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(5) Merller, M.; Alfheim, I. Atmos. Enuiron. 1980, 14, 83. (6) Pitts, J. N.,Jr.; Grosjean, D.; Mischke, T. M. Toxicol. Lett. 1977, 1, 65. (7) Talcott, R.; Wei, E. J. Natl. Cancer Inst. (U.S.) 1977,58, 449. (8) Tokiwa, H.; Kitamori, S.;Takahashi, K.; Ohnishi, Y. Mutat. Res. 1980, 77, 99. (9) Ames, B. N.; Durston, W. E.; Yamasaki, E.; Lee, F. D. Proc. Natl. Acad. Sei. U.S.A. 1973, 70, 2281. (10) Chrisp, C. E.; Fisher, G. G. Mutat. Res. 1980, 76, 143. (11) Hughes, T. J.; Pellizzari, E.; Little, L.; Sparacino, C.; Kolber, A. Mutat. Res. 1980, 76, 51. (12) Lbfroth, G. In "Health Effecta of Diesel Engines Emissions: Proceedings of an International Symposium"; Pepelko, W. E., Danner, R. M., Clarke, N.A., Eds.; Nov 1980; Vol. 1, EPA-600/9-8-057a. (13) Lbfroth, G. Environ. Sci. Res. 1981, 22, 319. (14) Ohnishi, Y.; Kachi, K.; Sato, K.; Tahara, I.; Takeyoshi, H.; Tokiwa, H. Mutat. Res. 1980, 77, 229. (15) Wang, Y. Y.; Rappaport, S. M.; Sawyer, R. F.; Talcott, R. E.; Wei, E. T. Cancer Lett. 1978, 5, 39. (16) Alfheim, I.; Merller, M. Sei. Total Environ. 1979, 13, 275. (17) Thrane, K. E.; Mikalsen, A. Atmos. Environ., in press. (18) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347. (19) Grimmer, G.; Bohnke, H. 2.Anal. Chem. 1972,261, 310. (20) Bjerrseth, A. Anal. Chin. Acta 1977, 94, 21. (21) Wei, E. T.; Wang, Y. Y.; Rappaport, S. M. J. Air Pollut. Control Assoc. 1980, 30, 267. (22) Talcott, R.; Harger, W. Mutat. Res. 1980, 79, 177. (23) Teranishi, K.; Hamada, K.; Watanabe, H. Mutat. Res. 1978, 56, 273. (24) Van Vaeck, L.; Van Cauwenberghe, K. Atmos. Environ. 1978, 12, 229. (25) Wynder, E. L.; Hoffmana, D. J. Air Pollut. Control Assoc. 1965, 15, 155. (26) Grimmer, G. IARC Sei. Publ. 1977, 16, 29.
samples. Our study indicates that samples collected at street level are qualitatively different even from those at roof level of the same location. The recovery of mutagens after chemical fractionation was lower in our study (48% in the presence of S9) than in the Japanese studies. This may be due to relatively unstable mutagenic compounds associated with car exhaust. Such compounds may degrade when exposed to strong acids or bases. Particulate matter from an area with heavy traffic has been fractionated and their carcinogenic effect tested by skin-painting experiments on mice (25). The results showed that the aromatic fraction was connected with carcinogenic properties while the acidic and oxygenated fractions were associated with promotor effects. Carcinogenic compounds in exhaust from gasoline- and diesel-driven cars have been reported. Grimmer (26)claims that PAHs together with alkyl-substituted PAHs were alone responsible for most of the carcinogenic effect in exhaust from gasoline. They found BaP to be responsible for 5-9% of this effect. On the basis of measurements on samples from street level, we found that the amount of BaP could account for only up to 2% of the mutagenic activity in day samples and up to 4% in night samples. Acknowledgments
We thank A. Osvik,A. K. B0, V. Opsahl, J. H. Wasseng, T. Olsen, and A. Bjerke for skillful technical assistance. Literature Cited (1) International Agency for Research on Cancer. IARC Monogr. Eval. Carcinog. Risk Chem. Man 1973, 3. (2) Daisy, J. M.; Kneip, T. J.; Hawryluk, I.; Mukai, F. Environ. Sei. Technol. 1980, 14, 1487. (3) Dehnen, W.; Pitz, N.;Tomingas, R. Cancer Lett. 1977,4, 5. (4) Commoner, B.; Madyastha, P.; Bronsdon, A.; Vithayathil, A. J. J. Toxicol. Emiron. Health 1978,4, 59.
Received for review May 18,1981. Revised manuscript received October 26, 1981. Accepted December 1,1981. This work was supported by the Norwegian State Pollution Control Authority.
Chronic Effects of a Coal Liquid on a Freshwater Alga, Selenasirum capricornuturn Robert H. Gray,* Robert W. Hanf, Dennls D. Dauble, and John R. Skalski
Ecological Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352 Static toxicity tests (EPA Bottle Test) were conducted to evaluate potential chronic effects of water-soluble fractions (WSFs) of a coal liquid on the freshwater alga, Selenastrum capricornutum. Tests measured population response during and after exposure (5-day exposure, 9-day recovery) to WSFs prepared by sequentially extracting a 2.9:l blend of solvent-refined coal (SRC-11) middle/heavy distillates with Columbia River water. WSFs from the first and fifth extractions represented a fresh mix and waterleached material, respectively. On the basis of dilution, water-leached material was less toxic than the first extraction. However, on the basis of chemical composition, water-leached material was more toxic than the first extraction, since lower concentrations of total carbon and total phenol inhibited algal recovery. Similar tests with a Prudhoe Bay crude and No. 6 fuel oil showed that these materials were less toxic than the SRC-I1liquid, primarily because of lower solubilities of their constituents in water.
interest in developing alternate sources of fuel. Because of vast national coal reserves, the US Department of Energy (DOE) is currently evaluating development of coal gasification and liquefaction processes. One option, the solvent-refined coal-I1 (SRC-11) process, hydrogenates pulverized coal under high temperature and pressure to produce liquid fuel products. Because the SRC-I1process may be commercialized, potential environmental effects of accidental spills of SRC-I1 liquids must be carefully assessed. Depending on the magnitude of potential detrimental effects, spill prevention and cleanup procedures may need to be developed. We utilized a modification of the standard EPA Bottle test to evaluate effects of water-soluble fractions (WSFs) of an SRC-I1liquid on the unicellular freshwater green alga, Selenastrum capricornutum. Relative toxicities of WSFs of a Prudhoe Bay crude and No. 6 fuel oil were also evaluated.
Introduction
Materials and Methods
Increasing energy demands coupled with rising prices and an unstable world oil market have stimulated Federal
The SRC-I1pilot plant is located in Fort Lewis, WA, and is operated by the Pittsburg and Midway Coal Mining Co.
0013-936X/82/0916-0225$01.25/0
0 1982 American Chemlcal Society
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for the US Department of Energy. Materials obtained from a pilot plant may or may not represent demonstration or commercial facility products. About 227 L (60 gal) of a 2.9:l blend of SRC-I1 middle/heavy distillates were obtained from the pilot plant and transported to our laboratories in four Teflon-lined stainless-steel drums, each containing about 57 L (15 gal) of material. Before shipment, all air in the drums was replaced with a nitrogen blanket and the drums were sealed to minimize chemical oxidation and avoid contamination. At our laboratory, equal amounts of material were removed from each of the four drums, mixed, and transferred to 1-gal brown glass bottles. All jugs were tightly sealed with Teflon-lined caps and stored in the dark at 4 "C until used. Prudhoe Bay crude oil was obtained from the Atlantic Richfield Co., Cherry Point Refinery, Ferndale, WA. No. 6 fuel oil was obtained from the Shell Oil Refiiery, Anacortes, WA. Reference oils were stored under similar conditions as the SRC-I1 liquid. Preparation of Test Solutions. An initial WSF was prepared by slowly stirring 300 mL of SRC-I1blend or oil with 29.7 L of filtered Columbia River water (pH 7.8, alkalinity 61 mg/L CaCO,, EDTA hardness 72 mg/L CaCO,, temperature 20 "C, dissolved oxygen 9.2 mg/L) in a 40-L glass carboy for 4 h. The resulting WSF was siphoned from near the bottom of the carboy after the mixture phase-separated for 1 h. A water-leached extraction of SRC-I1 liquid was prepared by siphoning all but 3 L (90% removal by volume) of the initial WSF and adding 27 L of fresh filtered Columbia River water to the remaining mostly insoluble fraction. Through a series of redilutions and remixes of the remaining insoluble materials over 5 days ( I ) , a final WSF (fifth extraction) was obtained for testing. When possible, tests were initiated immediately after the 1-h settling period. When testing was delayed, test solutions were placed in stoppered, 3.8-L (1-gal) glass bottles and stored in the dark at 4 OC. Total carbon (TC) concentration of WSFs was determined by direct aqueous injection in a Beckman 915B carbon analyzer. Phenols were estimated by the dye photometric method (2). Test Procedure. Cultures of S. capricornuturn (chlorococcales) were obtained from the US Environmental Protection Agency (EPA)Research Labs at Corvallis, Or, and were purified on agar plates. Cultures were maintained under test conditions (i.e., light, temperature, etc.) with new cultures started every 1 or 2 weeks. Test procedures were modified from those of Payne and Hall (3) and the recommendations of Miller et al. (4). We conducted three tests with dilutions of the initial SRC-I1 WSF, and four tests with dilutions of the fiith WSF. Tests were also conducted with similarly derived WSFs from a Prudhoe Bay crude oil and a No. 6 fuel oil. Each test consisted of at least three replicate 250-mL Erlenmeyer flasks containing one of five test material concentrations, and three replicate control flasks without test material for a minimum of 18 flasks. At test initiation, a measured amount of test material was added to sterile flasks and diluted to 50 mL with algal nutrient medium. After each flask was spiked with a known number of 7-16-day-old algal cells, they were randomly positioned in a controlled environment incubator under continuous fluorescent light at 24 f 2.0 "C for 120 h. Gas exchange within the flasks was promoted by restricting fluid volume to 20% of flask capacity and by swirling cultures at least once daily. Inverted 50-mL beakers were used as flask covers to prevent contamination. 226
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After 5 days, algal cells in each flask were enumerated with a Coulter electronic particle counter Model ZH (70fim aperture). Duplicate samples were also taken for chlorophyll a analysis. Equal numbers of cells from each flask were then introduced to fresh uncontaminated nutrient medium in new flasks, which were again randomly positioned, and incubated as before. Inoculation of equal cell numbers differs from the procedure of Payne and Hall (3) but allows a statistical evaluation, rather than a qualitative judgment, of population response. After 9 days, cell counts and chlorophyll a concentrations were determined to evaluate algal recovery. Statistical Analysis. Analysis of variance for a nested classification was used to analyze algal cell counts after exposure and recovery periods. Fisher's protected least significant difference (LSD) tests ( a = 0.05) were used to compare cell growth in controls with that in treatment groups. The lowest concentrations of test material observed to produce a significant reduction in algal growth compared to the controls, or lowest rejected concentration tested (LRCT) (5) for recovery and exposure periods were determined. Estimation of algistatic concentrations (concentrations that inhibit growth during exposure but not during recovery), as proposed by Payne and Hall (31, was inappropriate for out data because no linear relationship between In concentration and the In 5-day cell count/initial cell count (required to calculate the algistatic concentration) was observed. Assuming a monotonic decrease in cell growth with increasing toxicant concentration, sequential straight-line regression analyses were used to detect an algistatic concentration based on cell counts from the 9-day recovery period. At higher toxicant concentrations, cell growth during the recovery period approached but always exceeded zero. This asymptotic response formed the basis for our estimation of algistation concentration. Starting with cell counts from the three highest toxicant levels tested among replicate experiments, the parameters of the regression model
y,= a
+ pxi +
€,
where y,= 9-day cell count after recovery period for the ith toxicant concentration, x, = the ith toxicant concentration, and e, = normally distributed error terms, were estimated by the method of least squares. Additional data points at the next lower toxicant concentrations were sequentially added to the analysis to estimate the model parameters until the slope (p) was statistically ( a = 0.05) different from zero. The concentration where the slope first became significant was defined as the algistatic concentration. For example, in estimating the algistatic concentration for initial WSFs, we added data from the seventh and eighth toxicant levels to the analysis before the slope became significantly different from zero. The toxicant concentration associated with those cell counts was defined as the algistatic concentration. Data from three to four replicate tests were used to estimate algistatic concentrations of SRC-I1 WSFs. Results SRC-I1 WSF Tests. Observed LRCTs for cell counts in initial extract tests were dilutions containing 0.4-0.8% of the WSF during exposure. However, algal recovery was not inhibited until exposure dilutions contained 6.2-12.1 % of the WSF. That is, although cell counts were reduced at dilutions containing
