Structure-activity relationships for diorganotins, chlorinated benzenes, and chlorinated anilines established with bluegill sunfish BF-2 cells

FUNDAMENTAL

AND

APPLIED

Structure-Activity and Chlorinated

TOXICOLOGY

l&295-30

1 ( 1988)

Relationships for Diorganotins, Chlorinated Benzenes, Anilines Established with Bluegill Sunfish BF-2 Cells H. BABICH*,~

AND E. BORENEREUND*

*LaboratoryAnimalResearch Center, The Rockefeller University, 1230 YorkAvenue, New York, New York 10021; and TDepatiment of Biological Sciences, Yeshiva University, Stern College, 245 Lexington Avenue, New York, New York 10016 Received April 27,1987; accepted October 30, I987 Structure-Activity Relationships for Diorganotins, Chlorinated Benzenes, and Chlorinated Anilines Established with Bluegill Sunfish BF-2 Cells. BABICH, H., AND BORE~UND, E. (1988). Fundam. Appl. Toxicol. 10,295-301. The bluegill sunfish (Lepomis macrochirus) BF-2 cell line, propagated at 34°C served as target for evaluation of the acute toxicities of various classes of aquatic pollutants, using the neutral red cytotoxicity assay.For a series of chlorinated benzenes and anilines, the sequence of cytotoxicity was dependent on the degree of chlorination and on their hydrophobicity, as described by their logarithmic octanol/water partition coefficients (log P values). With increasing numbers of chlorine atoms in the ring structure or with increasing log P values, greater cytotoxicity was observed. For a series of diorganotins, the se.quence of cytotoxicity was dependent on the length of the carbon chain and upon their hydraphobicity, as described by Hansch rr parameters. Thus, increasing the chain length or increasing the Hansch T parameter resulted in greater cytotoxicity. Similar structure-activity relationships for these classesof test agents have been previously established using acute toxicity LC50 assays with aquatic species. The ability of the neutral red in vitro cytotoxicity assay,with cultured fish cells as the bioindicators, to mimic the acute toxicity data obtained from the LC50 assayssuggests its utility as a tool for preliminary screening (tier I testing) of aquatic pollutants. Q 1988 Society of Toxicology.

In 1984 the National Academy of Sciences estimated that in the United States approximately 66,000 chemicals were in commercial use, with 1000 new chemicals per year being introduced into commerce (NRC, 1984). Many of these chemicals enter into aquatic environments, whether unintentionally (e.g., in seepage from uncontrolled hazardous waste sites) or intentionally (e.g., in effluents from wastewater treatment facilities), and evoke a deleterious impact on the indigenous biota. Ecotoxicological data, however, are only available for less than 1000 of these chemicals (Richards and Shieh, 1986), with chemical manufacturers being lax in providing ecotoxicology data to the U.S. Environmental Protection Agency when submitting premanufacture notices (PMNs) for newly synthesized chemicals (LaFlamme, 1984). Many biological testing methodologies have been developed to evaluate the potential 295

hazard of xenobiotics to the aquatic biota, with the 96-hr LC50 acute toxicity assay with fish being the most commonly used test. Because of the volume of chemicals that require ecotoxicity data, of the need for the rapid generation of ecotoxicity data (such as that following an accidental spill), of the economics of preliminary toxicity testing in vivo, and of socioethical concerns to reduce the number of animals, including fish (Douglas et al., 1986), in acute toxicity testing, several methodologies have been developed for predicting the acute toxicity of aquatic pollutants to fish. Such assays have included the bacterial Microtox test, which measures the inhibition of luminescence of Photobacterium phosphoreum (Hermens et al., 1985), the immobilization of the ciliate protozoan, Tetrahymena pyriformis (Schultz et al., 1986), and the formulation of computer-derived predictions based on structure-activity relationships 0272-0590188 $3.00 Copyright 0 1988 by tbe Society ofToxicology. All rights of reproduction in any form reserved.

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AND BORENFREUND

(SARs) (Lipnick et al., 1985; Veith et al., 1985). Another approach has been the utilization of established fish cell lines in in vitro cytotoxicity assays of aquatic contaminants (Babich and Borenfreund, 1987a; Bols et al., 1985; Kocan et al., 1979; Rachlin and Perlmutter, 1968). This manuscript evaluated the applicability of the neutral red in vitro cytotoxicity assay with bluegill sunfish (Lepomis macrochirus) BF-2 cells for establishing SAR models of various classes of aquatic pollutants. The agents tested included benzenes, anilines, and organotins. Benzene and its chlorinated derivatives are used as solvents, degreasers, and lubricants and in the manufacturing of dyestuffs and herbicides; anilines are used primarily in the synthesis of dyestuffs; and diorganotins are used as heat stabilizers in plastics and triorganotins are employed as biocides (Sittig, 198 1). Many of the chemicals enter into aquatic environments and therein pose a threat to the biota (e.g., see Carlson and Kosian, 1987; Thompson et al., 1985). Although the BF-2 cell line has been used in previous studies with the neutral red assay (Babich et al., 1986; Babich and Borenfreund, 1987b), these assays were performed at incubation temperatures of 25-26°C i.e., the temperature regime at which optimal replication was reported to occur (see Wolf and Mann, 1980, for review). However, as noted by Kocan et al. ( 198 1) and later by Babich and Borenfreund (1987c), the BF-2 cell line apparently has adapted to tissue culture conditions, with optimal temperature for cell replication at 33-34°C. Furthermore, at this higher temperature, the BF-2 cell line exhibited enhanced sensitivity to chemical toxicants. Another aspect of this research was the evaluation of the use of the BF-2 cell line in the neutral red assay, now performed at 34°C. for the assessments of chemical toxicities, MATERIALS

AND

METHODS

Cell culture. The bluegill sunfish BF-2 cell line, obtained from the American Type Culture Collection, consists of

fibroblasts derived from the caudal trunk of fingerlings. The cells were confirmed to be free of mycoplasma by the Hoechst staining technique. Stock cultures were maintained in a humidified. 5.5% CO> atmosphere at 34°C in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 PgJrnl streptomycin, and 1.25 &ml amphotericin B. Cell cycle time was determined to be about 24 hr. For propagation, the cells were dissociated with a solution consisting of 0.8 g NaCI, 0.058 g NaHCO,, 0.04 g KCI, 0.1 g glucose, 0.05 g trypsin, and 0.02 g EDTA in 100 ml of deionized, distilled water. Neutral red assay. The neutral red assay, initially developed for use with mammalian cells (Borenfreund and Puemer, 1984) has been adapted for use with cultured fish cells. The principles of this assay, which are based on lysosomal membrane damage, have been previously described (Borenfreund and Puemer. 1985). Individual wells of a 96-well tissue culture microtiter plate were inoculated with 0.2 ml DMEM containing 2 X lo4 BF-2 cells. The plate was incubated at 34°C for 24 hr, after which the medium was removed, and the cells were refed with medium unamended (control) or amended with varied concentrations of test agents. Either 4 or 8 wells were used per concentration of test agent. After an additional 24 hr of incubation at 34°C the medium was removed and replaced with 0.2 ml DMEM containing 50 pg/ml neutral red. The neutral red-containing medium had been preincubated overnight at 37°C and centrifuged prior to use to remove fine precipitates of dye crystals. The plate was returned to the incubator for another 3 hr to allow for uptake of the vital dye into the lysosomes of viable, uninjured cells. Thereafter, the medium was removed and the cells were washed quickly with a fixative ( 1% formaldehyde- 1% CaClJ and then 0.2 ml of a solution of 1% acetic acid-50% ethanol was added to each well to extract the dye. After an additional 10 min at room temperature and rapid agitation on a microtiter plate shaker, the plate was transferred to a microtiter plate reader to measure absorbance of the extracted dye at 540 nm. Quantitation of the extracted dye has been shown to be linear with cell numbers, both by direct cell counts and by protein determination of cell populations. All experiments were performed at least five times and the relative cytotoxicities of the test agents were compared to control cultures by computing the concentration needed to reduce absorbance by 50%. using linear regression analysis. Such midpoint cytotoxicity determinations were designated NR50 values. Data for the doseresponse cytotoxicity curve were presented as the arithmetic mean + standard error of the mean. Tea agents. All test agents were dissolved in methanol, except for SnCh. 5HrO which was dissolved in ethanol. The solvents were used at a final concentration below their cytotoxic levels.

RESULTS Figure 1 demonstrates the types of doseresponse cytotoxicity curves that can be gen-

SARs ESTABLISHED

297

IN VITRO TABLE 1 MIDPOINT

SllCI~

CYTOTOXKITY (NR50) VALUES FOR A SERIES OF BUTYLATED TINS

PPi-

Tin compound

NR50 (mM)n

log Pb

SnCh BuSnCl, Bu2SnClz Bu,SnCl Bu$n

5.19 0.078 O.OcO83 0.00055 0.0101

0.35 1.49 2.60 3.90

60

\ 40

Bluegill

20

01111111111 0.1

I

IO

Concentration

loo

~poo

BF-2

l0,000

(pM)

FIG. 1. Comparative in vitro cytotoxicities of a series of butylated tins toward bluegill sunfish BF-2 cells. The data are expressed as the arithmetic means + SEM. SnQ, tin chloride; BuSnCls, butyltin trichloride; Bu2SnClz, dibutyltin dichloride; Bu,SnCl, tributyltin chloride, Bu,Sn, tetrabutyltin.

erated with the neutral red assay and shows the relative cytotoxicities to bluegill BF-2 cells of a series of butylated tins. The sequence of potency for this series was tributyltin chloride > dibutyltin dichloride > tetrabutyltin > butyltin trichloride > tin(W) chloride. The order of toxicity for this series of butylated tins did not appear to be a function of the degree of butylation or of the lipophilicity, as described by logarithmic octanol/water partition coefficients (log P), of the organotin molecules. Thus, tetrabutyltin, with a 1ogPvalue of 3.90, was less potent than tributyltin chloride and dibutyltin dichloride, with log P values of 2.60 and 1.49, respectively (Table 1). For a series of diorganotin salts, the sequence of cytotoxicity to bluegill BF-2 cells was dicyclohexyltin > dibutyltin > diphenyltin > dipropyltin > diethyltin > dimethyltin. For the alkyltins within this series, the longer the carbon chain length, the greater the cytotoxicity of the diorganotin. As log P values were not available for all the diorganotins that were tested, Hansch ?rparameters, which provide an index of the hydrophobicity of the organic ligands of the diorganotins, were used to establish a relationship between cytotoxicity and lipophilicity (Table 2). As noted in

’ Midpoint cytotoxicity, based on the 24hr neutral red WY. b Logarithmic octanol/water partition coefficient (Vighi and Calamari, 1985).

Fig. 2, there was a direct linear relationship between cytotoxicity and lipophilicity of the diorganotins, with the correlation coefficient being 0.958. The midpoint cytotoxicity values (i.e., NRSOs) for a series of chlorinated benzenes and anilines toward the BF-2 cells are presented in Table 3, which also lists the log P values for these test agents. The parent benzene molecule was the least cytotoxic, with potency increasing with the progressive incorporation of chlorine atoms into the aromatic ring structure. The sequence of cytotoxicity to the BF-2 cells was tetrachloro-

TABLE 2 MIDPOINT

CYTOTOXKTN (NR50) VALUES FOR A SERIES OF DIORGANOTINS

@MY

log Pb

Hansch ?r parameter’

39.89 17.10 11.58 1.22 0.83 0.25

-3.10 -1.40 1.90 1.49 -

0.56 1.02 1.55 1.96 2.13 2.51

NR50

Tin salt Dimethyltin Diethyltin Dipropyltin Diphenyltin Dibutyltin Dicyclohexyltin

a Midpoint cytotoxicity, based on the 24-hr neutral red ==Y. b Logarithmic octanol/water partition coefficient. ’ Hydrophobicity of the organic ligand attached to the tin (Laughlin et al., 1985).

298

BABICH AND BORENFREUND

Bluegill

BF-2

Diorganotins

-1

1

0 Hansch

2 7T

3

parameter

3luegill

FIG. 2. Midpoint cytotoxicity (NRSO) values for a series of diorganotins toward bluegill BF-2 cells as a function of their Hansch z parameters. Me*, dimethyltin dichloride; Etz, diethyltin dichloride; Prz, dipropyltin dichloride; BUM, dibutyltin dichloride; Phz, diphenyltin dichloride; cHexz, dicyclohexyltin dibromide.

> trichloro> dichloro- > monochloro> benzene. The presence of a chlorine atom was a more crucial determinant of cytotoxicity than was its ring position. Thus, whereas the trichlorobenzenes were more cytotoxic TABLE 3 MIDWINT CYTOTOXICITY (NR50) VALUES FOR A SERIES OF CHLORINATED BENZENES AND ANILINES

Test agent

NRSO (mM)’

log Pb

Aniline series Aniline 4-Chloroaniline 3,5-Dichloroaniline 2,4,6-Trichloroaniline

16.48 1.90 0.65 0.5 1

0.90 2.02 2.90 3.41

Benzene series Benzene Monochlorobenzene 1,2-Dichlorobenzene 1,3Dichlorobenzene I ,2,3-Trichlorobenzene 1,2,4-Trichlorobenzene 1,3,5-Trichlorobenzene 1,2,3,4-Tetrachlorobenzene

19.46 5.69 1.53 1.37 0.77 0.64 0.63 0.34

2.13 2.81 3.53 3.53 4.20 4.20 4.20 4.94

d Midpoint cytotoxicity, based on the 24-hr neutral red assay. b Logarithmic octanol/water partition coefficient for anilines (Hermens et al., 1984a) and benzenes (Hermens et al., 1984b).

Sunfish

BF-2

I

I

I

I

I

I

2

3

4

5

Log

P

FIG. 3. Midpoint cytotoxicity (NR50) values ofa series ofchlorinated benzenes and anilines towards bluegill BF2 cells as a function of their logarithmic octanol/water partition coefficients (log P). A, aniline; 4-CA, 4-chloroaniline; 3,5-CA, 3,5-dichloroaniline; 2,4,6-TCA, 2,4,6trichloroaniline; B, benzene; MCB, monochlorobenzene; 1,2-DCB, 1,2-dichlorobenzene; 1,3-DCB, 1.3dichlorobenzene; 1,2,3-TCB, 1,2,3-trichlorobenzene; 1,2,4-TCB, 1,2,4-trichlorobenzene; 1,3,5-TCB, 1,3,5trichlorobenzene; 1,2,3,4-TCB, 1,2,3,4-tetrachloroben-

than the dichlorobenzenes, the cytotoxicities of 1,2- and 1,3-dichlorobenzene were approximately equivalent, as were those of 1,2,3-, 1,2,4-, and 1,3,Michlorobenzene. The sequence of cytotoxicity to the BF-2 cells for the chlorinated benzenes and anilines was a function of the lipophilicity of the molecules, as described by their log P values. Figure 3 illustrates the linear relationship between cytotoxicities of the chlorinated benzenes and anilines and their log P values, with the negative slopes of the lines indicating that the higher the lipophilicity, the lower the NR50 value and the higher the cytotoxicity. The correlation coefficients were 0.985 and 0.965 for the chlorinated benzene and aniline series, respectively. DISCUSSION The test agents studied herein were selected because of the availability of data on their

SARs ESTABLISHED

acute in vivo toxicities, thereby permitting comparisons between the in vitro cy-totoxicity data generated for the bluegill sunfish BF-2 cells and in vivo acute toxicity data from the literature. The same sequence of the cytotoxicity of the butylated tins (i.e., Bu$nCl > Bu2SnClz > Bu$n > BuSnC& > SnCL+) observed in studies with the BF-2 cells has also been observed for the induction of nonenergy-dependent swelling of isolated rat liver mitochondria (Wulf and Byington, 1975), for the inhibition of bacterial dehydrogenase activity (Liu and Thomson, 1986), and for the 24-hr LC50 acute toxicity to Duphnia magna (Vighi and Calamari, 1985). The lack of correlation between either the degree of butylation or the lipophilicity of the molecules and their sequence of toxicity may be indicative of their differential mechanisms of acute toxicity. Thus, the acute toxicity of triorganotins is due to their inhibition of mitochondrial oxidative phosphorylation, whereas that of the diorganotins is due to their inhibition of the oxidation of cY-keto acids (Smith, 1978). The sequence of cytotoxicity for the diorganotins established with the bluegill BF-2 cells has been demonstrated in similar in vitro cytotoxicity studies with mammalian cell lines, i.e., BALB/c mouse 3T3 fibroblasts and mouse neuroblastoma Nza cells (Borenfreund and Babich, 1987). A similar sequence of potency for the diorganotins was demonstrated in vivo in 12&y LC50 toxicity tests with zoeae of the estuarine mud crab, Rhithropanopeus harrissi (Laughlin et al., 1985), and in 24-hr toxicity assays with D. magna (Vighi and Calamari, 1985) with in vivo toxicity being positively correlated with lipophilicity of the diorganotins, as described by their Hansch ?r parameters and their log P values, respectively. A similar correlation between cytotoxicity and lipophilicity was demonstrated herein with the BF-2 cells, as well as with the above-mentioned mammalian cell lines (Borenfreund and Babich, 1987). In previous studies with the bluegill BF-2 cells (Babich et al., 1986) and BALB/c mouse 3T3 (Borenfreund and Puemer, 1986) cells, the sequence of cytotoxicity for a series of diva-

IN VITRO

299

lent cationic metals was positively correlated with their chemical softness op parameters; similar correlations were attained with acute toxicity in vivo studies (Williams et al., 1982). The sequence of acute toxicities in vivo, based on LCSO assays, for a series of chlorinated benzenes to guppies (Konemann, 1981), fathead minnows (Hall et al., 1984; Veith et al., 1983), D. magna (Hermens et al., 1984a), and freshwater algae (Wong et al., 1984) and of chlorinated anilines to guppies (Hermens et al., 1984b) were functions of the degree of chlorination and of the lipophilicity of the molecules. Similar findings were demonstrated in this study using cultures of bluegill sunfish BF-2 cells as the bioindicators. Furthermore as noted by Hermens et al. ( 1984a,b) in the acute toxicity of LC50 studies with guppies, the chlorinated benzene and chlorinated aniline series should be classified as two distinct groupings, as the incorporation of an amino-moiety into the benzene ring apparently enhanced the toxicity of the aniline molecules. Similar observations were noted herein with the BF-2 cells. Thus, for molecules of comparable chlorination, aniline was more cytotoxic than benzene, 4chloroaniline more than monochlorobenzene, 3,5-dichloroaniline more than the dichlorobenzenes, and 2,4,6-trichloroaniline more than the trichlorobenzenes. Similar distinctions were noted when the comparisons were between molecules of similar lipophilicity. Thus, for example, 1,3-dichlorobenzene with a log Pvalue of 3.53 had an NR50 value of 1.37 mM, whereas 2,4,6-trichloroaniline with a slightly lower log P value of 3.41 and an NRSO value of 0.5 1 mM. In previous studies using either the bluegill BF-2 (Babich and Borenfreund, 1987b) or the mouse 3T3 (Babich and Borenfreund, 1987d) cells as the bioindicators with the neutral red assay, it was demonstrated that in vitro cytotoxicities of a series of chlorinated phenolics and toluenes were a function of their degree of chlorination and their lipophilicity. Similar SARs between acute toxicity in vivo and these physiochemical parameters of the phenolics and toluenes have been

300

BABICH AND BORENFREUND

established (Konemann, 198 1; Konemann and Munsch, 198 1; Lipnick et al., 1985). The studies presented herein with the neutral red assay using BF-2 cells as the bioindicaters, in conjunction with the previously mentioned studies (Babich et al., 1986; Babich and Borenfreund, 1987b-d), strongly suggest the utility of such an assay as a preliminary screening tool for assessments of chemical risks to aquatic environments. The vast number of chemicals that are already in commercial use (but, without preliminary ecotoxicity data), coupled with the numerous newly synthesized chemicals each year, suggests the applicability of the neutral red assay with BF2 cells for tier I testing of acute chemical toxicity. Such initial studies can be useful as preliminary range-finding screenings of test agents, in setting priorities for those chemicals that require further testing in vim, and for the prediction of acute toxicity based on SARs or on rankings of chemicals according to their acute toxicities. ACKNOWLEDGMENTS

BABICH, H., PUERNER, J. A.. AND BORENFREUND, E. (1986). In vitro cytotoxicity of metals to bluegill (BF2) cells. Arch. Environ. Contam. Toxicol. l&31-37. BOLS, N. C.. BOLISKA, S. A.. DIXON, D. G., HODSON, P. V., AND KAISER, K. L. E. ( 1985). The use of fish cell cultures as an indication of contaminant toxicity to fish. Aquat. Toxicol. 6, 147-15.5. BORENFREUND,E., AND BABICH H. (I 987). In vitrocytotoxicity of heavy metals, acrylamide, and organotin salts to neural cells and fibroblasts. Cell Biol. Toxicol. 3,63-73.

BORENFREUND, E., AND PUERNER, J. A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays(HTD/NR-90). J. Tissue Cult. Methods 9,7-9. BORENFREUND, E., AND PUERNER, J. A. (1985). Toxicity determined in vitro by morphological alteration and neutral red absorption. To,xicol. Lett. 24, 119124.

BORENFREUND, E., AND PUERNER, J. A. (1986). Cytotoxicity of metals, metal-metal and metal-chelator combinations assayed in vitro. Toxicology 39, I2 I 134.

CARLSON, A. R., AND KOSIAN, P. A. (1987). Toxicity of chlorinated benzenes to fathead minnows (Pimephales promelas). Arch. Environ. Contam. Toxicol. 16, 129135. DOUGLAS, M. T., CHANTER, D. 0.. PELL, I. B., AND BURNEY, G. M. (1986). A proposal for the reduction of animal numbers required for the acute toxicity to fish test (LC50 determination). .4quat. Toxicol. 8,243249.

Gratitude is expressed to Dr. Frederick Brinckman, U.S. Department of Commerce, for supplying the tin compounds and the U.S. EPA Repository for Toxic and Hazardous Materials, Cinncinati, Ohio, for supplying many of the chlorinated benzenes and anilines. Portions ofthis research were supported, in part, by Grant 8 13760 from the U.S. Environmental Protection Agency. The views expressed are not necessarily those of the Agency.

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BABICH, H., AND BORENFREUND,E. (1987d). Structureactivity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxicol. In Vitro 1,3-9.

HALL, L. H., KIER, L. B.. AND PHIPPS,G. (1984). Structure-activity relationship studies on the toxicities of benzene derivatives. I. An additivity model. Environ. To.xicol. Chem. 3,355-365. HERMENS, J., BUSSER, F., LEEUWANGH, P., AND MUSCH, A. (1985). Quantitative structure-activity relationships and mixture toxicity of organic chemicals in Photobacterium phosphoreum: The Microtox test. Ecotoxicol. Environ. Saf 9, 17-25. HERMENS, J., CANTON, H., JANSSEN, P., AND DE JONG, R. (I 984a). Quantitative structure-activity relationships and toxicity studies of mixtures ofchemicals with anaesthetic potency: Acute lethal and sublethal toxicity to Daphnia magna. Aquat. Toxicol. 5, 143- 154. HERMENS, J., LEEUWANGH, P.. AND MUSCH, A. (1984b). Quantitative structure-activity relationships and mixture toxicity studies of chloro- and alkyl anilines at an acute lethal toxicity level to the guppy (Poecilia reticulata). Ecotoxicol. Environ. Saf 8,388-394. KOCAN, R. M., LANDOLT, M. L., BOND, J., AND BENDITT, E. P. ( I98 I). In vitro effect of some mutagens/ carcinogens on cultured fish cells. .4rch. Environ. Contam. Toxicol. 10,663-67 1. KOCAN, R. M., LANDOLT, M. L., AND SABO, K. M. ( 1979). In vitro toxicity of eight mutagens/carcinogens for three fish cell lines. Bull. Environ. Contam. Tox-icol. 23,269-274.

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SMITH, P. J. (1978). Structure/Activity Relationships for Di- and Tri-Organotin Compounds. International Tin Research Institute, Middlesex, England. THOMPSON, J. A. J., SHEFFER, M. G., PIERCE, R. C., CHAU, Y. K., COONEY, J. J., CULLEN, W. R., AND MAGUIRE, R. J. (1985). Organotin Compounds in the Aquatic Environment. National Research Council Canada, Ottawa, Canada. VEITH, G. D., CALL, D. J., AND BROOKE, L. T. (1983). Structure-activity relationships for the fathead minnow, Pimphales promelas: Narcotic industrial chemicals. Canad. J. Fish. Aquat. Sci. 40,743-748. VEITH, G. D., DEFOE, D., AND KNUTH, M. (1985). Structure-activity relationships for screening organic chemicals for potential ecotoxicity effects.DrugMetabol. Rev. l&1295-1303. VIGHI, M., AND CALAMARI, D. (1985). QSARs for orgynotin compounds on Daphnia magna. Chemosphere 14,1925-1932.

WALSH, G. E., MCLAUGHLIN, L. L., LORES, E. M., LQUIE, M. K., AND DEANS, C. H. (1985). Effects of organotins on growth and survival of two marine diatoms, Skeletonema costatum and Thalassiosira pseudonana. Chemosphere 14,383-392. WILLIAMS, M. W., HOESCHELE, J. D., TURNER, J. E., JACOBSON, K. B., CHRISTIE, N. T., PATON, C. L., SMITH, L. H., WITSCHI, H. R., AND LEE, E. H. (1982). Chemical softnessand acute metal toxicity in mice and Drosophila. Toxicol. Appl. Pharmacol. 63,46 l-469. WOLF, K., AND MANN, J. A. (1980). Poikilotherm veterbrate cell lines and viruses: A current listing for fishes. In Vitro 16, 168-179. WONG, P. T. S., CHAU, Y. K., KRAMAR, O., AND BENGERT, G. A. (1982). Structure-activity relationship of tin compounds on algae. Canad. J. Fish. Aquat. Sci. 39,482-488.

WONG, P. T. S., CHAU, Y. K., RHAMEY, J. S., AND DOCKER, M. (1984). Relationship between water solubility of chlorobenzenes and their effectson a freshwater green alga. Chemosphere 13,99 l-996. WULF, R. G., AND BYINGTON, K. H. ( 1975). On the structure activity relationships and mechanism of organotin induced, nonenergy dependent swelling of liver mitochondria. Arch. B&hem. Biophys. 167, 176-185.