Fathead minnow FHM cells for use in in vitro cytotoxicity assays of aquatic pollutants

ECOTOXICOLOGY

AND ENVIRONMENTAL

SAFETY

14,78-87 (1987)

Fathead Minnow FHM Cells for Use in in Vitro Cytotoxicity Assays of Aquatic Pollutants H. BABICH AND E. BORENFREUND LaboratoryAnimal ResearchCenter,TheRockefellerUniversity, 1230 YorkAvenue,New York,New York, 100214399 ReceivedFebruary 9.1987 The suitability ofthe fathead minnow (FHM) epithelial cell line for use as the target (indicator) system in in vitro cytotoxicity assays was evaluated using several endpoints. The organometal diethyltin dichloride served as the representative test agent. The concentration of diethyltin dichloride which resulted in a midpoint toxicity was 3.5 &4 in a 3day cell growth assay, 3.8 Min the 24-hr neutral red assay, and 16.5 r&fin a 4-hr cell detachment assay.The neutral red assay was used to compare the relative sensitivities of the FHM cells (exposed at 34’C) with those of bluegill sunfish (BF-2) cells, a fibroblastic cell culture (exposed at 26’C), in the presence of different classes of test agents frequently occurring as aquatic pollutants. For both fish species the sequence of potencies of the test agents was in the order of organometals > pesticides N polychlorinated biphenyls > polynuclear aromatic hydrocarbons z phenolics. Overall, the FHM cells were more sensitive than were the BF-2 cells. However, there was a better correlation between the in vitro cytotoxicity data for the BF-2 cell culture and LCW data for bluegill sun6s.h than between similar data for the FHM cell line and fathead minnows. Q 1987 Academic PIES, 1~.

INTRODUCTION Although the need for adequate testing of chemicals is recognized, the high costs of such tests-especially those using animal models, the need for the rapid generation of data, and the volume of chemicals that require toxicological testing-have directed much effort to develop short-term in vitro assays. One such approach has been the designing of in vitro cytotoxicity tests using cultured mammalian cells to assay the acute toxicities of those chemicals of concern to human health. The use of short-term cytotoxicity assays for the initial screening of chemicals not only aids in establishing priorities for the selection of chemicals that should be tested further in vivo, but also accelerates the time in which potential toxicants can be evaluated. Rachlin and Perlmutter (1968) were the investigators who initially suggested the use of cultured fish cells in cytotoxicity assays for the screening of the relative acute toxicities to fish of aquatic pollutants. Later studies by Kocan et al. (1979), Marion and Denizeau (1983a,b), and Bols et al. (1985) using RTG-2 cells, a fibroblastic cell line derived from rainbow trout, and by Babich et al. ( 1986a,b) and Babich and Borenfreund ( 1987a,b) using BF-2 cells, a fibroblastic cell line derived from bluegill sunfish, have further developed and applied in vitro cytotoxicity assays with varied endpoints for assessments of the acute toxicities of aquatic pollutants to fish. With fish exposed to water-borne toxicants, the epithelial cells of the gill tissue are the initial cell types encountered by chemical contaminants in the aquatic environment. The studies reported herein utilized FHM cells, which are an epithelioid cell line derived from the skin of fathead minnows (Pimephales promelas), for cytotoxic0147-6513187 $3.00 Copyright 0 1987 by Academic F’ms, Inc. AU rigb of mpmduction in any form reservd

78

FHM CELL LINE: CYTOTOXICITY

ASSAY

79

ity assays. There is only limited information on the suitability of this cell line for use in in vitro cytotoxicity assays, with Rachlin and Perlmutter (1968) and Boume and Jones (1973) using the reduction in the mitotic index as the cytotoxicity endpoint. However, the use of such an endpoint is tedious and time-consuming, as it requires microscopic enumeration of cells in mitosis versus the total cell population and is, therefore, not suitable for screening large numbers of test agents. MATERIALS

AND

METHODS

Cell Lines The fathead minnow FHM (American Type Culture Collection, CCL 42) and the bluegill sunfish BF-2 (ATCC, CCL 91) cell lines were maintained in a humidified, 5.5% COz atmosphere in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin G, 100 Ilg/ml streptomycin, and 1.25 &ml fungizone. Unless otherwise stated, all incubation steps were at 34°C for the FHM and at 26°C for the BF-2 cells. Solutions and Test Agents The FHM cells were dissociated with a solution consisting of 0.05 g trypsin and 0.02 g EDTA in 100 ml phosphate-buffered saline (PBS) and the BF-2 cells with a solution containing 0.8 g NaCl, 0.058 g NaHCOS, 0.04 g KCl, 0.1 g dextrose, 0.05 g trypsin, and 0.02 g EDTA in 100 ml of deionized, distilled water. Stock solutions of test agents were prepared in methanol, but those of methylmercuric chloride in DMSO. Temperature-Replication

Study

A quantity of 1 X lo5 FHM or BF-2 cells was seeded into 35-mm tissue culture dishes containing 2 ml of complete DMEM and incubated at either 26 or 34°C for up to 4 days. At daily intervals, four dishes were sacrificed and the cells were counted with a hemacytometer. Cytotoxicity Assays (a) Neutral red assay. The neutral red (NR) cytotoxicity assay quantifies the number of viable, uninjured cells after their exposure to toxicants and is based on the uptake and subsequent lysosomal accumulation of the supravital dye, neutral red (Borenfreund and Puemer, 1984). Individual wells of a 96-well tissue culture microtiter plate were inoculated with 0.2 ml complete medium containing 2 X lo4 FHM or BF-2 cells and incubated for 24 hr. The medium was then removed and the celIs were refed with medium unamended (control) or amended with varied concentrations of test agent. Alter another 24 hr of incubation, the medium was removed and replaced with medium containing 50 &ml NR. The NR-containing medium had been preincubated overnight at 37°C and was centrifuged prior to use to remove fine precipitates of dye crystals. The microtiter plate was returned to the incubator for an additional 3 hr to allow for the uptake of the dye into the lysosomes of viable, uninjured cells. Thereafter, the medium was removed and the cells were quickly washed with 0.2 ml of a fixative ( 1% formaldehyde- 1% CaCl*) and then 0.2 ml of a solution of 1% acetic

80

BABICH

AND BORENFREIJND

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 microplate reader equipped with a 540-nm filter to measure ab sorbance of the extracted dye. (b) Cell protein assay. The cell protein assay, adapted from Shopsis and Eng ( 1985), is a measurement of the total cellular protein after incubation in medium containing toxicants. FHM cells (9 X 103) suspended in 0.2 ml of complete medium were inoculated into wells of a 96-well microtiter plate and incubated for 24 hr, after which the medium was replaced with fresh medium without/with test agents. After 3 days of growth at 34”C, the medium was removed and the cells were washed three times with PBS and lysed with 50 ~1 of 0.1 N NaOH. The plate was then incubated for 3 hr at 37°C and, thereafter, 0.2 ml of 1:3 Coomassie bluedistilled water solution was added to each well. After an additional 20 min of incubation at room temperature, absorbance was measured with a microplate reader, using 405- and 630-nm filters for the reference and absorption wavelengths, respectively. Serial dilutions of 1 to 10 &well bovine serum albumin dissolved in 0.1 N NaOH were used for the protein standard. (c) Cell detachmentassay.The cell detachment assay is based on the detachment of cells from a substratum upon their incubation in toxicant-amended medium (Reinhardt et al., 1985). Individual wells of a 96-well microtiter plate were inoculated with 1 X lo4 FHM cells and incubated overnight at 34”C, after which the medium was replaced with fresh medium without/with toxic agents. After 4 hr of incubation at 34”C, the medium was removed, the cells were washed three times with PBS, and total cellular protein was determined as described above.

Statistics All experiments were performed at least four times, with from four to eight wells per concentration of test agent. Midpoint toxicity values were computed by linear regression of the data, presented as percentage of control. The data were presented graphically as the arithmetic means + SE. RESULTS The FHM cells, derived from the skin, are epithelioid (Fig. 1). As the reduction in mitotic index was the only in vitro cytotoxicity assay that has previously been used with FHM cells (Rachlin and Perlmutter, 1968; Boume and Jones, 1973), initial studies were performed to evaluate the response of FHM cells in other in vitro cytotoxicity assays. Diethyltin dichloride was used as the representative test agent, in light of the current concern of aquatic contamination by organotins (Thompson et al., 1985). The concentration of diethyltin dichloride causing midpoint cytotoxicity after a 24hr exposure was 3.8 j&in the NR assay (Fig. 2). In a 3&y cell protein assay and a 4hr cell detachment assay, the midpoint toxicity value was 3.5 and 16.5 adiethyltin dichloride, respectively (Fig. 2). Using the NR assay, studies then evaluated the comparative sensitivities of the FHM and bluegill sunfish BF-2 (a fibroblastic cell line (Fig. 3)) cells to several classes of environmental toxicants. Exposures for the FHM cells were at 34°C and for the BF-2 at 26”C, i.e., the temperatures recommended for optimal growth conditions (see review by Wolf and Mann, 1980). For both cell types, the order of toxicity for the

FHM CELL LINE: CYTOTOXICITY

ASSAY

FIG. 1. FHM cells, derived from the skin of fathead minnows, were propagated at 34°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Fixed in methanol and stained with Giemsa. X260.

different classes of test agents was in the order of organometals > chlorinated pesticides N polychlorinated biphenyls > polynuclear aromatic hydrocarbons > phenolies. The correlation coefficient was 0.988 when the 22 test agents were compared by ranking their potencies (NR50 values, in a) to the FHM and BF-2 cell lines. However, for each class of test agent, the FHM cells were more sensitive than the BF-2 cells (Table 1). As the temperature of exposure to toxicants influences the in vivo response of fish (Macek et al., 1969), studies were conducted to compare the relative sensitivities of the FHM and BF-2 cells when incubated at similar temperatures. Preliminary studies showed that both cell types replicate at 26 and 34”C, with the doubling time for the FHM cells being approximately 2 days at 26°C and 1.5 days at 34°C and for the BF2 cells being approximately 2 days and 1 day at 26 and 34”C, respectively (Fig. 4). This enhanced effect on replication of the BF-2 cells when at 34’C was not expected, as the optimal growth temperature for the BF-2 cells has been reported at 25°C (see the review by Wolf and Mann, 1980). Both the FHM and BF-2 cells were more sensitive to diethyltin dichloride and 4,4’DDE when exposed at 34°C rather than at 26°C (Figs. 5 and 6). The NRH, value (midpoint toxicity in the NR assay) was 70.8 and 17.0 @ diethyltin dichloride for the BF-2 cells exposed at 26 and 34’C, respectively, and was 7.3 and 3.8 adiethyltin dichloride for the FHM cells at 26 and 34”C, respectively. With 4,4’-DDE, the NRSo value, in PAI, was 240.1 and 142.6 for the BF-2 cells exposed at 26 and 34°C respectively and was 157.8 and 96.9 for the FHM cells at 26 and 34°C. Although the FHM

82

BABICH

AND BORENFREUND

Fathead Minnow

FHM:34T

4-t-k Detachment Assay

72-Hr

a

0

2

4

6

8

IO

0 Diethyltm

IO dichloride

Cell Protein

20 ipM)

30

40

5 1

FIG. 2. In vitro cytotoxicity of diethyltin dichloride to FHM cells as determined in the 24-hr neutral red assay, the 4-hr cell detachment assay, and the 724~ cell protein assay. The data are expressed as the arithmetic mean percentage of control k SE.

cells retained their greater sensitivity to the test agents compared to the BF-2 cells, these differences in sensitivity were lessened when compared at equivalent temperatures of exposure.

FIG. 3. BF-2 cells, derived from the caudal trunk of bluegill sunfish fingerlings, were propagated at 26% in Dulbecco’s modified Eagle’s medium supplemented with 10% fe.tal calf serum. Fixed in methanol and stained with Giemsa. X200.

83

FHM CELL LINE: CYTOTOXICITY ASSAY TABLE 1 COMPARATIVE

in V’ifro CYTOTOXICITIES, AS DETERMINED BY THE NEUTRAL

REDASSAY,OFCHEMICALTFSTAGENTSTO BLUEGILL SUNFISH BF-2 AND TO FATHEAD MINNOW FHM CELLS

Test agent

BF-2 (26°C)

Organometal Methylmercuric chloride Diethyltin dichloride Dimethyltin dichloride Chlorinated pesticide Chlordane Heptachlor Aldrin

4,4’-DDD 4,4’-DDT 4,4’-DDE Polychlorinated biphenyl* Aroclor 1254 Aroclor 1260 Aroclor 1242 Aroclor 1232 Polynuclear aromatic hydrocarbon Acenaphthylene 3-Methylcholanthrene Benzo[a]pyrene Phenolic 2,4,6-Trichlorophenol 2,CDichlorophenol 2Chlorophenol 2,CDimethylphenol 3-Methylphenol (m-cresol) Phenol

FHM (34°C)

4.2 70.8 368.0

2.7 3.8 29.8

82.0 125.7 192.4 199.0 234.2 240.1

56.4 67.8 66.7 71.6 96.9

138.8 167.7 177.4 268.3

89.9 109.1 124.9 155.7

242.6 344.3’ 364.5’

142.9 164.2 182.5

480.0 5 10.0 2390.0 2390.0 5250.0 6900.0

730.0 450.0

55.1

1130.0

1800.0 2890.0 4200.0

LINRSo = midpoint cytotoxicity value in the netural red assay, as compared to untreated control cultures. *Average molecularweightsfor Aroclor 1232,1242,1254, and 1260 are221,261,327, and 372 (Safe, 1984). ’ Extrapolated value.

The degree to which the in vitro cytotoxicity data for the FHM cells were reflective of the in vivo response of fathead minnows to acute exposures with similar toxicants was evaluated only for the phenolics and pesticides, as there was either no or only insufficient 96-hr LCSOdata in the literature on the response of fathead minnows to organometals (EPA, 198Ob), polynuclear aromatic hydrocarbons (EPA, 198Oc), and polychlorinated biphenyls (EPA, 1980d) to serve for comparisons. Similar correlations are also presented for the BF-2 in vitro data and LCSOdata for bluegill sunfish. Because of the mediating influence of temperature (Macek et al., 1969) and biotic factors (e.g., the stage of development of the test species (Nebeker et al., 1974)) on the 96-hr LCSOresponse of fish, the in vivo data that are presented in Table. 2 are, for the most part, from investigations that were performed under equivalent exposure

84

BABICH

AND BORENFREUND

i-’ Bluegill Sunfish BF-2

34T

26T

0123401234

Days

FIG. 4. Influence of temperature on the replication of FHM and BF-2 cells. The data are expressed as the arithmetic means k SE.

conditions with both species of fish. There was a better correlation between the in vitro/in vivo data for the BF-2/bluegill sunfish comparison than for the FHM/fathead minnow comparison. The correlation coefficient was 0.988 for the comparison between the in vitro toxicities of the pesticides and phenolics to the BF-2 cells with their in vivo acute toxicities to bluegill sunfish, a similar comparison for the FHM in vitro data with the fathead minnow in vivo data yielded a correlation coefficient of 0.835. DISCUSSION The objectives of this research were to evaluate the suitability of the FHM cell line for use in in vitro cytotoxicity assays and to ascertain the extent of correlation between the relative toxicities of the test agents in vitro with published in vivo 96-hr LCsO data with fathead minnows. Apparently, the FHM cells were suitable for use in the neutral red, cell protein, and cell detachment assays. However, the FHM cells did not form colonies when seeded at low density, thereby preventing their use in a colony-forma-

lrnh- 24-Hr -1 Ii i

Q

Neutral

26”f

20

40 Dlethyltm

Red Assay

‘*..

60 dlchlorlde

BO (pM)

IOI

FIG. 5. Influence of temperature on the toxicity of diethyltin dichloride, as determined with the neutral red assay, to FHM and BE2 cells. The data are expressed as the arithmetic mean percentage of control + SE.

FHM

B 4

CELL

LINE:

“0

80

CYTOTOXICITY

160 4,4,-DDE

85

ASSAY

320

240 QLMI

400

FIG. 6. Influence of temperature on the toxicity of 4$-DDE, as determined with the neutral red assay, to FHM and BF-2 cells. The data are expressed as the arithmetic mean percentage of control f SE.

tion assay. With regard to the correlation of the in vitro toxicity data with in vivo acute toxicity data, it would appear that a better correlation occurred between the BF-2/bluegill sunfish comparison than between the FHM/fathead minnow comparison. This may be a reflection of the greater xenobiotic-metabolizing capabilities of the BF-2 than of the FHM cell line (Thornton et al., 1982) thereby enabling the former cells to more closely mimic the metabolic capacities of the intact animal. In

TABLE COMPARATIVE

ACUTE SUNFISH

Chemical

TOXICITIES, IN AND FATHEAD

&ml,

2 OF ORGANIC

MINNOW,

CHEMICAL~

BF-2 cells Bluegill sunfish FHM cells (24hr NR& (24-hr NR5,,) (96-hr L&J In vitro

Pesticide Chlordane Heptachlor Aldrin 4,4’-DDT Phenolic 2,4-Dichlorophenol 2,4,6-Trichlorophenol 2-Chlorophenol 2,CDimethylphenol Phenol 3-Methylphenol a Henderson et al. (1959). ’ Buccafusco et al. ( 1981). ‘Phippsetul.(1981). eDekseve et al. ( I98 1). ‘U.S. EPA (1980). g Pickering and Henderson ( 1966).

33.6 46.9 70.2 83.0 83.1 94.8 307.3 368.5 652.1 657.7

TO BLUEGILL

in Vitro AND in Vivo

In vivo 0.022” 0.019” 0.013” 0.016” 2.06 0.32’ 6.66 7.8’ 13.5’; 23.9f -

In vitro 23.1 25.3 24.4 25.4 73.5 144.3 145.3 277.5 395.2 312.5

Fathead minnow (96-hr LC& Zn vivo 0.052” 0.094” 0.033” 0.032” 8.2’ 9.2” 12.0f; 14.5d 17.0c 24.9d; 28.5’; 34.4/ 55.9d

86

BABICH

AND BORENFREUND

previous studies we also noted good correlations between the relative cytotoxicities of the cationic metals (Babich et al., 1986a), chlorinated toluenes (Babich and Borenfreund, 1987c), and a broader spectrum of chlorinated phenolics (Babich and Borenfreund, 1987b) to BF-2 cells and their acute toxicities toward bluegill sunfish. Both the FHM and BF-2 cells were more sensitive to the test agents when the exposures were at 34°C rather than at 26°C. This greater sensitivity to the test agents at the higher temperatures of exposure was consistent with similar acute toxicity studies (i.e., 96-hr LCsO tests) with bluegill sunfish (Macek et al., 1969) and fathead minnows (Ruesink and Smith, 1975). CONCLUSION The fathead minnow FHM cell line was suitable for use in the neutral red, cell protein, and cell detachment assays, but not in a colony-formation assay, and was more sensitive to the test agents than was the BF-2 cells. The better correlation between the in vitro/in vivo toxicity data for the BF-2/bluegill sun&h comparison than between the FHM/fathead minnow comparison, however suggests that the former cell line be the more suitable for screening the toxicity of aquatic pollutants. Furthermore, in an earlier study we compared the suitability of the BF-2 and rainbow trout RTG-2 cell lines for use in in vitro cytotoxicity assays and concluded that the slow rate of replication of the RTG-2 cells (optimal growth at 2o’C) made the BF-2 cell line a better candidate for use in in vitro cytotoxicity assays (Babich et al., 1986b). ACKNOWLEDGMENTS This research was supported, in part, by Revlon, Inc., and by Schering Corp. Gratitude is also expressed to the U.S. EPA Repository for Toxic and Hazardous Materials, Cincinnati, Ohio, and to Dr. Frederick Brinckman, U.S. Department of Commerce, for supplying some of the test agents.

REFERENCES BABICH, H., AND BORENFREUND, E. (1987a). In vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells. Environ. Res. 42,229-237. BABICH, H., AND BORENFREUND, E. (1987b). Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxicol. in Vitro 1,3-9. BABICH, H., AND B~RENFREUND, E. (1987~). In vitro cytotoxicity of polychlorinated biphenyls (PCBs) and toluenes to cultured bluegill sunfish BF-2 cells. In Aquatic Toxicology and Hazard Evaluation, 10th Symposium. American Society for Testing and Materials, Philadelphia, in press. BABICH, H., PIJERNER, J. A., AND BORENFREUND, E. (1986a). In vitro cytotoxicity of metals to bluegill (BF-2) cells. Arch. Environ. Contam. Toxicol. 15,31-37. BABICH, H., SHOPSIS,C., ANDBORENFREUND, E. (1986b). In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines. Ecotoxicol. Environ. Saj 11,91-99. BOLS, N. C., B~LISKA, 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- 155. B~RENFREUND, E., ANDPuERNER, J. A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Methods 9,7-9. E%OURNE, E. W., AND JONES, R. W. (1973). Effects of 7,12dimethyl-benx[a]anthracene (DMBA) in fish cells in vitro. Trans. Amer. Microsc. Sot. 92,140-142. Bucc~~sco, R. J., ELLS, S. J., AND LEBLANC, G. A. (1981). Acute toxicity of priority pollutants to bluegill (Lepomis macrochirus). Bull. Environ. Contam. Toxicol. 26,446-I52. DEGRAEVE, G. M., GEIGER, D. L., MEYER, J. S., AND BERGMAN, H. L. (1980). Acute and embryo-larval toxicity of phenolic compounds to the aquatic biota. Arch. Environ. Contam. Toxicol. 9,557-568.

FHM CELL LINE: CYTOTOXICITY

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87

Environmental Protection Agency (EPA) (1980a). Ambient Water Quality Criteria for Phenol. EPA 4401 5-80-066. Office of Water Regulations and Standards, U.S. EPA, Washington, DC. Environmental Protection Agency (EPA) (198Ob). Ambient Water Quality Criteriafor Mercury. EPA 4401 5-80-058. Office of Water Regulations and Standards, U.S. EPA, Washington, DC. Environmental Protection Agency ( 198Oc). Ambient Water Quality Criteria for Polynuclear Aromatic Hydrocarbons. EPA 440/5-80-069. Office of Water Regulations and Standards, U.S. EPA, Washington, DC. Environmental Protection Agency (198Od). Ambient Water Quality CriteriaforPoIychlorinatedBiphenyls. EPA 440/5-80-068. Office of Water Regulations and Standards, U.S. EPA, Washington, DC. HENDERSON, C., PICKERING, Q. H., AND TARZWELL, C. M. (1959). Relative toxicity of ten chlorinated hydrocarbon insecticides to four species of tkh. Trans. Amer. Fish. Sot. 88,23-32. KOCAN, R. M., LANDOLT, M. L., BOND, J., AND BENEDITT, E. P. (198 1). In vitro effect of some mutagens/ carcinogens on cultured fish cells. Arch. Environ. Contam. Toxicol. 10,663-67 1. KOCAN, R. M., LANLIOLT, M. L., AND SAEXO,K M. (1979). In vitro toxicity of eight mutagens/carcinogens for three fish cell lines. Bull. Environ. Contam. Toxicol. 23,269-274. MACEK, K. J., HUTCHINSON, C., AND COPE, 0. B. ( 1969). The effects of temperature on the susceptibility of bluegills and rainbow trout to selected pesticides. Bull. Environ. Contam. Toxicol. 4,174- 183. MARION, M., AND DENIZEAU, F. (1983a). Rainbow trout and human cells in culture for the evaluation of the toxicity of aquatic pollutants. A study with lead. Aquat. ToxicoZ.3,47-60. MARION, M., AND DENIZEAU, F. (1983b). Rainbow trout and human cells in culture for the evaluation of the toxicity of aquatic polhrtants. A study with cadmium. Aquat. Toxicol. 3,329-343. NEBEKER, A. V., PUGLISIS, F. A., AND DEFOE, D. L. (1974). Effect ofpolychlorinated biphenyl compounds on survival and reproduction of the fathead minnow and tla@ish. Trans. Amer. Fish. Sot. 103, 562568. PHIPPS, G. L., HOLCOMBE, G. W., AND FLANDT, J. T. (198 1). Acute toxicity of phenol and substituted phenols to the fathead minnow. Bull, Environ. Contam. Toxicol. 26,585-593. PICKERING, Q. H., ANDHENDERSON, C. (1966). Acute toxicity of some important petrochemicals to fish. J. Water Pollut. Control Fed. 38, 1419-1429. RACHLIN, J. W., AND PERLMU~R, A. (1968). Fish cells in culture for study of aquatic pollutants. Water Res. 2,409-4 14. REINHARDT, C. A., ~ELLI, D. A., AND SANDVOLD, M. (1985). Cell detachment and growth of fibroblasts as parameters for cytotoxicity of inorganic metal salts in vitro. Cell Biol. Toxicol. 1,33-43. RUESINK, R. G., AND SMITH, L. L. (1975). The relationship of the 96-hr LCm to the lethal threshold concentration of hexavalent chromium, phenol and sodium pentachlorophenate for fathead minnow (Pimephales promelas Ratksque). Trans. Amer. Fish. Sot. 104,567-570. SAFE, S. (1984). Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs): Biochemistry, toxicology, and mechanism of action. CRC Crit. Rev. Toxicol. 13,3 19-393. SHOPSIS,C., ANDENG, B. ( 1985). Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicol. Lett. 26, l-8. THOMPSON, J. A. J., SHEFFER,M. G., PIERCE, R. C., CHAN, Y. X., COONEY, J. J., CULLEN, W. R., AND MAQUIRE, R. J. (1985). Organotin Compounds in the Aquatic Environment. National Research Council Canada, Ottawa. THORNTON, S. C., DIAIUIOND, L., AND BAIRD, W. M. (1982). Metabolism of benzo[a]pyrene by fish cells in culture. J. Toxicol. Environ. Health 10, 157-167. WOLF, K., AND MANN, J. A. (1980). Poikilotherm vertebrate cell lines and viruses: A current listing for fishes. In Vitro 16, 168-179.