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(24) Nesmeyanov, A. N.; Freidlina, R. K. H. Zakharkin, L. I. Dokl. Akad. Nauk. SSSR 1954,97,91-94; Chem. Abstr. 1955,49,8793. (25) V'yunov, K. A; Zhukova, T. I.; Sochilin, E. G.; Smorygo, N. A. Zh. Org. Khim. 1976,11, 2331-2335. (26) V'yunov, K. A.; Garabadzhiu, A. W.; Zhukova, T. I.; Zhi. linskaya, T. D.; Sochilin, E, G. Zh.Org. Khim. 1978, 14, 1187-1193. (27) Delorenzo, F.; Staiao, N.; Silengo, L.; Cortese, R. Cancer Res. 1978, 38, 13-15. (28) Rosen, J. D.; Segall, Y.; Casida, J. E. Mutat. Res. 1980, 78, 113-119.
(29) Douglas, G. R.; Nestman, E. R.; Grant, C. E.; Bell, R. D. L.; Wytsma, J. M.; Kowbel, D. J. Mutat. Res. 1981, 85, 45-56. (30) Sikka, H.C.; Florczky, P. J . Agric. Food Chem. 1978,26, 146-148. (31) Coleman, W. E. US.Environmental Protection Agency Health Effects Research Laboratory, personal communication, 1981. Received for review May 3, 1982. Accepted January 24, 1983. This work was supported by U.S.Environmental Protection Agency Grants CR806872 and CR808603.
Combined Bioassay-Chemical Fractionation Scheme for the Determination and Ranking of Toxic Chemicals in Sediments Martln R. Samoiloff,*t James Bell,$ Detlef A. Birkholz,$G. R. Barry Webster,§ Evelyn G. Arnott," Rock Pulak," and Alicia MadridL Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, Environmental Protection Service, Western and Northern Region, Northern Forest Research Centre, Edmonton, Alberta, Canada, Pesticide Research Laboratory, Department of Soil Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, and Manitoba Research Council, Industrial Technology Centre, Winnipeg, Manltoba, Canada R2J 3T4
A protocol for the chemical fractionation of sediments and biological testing of these fractions has been developed. Fractions obtained were directly tested for toxicity by using both the Salmonella typhimurium test and the Panagrellus rediviuus test. When applied to sediments from Tobin Lake, Saskatchewan, this method demonstrated that the major toxic constituents of the sediment were neutral compounds that eluate from Florisil columns by 1:l hexane-dichloromethane. This most toxic fraction contained none of the priority toxic chemicals. These testa demonstrate agreement between the two biological assay systems.
Introduction The conventional approach to environmental toxicology involves parallel but distinct "arms length" activities of chemists on the one hand and biologists and toxicologists on the other hand. The chemist establishes what chemicals are present in a particular contaminated environment, while the biologist or toxicologist establishes the toxic effects of individual chemical species. Much of environmental toxicology involves the determination of the presence and levels of previously established toxic chemical species, usually detected one chemical or chemical family at a time. However, real aquatic ecosystems are seldom contaminated with a single chemical issuing from a single source. Rather, there is an extensive multiplicity of compounds and sources contaminating most aquatic systems. Any system in close proximity to human activity will contain literally thousands of contaminating chemicals and their byproducts. The major problem in such systems is to determine which specific components pose the greatest long- and short-term risks to biological systems, including man. The question of what chemicals are present is secondary; the primary objective is to establish which compounds in a particular system pose the greatest risk. 'Department of Zoology, University of Manitoba. Northern Forest Research Centre. Department of Soil Science, University of Manitoba. Industrial Technology Centre.
*
Toward this objective, we are developing methods involving chemical fractionation of environmental samples, coupled with biological testing of the fractions, as a means of establishing which components of the contaminated system produce the greatest toxic effects. Biological assays are utilized as an analytical method of establishing the risk potential of each fraction. The initial methods involve (1)the extraction of contaminants from environmental samples, (2) the fractionation of the extracts on the basis of differential solubility, (3) the bioassay of each fraction to ascertain the relative toxicity of each, and (4) the identification of the components of the major toxic fraction. Ultimately, a series of subfractionations of those fractions determined by this method to be the most toxic would be utilized to pinpoint the exact chemicals producing the greatest risk. We are using a protocol in which biological assays of broad classes of chemical fractions from sediments function as analytical means to localize and rank potential risk. Two bioassay methods are utilized in this study. The Salmonella typhimurium test (1,2)was used as a standard indicator of mutagenesis. A developmental assay (3) using the nematode Panagrellus rediuivus was used to detect lethal, semilethal, developmental, and mutagenic effects. These bioassays, as adjuncts to chemical fractionation, have permitted us to establish the characteristics of the major contaminants in sediments from two sites on Tobin Lake, Saskatchewan. We have selected as sources of contaminants sediment samples from Tobin Lake, on the Saskatchewan River. The Saskatchewan River, upstream of Tobin Lake, is exposed to contaminants from a broad spectrum of human activities: agriculture, mining, petrochemical industries, pulp and paper mills, and municipalities. Tobin Lake represents a major sink for these contaminants. From this extensive range of sources of contamination, we are attempting to focus on those compounds that represent the greatest potential risk. In this report, we present the methodologies and initial toxicity determinations. The work reported here represents only a single component of the Tobin Lake project. The other two components of the study involve the bio-
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logical determination of in situ toxic effects in Tobin Lake sediments, being performed by W. F. Warwick and coworkers, and detailed chemical analyses of Tobin Lake sediments by one of us (G.R.B.W). The total thrust of the Tobin Lake project is to utilize this three-pronged approach ranging from strictly chemical to strictly field biological to establish the precise causality of the ecotoxicity in Tobin Lake.
Materials and Methods Sampling Program. The Saskatchewan River system extends from Alberta, through Saskatchewan, into Lake Manitoba. Tobin Lake, near the Manitoba-Saskatchewan border, is a settling basin for the entire North Saskatchewan River and the South Saskatchewan River downstream of Lake Diefenbaker. The initial studies reported here were made with sediment samples taken from Tobin Lake at the sites designated Jackfish Point and Channel. Jackfish Point is situated approximately 8 km downstream from the town of Nipawin, Saskatchewan. The site designated Channel is situated approximately 12 km downstream from Jackfish Point. Both sites are approximately in midchannel at sites where sediment deposition would occur. Sediment samples were collected by Eckman dredge and temporarily held in polyethylene bags. These samples were quickly transferred to precleaned tin-plated cans. Samples in the cans were frozen and stored at -40 "C prior to analysis. Chemical Extraction Procedures. Samples were thawed at room temperature, lyophilized, and then homogenized by placing in a soil roller for 4 h. Each sample was divided into five replicate portions, each weighing approximately 40 g. Each replicate was rehydrated with organic-free water to raise its moisture content to 25% and then extracted following the protocol described below. (1)The rehydrated replicate was Soxhlet extracted in 1:l acetone-hexane for 24 h. The extracted sediment was retained for further extraction (point 5 below). (2) The acetoneheme extract was preconcentrated and partitioned into organic-free water. The water was adjusted to pH 7.0 and extracted with dichloromethane. The aqueous phase was retained for further extraction (point 4 below). (3) The dichloromethane extract, containing neutral compounds, was dried by passage through a column of anhydrous sodium sulfate and preconcentrated, and the dichloromethane was evaporated following addition of 10 mL of hexane. This hexane solution was applied to a 10-g Florisil column (5% water deactivated). (a) Material eluted from the Florisil column by 160 mL of hexane was termed fraction 1. (b) Material eluted from the Florisil column by 160 mL of 1:l hexane-dichloromethane was designated fraction 2. (c) Material eluted from the Florisil column by 200 mL of dichloromethane was designated fraction 3. (d) Material eluted from the Florisil column by 200 mL of methanol was designated fraction 4. (4) The aqueous phase of the original dichloromethane extraction (step 2) was further fractionated as follows: (a) The aqueous phase was adjusted to pH >11and extracted with dichloromethane. The dichloromethanewas removed, and the extracted material, containing strong bases, was designated fraction 5. (b) The aqueous phase was adjusted to pH
