic Press. Inc.
INTRODUCTION At a grassland site close to an antimony smelter elevated concentrations of antimony were found in soil, vegetation, and animals (Ainsworth et al., 1990a,b). In grass over 200 mg kg-’ was present on several occasions and in the liver tissue of the herbivore Microtus agrestis a mean of 0.30 mg kg-‘. The grass appeared to be contaminated by surface deposits, probably of antimony oxides, rather than by uptake from the soil (Ainsworth et al., 1990a). Antimony is known to cause a number of toxic effects in animals, including cardiac damage (Girgis et al., 1970), suppression of weight gain (Hiraoka, 1986), shortened lifespan (Schroeder et al., 1968, 1970), and damage to liver, thyroid, and kidneys (Osintseva et al., 1966). However, such studies have been undertaken with regard to medicinal uses of antimony or occupational exposure in industry. The animal species, chemical form, and amount of antimony and route of administration used in these experiments mean that they are not directly relevant to possible effects on wild animal populations in antimony-contaminated environments. The experiments described here were undertaken to investigate whether the elevated concentrations of antimony found in the organs of M. ugrestis caught in the wild were sufficient to cause harmful effects, and to study the uptake and retention of ingested antimony. MATERIALS
AND
METHODS
All the animals used in the experiments were bred in the laboratory under conditions recommended by Baker and Clarke (1985), except that a commercial rat and mouse r To whom correspondence should be addressed at present address: Imperial College of Science, Technology & Medicine, Silwood Park, Ascot, Berkshire, England SLS 7PY. I65
0147-6513/9l
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
166
AINSWORTH,
COOKE, AND JOHNSON
diet was supplied instead of oats and hay. Animals were usually transferred to experimental diets at an age of 35 to 40 days. Experimental diets were prepared by grinding up the normal food, mixing in the appropriate amount of antimony trioxide powder, then repelleting the mixture including 3% (dry weight) methyl cellulose as a binder. A control diet prepared in the same way, but without added antimony, had an antimony concentration of less than 0.025 mg kg-‘. Antimony was added to the diet as the trioxide. In the first experiment voles were supplied with control diet, a 500 mg Sb kg-’ diet or a 6700 mg Sb kg-’ diet for 21 days, after which the antimony concentrations in liver, lung, and kidney were measured, using hydride-generation atomic absorption spectrophotometry as previously developed for voles caught in the wild (Ainsworth et al., 1990b). For the second experiment the 6700 mg Sb kg-’ diet was supplied to 10 voles for 7 days. Half of the voles were sacrificed at this stage; those remaining, were transferred to the control diet for a further 7 days before sacrifice. In the third and largest experiment voles were supplied with the 500 mg Sb kg-’ diet for 30,40,50, or 60 days, or fed on the control diet for the full 60 days. Additional groups were fed on the 500 mg Sb kg-’ diet for 30 days, then transferred to the control diet for a further 5, 10, or 15 days. A final experiment was performed in which six voles were fed on a diet containing 20,000 mg Sb kg-’ for 12 days, and another five voles received the control diet for the same period. Kidneys and portions of the livers of these animals were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, then postfixed in 1% osmium tetroxide. The tissues were then sectioned to 60-90 nm poststained with uranyl acetate and lead citrate and examined by transmission electron microscopy. The antimony contents of the remaining part of each liver and the whole of the lungs were determined as in the previous experiments. RESULTS In the first experiment voles receiving the 500 mg Sb kg-’ diet had higher organ antimony concentrations than voles in the control group (Table 1). Antimony concentrations in the 500 mg Sb kg-’ diet group followed the order liver > kidney > lung, and the differences between these organs were significant using the Mann-Whitney U test (P < 0.05 for liver and kidney, P < 0.001 for kidney and lung). The order of TABLE
1
ANTIMONY CONCENTRATIONS IN ORGANSAFTER2 1 DAYSCONSUMPTION OFEXPERIMENTAL DIETS Antimony concentration in diet (mg Sb kg-‘)
Liver Lung Kidney
c.02 ~06 c.05
1.25 0.20 0.49
500
6700
(7)
(4)
It 0.15 + 0.02
+ 0.10
8.58-t 2.26 + 0.56
2.44
5.18 f 0.72
6700*
(1) 22.3 37.6 44.8
Note. Figures aremeans +-standard error,except6700*,whichwasapregnant animalincludedaccidentally. Numbersof animals in parentheses.
TOXICITY
OF ANTIMONY
IN THE VOLE
167
organ antimony concentrations was the same in voles receiving the 6700 mg Sb kg-’ diet, except for one pregnant female, included accidentally, which is shown separately in Table 1. Two healthy young were produced, and these had liver antimony concentrations of 2.0 and 1.4 mg Sb kg-’ when 7 days old at the end of the experiment. In the second experiment the liver and lung antimony concentrations after 7 days feeding on the 6700 mg Sb kg-’ diet were not significantly different from those found in voles which had fed on the same diet for 2 1 days in the first experiment (Table 2). Kidney antimony concentrations, however, were significantly lower than those in the first experiment. Antimony concentrations in all three organs were much lower in the voles transferred to the control diet for 7 days than in those sacrificed immediately after 7 days feeding on the 6700 mg Sb kg-’ diet. Assuming that the organ antimony concentrations of the two groups were similar after the first 7 days, it appears that antimony was cleared rapidly after intake from the diet ceased. Organ antimony concentrations of voles in the longer-term experiment also followed the order liver > kidney > lung when the animals had received the 500 mg Sb kg-’ diet continuously (Table 3). The only significant differences between organ antimony concentrations after different lengths of time feeding on the 500 mg Sb kg-’ diet were that the mean liver antimony concentration after 40 days was lower than the concentrations after 30 and 50 days (P c 0.05), and the kidney antimony concentration after 50 days was higher than at any of the other times. There was no trend of increasing antimony concentration with greater length of time receiving the 500 mg Sb kg-’ diet for any of the organs. The groups of voles transferred to the control diet after 30 days receiving the 500 mg Sb kg-’ diet showed rapid clearance of antimony from all the organs analyzed, although the rate of clearance from lung and kidney was uncertain because some of the concentrations in these organs were too low to be determined precisely. All the voles appeared to be healthy throughout the 60day experiment. The appearance of the internal organs of voles receiving the 500 mg Sb kg-’ diet was indistinguishable from that of the control voles. Wet and dry weights of the liver, kidney, and lungs of animals receiving the 500 mg Sb kg-’ diet were not significantly different from those of control animals. The six voles fed on the 20,000 mg Sb kg-’ diet for 12 days had very high antimony concentrations in liver and lungs: 23.9 mg kg-’ in the liver and 10.5 mg kg-’ in lungs, with standard errors of 1.5 and 4.3, respectively. Despite this they remained apparently
TABLE
2
ORGANANTIMONYCONCENTRATIONS IN ANUPTAKE/CLEARANCE EXPERIMENT (mg Sb kg-‘) 7
days antimony diet
Liver Lung Kidney
+ k 1.49 +
7.63 1.83
1.24 0.39 0.32
7
days antimoy diet then 7 dayscontrol diet 1.02 0.12 0.20
k 0.18 + 0.03 +- 0.05
Note.Figuresaremeansf standarderror.Fiveanimalswereusedin each treatment.Antimonydiet contained 6700 mg Sb kg-‘.
168
AINSWORTH,
COOKE, AND JOHNSON TABLE
3
ORGANANTIMONYCONCENTRATJONS JNFIELDVOLESAFTERDJFFERENTPERJODSOF ADMJNISTRATJON OF HJGHANTJMONYAND/ORCONTROLDIETS (High antimony
diet = 500 mg kg-‘)
Number of days receiving diet
n
Liver
Lung
Kidney
30 40 50 60 60 (control) 30 (+5)
8 10 8 8 8 4
1.52 + 0.12 1.27 t- 0.05 1.78 + 0.08 1.45 + 0.11 co.02 1.05 k 0.21
0.48 +- 0.04 0.47 -t 0.05 0.97 k 0.13 0.60 + 0.08 co.05 0.07 Ifr 0.02
30 (+lo)
8
0.56 _+ 0.03
30 (+5)
8
0.37 + 0.03
0.27 k 0.07 0.37 + 0.07 0.35 f 0.04 0.38 +- 0.16 <0.06 <0.04, x0.04, <0.08, 0.04 0.07,0.06, ~0.05, <0.05, <0.06, <0.05, <0.05, <0.06 0.13,0.09,0.08,0.04, 0.04, co.07, co.05, co.05
0.04, 0.09, 0.06, 0.03, 0.04, <0.03, <0.03, co.03 0.07,0.14, <0.04,0.05, <0.04, 0.22, 0.02, 0.03
Note. Figures in parentheses show number of days administration of control diet after antimony diet. Concentrations are mean mg Sb kg-’ -+_standard error, except where some organs were below limit of detection. No antimony was detected in any organs of control animals. In treatments where antimony was detectable in only some of the organs each animal is shown individually.
healthy. No histological changes compared to control group voles could be observed in the sections of liver and kidney examined by electron microscopy. DISCUSSION It seems from the experiments described here that when field voles are supplied with high-antimony diets the concentrations in liver, lung, and kidney rapidly reach an equilibrium as continuing absorption is balanced by excretion. Progressive increases in the antimony concentrations in these organs over a longer period of time do not seem to occur. Since whole-body analyses were not performed it is of course possible that progressive accumulation of antimony did occur in other organs. A general pattern of rapid establishment of an equilibrium with dietary intakes was, however, also found on a whole-body basis by Gerber et al. (1982) in laboratory mice fed antimony trichloride. The antimony concentrations in liver, lung, and kidney were very low in all the experiments compared to the concentration in the diet. Since these organs have often been found to contain high concentrations of antimony relative to other organs (Felicetti et al., 1974; Hiraoka, 1986), it would seem that antimony is not concentrated by voles compared to their diet. The order of organ antimony concentrations in these experiments was consistently liver > kidney > lung. This contrasts with the situation found in wild voles living close to an antimony smelter (Ainsworth et al., 1990b) in which liver and lung concentrations were similar to each other, and both were higher than the kidney concentration. The most likely explanation of this is that in the field some antimony-containing
TOXICITY
OF
ANTIMONY
IN
THE
VOLE
169
particles are inhaled and retained in the lungs, whereas in the laboratory, with the antimony bound in pellets of food, inhalation should be negligible. This explanation is supported by several studies which have found long-term retention in the lungs of particles containing antimony (McCallum et al., 1970; Gerhardsson et al., 1982; Leffler
et al., 1984). The antimony concentration in the estimated diet of the field population at the time of trapping in May was approximately 150 mg kg-’ (Ainsworth et al., 1990b). The 500 mg Sb kg-’ diet was intended to provide an antimony intake equivalent to the highest intake likely to occur in the field, based on concentrations found in grass samples taken in winter. Lung antimony concentrations were similar in laboratory voles in the 60-day (500 mg Sb kg-’ diet) experiment and in wild-caught voles, but liver concentrations were approximately five times lower, and kidney concentrations three times lower in animals caught in the wild. The higher lung antimony concentrations in relation to dietary intake in the field are probably due to inhalation as concluded above. The relationships between kidney and liver antimony concentrations and antimony concentration in the diet were similar in both laboratory and wildcaught voles. It could be suggested on the basis of these results that inhalation of antimony in the field, while increasing the lung concentration, does not have much effect on the antimony concentration in liver and kidney, and therefore probably has little effect on overall body burden. However, in view of the uncertainty in the estimate of dietary antimony intake in the field it is also possible that the apparent similarity in the relationship of dietary intake to liver and kidney concentrations in the field and laboratory is coincidental. It should also be remembered that the voles in the laboratory experiments had not received high antimony diets until 35-40 days old, whereas the animals in the field would have had continuous exposure. For this reason comparisons between the field and laboratory animals should be treated with caution. An exception to the general pattern of organ concentrations in the laboratory experiments was the female which gave birth to two offspring during the first experiment. In this animal, the organ concentrations, especially in lung and kidney, were many times higher than in the other voles receiving the same diet, and the order was kidney > lung > liver. Gerber et al. (1982) found a similar increase in the antimony content of pregnant mice: the whole body content (excluding intestinal content) was approximately nine times higher than that of nonpregnant mice receiving the same diet. It is known that antimony can reduce reproductive success in rats (Belyaeva, 1967) and it appears that the effect of antimony on reproduction and on health of the young deserves further study. No effects of the high antimony diet were observed in the 60-day experiment, even though the liver antimony concentrations produced were approximately 4-5 times higher than in voles trapped at a highly contaminated site. Furthermore, no adverse effects were observed following the shorter-term administration of a 20,000 mg Sb kg-’ diet, a concentration which might be briefly present in the field following an exceptional release from the smelter. From the experiments reported here it seems that no harmful effects of antimony are likely to occur in the voles inhabiting grassland close to the antimony smelter, although further work on possible reproductive effects is required. It should be noted that the voles used in the laboratory were kept in a very stable environment and supplied ad libitum with a highly nutritious diet. In these circumstances they may have been able to tolerate higher body burdens of antimony than would have been possible under field conditions.
170
AINSWORTH,
COOKE, AND JOHNSON
CONCLUSIONS 1. Administration of a diet containing 500 mg Sb kg-’ for up to 60 days produced no obvious harmful effects in field voles. 2. The antimony concentrations in liver, lung, and kidney tissue appeared to reach a plateau after approximately 30 days feeding on the above diet. 3. Consumption of a diet containing 20,000 mg Sb kg-’ for 12 days did not appear to be harmful. 4. Comparison of voles in the experiments with those from a contaminated field site suggested that inhalation may be an additional route of antimony intake in the field. 5. Insofar as the laboratory results can be applied to the field population, it does not seem that the level of antimony at the contaminated site is likely to be harmful. ACKNOWLEDGMENTS The research described here was undertaken while one of us (N.A.) was in receipt of an N.E.R.C. studentship. Acknowledgement is made of site accessgranted by Cookson plc and of site accessand financial assistance provided by Tyne Tunnels, Ltd. S. J. Evans of Liverpool University is thanked for his assistance with the analysis.
REFERENCES AINSWORTH, N., COOKE, J. A., AND JOHNSON, M. S. (1990a). Distribution of antimony in contaminated grassland. 1. Vegetation and soils. Environ. PoNut. 65,65-78. AINSWORTH, N., COOKE, J. A., AND JOHNSON,M. S. (1990b). Distribution of antimony in contaminated grassland. 2. Small mammals and invertebrates. Environ. Pollut. 65, 79-87. BAKER, J., AND CLARKE, J. R. (1985). The field vole and the bank vole. In UFAW Handbook (T. Poole, Ed.), Churchill Livingstone, London. BELYAEVA, A. P. (1967). An experimental study of the effect of antimony on the breeding of progeny. Tr. Kirg.
Med.
Inst. 43, 94-95.
FELICETTI, S. A., THOMAS, R. G., AND MCCLELLAN, R. 0. (1974). Amer. Industr. Hyg. Ass. J. 35, 292300. GERBER, G. B., MAES, J., AND EYKENS, B. (1982). Transfer of antimony and arsenic to the developing organism. Arch. Toxicol. 49, 159-168. GERHARDSSON, L., BRUNE, D., NORDBERG, G. F., AND WESTER, P. 0. (1982). Antimony in lung, liver and kidney tissue from deceased smelter workers. Stand. J. Work Environ. Health 8,20 l-208. GIRGIS, N. I., KHAYYAL, M. T., MCCONNEL, E., AND NORTON, J. (1970). Penicillamine as an adjuvant to antimonial therapy, effect on electrocardiographic changes in dogs. East Afi. Med. J. 47, 576-58 1, HIRAOKA, N. (1986). The toxicity and organ distribution of antimony after chronic administration to rats. J. Kyoto
Prefect.
Univ.
Med.
95,997-1017.
LEFFLER, P., GERHARDSSON,L., BRUNE, D., AND NORDBERG, G. F. (1984). Lung retention of antimony and arsenic in hamsters after the intratracheal instillation of industrial dust. Stand. J. Work Environ. Health
10,245-25
1.
MCCALLUM, R. I., DAY, M. J., UNDERHILL, J., AND AIRD, E. G. A. (1970). Measurement of antimony oxide dust in human lungs in vivo by X-ray spectrophotometry. Inhal. Part. Vap. 2,6 1 l-6 19. GSINTSEVA, V. P., BONASHEVSKAYA, T. I., AND ARZAMESTEV, E. V. (1966). Effect of trivalent antimony on the organism of experimental animals. Arkh. Patol. 28,60-64. SCHROEDER,H. A., MITCHENER, M., BALASSA, J., KANISAWA, M., AND NASON, A. P. (1968). Zirconium, niobium, antimony and fluorine in mice: Effects on growth, survival and tissue levels. J. Nutrition 95, 95-101. SCHROEDER,H. A., MITCHENER, M., AND NASON, A. P. (1970). Zirconium, niobium, antimony, vanadium and lead in rats: Life-term studies. J. Nutrition 100, 59-68.
AND
ENVIRONMENTAL
Behavior
of Biology,
21,16.5-
170( 199I)
and Toxicity of Antimony in the Short-Tailed Field Vole (Microtus agrestis)
N.AINSWORTH,’ School
SAFETY
J.A. COOKE,AND M.S. JOHNSON*
Sunderland Polytechnic, and Evolutionary Biology, Received
Sunderland, England: Liverpool University, November
and *Department qf Environmental Liverpool, England
23. I989
Laboratory experiments were undertaken to study uptake and retention of antimony and to investigate whether the elevated organ antimony concentrations found previously in a population of Microfus agrestis at a contaminated site could cause harmful effects.Antimony trioxide in the diet produced elevated organ concentrations, but even in a 60-day experiment no harmful effects were evident. An equilibrium between uptake and excretion of antimony seemed to be rapidly established and progressive increases in organ concentrations did not occur. When dietary intake was terminated antimony was rapidly cleared. Comparison of findings from the laboratory and field suggested that inhalation was an additional route of intake in the field. It seems that present levels of antimony are unlikely to cause toxic effects in the wild population. o 1991 Academic Press. Inc.
INTRODUCTION At a grassland site close to an antimony smelter elevated concentrations of antimony were found in soil, vegetation, and animals (Ainsworth et al., 1990a,b). In grass over 200 mg kg-’ was present on several occasions and in the liver tissue of the herbivore Microtus agrestis a mean of 0.30 mg kg-‘. The grass appeared to be contaminated by surface deposits, probably of antimony oxides, rather than by uptake from the soil (Ainsworth et al., 1990a). Antimony is known to cause a number of toxic effects in animals, including cardiac damage (Girgis et al., 1970), suppression of weight gain (Hiraoka, 1986), shortened lifespan (Schroeder et al., 1968, 1970), and damage to liver, thyroid, and kidneys (Osintseva et al., 1966). However, such studies have been undertaken with regard to medicinal uses of antimony or occupational exposure in industry. The animal species, chemical form, and amount of antimony and route of administration used in these experiments mean that they are not directly relevant to possible effects on wild animal populations in antimony-contaminated environments. The experiments described here were undertaken to investigate whether the elevated concentrations of antimony found in the organs of M. ugrestis caught in the wild were sufficient to cause harmful effects, and to study the uptake and retention of ingested antimony. MATERIALS
AND
METHODS
All the animals used in the experiments were bred in the laboratory under conditions recommended by Baker and Clarke (1985), except that a commercial rat and mouse r To whom correspondence should be addressed at present address: Imperial College of Science, Technology & Medicine, Silwood Park, Ascot, Berkshire, England SLS 7PY. I65
0147-6513/9l
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
166
AINSWORTH,
COOKE, AND JOHNSON
diet was supplied instead of oats and hay. Animals were usually transferred to experimental diets at an age of 35 to 40 days. Experimental diets were prepared by grinding up the normal food, mixing in the appropriate amount of antimony trioxide powder, then repelleting the mixture including 3% (dry weight) methyl cellulose as a binder. A control diet prepared in the same way, but without added antimony, had an antimony concentration of less than 0.025 mg kg-‘. Antimony was added to the diet as the trioxide. In the first experiment voles were supplied with control diet, a 500 mg Sb kg-’ diet or a 6700 mg Sb kg-’ diet for 21 days, after which the antimony concentrations in liver, lung, and kidney were measured, using hydride-generation atomic absorption spectrophotometry as previously developed for voles caught in the wild (Ainsworth et al., 1990b). For the second experiment the 6700 mg Sb kg-’ diet was supplied to 10 voles for 7 days. Half of the voles were sacrificed at this stage; those remaining, were transferred to the control diet for a further 7 days before sacrifice. In the third and largest experiment voles were supplied with the 500 mg Sb kg-’ diet for 30,40,50, or 60 days, or fed on the control diet for the full 60 days. Additional groups were fed on the 500 mg Sb kg-’ diet for 30 days, then transferred to the control diet for a further 5, 10, or 15 days. A final experiment was performed in which six voles were fed on a diet containing 20,000 mg Sb kg-’ for 12 days, and another five voles received the control diet for the same period. Kidneys and portions of the livers of these animals were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, then postfixed in 1% osmium tetroxide. The tissues were then sectioned to 60-90 nm poststained with uranyl acetate and lead citrate and examined by transmission electron microscopy. The antimony contents of the remaining part of each liver and the whole of the lungs were determined as in the previous experiments. RESULTS In the first experiment voles receiving the 500 mg Sb kg-’ diet had higher organ antimony concentrations than voles in the control group (Table 1). Antimony concentrations in the 500 mg Sb kg-’ diet group followed the order liver > kidney > lung, and the differences between these organs were significant using the Mann-Whitney U test (P < 0.05 for liver and kidney, P < 0.001 for kidney and lung). The order of TABLE
1
ANTIMONY CONCENTRATIONS IN ORGANSAFTER2 1 DAYSCONSUMPTION OFEXPERIMENTAL DIETS Antimony concentration in diet (mg Sb kg-‘)
Liver Lung Kidney
c.02 ~06 c.05
1.25 0.20 0.49
500
6700
(7)
(4)
It 0.15 + 0.02
+ 0.10
8.58-t 2.26 + 0.56
2.44
5.18 f 0.72
6700*
(1) 22.3 37.6 44.8
Note. Figures aremeans +-standard error,except6700*,whichwasapregnant animalincludedaccidentally. Numbersof animals in parentheses.
TOXICITY
OF ANTIMONY
IN THE VOLE
167
organ antimony concentrations was the same in voles receiving the 6700 mg Sb kg-’ diet, except for one pregnant female, included accidentally, which is shown separately in Table 1. Two healthy young were produced, and these had liver antimony concentrations of 2.0 and 1.4 mg Sb kg-’ when 7 days old at the end of the experiment. In the second experiment the liver and lung antimony concentrations after 7 days feeding on the 6700 mg Sb kg-’ diet were not significantly different from those found in voles which had fed on the same diet for 2 1 days in the first experiment (Table 2). Kidney antimony concentrations, however, were significantly lower than those in the first experiment. Antimony concentrations in all three organs were much lower in the voles transferred to the control diet for 7 days than in those sacrificed immediately after 7 days feeding on the 6700 mg Sb kg-’ diet. Assuming that the organ antimony concentrations of the two groups were similar after the first 7 days, it appears that antimony was cleared rapidly after intake from the diet ceased. Organ antimony concentrations of voles in the longer-term experiment also followed the order liver > kidney > lung when the animals had received the 500 mg Sb kg-’ diet continuously (Table 3). The only significant differences between organ antimony concentrations after different lengths of time feeding on the 500 mg Sb kg-’ diet were that the mean liver antimony concentration after 40 days was lower than the concentrations after 30 and 50 days (P c 0.05), and the kidney antimony concentration after 50 days was higher than at any of the other times. There was no trend of increasing antimony concentration with greater length of time receiving the 500 mg Sb kg-’ diet for any of the organs. The groups of voles transferred to the control diet after 30 days receiving the 500 mg Sb kg-’ diet showed rapid clearance of antimony from all the organs analyzed, although the rate of clearance from lung and kidney was uncertain because some of the concentrations in these organs were too low to be determined precisely. All the voles appeared to be healthy throughout the 60day experiment. The appearance of the internal organs of voles receiving the 500 mg Sb kg-’ diet was indistinguishable from that of the control voles. Wet and dry weights of the liver, kidney, and lungs of animals receiving the 500 mg Sb kg-’ diet were not significantly different from those of control animals. The six voles fed on the 20,000 mg Sb kg-’ diet for 12 days had very high antimony concentrations in liver and lungs: 23.9 mg kg-’ in the liver and 10.5 mg kg-’ in lungs, with standard errors of 1.5 and 4.3, respectively. Despite this they remained apparently
TABLE
2
ORGANANTIMONYCONCENTRATIONS IN ANUPTAKE/CLEARANCE EXPERIMENT (mg Sb kg-‘) 7
days antimony diet
Liver Lung Kidney
+ k 1.49 +
7.63 1.83
1.24 0.39 0.32
7
days antimoy diet then 7 dayscontrol diet 1.02 0.12 0.20
k 0.18 + 0.03 +- 0.05
Note.Figuresaremeansf standarderror.Fiveanimalswereusedin each treatment.Antimonydiet contained 6700 mg Sb kg-‘.
168
AINSWORTH,
COOKE, AND JOHNSON TABLE
3
ORGANANTIMONYCONCENTRATJONS JNFIELDVOLESAFTERDJFFERENTPERJODSOF ADMJNISTRATJON OF HJGHANTJMONYAND/ORCONTROLDIETS (High antimony
diet = 500 mg kg-‘)
Number of days receiving diet
n
Liver
Lung
Kidney
30 40 50 60 60 (control) 30 (+5)
8 10 8 8 8 4
1.52 + 0.12 1.27 t- 0.05 1.78 + 0.08 1.45 + 0.11 co.02 1.05 k 0.21
0.48 +- 0.04 0.47 -t 0.05 0.97 k 0.13 0.60 + 0.08 co.05 0.07 Ifr 0.02
30 (+lo)
8
0.56 _+ 0.03
30 (+5)
8
0.37 + 0.03
0.27 k 0.07 0.37 + 0.07 0.35 f 0.04 0.38 +- 0.16 <0.06 <0.04, x0.04, <0.08, 0.04 0.07,0.06, ~0.05, <0.05, <0.06, <0.05, <0.05, <0.06 0.13,0.09,0.08,0.04, 0.04, co.07, co.05, co.05
0.04, 0.09, 0.06, 0.03, 0.04, <0.03, <0.03, co.03 0.07,0.14, <0.04,0.05, <0.04, 0.22, 0.02, 0.03
Note. Figures in parentheses show number of days administration of control diet after antimony diet. Concentrations are mean mg Sb kg-’ -+_standard error, except where some organs were below limit of detection. No antimony was detected in any organs of control animals. In treatments where antimony was detectable in only some of the organs each animal is shown individually.
healthy. No histological changes compared to control group voles could be observed in the sections of liver and kidney examined by electron microscopy. DISCUSSION It seems from the experiments described here that when field voles are supplied with high-antimony diets the concentrations in liver, lung, and kidney rapidly reach an equilibrium as continuing absorption is balanced by excretion. Progressive increases in the antimony concentrations in these organs over a longer period of time do not seem to occur. Since whole-body analyses were not performed it is of course possible that progressive accumulation of antimony did occur in other organs. A general pattern of rapid establishment of an equilibrium with dietary intakes was, however, also found on a whole-body basis by Gerber et al. (1982) in laboratory mice fed antimony trichloride. The antimony concentrations in liver, lung, and kidney were very low in all the experiments compared to the concentration in the diet. Since these organs have often been found to contain high concentrations of antimony relative to other organs (Felicetti et al., 1974; Hiraoka, 1986), it would seem that antimony is not concentrated by voles compared to their diet. The order of organ antimony concentrations in these experiments was consistently liver > kidney > lung. This contrasts with the situation found in wild voles living close to an antimony smelter (Ainsworth et al., 1990b) in which liver and lung concentrations were similar to each other, and both were higher than the kidney concentration. The most likely explanation of this is that in the field some antimony-containing
TOXICITY
OF
ANTIMONY
IN
THE
VOLE
169
particles are inhaled and retained in the lungs, whereas in the laboratory, with the antimony bound in pellets of food, inhalation should be negligible. This explanation is supported by several studies which have found long-term retention in the lungs of particles containing antimony (McCallum et al., 1970; Gerhardsson et al., 1982; Leffler
et al., 1984). The antimony concentration in the estimated diet of the field population at the time of trapping in May was approximately 150 mg kg-’ (Ainsworth et al., 1990b). The 500 mg Sb kg-’ diet was intended to provide an antimony intake equivalent to the highest intake likely to occur in the field, based on concentrations found in grass samples taken in winter. Lung antimony concentrations were similar in laboratory voles in the 60-day (500 mg Sb kg-’ diet) experiment and in wild-caught voles, but liver concentrations were approximately five times lower, and kidney concentrations three times lower in animals caught in the wild. The higher lung antimony concentrations in relation to dietary intake in the field are probably due to inhalation as concluded above. The relationships between kidney and liver antimony concentrations and antimony concentration in the diet were similar in both laboratory and wildcaught voles. It could be suggested on the basis of these results that inhalation of antimony in the field, while increasing the lung concentration, does not have much effect on the antimony concentration in liver and kidney, and therefore probably has little effect on overall body burden. However, in view of the uncertainty in the estimate of dietary antimony intake in the field it is also possible that the apparent similarity in the relationship of dietary intake to liver and kidney concentrations in the field and laboratory is coincidental. It should also be remembered that the voles in the laboratory experiments had not received high antimony diets until 35-40 days old, whereas the animals in the field would have had continuous exposure. For this reason comparisons between the field and laboratory animals should be treated with caution. An exception to the general pattern of organ concentrations in the laboratory experiments was the female which gave birth to two offspring during the first experiment. In this animal, the organ concentrations, especially in lung and kidney, were many times higher than in the other voles receiving the same diet, and the order was kidney > lung > liver. Gerber et al. (1982) found a similar increase in the antimony content of pregnant mice: the whole body content (excluding intestinal content) was approximately nine times higher than that of nonpregnant mice receiving the same diet. It is known that antimony can reduce reproductive success in rats (Belyaeva, 1967) and it appears that the effect of antimony on reproduction and on health of the young deserves further study. No effects of the high antimony diet were observed in the 60-day experiment, even though the liver antimony concentrations produced were approximately 4-5 times higher than in voles trapped at a highly contaminated site. Furthermore, no adverse effects were observed following the shorter-term administration of a 20,000 mg Sb kg-’ diet, a concentration which might be briefly present in the field following an exceptional release from the smelter. From the experiments reported here it seems that no harmful effects of antimony are likely to occur in the voles inhabiting grassland close to the antimony smelter, although further work on possible reproductive effects is required. It should be noted that the voles used in the laboratory were kept in a very stable environment and supplied ad libitum with a highly nutritious diet. In these circumstances they may have been able to tolerate higher body burdens of antimony than would have been possible under field conditions.
170
AINSWORTH,
COOKE, AND JOHNSON
CONCLUSIONS 1. Administration of a diet containing 500 mg Sb kg-’ for up to 60 days produced no obvious harmful effects in field voles. 2. The antimony concentrations in liver, lung, and kidney tissue appeared to reach a plateau after approximately 30 days feeding on the above diet. 3. Consumption of a diet containing 20,000 mg Sb kg-’ for 12 days did not appear to be harmful. 4. Comparison of voles in the experiments with those from a contaminated field site suggested that inhalation may be an additional route of antimony intake in the field. 5. Insofar as the laboratory results can be applied to the field population, it does not seem that the level of antimony at the contaminated site is likely to be harmful. ACKNOWLEDGMENTS The research described here was undertaken while one of us (N.A.) was in receipt of an N.E.R.C. studentship. Acknowledgement is made of site accessgranted by Cookson plc and of site accessand financial assistance provided by Tyne Tunnels, Ltd. S. J. Evans of Liverpool University is thanked for his assistance with the analysis.
REFERENCES AINSWORTH, N., COOKE, J. A., AND JOHNSON, M. S. (1990a). Distribution of antimony in contaminated grassland. 1. Vegetation and soils. Environ. PoNut. 65,65-78. AINSWORTH, N., COOKE, J. A., AND JOHNSON,M. S. (1990b). Distribution of antimony in contaminated grassland. 2. Small mammals and invertebrates. Environ. Pollut. 65, 79-87. BAKER, J., AND CLARKE, J. R. (1985). The field vole and the bank vole. In UFAW Handbook (T. Poole, Ed.), Churchill Livingstone, London. BELYAEVA, A. P. (1967). An experimental study of the effect of antimony on the breeding of progeny. Tr. Kirg.
Med.
Inst. 43, 94-95.
FELICETTI, S. A., THOMAS, R. G., AND MCCLELLAN, R. 0. (1974). Amer. Industr. Hyg. Ass. J. 35, 292300. GERBER, G. B., MAES, J., AND EYKENS, B. (1982). Transfer of antimony and arsenic to the developing organism. Arch. Toxicol. 49, 159-168. GERHARDSSON, L., BRUNE, D., NORDBERG, G. F., AND WESTER, P. 0. (1982). Antimony in lung, liver and kidney tissue from deceased smelter workers. Stand. J. Work Environ. Health 8,20 l-208. GIRGIS, N. I., KHAYYAL, M. T., MCCONNEL, E., AND NORTON, J. (1970). Penicillamine as an adjuvant to antimonial therapy, effect on electrocardiographic changes in dogs. East Afi. Med. J. 47, 576-58 1, HIRAOKA, N. (1986). The toxicity and organ distribution of antimony after chronic administration to rats. J. Kyoto
Prefect.
Univ.
Med.
95,997-1017.
LEFFLER, P., GERHARDSSON,L., BRUNE, D., AND NORDBERG, G. F. (1984). Lung retention of antimony and arsenic in hamsters after the intratracheal instillation of industrial dust. Stand. J. Work Environ. Health
10,245-25
1.
MCCALLUM, R. I., DAY, M. J., UNDERHILL, J., AND AIRD, E. G. A. (1970). Measurement of antimony oxide dust in human lungs in vivo by X-ray spectrophotometry. Inhal. Part. Vap. 2,6 1 l-6 19. GSINTSEVA, V. P., BONASHEVSKAYA, T. I., AND ARZAMESTEV, E. V. (1966). Effect of trivalent antimony on the organism of experimental animals. Arkh. Patol. 28,60-64. SCHROEDER,H. A., MITCHENER, M., BALASSA, J., KANISAWA, M., AND NASON, A. P. (1968). Zirconium, niobium, antimony and fluorine in mice: Effects on growth, survival and tissue levels. J. Nutrition 95, 95-101. SCHROEDER,H. A., MITCHENER, M., AND NASON, A. P. (1970). Zirconium, niobium, antimony, vanadium and lead in rats: Life-term studies. J. Nutrition 100, 59-68.