United States Environmental Protection Agency Environmental Research Laboratory Duluth MN 55804 Research and Development EPA-600/S3-84-009 Feb. 1984 &EPA Project Summary Aquatic Toxicity Tests to Characterize the Hazard of Volatile Organic Chemicals in Water: AToxicity Data Summary N. Ahmad, D. Benoit, L. Brooke, D. Call, A. Carlson, D. DeFoe, J. Huot, A. Moriarity, J. Richter, P. Shubat, G. Veith, and C. Walbridge This summary presents acute and chronic toxicity test data and bioconcen- tration factors compiled over a two- year period on fish and invertebrates exposed to several representative chem- icals from five chemical classes (chlor- inated ethanes, chlorinated benzenes, chlorinated ethylenes, chlorinated di- enes, and chlorinated propanes). The fathead minnows and Daphnia were quite similar in their sensitivities (acute and chronic) to each chemical class, while the rainbow trout were considerably more sensitive to all class- es during acute tests, except for the chlorinated diene exposures, where they were more resistant. The ranking of acute and chronic sensitivity was generally the same for each chemical within each class of chemicals for all three species tested. Both the acute and chronic toxicity of all chemicals within a class increased as the number of chlorines in the chemical structure increased. Bioconcentration factors for fathead minnows were determined for four of the chemical classes tested. Hexachlo- robenzene was bioconcentrated the most (23,000x), and tetrachloroethane was bioconcentrated the least (8x). Again, as with the toxicity experiments, the greater the number of chlorines on the molecule the greater the bioconcen- tration within each class of chemicals. This Project Summary was developed by EPA's Environmental Research Labo- ratory. Duluth, MN. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report order- ing information at back). Introduction The objectives of this study were to evaluate the use of sensitive aquatic toxicity tests in determining the relative hazard of volatile organic compounds; and to compare the sensitivities of three aquatic species to several homologous series of organic chemicals. Investiga- tions were divided into two major areas. First, preliminary studies were conducted to determine similarities and differences in metabolism of selected xenobiotics between aquatic and mammalian organ- isms. The second phase, reported here, represents the largest effort, which was to develop methods for testing volatile chemicals and to determine the differ- ences in toxicity between daphnids (Daph- nia magna) and fish to selected volatile chemicals. Both acute and chronic toxicitv tests were completed with fathead minnows (Pimephales promelas) and daphnids. Only acute tests were performed using rainbow trout (Salmo gairdneri). Test chemicals were selected from the follow- ing classes of compounds: chlorinated ethanes; chlorinated benzenes; chlori- nated ethylenes; chlorinated dienes and chlorinated propanes. Bioconcentration factors for fathead minnows exposed to ------- 10 chemicals from the above classes were also determined. The full report represents an overview of the results from these investigations. Most data have been or are scheduled to be published in scientific journals. Methods and Procedures Bioassay-Chemical Routine toxicity test met hods and chem- ical procedures followed closely those described by Mount and Brungs (1967), APHA(1975),USEPA(1975), Phippsetal (1981), and Benoit et al (1982) All tests, except for the static Daphnia exposures, were performed using propor- tional diluters. Lake Superior water was used for all tests. Nominal water temper- atures were 12, 20, and 25° C for rainbow trout, Daphnia, and fathead minnows, respectively Acute tests were for 96 hrs forfatheadsand rainbowtroutand48 hrs for Daphnia. Chronic fish (embryo-larval) tests were conducted for 32 days, and chronic Daphnia tests were of 28-day duration Chemical characteristics of the control and experimental waters were as follows: dissolved oxygen 7.0-9.6 mg/l; pH 6.7- 7.6, total hardness (as CaCO3) 43-57 mg/l, and alkalinity 35-53 mg/l Several statistical procedures (Spearman-Karber, ANOV, Probit analyses) were used to test the significance of the results. Chemicals selected for testing ranged in purity from 95-99 percent Solvents used in the extraction process were of glass-distilled, gas-chromatography grade. The effectiveness of the extraction procedure was examined by determining the percent recovery of a known amount of each chemical in the experimental water. All toxicant concentrations in water and tissues were analyzed by gas chromatography Further description of these analytical procedures, according to chemical, appear in the full report. Development of Early Life Stage, Mini-Diluter Apparatus In order to successfully and safely test volatile chemicals, it was necessary to develop specialized exposure systems and methods for monitoring air and water leaving these systems. Interest in develop- ing fish early life-stage (ELS)toxicity tests led to the design of a compact continuous flow mini-diluter exposure system which accurately delivered as little as 3 liters of test water per hour to each of five concentrations plus a control This system can be used to test the effects of either single chemicals or treated complex effluents on young fish in the laboratory or in the field. The small ELS test appa- ratus takes less space and requires smaller volumes of test water, which is a critical factor when effluents are shipped to the laboratory or on-site toxicity tests are conducted. Smaller volumes of test water also reduce filtration costs for the removal of hazardous test chemicals before discharging waste water to the sewer. The ELS test system has been tested and evaluated in the laboratory and on- site in a mobile trailer (Benoit et al., 1982). This apparatus has been used to conduct fathead minnow (Pimephales promelas) ELS exposures to various toxi- cants including volatile organic com- pounds, metals, pesticides, and treated complex effluents from metal plating, oil refinery, and sewage treatment plants The system has also been successfully used for testing macromvertebrates. All embryo-larval fish tests described herein were conducted in this mini-diluter sys- tem. Summary and Conclusions It has long been known that aquatic animals are extremely sensitive to chemi- cals at very low /ug/L to mg/L concentra- tions in the aqueous environment. This knowledge led us to the position of evaluating both acute and chronictoxicity tests with several sensitive aquatic spe- cies in an effort to deter mi net he range of sensitivities and the possible application of the data screening out chemicals, which after short inexpensive tests with selected aquatic species are shown to be extremely toxic, and/or highly bioaccumu- lable Phase one of these studies was de- signedto provide preliminary information on the metabolic capabilities of rainbow trout and daphnids These studies, coupled with earlier studies on mixed function oxidase activity in mammals and other animals, indicated the metabolic systems to be similar qualitatively, therefore, the mechanisms leading to toxicity and neo- plasia, for example, are presumed to be similar in all organisms. Phase two was to determine the acute and chronic toxicity of five classes of chlorinated organic compounds to selected fish and inverte- brate animals In addition, the bioconcen- tration potential of these chemicals was measured to determine possible food- chain problems involving man. Daphnia showed generally the same order of acute and chronic sensitivity as the fish for all classes of chemicals tested. A comparison of species acute sensitivities (Table 1) indicated that Daph- nia were slightly more resistant in mosl cases than the fathead minnow, whereas the rainbow trout was considerably more sensitive than either the fathead minnow or Daphnia to all compounds excep! hexachlorobutadiene One of the more interesting findings ol the acute studies was that the toxicity ol the ethanes, benzenes, and ethylenes increased as the number of chlorines on the molecule increased. This was true for both fathead minnows and Daphnia (Table 1). These data indicate that either fathead minnows or Daphnia would provide es- sentially the same acute values for these particular chemicals. These chemicals are not considered to be very toxic tc aquatic species, since their 96-hr LC50s are one to two orders of magnitude above those environmental chemicals consid- ered as extremely toxic. Fifteen chronic toxicity tests with fish were also conducted on chemicals in the five chemical classes. The order of sensi- tivity for all chemicals tested chronically (Table 2) was the same as that established for acute toxicity (Table 1). Six chronic values were also determined for Daphnia and in most cases the sensitivity was similar except for the ethanes, where there seemed to be considerable variation between the fathead and Daphnia results. Again, chronic toxicity increased consid- erably for both species as the number of chlorines on the molecule increased (Table 2). The bioconcentration potentials of these chemicals were determined by establishing a bioconcentration factor (BCF) Cp.sh/Cwater) for fathead minnows exposed for 32 days to each chemical during an early life-stage toxicity test. These BCFs were then compared to BCF values for other species of fish found in the literature (Table 3). In the two classes where comparisons were possible (chlori- nated benzenes and ethanes), bioconcen- tration as well as toxicity increased as the number of chlorines on the molecule increased. The literature values for other fathead minnow studies as well as studies with bluegill, sunfish (Lepomis macro- chirus), and guppies (Poecilia reticulata] (Table 3) all agree very closely with the BCFs generated here. This indicates that age, size, and species of fish have little effect on the BCF generated over a 32-day period of water exposures. Based on BCF values, hexachlorobenzene, hexachloro- butadiene, and 1,2,3,4-tetrachloroben- zene are the chemicals from the groups ------- Table 1. Summary of Acute Toxicity Data for Fathead Minnows, Rainbow Trout, and Daphnia Compound Fathead Minnow 96-hr LCSO fmg/IJ Rainbow Trout 96-hr LCSO (mg/lj Daphnia 48-hr LCSO (mg/l) Chlorinated Ethanes Hexach/oroethane Pentachloroethane 1,1,2,2-Tetrachloroethane 1,1,2-Tnchloroethane 1,2-Dichloroethane Chlorinated Benzenes Hexachlorobenzene Pentachlorobenzene 1,2,3,4- Tetrachlorobenzene 1,2,4 • Tnchlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene Chlorinated Ethylenes Tetrachloroethylene 1,1,2-Tnchloroethylene Chlorinated Propanes 1,2-Dichloropropane 1,3-Dichloropropane Chlorinated Butadienes Hexachlorobutadiene 1.53 730 2030 81 70 7 77 SO 1 07 •276 779 4 re 1350 44 10 139 30 131 10 0.10 084 1.52 1.61 1.12 499 290 732 62.10 186.0 268.0 2.09 7.43 18 10 032 "Not tested "Not toxic at water saturation Table 2. Summary of Fathead Minnow and Daphnia Chronic Toxicity Data Compound Fathead Minnow 32-day (ELS) MA TC Daphnia 28-day" Chronic Chlorinated Ethanes Hexach/oroethane Pentachloroethane 1,1,2,2-Tetrachloroethane 1,1,2-Trichloroethane 1,2 -Dichloroethane Chlorinated Benzenes Hexachlorobenzene Pentachlorobenzene 1,2,3,4- Tetrachlorobenzene 1,2,4-Tnchlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene Chlorinated Ethylenes Tetrachloroethylene 1,1,2-Tnchloroethylene Chlorinated Propanes 1,2-Dichloropropane 1,3-Dichloropropane Chlorinated Butadienes Hexachlorobutadiene 69-207 900-1,400 1,40O-4,OOO 6,000-14,800 29,000-59,000 4.76" 549" 245-412 499-1,008 1.000-2,267 565-1,040 500-1,400 6,000-11,000 8,000-16.000 65-13.0 6,850-14,4OO 13,200-26,000 10,600-20,700 363-694 689-1,450 505-1.110 "Effect—no effect concentrations "Water saturation—no effects noted tested that pose the greatest bioconcen- tration threat to the environment. The acute toxicity tests run with both fish and invertebrates established a rela- tive order of toxicity for the individual chemicals that was nearly identical to the order seen in the more sensitive chronic exposures. Therefore, the short 96-hr LCSO fish exposures or the 48-hr Daphnia exposures could be used to establish a ranking of chemicals found in water that will permit the researcher to initially concentrate the expensive chronic testing on the more toxic materials The Daphnia acute test may be better than a fish acute test, since it takes only 48-hrs and does not require the more difficult flow-thro ugh system required for a fish 96-hour LC50 estimate, yet it gives the same relative order of chemical sensitivity for these classes of chemicals (Tables 1 and 2). Early life-stage toxicity tests with fish (32-days) and/or 28-day Daphnia chron- ics would provide a more sensitive test and the fish exposures would also allow the determination of a BCF, which would provide further information on the envi- ronmental impact of a chemical. The usefulness of aquatic tests as an early warning system for toxic chemicals in these classes may be somewhat limit- ed, because of their low toxicity and low ambient water concentrations. However, this approach for more toxic chemicals has considerable promise as an early warning system for higher animals includ- ing man. References American Public Health Association, A- merican Water Works Association, and Water Pollution Control Federation. 1975. Standard methods for the exami- nation of water and wastewater 14th ed. American Public Health Associa- tion, Washington, DC 20036. Benoit, D. A., V. R Mattson, and D L. Olson 1982. A continuous-flow mini- diluter system for toxicity testing. Water Res 16-457-464. Mount, D I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Res., Vol 1, pp 21-29. Phipps, G. L., G. W. Holcombe, and J. T. Fiandt. 1981. Acute toxicity of phenol and substituted phenols to the fathead minnow. Bull. Environ. Contam. Toxicol. 26:585-593. U.S. Environmental Protection Agency, Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. ------- Table 3. A Comparison of Bioconcentration Factors for Chemicals Tested in Present Study in Fathead Minnows vs. Other Species of Fish in Other Studies Chemicals Present Study Fathead Minnows ° BCF Log BCF Literature Values Fathead Minnow* Bluegilf Guppyc Chlorinated Ethanes Hexach/oroethane Pentachloroethane 1 ', 1 ,2,2-Tetrachloroethane Chlorinated Ethylenes Tetrachloroethylene Chlorinated Butadienes Hexachloro- 1,3-butadiene Chlorinated Benzenes Hexachlorobenzene 1 .2,3,4- Tetrachlorobenzene 1 ,2,4- Trichlorobenzene 1 , 3-Dichlorobenzene 1 ,4-Dichlorobenzene 703.4 60.4 8.1 61.5 6,988.6 23,391 5 2,567.3 398.5 97.9 112.4 285 1.78 0.91 1.79 3.84 4.37 3.41 260 1.99 205 138 68 8 49 - 21,878 1,820 1,698 66 60 - - - - 14,454 3.631 646 91 ^32-day exposure ELS toxicity test. ^30-day old fish exposed for 30 days. 1Adult fish exposed for 30 days. Standard practice for conducting acute toxicity tests with fishes and macro- invertebrates, and amphibians. EPA- 660/3-75-009. Duluth, MN. 67 p. N. Ahmad, J. Huot, and A. Moriarity are with the University of Wisconsin, Superior, Wl 54880; the EPA authors D. Benoit, L. Brooke. D. Call, A. Carlson, D. Defoe, J. Richter. P. Shubat, G. Ve/th, and C. Walbridge are with the Environmental Research Laboratory, Duluth, MN 55804. J. M. McKim and R. A. Drummond are the Co-Project Coordinators (see below). The complete report, entitled "Aquatic Toxicity Tests to Characterize the Hazard of Volatile Organic Chemicals in Water: A Toxicity Data Summary," (Order No. PB 84-141 506; Cost: $13.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The Co-Project Coordinators can be contacted at: Environmental Research Laboratory U.S. Environmental Protection Agency 6201 Congdon Blvd. Duluth, MN 558O4 AUS GOVERNMENT PRINTING OFFICE 1984-759-015/7314 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 BULK RATE POSTAGE & FEES PA EPA PERMIT No G-35 Official Business Penalty for Private Use $300 ------- |