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Lethal body burden of triphenyltin chloride in fish: Preliminary results
0306-4492/91 $3.00+0.00 PergamonPressplc
Camp. Biochem.Physiol. Vol.IOOC,No. l/Z, pp.59-60,1991 Printed inGreatBritain
LETHAL
BODY BURDEN OF TRIPHENYLTIN CHLORIDE IN FISH: PRELIMINARY RESULTS J. WIEKE TAS, WILLEM SEINENand ANTOON OPPERHUIZEN
Research Institute of Toxicology, Environmental Toxicology Section, University of Utrecht, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands (Received 1 October 1990) Abstract-l. Guppies exposed to several triphenyltin chloride (TPTC) concentrations in water died as soon as a body burden of 20 k 10 nmol/g fish was reached. 2. Accumulation of TPTC during exposure in acute toxicity experiments can be predicted by using the kinetic parameters of TPTC. 3. The lethal body burden is two orders of magnitude lower than for narcotic organic compounds such as chlorobenzenes.
INTRODUCTION
static, semi-static, and continuous-flow systems. Experimental condition also affects the time required to reach steady-state. For example, in a static system, with a high fish density, steady-state will be reached more rapidly than in the other systems because of the declining aqueous concentration. Consequently, LC~~S obtained by using different test systems are not necessarily the same; after a certain time, a steadystate may be obtained in one test system while this is not the case in another. For these reasons a variation in LC~~ values may be observed and the use of internal body concentrations may be preferred as a toxicity parameter for xenobiotics (Van Hoogen and Opperhuizen, 1988). Prior to this, no published LC~,,studies have reported internal body burdens for organotins. In the case of chlorinated benzenes it has been found that, after exposure, fish die when the body burden of these narcotic compounds reaches 2-2.5 pmol/g (Van Hoogen and Opperhuizen, 1988). This body burden was independent of the toxicant concentration in the aqueous phase, and the chemical structure of the compound. By combining lethal body burdens and a first-order bioconcentration model, LC~,,Smay be predicted for one species under different experimental conditions. Also, relationships between the exposure required to cause toxic effects in organisms and the exposure concentrations, can be predicted. In the present study the lethal body burden for triphenyltin chloride (TPTC) was investigated, by exposing guppies to several aqueous concentrations. The measured lethal body burdens were compared to calculated values, using first order uptake and elimination kinetics.
Organotin compounds have been widely introduced into the aquatic environment as biocides. For example tributyltin (TBT) is used in antifouling paints and triphenyltin (TPT) as a fungicide in agriculture (Bock, 1979). Therefore, many toxicity tests (LC~~S)have been carried out with organotins, using various aquatic species and experimental conditions. A wide range of ~q,,s for organotins has been published. For TPT LC~,,S range from 0.008 mg/l (96 hr) for the harpacticoid (Linden et al., 1979) to 0.4 mg/l for the mosquitofish (24 hr, LC,& (Schaefer et al., 1982) and bleak (96 hr) (Linden et al., 1979). For TBT LC~~S range from 0.0007 for the copepod (4 hr) (Hall and Pinkney, 1985), to 0.4 mg/l for the goldfish (24 hr) (Gras and Rioux, 1965). Most of the reported LC~~S were based on nominal concentrations in the water, although in some cases levels were verified by GC or AAS analysis. Limitations and assumptions in LC~,,testing have been reported previously (McCarty et af., 1985; Rand and Petrocelli, 1985; Van Hoogen and Opperhuizen, 1988). One of the primary assumptions in the use of ~q,, is that a steady-state between the aqueous concentration of the test compound and the concentration in the fish is obtained during the exposure period. However, the time required to obtain steadystate is dependent on the uptake and elimination rate constants (Banerjee et al., 1984). In some toxicity tests with organotins it is likely that steady-state is not reached between the aqueous concentration and the concentration in the fish. An example is TPT, since the elimination rate constant is very low (Tas et al., 1990). When steady-state is not reached, extrapolation from the aqueous concentration to the tissue concentration using only the bioconcentration factor is not valid. In this case, the LC, does not reflect the actual concentration in the organism causing the lethal effect. In fact, the LC~~ will probably be an overestimate of lethal internal concentrations which would be obtained at longer exposure times. An additional complicating factor is the different experimental conditions used to determine LC~~: CBP Iooc,l-2-a
THEORY
The uptake and elimination can be described by a first order exchange process (Branson et al., 1975): dG -=k,C,-k,C, dt 59
J. W. TASet al.
60
where k, and k, are the uptake and elimination rate constants and Cr and C, are the concentrations of the test compound in fish and water, respectively. According to the integrated equation 1, the relationship between LC,,, exposure time and lethal body concentrations may be expressed by (Neely, 1984): C
Lcso at time t = (k, ,k2) tldye _k2,).
(2)
Table I. Lethal body burdens of triphenyltin in WPPY CW (me/Q
I dead* (br)
C, dead (nmolk)
2 0.4 0.08
1.5 13 42
175 14 24k 13 25+ 13
MATERIALS
AND METHODS
Triphenyltin chloride (Merck) was recrystallized from toluene such that colorless crystals were obtained. It was coated on Chromosorb and solubilized in water with a generator column. This represented the highest undiluted concentration, To obtain lower concentrations, dilutions of 5 and 25 times were prepared. A one-liter aquarium was used. Guppies (5-7) ranging in weight from 115to 315 mg, with an average lipid concentration of 2.6 &- 1.2% were placed in it. The water, containing 2.0, 0.4, and 0.08 mg/l TPTC, in turn, was pumped through the aquarium at a flow rate of SO-100 ml/hr. Fish were exposed until death and the time required for this effect was recorded. Dead fish were removed from the aquarium, rinsed with water, blotted dry and weighed. Analysis of fish for TPTC was carried out according to Tas and Opperhuizen (submitted). The method consists of the following steps: single fish were homogenized with water, refluxed in a hexane: water (I : 1) mixture, and, after centrifugation, the hexane layer was separated and reduced in volume. The organotin in the hexane layer was methylated with methyl magnesium chloride (20% in THF, Merck), and a clean-up was carried out with a Florisil column. The organotin was detected with a gas chromatograph equipped with a flame photometric detector (GC-FPD). Triple water samples of the highest concentration (2 mg/l) were taken when the fish were placed into the aquarium, and at the time of death. These samples were extracted with hexane, methylated and detected with GC-FPD. RESULTS AND DISCUSSION
The highest TPTC concentration in the water was 2.06 and 1.97 mg/l after the fish died, values which were in good agreement with the expected levels, and indicated little loss of TPTC from the water column. Water samples of the other concentrations were not taken, but according to the dilution, they will be referred to as 0.4 and 0.08 mg/l TPTC. The fish exposed to 2,0.4 and 0.08 mg/l TPTC died after 1.5, between 8 and 18, and between 37 and 47 hr, respectively. All fish contained between 17 and 25 nmol/g TPTC (Table 1). It was found for triphenyltin hydroxide (TPTH) that the uptake and elimination rate constants, for guppies of approximately the same size, are approximately 50 ml/g.day and 0.014 day-‘, respectively (Tas et al., 1990). Using these values, together with the approximate aqueous concentrations and the time of death, an estimation of the lethal tissue concentration can be made (equation 2). According to this calculation, the fish should contain 24 + 5 nmol/g (Table l), which corresponds very well with the experimental value.
26 28 18
‘Average time, see text. tLcw at time f =
Equation 2 assumes that fish die when the lethal body concentration in the organism is reached, regardless of exposure time or exposure conditions.
Gt (nmolid
k, = 50 ml/g.day:
C,, dead (k,/k,)(l
-d2’)
k, = 0.014 day-‘:
f= t
dead;
LCSO=e,.
The lethal body burden for triphenyltin is two orders of magnitude lower than for narcotic compounds such as chlorinated aromatic hydrocarbons. It can be speculated that this is due to the higher reactivity of the organotin compound, which exhibits some specific toxicity. Acknowledgements-This
work was supported by the Institute for Inland Water Management and Waste Water Treatment, Ministry of Transport and Public Works, The Netherlands. A.O. would like to thank the Royal Dutch Academy of Sciences for their financial support. REFERENCES
Banerjee S., Sugatt R. H. and O’Grady D. P. (1984) A simple method for determining bioconcentration parameters of hydrophobic compounds. Environ. Sci. Technol. 18, 79-81.
Branson D. R., Blau G. E., Alexander H. C. and Neely W. B. (1975) Bioconcentration of 2,2’,4,4’-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Am. Fish Sot. 104, 7855792. Gras G. and Rioux J. A. (1965) Relation entre la structure chimique et l’activitt insecticide des composes organiques de l’itain (essai sur les larves de Culex pipiens L.) Arch. Inst. Pasteur Tunis 42, 9-22.
Hall L. W. Jr. and Pinkney A. E. (1985) Acute and sublethal effects of organotin compounds on aquatic biota: an interpretative literature evaluation. CRC Crit. Rev. Toxicol. 14, 159-209. Linden E., Bengtsson B. E., Svanberg 0. and Sundstrom G. (1979) The acute toxicity of 78 chemicals and pesticide formulations against two brackish water organisms, the bleak (Alburnis alburnis) and the harpacticoid Nitocra spinipes. Chemosphere, 11, 843-851. McCarty L. S., Hodson P. V., Craig G. R. and Kaiser K. L. E. (1985) The use of quantitative structure-activity relationships to predict the acute and chronic toxicities of organic chemicals to fish. Environ. Toxicol. Chem. 4, 595-606.
Neely W. B. (1984) An analysis of aquatic toxicity data: water solubility and acute LC~ fish data. Chemosphere 7, 813-819.
Rand G. and Petrocelli S. (Editors) (1985) Fundamentals of Aquatic Toxicology:
Methods and Applications. 666
p.
Hemisphere, Washington, DC. Schaefer C. H., Miura T., Dupras E. F., Jr. and Wilder W. H. (1982) Environmental impact of the fungicide triphenyltin hydroxide after application to rice fields. J. Econ. Entomol. 74, 597-600.
Tas J. W., Opperhuizen A. and Seinen W. (1990) Uptake and elimination kinetics of triphenyltin hydroxide by two fish species. Toxicol. Environ. Chem. 28, 129-141. Tas J. W. and Opperhuizen A. (1991) Analysis of triorganotin compounds in fish. Mar. Environ. Res. (Submitted). Van Hoogen G. and Opperhuizen A. (1988) Toxicokinetics of chlorobenzenes in fish. Environ. Toxicol. Chem. 7, 213-219.
Camp. Biochem.Physiol. Vol.IOOC,No. l/Z, pp.59-60,1991 Printed inGreatBritain
LETHAL
BODY BURDEN OF TRIPHENYLTIN CHLORIDE IN FISH: PRELIMINARY RESULTS J. WIEKE TAS, WILLEM SEINENand ANTOON OPPERHUIZEN
Research Institute of Toxicology, Environmental Toxicology Section, University of Utrecht, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands (Received 1 October 1990) Abstract-l. Guppies exposed to several triphenyltin chloride (TPTC) concentrations in water died as soon as a body burden of 20 k 10 nmol/g fish was reached. 2. Accumulation of TPTC during exposure in acute toxicity experiments can be predicted by using the kinetic parameters of TPTC. 3. The lethal body burden is two orders of magnitude lower than for narcotic organic compounds such as chlorobenzenes.
INTRODUCTION
static, semi-static, and continuous-flow systems. Experimental condition also affects the time required to reach steady-state. For example, in a static system, with a high fish density, steady-state will be reached more rapidly than in the other systems because of the declining aqueous concentration. Consequently, LC~~S obtained by using different test systems are not necessarily the same; after a certain time, a steadystate may be obtained in one test system while this is not the case in another. For these reasons a variation in LC~~ values may be observed and the use of internal body concentrations may be preferred as a toxicity parameter for xenobiotics (Van Hoogen and Opperhuizen, 1988). Prior to this, no published LC~,,studies have reported internal body burdens for organotins. In the case of chlorinated benzenes it has been found that, after exposure, fish die when the body burden of these narcotic compounds reaches 2-2.5 pmol/g (Van Hoogen and Opperhuizen, 1988). This body burden was independent of the toxicant concentration in the aqueous phase, and the chemical structure of the compound. By combining lethal body burdens and a first-order bioconcentration model, LC~,,Smay be predicted for one species under different experimental conditions. Also, relationships between the exposure required to cause toxic effects in organisms and the exposure concentrations, can be predicted. In the present study the lethal body burden for triphenyltin chloride (TPTC) was investigated, by exposing guppies to several aqueous concentrations. The measured lethal body burdens were compared to calculated values, using first order uptake and elimination kinetics.
Organotin compounds have been widely introduced into the aquatic environment as biocides. For example tributyltin (TBT) is used in antifouling paints and triphenyltin (TPT) as a fungicide in agriculture (Bock, 1979). Therefore, many toxicity tests (LC~~S)have been carried out with organotins, using various aquatic species and experimental conditions. A wide range of ~q,,s for organotins has been published. For TPT LC~,,S range from 0.008 mg/l (96 hr) for the harpacticoid (Linden et al., 1979) to 0.4 mg/l for the mosquitofish (24 hr, LC,& (Schaefer et al., 1982) and bleak (96 hr) (Linden et al., 1979). For TBT LC~~S range from 0.0007 for the copepod (4 hr) (Hall and Pinkney, 1985), to 0.4 mg/l for the goldfish (24 hr) (Gras and Rioux, 1965). Most of the reported LC~~S were based on nominal concentrations in the water, although in some cases levels were verified by GC or AAS analysis. Limitations and assumptions in LC~,,testing have been reported previously (McCarty et af., 1985; Rand and Petrocelli, 1985; Van Hoogen and Opperhuizen, 1988). One of the primary assumptions in the use of ~q,, is that a steady-state between the aqueous concentration of the test compound and the concentration in the fish is obtained during the exposure period. However, the time required to obtain steadystate is dependent on the uptake and elimination rate constants (Banerjee et al., 1984). In some toxicity tests with organotins it is likely that steady-state is not reached between the aqueous concentration and the concentration in the fish. An example is TPT, since the elimination rate constant is very low (Tas et al., 1990). When steady-state is not reached, extrapolation from the aqueous concentration to the tissue concentration using only the bioconcentration factor is not valid. In this case, the LC, does not reflect the actual concentration in the organism causing the lethal effect. In fact, the LC~~ will probably be an overestimate of lethal internal concentrations which would be obtained at longer exposure times. An additional complicating factor is the different experimental conditions used to determine LC~~: CBP Iooc,l-2-a
THEORY
The uptake and elimination can be described by a first order exchange process (Branson et al., 1975): dG -=k,C,-k,C, dt 59
J. W. TASet al.
60
where k, and k, are the uptake and elimination rate constants and Cr and C, are the concentrations of the test compound in fish and water, respectively. According to the integrated equation 1, the relationship between LC,,, exposure time and lethal body concentrations may be expressed by (Neely, 1984): C
Lcso at time t = (k, ,k2) tldye _k2,).
(2)
Table I. Lethal body burdens of triphenyltin in WPPY CW (me/Q
I dead* (br)
C, dead (nmolk)
2 0.4 0.08
1.5 13 42
175 14 24k 13 25+ 13
MATERIALS
AND METHODS
Triphenyltin chloride (Merck) was recrystallized from toluene such that colorless crystals were obtained. It was coated on Chromosorb and solubilized in water with a generator column. This represented the highest undiluted concentration, To obtain lower concentrations, dilutions of 5 and 25 times were prepared. A one-liter aquarium was used. Guppies (5-7) ranging in weight from 115to 315 mg, with an average lipid concentration of 2.6 &- 1.2% were placed in it. The water, containing 2.0, 0.4, and 0.08 mg/l TPTC, in turn, was pumped through the aquarium at a flow rate of SO-100 ml/hr. Fish were exposed until death and the time required for this effect was recorded. Dead fish were removed from the aquarium, rinsed with water, blotted dry and weighed. Analysis of fish for TPTC was carried out according to Tas and Opperhuizen (submitted). The method consists of the following steps: single fish were homogenized with water, refluxed in a hexane: water (I : 1) mixture, and, after centrifugation, the hexane layer was separated and reduced in volume. The organotin in the hexane layer was methylated with methyl magnesium chloride (20% in THF, Merck), and a clean-up was carried out with a Florisil column. The organotin was detected with a gas chromatograph equipped with a flame photometric detector (GC-FPD). Triple water samples of the highest concentration (2 mg/l) were taken when the fish were placed into the aquarium, and at the time of death. These samples were extracted with hexane, methylated and detected with GC-FPD. RESULTS AND DISCUSSION
The highest TPTC concentration in the water was 2.06 and 1.97 mg/l after the fish died, values which were in good agreement with the expected levels, and indicated little loss of TPTC from the water column. Water samples of the other concentrations were not taken, but according to the dilution, they will be referred to as 0.4 and 0.08 mg/l TPTC. The fish exposed to 2,0.4 and 0.08 mg/l TPTC died after 1.5, between 8 and 18, and between 37 and 47 hr, respectively. All fish contained between 17 and 25 nmol/g TPTC (Table 1). It was found for triphenyltin hydroxide (TPTH) that the uptake and elimination rate constants, for guppies of approximately the same size, are approximately 50 ml/g.day and 0.014 day-‘, respectively (Tas et al., 1990). Using these values, together with the approximate aqueous concentrations and the time of death, an estimation of the lethal tissue concentration can be made (equation 2). According to this calculation, the fish should contain 24 + 5 nmol/g (Table l), which corresponds very well with the experimental value.
26 28 18
‘Average time, see text. tLcw at time f =
Equation 2 assumes that fish die when the lethal body concentration in the organism is reached, regardless of exposure time or exposure conditions.
Gt (nmolid
k, = 50 ml/g.day:
C,, dead (k,/k,)(l
-d2’)
k, = 0.014 day-‘:
f= t
dead;
LCSO=e,.
The lethal body burden for triphenyltin is two orders of magnitude lower than for narcotic compounds such as chlorinated aromatic hydrocarbons. It can be speculated that this is due to the higher reactivity of the organotin compound, which exhibits some specific toxicity. Acknowledgements-This
work was supported by the Institute for Inland Water Management and Waste Water Treatment, Ministry of Transport and Public Works, The Netherlands. A.O. would like to thank the Royal Dutch Academy of Sciences for their financial support. REFERENCES
Banerjee S., Sugatt R. H. and O’Grady D. P. (1984) A simple method for determining bioconcentration parameters of hydrophobic compounds. Environ. Sci. Technol. 18, 79-81.
Branson D. R., Blau G. E., Alexander H. C. and Neely W. B. (1975) Bioconcentration of 2,2’,4,4’-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test. Trans. Am. Fish Sot. 104, 7855792. Gras G. and Rioux J. A. (1965) Relation entre la structure chimique et l’activitt insecticide des composes organiques de l’itain (essai sur les larves de Culex pipiens L.) Arch. Inst. Pasteur Tunis 42, 9-22.
Hall L. W. Jr. and Pinkney A. E. (1985) Acute and sublethal effects of organotin compounds on aquatic biota: an interpretative literature evaluation. CRC Crit. Rev. Toxicol. 14, 159-209. Linden E., Bengtsson B. E., Svanberg 0. and Sundstrom G. (1979) The acute toxicity of 78 chemicals and pesticide formulations against two brackish water organisms, the bleak (Alburnis alburnis) and the harpacticoid Nitocra spinipes. Chemosphere, 11, 843-851. McCarty L. S., Hodson P. V., Craig G. R. and Kaiser K. L. E. (1985) The use of quantitative structure-activity relationships to predict the acute and chronic toxicities of organic chemicals to fish. Environ. Toxicol. Chem. 4, 595-606.
Neely W. B. (1984) An analysis of aquatic toxicity data: water solubility and acute LC~ fish data. Chemosphere 7, 813-819.
Rand G. and Petrocelli S. (Editors) (1985) Fundamentals of Aquatic Toxicology:
Methods and Applications. 666
p.
Hemisphere, Washington, DC. Schaefer C. H., Miura T., Dupras E. F., Jr. and Wilder W. H. (1982) Environmental impact of the fungicide triphenyltin hydroxide after application to rice fields. J. Econ. Entomol. 74, 597-600.
Tas J. W., Opperhuizen A. and Seinen W. (1990) Uptake and elimination kinetics of triphenyltin hydroxide by two fish species. Toxicol. Environ. Chem. 28, 129-141. Tas J. W. and Opperhuizen A. (1991) Analysis of triorganotin compounds in fish. Mar. Environ. Res. (Submitted). Van Hoogen G. and Opperhuizen A. (1988) Toxicokinetics of chlorobenzenes in fish. Environ. Toxicol. Chem. 7, 213-219.