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Mutagenicity of Sediments from the Detroit River
J. Great Lakes Res. 17(3):314-321 Internat. Assoc. Great Lakes Res., 1991
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
Alexander E. Maccubbin l •• , Noreen Ersing l , and Mary Ellen Frank Department of Experimental Biology Roswell Park Cancer Institute Elm and Carlton Streets Buffalo, New York 14263
ABSTRACT. Sediment samples from 30 sites in the lower Detroit River and one site in Lake Michigan were extracted with organic solvent and were tested for mutagenicity in the Ames test. Without metabolic activation, sediment samples were either non-mutagenic or weakly mutagenic. With metabolic activation, mutagenic responses (defined as doubling of the spontaneous mutation rate at one or more concentrations of organic extract) were observed in 16 of 31 samples. Mutation rates ranged from 20 to 370 histidine positive revertants per mg of organic extract. These results demonstrate the existence of compounds in sediments of the Detroit River that can be activated to mutagens. The sediments thus are a potential source of mutagenic and possibly carcinogenic compounds for fish and other organisms. INDEX WORDS: Toxic substances, Detroit River, sediments, bioassay, mutagen.
INTRODUCTION
contaminated waterways has received much attention in the past few years. There have been a number of studies describing toxic effects of contaminated sediments. Thornley and Hamdy (1984, 1985) analyzed macrobenthos of the Detroit River and neighboring waters and demonstrated that pollution in the Detroit River significantly altered benthic assemblages. In laboratory studies, chemicals bound to sediments from the Detroit River have been found to be toxic to phytoplankton (Munawar et al. 1983, 1985) and bacteria (Giesy et al. 1988). Several other studies have demonstrated the presence of contaminants or metabolites of contaminants in ducks (Smith et al. 1985), gulls (Struger et al. 1985), clams (Pugsley et al. 1985), and fish (Suns et al. 1985, Maccubbin et al. 1990). In addition, chemical-DNA adducts have been observed in liver tissue from fish collected from the Detroit River (Dunn et al. 1987, Maccubbin et al. 1990) and tumors have been found in several species of fish (Maccubbin et al. 1987, 1990; Maccubbin and Ersing 1990). The detection of chemical-DNA adducts provides a direct measurement of exposure to genotoxic agents (Dunn 1990). Moreover, some of the chemicals found in the sediments of the Detroit River are known genotoxins [see Kraybill (1976) for a discussion of carcinogens in the aquatic envi-
Waterways, such as the Detroit River, that flow through urban areas receive chemical discharges from industrial, municipal, and agricultural sources. Many of the chemicals contained in these discharges are hydrophobic in nature and are sequestered by sediment particles that eventually settle in deposition zones within the waterway (Furlong et al. 1988). In addition to being hydrophobic, some sediment-bound chemicals may resist microbial degradation and may accumulate in high concentrations in sediment or biota (Alexander 1981, Furlong et al. 1988). Within the Detroit River system, sediments have been found to be contaminated with a wide array of chemicals including organochlorine pesticides, heavy metals, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons (Fallon and Horvath 1985, Hamdy and Post 1985, Kaiser et al. 1985, Lum and Gammon 1985, Maguire et al. 1985, Platford et al. 1985, Furlong et al. 1988). The potential effect of such sediment-bound chemicals on the biota of I Current
Address: Department of Experimental Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 aTo whom reprint requests should be addressed.
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MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
ronment]. Although the liver tumors observed in fish from the Detroit River may have multiple causes, many of the fish are bottom dwelling/ feeding species that potentially could be exposed to sediments containing genotoxic chemicals. The Salmonella/microsome mutagenicity assay or the Ames test (Ames et 01. 1975) has been widely used to analyze chemicals for their mutagenic potential. In most cases, pure chemicals have been analyzed. However, the Ames test also has been used to analyze complex mixtures of chemicals extracted from a variety of sources such as pulpmill effluents (Douglas et 01. 1980, Kinae et 01. 1981, Kamra et 01. 1983), petroleum refinery effluents (Metcalfe et 01. 1985), and wastes from the wood-preserving industry (Donnelly et 01. 1987). In addition the Ames test has been used to analyze organic solvent extracts of sediments collected from chemically contaminated rivers (Black et 01. 1980; Sato et 01. 1983, 1985; West et 01. 1986a, 1986b, 1988; Pittinger et 01. 1987; Fabacher et 01. 1988). We have used the Ames test to screen sediment samples for mutagenic potential as a measure of genotoxicity and in this report we describe the results of our studies on sediments from the Detroit River. MATERIALS AND METHODS
Sampling Sediments were collected from 30 stations in the lower Detroit River (Fig. 1). Multiple grab samples were placed in large tubs and were thoroughly mixed: subsamples were then placed in prewashed (nitric acid followed by hexane) jars which were sealed. Samples were kept on ice in the field and, upon returning to the laboratory, were stored at 4°C prior to extraction. Samples were usually processed and extracted within 1 week after collection. Uncontaminated reference sediment from Lake Michigan (LM), collected 10 km offshore from Bridgman, Michigan at a water depth of 40 meters, was generously provided by Dr. David White, Murray State University.
+ N
FIG. 1.
Location of sediment sampling stations.
Sample Preparation Samples were warmed to room temperature and then were air-dried for 24-48 hours in a fumehood prior to extraction. Air-dried sediment (60-100 g) was ground into small pieces and was placed in a pre-weighed cellulose thimble which was placed in a Soxhlet extraction unit. The sediment samples
were then extracted with dichloromethane for 16 hours. After extraction, the solvent was reduced to about 40 mL and triplicate I-mL aliquots were removed to determine the organic residue content (Black 1983). After residue content was determined, an appropriate amount of extract was
MACCUBBIN et al.
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solvent-exchanged into dimethyl sulfoxide (DMSO, 99+0,10 Sigma Chemical Co., St. Louis, MO) to give a final residue concentration of 10 mg/mL and was designated as the organic extract. A subsample of each sediment was also taken to determine moisture content and loss on ignition (Black 1965). Salmonella/Microsome Assay Prior to testing in the Ames test, organic extracts were diluted such that 1,000 p,g, 600 p,g, 200 p,g, 100 p,g, and 60 p,g of residue were tested for each sample. Each organic extract was tested for mutagenicity with and without metabolic activation using the standard plate incorporation protocol (Maron and Ames 1983). When testing for mutagenicity without metabolic activation, 100 p,L of organic extract was mixed with 100 p,L of an overnight culture of bacteria and 2 mL of melted agar containing 5 mM histidine and biotin. The molten top agar was then poured onto a minimal glucose agar base plate and incubated at 37°C for 2 days. The existence of compounds requiring metabolic activation was evaluated by adding 0.5 mL S9 mix to 2 mL top agar plus 100 p,L organic extract and 100 p,L bacterial culture. The S9 mix contained phosphate buffer, rat liver homogenate (Litton Bionetics, Charleston SC), and cofactors as described for high S9 mix by Maron and Ames (1983). Tester strain TA98 (a gift from Dr. B. N. Ames, University of California, Berkley) was used for all Ames tests. Plates with DMSO only or DMSO plus S9 mix were included to evaluate spontaneous mutation rates. Positive controls included daunomycin (99 + % pure, Adria Laboratories, Columbus, OH) and benzo(a)pyrene (Gold label 99 + % pure, Aldrich, Milwaukee, WI). Each dilution of extract and controls were assayed in triplicate. After incubation, the number of revertant colonies were counted (His+ revertants). Dose response data were analyzed using the 2-fold rule to determine mutagenicity (Chu et al. 1981). Those samples that caused doubling of His + revertants above controls at one or more dose were analyzed to determine His + revertants/mg residue. His + revertants/mg residue were determined from linear segments of dose response curves by linear regression analysis and converted to His + revertants/g dry weight sediment based on the residue content of each sediment.
TABLE 1. Characterization of sediments assayed in the Ames Test. 070
Station LM 82 83 25 25A III 77
110 30UP 30CR 30 30AC 104 34 104
107 112 41 42 43 44A 113 45 47
114 49 51 52 53 54 59A
Loss on Ignition b
Extractablec
NDd
2.9
14.8 32.7 41.5 31.8 43.6 42.0 50.5 6504 60.8 61.2 50.3 37.2 40.9 37.7 62.8
ND
0.05 0.04 0.07 0.18 0.09 0.79 0.21 0.85 1.20 1048 0.84 0.34 0.26 1.77 0.70 1.57 1.86 0042 2.27 0.67 0.06 0.21 0.32 0.17 0.39 0.23 0.05 0.02 0044
Moisture a
ND 3404 49.3 42.1
ND 30.6 37.8
ND 5704 35.1
ND 1604 45.3 35.1 32.6
3.6 5.1 2.9 9.5 5.8 12.2 13.7 12.5
10.9 5.9 5.7 9.0 6.0 12.1 9.7 5.8 12.1 11.6 104 4.5 8.7 2.8 15.1 4.3 1.5 2.5 6.9 5.5 3.3
0.14 0.20
aMoisture content was determined by weighing a sediment sample before and after drying at 60°C. bLoss on ignition is expressed as percent of dry weight of original sediment. cDichloromethane extractable material expressed as percent of dry weight of original sample. dND = not determined. All values are the mean of triplicate analyses.
RESULTS The moisture content, loss on ignition, and dichloromethane extractable material in the sediment samples are summarized in Table 1. The moisture content varied from 14.80,10 to 65.4%. Dichloromethane extractable material ranged from 0.04% for station 82 to 2.27% for station 42. The loss on ignition for each station although higher than the extractable material was linearly correlated with extractable material (correlation coefficient = .7181, P > 99.9, n = 30). All 31 sediment samples were assayed for muta-
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
genicity using tester strain TA98. Based on dose response curves, stations were placed into one of four classifications of mutagenic potential. Strongly mutagenic - those stations having an increase in His + revertants with increased concentration of organic extract and a doubling of the spontaneous reversion rate at two or more doses; mutagenic-stations exhibiting a dose response in His + revertants and a doubling at one dose; weakly mutagenic-stations exhibiting a dose response without a doubling of the spontaneous rate and non-mutagenic-stations have no response at the concentrations of organic residue tested. Examples of dose response curves typical for each classification are given in Figure 2. Without S9 activation, all of the organic extracts tested were weakly or non-mutagenic at doses that were nontoxic to the bacterial tester strain (data not shown). In general, without S9 treatment, organic extracts were toxic at higher doses tested (200 /lg/plate and greater) as evidenced by reduced His + revertants and depletion of the background lawn (Maron and Ames 1983). Thus, toxicity prevented testing for mutagenicity at doses greater than 200 /lg/plate. In contrast, the results of Ames tests with metabolic activation demonstrated that organic extracts of sediment from all but three of the stations caused increases in His + revertants. Of the 31 sediment samples tested, 16 stations were classified as either mutagenic or strongly mutagenic. His + revertants/mg of organic extract for these 16 stations were calculated from linear portions of dose response curves. His+ revertants/mg organic extract ranged from 20-370 (Table 2). His+ revertants/g of sediment were also determined from the His + revertants/mg of organic extract and the amount of extractable material per g of sediment. His + revertants/g ranged from 80-2,600 (Table 2). Toxicity was also observed at higher doses of organic extract incubated with S9. However, in general, toxicity was not observed until the levels of extract exceeded 600 /lg/plate. DISCUSSION
More than half (16/31) of the sediments analyzed in this study contained organic solvent extractable chemicals that could be activated to mutagens that caused two-fold or greater increases in His + revertants above spontaneous levels in the Ames test. This is not surprising since high levels of organic contaminants have been detected in sediments
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from the study area (Furlong et at. 1988). All but one of the stations that had mutagenic sediments were found in the Trenton Channel between Grosse lIe and the mainland. This stretch of the Detroit River is a depositional zone and several industries discharge wastewater into the channel (Furlong et at. 1988). The nature of the chemicals causing mutagenicity and the source of these chemicals is unknown at the present time. Additional studies will be required to determine the exact chemicals and potential sources. Although comparisons of our results with results from other studies ae difficult because of differences in sample preparation techniques, qualitative and quantitative differences in sediment contaminants and variations in the testing protocol, certain generalities can be made about the mutagenic potential of sediments from the Detroit River. In general, the sediments we tested had a lower mutagenic potential than that found in the Buffalo River (New York) or the Grand Calumet River (Indiana). For example, the highest levels of His + revertants/g of sediment in the Buffalo River were found to range from 2,898-12,000 (Black et at. 1980, Maccubbin 1986, Ersing 1987) when tested with metabolic activation. Similarly, sediments from ten stations in the Grand Calumet River had an average mutation rate of 23,500 His + revertants/g (Maccubbin 1991). These values are higher than the highest values we observed in the Detroit River. Moreover, sediments from both the Buffalo and Grand Calumet rivers contained direct-acting mutagens as determined by mutagenic responses in the Ames test without metabolic activation (Maccubbin 1986, 1991; Ersing 1987). In the present study, no direct-acting mutagens were observed. In contrast to studies in the Buffalo and Grand Calumet rivers, sediments from the Black River (Ohio) were less mutagenic than Detroit River sediments. Black River sediment samples caused approximately 200 His+ revertants/g with metabolic activation (Maccubbin 1986). However, in the case of the Black River, it should be pointed out that fractionation of organic extracts resulted in higher reversion rates (Maccubbin 1986; West et at. 1986a, 1986b, 1988), suggesting the existance of compounds in the unfractionated organic extract that inhibited or suppressed mutagenesis. The values for reversion rates described in this report may represent minimum estimates of the mutagenic potential of chemicals contained in Detroit River sediments. As mentioned above, the
FIG. 2. Examples of dose response curves used for classifying the mutagenic potential of sediments from the Detroit River. A) Non-mutagenic response over the dose range tested. B) Weakly mutagenic response as determined by increases in His+ revertants that were not twice the spontaneous reversion rate. C) Mutagenic response as determined by an increase in His+ revertants that was more than twice the spontaneous rate at one dose (600 p,g). D) Strongly mutagenic as determined by increases in His+ revertants that were more than twice the spontaneous at more than one dose (200-1,000 p,g). Organic extracts were tested in strain TA98 with metabolic activation. All points are the mean of three plates per level of residue and the lines through the points are one standard deviation.
mutagenic response can be suppressed in the presence of certain compounds. For example, complex mixtures of aromatic compounds from coalderived oil (Haugen and Peak 1983) and petroleum distillates (Carver et at. 1985) inhibited the mutagenic response to known mutagens. This inhibition was presumably caused by partial inactivation of enzymes in the S9 mix that are needed to activate some mutagens. An additional source of suppression of mutagenic response could come from chemicals in the crude extract that are toxic or inhibitory to the tester strain used. Our results demonstrated that there were substantial amounts
of toxic materials in the crude extracts a evidenced by depleted background lawns of bacteria at high residue concentrations. This toxicity was usually reduced with S9 treatment, suggesting some of the toxic materials were metabolized. However, in several cases, toxicity of extracts appeared to block the mutagenic response. Support for this suggestion comes from preliminary studies in which the mutagenicity data were examined by a computer model that accounts for toxicity (Stead et at. 1981). Analysis of the data using this model demonstrated that, with the exception of stations LM and 83, with metabolic activation, doses at which reversion
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER TABLE 2. River"
Mutagenicity ofsediments from the Detroit
His+ His+ Linear Correlation Revertants/ge Coefficientd Station b Revertants/mg c 25 30CR 34 41 43 44A 45 47 51 77 82 105 110 111 112 113
160 30 30 120 170 230 100 370 260 160 210 20 150 190 140 350
.81 .75 .67 .82 .91 .92 .60 .93 .83 .76 .72
.54 .87 .75 .88 .95
288 444 531 504 1,139 138 320 629 130 336 84 140 1,275 1,501 2,604 735
aTester strain TA98 of Salmonella typhimurium was used with S9. Solvent control: 26 ± 8 His + revertants. Positive control (Benzo[a]pyrene): 112±30 His+ revertants. bStation numbers are from Figure I. cHis + revertants/mg organic extract was calculated from the linear portion of dose response curves by linear regression analysis of net His+ revertants and p.g of organic extract. dCorreiation coefficients were significant at p > .95 for all stations except 45 and 105 which were significant at p > .90. eHis + revertants/g sediment were determined from the His +/mg organic extract and the 070 extractable material in a gram of sediment.
rates doubled could be calculated for Detroit River sediments (Parkerton, Maccubbin, and DiToro, unpublished). Moreover, similar analysis of results of Ames tests without metabolic activation revealed doses of organic extract that caused doubling of background reversion rates for about 50070 of the stations (Parkerton et al. unpublished). Finally, we only tested the mutagenic potential of the sediment extracts with one tester strain, TA98. TA98 detects frameshift mutagens and is widely used in general mutagenicity testing (Maron and Ames 1983). However, it is possible that some frameshift mutagens can be missed by TA98 and the use of an additional strain, e.g., TA97, would increase detection of this type of mutagen. In addition, it is possible that the other mutagens could be detected by assaying with different tester strains TAlOO (for base-pair mutagens) or TA102 (for oxidative mutagens) (Maron and Ames 1983). Although the results of the Ames tests on Detroit River sediments may underestimate the mutagenic potential, they provide a rapid way to
319
get relative measures of mutagenicity. The exact cause of the mutagenic response warrants further experimentation and results of the Ames test can be used to choose sediments for more extensive analysis. In addition, the Ames test data can be combined with other data from chemical analysis, acute toxicity testing, and so forth to form the basis of a ranking system for sites with contaminated sediments (Kreis and Woodring 1988). This ranking system may prove to be a valuable tool for ecosystem managers in deciding what action to take on contaminated sediments and to determine priorities for remediation of polluted waterways . ACKNOWLEDGMENTS
This work was supported by cooperative agreement CR812575 between the U.S. Environmental Protection Agency and Roswell Park Cancer Institute. We thank Karen Schrader for her assistance in preparing the manuscript. REFERENCES Alexander, M. 1981. Biodegradation of chemicals of environmental concern. Science 211: 132-138. Ames, B. N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31:347-364. Black, C. A. 1965. Methods of Soil Analysis, Vol. 9 (Parts 1 and 2). Madison: American Society of Agronomy. Black, J. J. 1983. Field and laboratory studies of environmental carcinogenesis in Niagara River fish. J. Great Lakes Res. 9:326-334. _ _ _ _ , Holmes, M., Dymerski, P. P., and Zapisek, W. F. 1980. Fish tumor pathology and aromatic hydrocarbon pollution in a Great Lakes estuary. In Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment, B. K. Afgan and D. Mackay, eds., pp. 559-565. Environmental Science Research. Carver, '1. H., Machado, M. L., and MacGregor, J. A. 1985. Petroleum distillates suppress in vitro metabolic activation: Higher [S-9] required in the Salmonella/microsome mutagenicity assay. Environ. Mutagen. 7:369-379. Chu, K. C., Patel, K. M., Lin, A. H., Tarone, R. E., Linhart, M. S., and Dunkel, V. C. 1981. Evaluating statistical analyses and reproducibility of microbial mutagenicity assays. Mutation Res. 85: 119-132. Donnelly, K. C., Brown, K. W., and Kampbell, D. 1987. Chemical and biological characterization of hazardous industrial waste. 1. Procaryotic bioaasays and chemical analysis of a wood-preserving bottomsediment waste. Mutation Res. 180:31-42.
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Douglas, G. R., Nestmann, E. R., Betts, J. L., Mueller, J. C., Lee E. G.-H, Stich, H. E, San, R. H. C., Brouzes, R. P., Chmelanskas, A. L., Paavilla, H. D. and Walden, C. C. 1980. Mutagenic activity in pulp mill effluents. In Water Chlorination: Environmental Impact and Health Effects, Vol. 3, ed. R. L. Jolley, W. A. Brungs and R. B. Cumming, pp. 865-880, Ann Arbor Science. Dunn, B. P. 1990. DNA-carcinogen adducts in fish as a tool for measuring the effective biological dose of aquatic carcinogens. In In Situ Evaluations of Biological Hazards of Environmental Pollutants, ed. S. S. Sandhu et al. pp. 177-181, Plenum Press, NY. _ _ _ _ , Black, J. J., and Maccubbin, A. E. 1987. 3zP-postlabeling analysis of aromatic DNA adducts in fish from polluted areas. Cancer Res. 47:6543-6548. Ersing, N. 1987. Mutagenic and carcinogenic potential of chemically contaminated sediments from the Buffalo River. M.S. thesis, University of Buffalo, Buffalo, NY. Fabacher, D. L., Schmitt, C. J., Besser, J. M., and Mac, M. J. 1988. Chemical characterization and mutagenic properties of polycyclic aromatic compounds in sediment from tributaries of the Great Lakes (USA). Environ. Toxico/. Chem. 7:529-544. Fallon, M. E., and Horvath, E J. 1985. Preliminary assessment of contaminants in soft sediments of the Detroit River. J. Great Lakes Res. 11 :373-378. Furlong, E. T., Carter, D. S., and Hites, R. A. 1988. Organic chemical contaminants in sediments from the Trenton Channel of the Detroit River, Michigan. J. Great Lakes Res. 14:489-501. Giesy, J. P., Rosiu, C. J., Graney, R. L., Newsted, J. L., Benda, A., Kreis, R. G. Jr., and Horvath, F. J. 1988. Toxicity of Detroit River sediment interstitial water to the bacterium Photobacterium phosphoreum. J. Great Lakes Res. 14:502-513. Hamdy, Y., and Post, L. 1985. Distribution of mercury, trace organics and other heavy metals in Detroit River sediments. J. Great Lakes Res. 11 :353-365. Haugen, D. A., and Peak, M. J. 1983. Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation. Mutation Res. 116:257-267. Kaiser, K. L. E., Comba, M. E., Hunter, H., Maguire, R. J., Tkacz, R. J., and Platford, R. F. 1985. Trace organic contaminants in the Detroit River. J. Great Lakes Res. 11 :386-399. Kamra, O. P., Nestmann, E. R., Douglas, G. R., Kowbel, D. J. and Harrington, T. R. 1983. Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays. Mutation Res. 118:269-276. Kinae, N., Hashizume, T., Makita, T., Tomita, I., Kimua, I., and Kanamori, H. 1981. Studies on the toxicity of pulp and paper mill effluents. I. Muta-
genicity of the sediment samples derived from Kraft paper mills. Water Res. 15:17-24. Kraybill, H. F. 1976. Distribution of chemical carcinogens in aquatic environments. Prog. Exp. Tumor Res. 20:3-34. Kreis, R. G., Jr., and Woodring, D. 1988. Development of a sediment action index for the Great Lakes: The lower Detroit River as a pilot application. Abstract Internat. Assoc. Great Lakes Res. 31st Conference, Hamilton, Ontario, May 1988. Lum, K. R., and Gammon, K. L. 1985. Geochemical availability of some trace and major elements in surficial sediments of the Detroit River and western Lake Erie. J. Great Lakes Res. 11 :328-338. Maccubbin, A. E. 1986. Mutagenicity of sediments from the Great Lakes ecosystem. Abstracts, 29th Conference on Great Lakes Research, p. 58. International Association for Great Lakes Research. _ _ _ _ . 1991. Mutagenic potential of sediments from the Grand Calumet River. Bull. Environ. Contam. Toxicol., in press. _ _ _ _ , and Ersing, N. 1990. Tumors in fish from the Detroit River. Hydrobiologia, in press. _ _ _ _ , Black, J. J., and Harshbarger, J. C. 1987. A case report of hepatocellular neoplasia in bowfin, Amia calva. J. of Fish Diseases 10:329-331. _ _ _ _ , Black, J. J., and Dunn, B. P. 1990. 32p_ postlabeling detection of DNA adducts in fish from chemically contaminated waterways. Science of the Total Environ. 94:89-104. Maguire, R. J., Tkacz, R. J., and Sartor, D. L. 1985. Butyltin species and inorganic tin in water and sediment of the Detroit and St. Clair Rivers. J. Great Lakes Res. 11 :320-327. Maron, D. M., and Ames, B. N. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215. Metcalfe, C. D., Sonstegard, R. A., and Quilliam, M. A. 1985. Genotoxic activity of particulate material in petroleum refinery effluents. Bull. Environ. Contam. Toxicol. 35:240-248. Munawar, M., Mudroch, A., Munawar, I. F., and Thomas, R. L. 1983. The impact of sedimentassociated contaminants from the Niagara River mouth on various size assemblages of phytoplankton. J. Great Lakes Res. 9:303-313. _ _ _ _ , Thomas, R. L., Norwood, W., and Mudroch, A. 1985. Toxicity of Detroit River sediment-bound contaminants to ultraplankton. J. Great Lakes Res. 11 :264-274. Pittinger, C. A., Buikema, A. L. Jr., and Falkinham, J. O. III. 1987. In situ variations in oyster mutagenicity and tissue concentrations of polycyclic aromatic hydrocarbons. Environ. Toxicol. and Chem. 6:51-60. Platford, R. E, Maguire, R. J., Tkacz, R. J., Comba, M. E., and Kaiser, K. L. E. 1985. Distribution of
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER hydrocarbons and chlorinated hydrocarbons in various phases of the Detroit River. J. Great Lakes Res. 11:379-385. Pugsley, C. W., Hebert, P. D. N., Wood, G. W., Brotea, G., and Obal, T. W. 1985. Distribution of contaminants in clams and sediments from the HuronErie corridor. I-PCBS and octachlorostyrene. J. Great Lakes Res. 11 :275-289. Sato, T., Momma, T., Ose, Y., Ishikawa, T., and Kato, K. 1983. Mutagenicity of Nagara River sediments. Mutation Res. 118:257-267. _ _ _ _ , Kato, K., Ose, Y., Nagose, H., and Ishikawa, T. 1985. Nitroarenes in Suimon River sediment. Mutation Res. 157:135-143. Smith, V. E., Spurr, J. M., Filkins, J. C., and Jones, J. J. 1985. Organochlorine contaminants of wintering ducks foraging on Detroit River sediments. J. Great Lakes Res. 11:231-246. Stead, A. G., Hasselblad, V., Creason, J. P., and Claxton, L. 1981. Modeling the Ames test. Mutation Res. 85:13-27. Struger, J., Weseloh, D. V., Hallett, D. J., and Mineau, P. 1985. Organochlorine contaminants in herring gull eggs from the Detroit and Niagara Rivers and Saginaw Bay (1978-1982): Contaminant discriminants. J. Great Lakes Res. 11:223-230.
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Suns, K. Crawford, G., and Russell, D. 1985. Organochlorine and mercury residues in young-ofthe-year spottail shiners from the Detroit River, Lake St. Clair, and Lake Erie. J. Great Lakes Res. 11:347-352. Thornley, S., and Hamdy, Y. 1984. An assessment of the bottom fauna and sediments of the Detroit River. Ontario Ministry of the Environment Report, Toronto, Ontario. _ _ _ _ , and Hamdy, Y. 1985. Macrozoobenthos of the Detroit and St. Clair Rivers with comparisons to neighboring waters. J. Great Lakes Res. 11:290-296. West, W. R., Smith, P. A., Booth, G. M., Wise, S. A., and Lee, M. L. 1986a. Determination of genotoxic polycyclic aromatic hydrocarbons in a sediment from the Black River (Ohio). Arch. Environ. Contam. Toxieo!. 15:241-249. _ _ _ _ , Smith, P. A., Booth, G. M., and Lee, M. L. 1986b. Determination and genotoxicity of nitrogen heterocycles in a sediment from the Black River. Environ. Toxieo!. and Chem. 5:511-519. _ _ _ _ , Smith, P. A., Booth, G. M., and Lee, M. L. 1988. Isolation and detection of genotoxic components in a Black River sediment. Environ. Sci. Teehno/. 22:224-228.
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
Alexander E. Maccubbin l •• , Noreen Ersing l , and Mary Ellen Frank Department of Experimental Biology Roswell Park Cancer Institute Elm and Carlton Streets Buffalo, New York 14263
ABSTRACT. Sediment samples from 30 sites in the lower Detroit River and one site in Lake Michigan were extracted with organic solvent and were tested for mutagenicity in the Ames test. Without metabolic activation, sediment samples were either non-mutagenic or weakly mutagenic. With metabolic activation, mutagenic responses (defined as doubling of the spontaneous mutation rate at one or more concentrations of organic extract) were observed in 16 of 31 samples. Mutation rates ranged from 20 to 370 histidine positive revertants per mg of organic extract. These results demonstrate the existence of compounds in sediments of the Detroit River that can be activated to mutagens. The sediments thus are a potential source of mutagenic and possibly carcinogenic compounds for fish and other organisms. INDEX WORDS: Toxic substances, Detroit River, sediments, bioassay, mutagen.
INTRODUCTION
contaminated waterways has received much attention in the past few years. There have been a number of studies describing toxic effects of contaminated sediments. Thornley and Hamdy (1984, 1985) analyzed macrobenthos of the Detroit River and neighboring waters and demonstrated that pollution in the Detroit River significantly altered benthic assemblages. In laboratory studies, chemicals bound to sediments from the Detroit River have been found to be toxic to phytoplankton (Munawar et al. 1983, 1985) and bacteria (Giesy et al. 1988). Several other studies have demonstrated the presence of contaminants or metabolites of contaminants in ducks (Smith et al. 1985), gulls (Struger et al. 1985), clams (Pugsley et al. 1985), and fish (Suns et al. 1985, Maccubbin et al. 1990). In addition, chemical-DNA adducts have been observed in liver tissue from fish collected from the Detroit River (Dunn et al. 1987, Maccubbin et al. 1990) and tumors have been found in several species of fish (Maccubbin et al. 1987, 1990; Maccubbin and Ersing 1990). The detection of chemical-DNA adducts provides a direct measurement of exposure to genotoxic agents (Dunn 1990). Moreover, some of the chemicals found in the sediments of the Detroit River are known genotoxins [see Kraybill (1976) for a discussion of carcinogens in the aquatic envi-
Waterways, such as the Detroit River, that flow through urban areas receive chemical discharges from industrial, municipal, and agricultural sources. Many of the chemicals contained in these discharges are hydrophobic in nature and are sequestered by sediment particles that eventually settle in deposition zones within the waterway (Furlong et al. 1988). In addition to being hydrophobic, some sediment-bound chemicals may resist microbial degradation and may accumulate in high concentrations in sediment or biota (Alexander 1981, Furlong et al. 1988). Within the Detroit River system, sediments have been found to be contaminated with a wide array of chemicals including organochlorine pesticides, heavy metals, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons (Fallon and Horvath 1985, Hamdy and Post 1985, Kaiser et al. 1985, Lum and Gammon 1985, Maguire et al. 1985, Platford et al. 1985, Furlong et al. 1988). The potential effect of such sediment-bound chemicals on the biota of I Current
Address: Department of Experimental Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263 aTo whom reprint requests should be addressed.
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MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
ronment]. Although the liver tumors observed in fish from the Detroit River may have multiple causes, many of the fish are bottom dwelling/ feeding species that potentially could be exposed to sediments containing genotoxic chemicals. The Salmonella/microsome mutagenicity assay or the Ames test (Ames et 01. 1975) has been widely used to analyze chemicals for their mutagenic potential. In most cases, pure chemicals have been analyzed. However, the Ames test also has been used to analyze complex mixtures of chemicals extracted from a variety of sources such as pulpmill effluents (Douglas et 01. 1980, Kinae et 01. 1981, Kamra et 01. 1983), petroleum refinery effluents (Metcalfe et 01. 1985), and wastes from the wood-preserving industry (Donnelly et 01. 1987). In addition the Ames test has been used to analyze organic solvent extracts of sediments collected from chemically contaminated rivers (Black et 01. 1980; Sato et 01. 1983, 1985; West et 01. 1986a, 1986b, 1988; Pittinger et 01. 1987; Fabacher et 01. 1988). We have used the Ames test to screen sediment samples for mutagenic potential as a measure of genotoxicity and in this report we describe the results of our studies on sediments from the Detroit River. MATERIALS AND METHODS
Sampling Sediments were collected from 30 stations in the lower Detroit River (Fig. 1). Multiple grab samples were placed in large tubs and were thoroughly mixed: subsamples were then placed in prewashed (nitric acid followed by hexane) jars which were sealed. Samples were kept on ice in the field and, upon returning to the laboratory, were stored at 4°C prior to extraction. Samples were usually processed and extracted within 1 week after collection. Uncontaminated reference sediment from Lake Michigan (LM), collected 10 km offshore from Bridgman, Michigan at a water depth of 40 meters, was generously provided by Dr. David White, Murray State University.
+ N
FIG. 1.
Location of sediment sampling stations.
Sample Preparation Samples were warmed to room temperature and then were air-dried for 24-48 hours in a fumehood prior to extraction. Air-dried sediment (60-100 g) was ground into small pieces and was placed in a pre-weighed cellulose thimble which was placed in a Soxhlet extraction unit. The sediment samples
were then extracted with dichloromethane for 16 hours. After extraction, the solvent was reduced to about 40 mL and triplicate I-mL aliquots were removed to determine the organic residue content (Black 1983). After residue content was determined, an appropriate amount of extract was
MACCUBBIN et al.
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solvent-exchanged into dimethyl sulfoxide (DMSO, 99+0,10 Sigma Chemical Co., St. Louis, MO) to give a final residue concentration of 10 mg/mL and was designated as the organic extract. A subsample of each sediment was also taken to determine moisture content and loss on ignition (Black 1965). Salmonella/Microsome Assay Prior to testing in the Ames test, organic extracts were diluted such that 1,000 p,g, 600 p,g, 200 p,g, 100 p,g, and 60 p,g of residue were tested for each sample. Each organic extract was tested for mutagenicity with and without metabolic activation using the standard plate incorporation protocol (Maron and Ames 1983). When testing for mutagenicity without metabolic activation, 100 p,L of organic extract was mixed with 100 p,L of an overnight culture of bacteria and 2 mL of melted agar containing 5 mM histidine and biotin. The molten top agar was then poured onto a minimal glucose agar base plate and incubated at 37°C for 2 days. The existence of compounds requiring metabolic activation was evaluated by adding 0.5 mL S9 mix to 2 mL top agar plus 100 p,L organic extract and 100 p,L bacterial culture. The S9 mix contained phosphate buffer, rat liver homogenate (Litton Bionetics, Charleston SC), and cofactors as described for high S9 mix by Maron and Ames (1983). Tester strain TA98 (a gift from Dr. B. N. Ames, University of California, Berkley) was used for all Ames tests. Plates with DMSO only or DMSO plus S9 mix were included to evaluate spontaneous mutation rates. Positive controls included daunomycin (99 + % pure, Adria Laboratories, Columbus, OH) and benzo(a)pyrene (Gold label 99 + % pure, Aldrich, Milwaukee, WI). Each dilution of extract and controls were assayed in triplicate. After incubation, the number of revertant colonies were counted (His+ revertants). Dose response data were analyzed using the 2-fold rule to determine mutagenicity (Chu et al. 1981). Those samples that caused doubling of His + revertants above controls at one or more dose were analyzed to determine His + revertants/mg residue. His + revertants/mg residue were determined from linear segments of dose response curves by linear regression analysis and converted to His + revertants/g dry weight sediment based on the residue content of each sediment.
TABLE 1. Characterization of sediments assayed in the Ames Test. 070
Station LM 82 83 25 25A III 77
110 30UP 30CR 30 30AC 104 34 104
107 112 41 42 43 44A 113 45 47
114 49 51 52 53 54 59A
Loss on Ignition b
Extractablec
NDd
2.9
14.8 32.7 41.5 31.8 43.6 42.0 50.5 6504 60.8 61.2 50.3 37.2 40.9 37.7 62.8
ND
0.05 0.04 0.07 0.18 0.09 0.79 0.21 0.85 1.20 1048 0.84 0.34 0.26 1.77 0.70 1.57 1.86 0042 2.27 0.67 0.06 0.21 0.32 0.17 0.39 0.23 0.05 0.02 0044
Moisture a
ND 3404 49.3 42.1
ND 30.6 37.8
ND 5704 35.1
ND 1604 45.3 35.1 32.6
3.6 5.1 2.9 9.5 5.8 12.2 13.7 12.5
10.9 5.9 5.7 9.0 6.0 12.1 9.7 5.8 12.1 11.6 104 4.5 8.7 2.8 15.1 4.3 1.5 2.5 6.9 5.5 3.3
0.14 0.20
aMoisture content was determined by weighing a sediment sample before and after drying at 60°C. bLoss on ignition is expressed as percent of dry weight of original sediment. cDichloromethane extractable material expressed as percent of dry weight of original sample. dND = not determined. All values are the mean of triplicate analyses.
RESULTS The moisture content, loss on ignition, and dichloromethane extractable material in the sediment samples are summarized in Table 1. The moisture content varied from 14.80,10 to 65.4%. Dichloromethane extractable material ranged from 0.04% for station 82 to 2.27% for station 42. The loss on ignition for each station although higher than the extractable material was linearly correlated with extractable material (correlation coefficient = .7181, P > 99.9, n = 30). All 31 sediment samples were assayed for muta-
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER
genicity using tester strain TA98. Based on dose response curves, stations were placed into one of four classifications of mutagenic potential. Strongly mutagenic - those stations having an increase in His + revertants with increased concentration of organic extract and a doubling of the spontaneous reversion rate at two or more doses; mutagenic-stations exhibiting a dose response in His + revertants and a doubling at one dose; weakly mutagenic-stations exhibiting a dose response without a doubling of the spontaneous rate and non-mutagenic-stations have no response at the concentrations of organic residue tested. Examples of dose response curves typical for each classification are given in Figure 2. Without S9 activation, all of the organic extracts tested were weakly or non-mutagenic at doses that were nontoxic to the bacterial tester strain (data not shown). In general, without S9 treatment, organic extracts were toxic at higher doses tested (200 /lg/plate and greater) as evidenced by reduced His + revertants and depletion of the background lawn (Maron and Ames 1983). Thus, toxicity prevented testing for mutagenicity at doses greater than 200 /lg/plate. In contrast, the results of Ames tests with metabolic activation demonstrated that organic extracts of sediment from all but three of the stations caused increases in His + revertants. Of the 31 sediment samples tested, 16 stations were classified as either mutagenic or strongly mutagenic. His + revertants/mg of organic extract for these 16 stations were calculated from linear portions of dose response curves. His+ revertants/mg organic extract ranged from 20-370 (Table 2). His+ revertants/g of sediment were also determined from the His + revertants/mg of organic extract and the amount of extractable material per g of sediment. His + revertants/g ranged from 80-2,600 (Table 2). Toxicity was also observed at higher doses of organic extract incubated with S9. However, in general, toxicity was not observed until the levels of extract exceeded 600 /lg/plate. DISCUSSION
More than half (16/31) of the sediments analyzed in this study contained organic solvent extractable chemicals that could be activated to mutagens that caused two-fold or greater increases in His + revertants above spontaneous levels in the Ames test. This is not surprising since high levels of organic contaminants have been detected in sediments
317
from the study area (Furlong et at. 1988). All but one of the stations that had mutagenic sediments were found in the Trenton Channel between Grosse lIe and the mainland. This stretch of the Detroit River is a depositional zone and several industries discharge wastewater into the channel (Furlong et at. 1988). The nature of the chemicals causing mutagenicity and the source of these chemicals is unknown at the present time. Additional studies will be required to determine the exact chemicals and potential sources. Although comparisons of our results with results from other studies ae difficult because of differences in sample preparation techniques, qualitative and quantitative differences in sediment contaminants and variations in the testing protocol, certain generalities can be made about the mutagenic potential of sediments from the Detroit River. In general, the sediments we tested had a lower mutagenic potential than that found in the Buffalo River (New York) or the Grand Calumet River (Indiana). For example, the highest levels of His + revertants/g of sediment in the Buffalo River were found to range from 2,898-12,000 (Black et at. 1980, Maccubbin 1986, Ersing 1987) when tested with metabolic activation. Similarly, sediments from ten stations in the Grand Calumet River had an average mutation rate of 23,500 His + revertants/g (Maccubbin 1991). These values are higher than the highest values we observed in the Detroit River. Moreover, sediments from both the Buffalo and Grand Calumet rivers contained direct-acting mutagens as determined by mutagenic responses in the Ames test without metabolic activation (Maccubbin 1986, 1991; Ersing 1987). In the present study, no direct-acting mutagens were observed. In contrast to studies in the Buffalo and Grand Calumet rivers, sediments from the Black River (Ohio) were less mutagenic than Detroit River sediments. Black River sediment samples caused approximately 200 His+ revertants/g with metabolic activation (Maccubbin 1986). However, in the case of the Black River, it should be pointed out that fractionation of organic extracts resulted in higher reversion rates (Maccubbin 1986; West et at. 1986a, 1986b, 1988), suggesting the existance of compounds in the unfractionated organic extract that inhibited or suppressed mutagenesis. The values for reversion rates described in this report may represent minimum estimates of the mutagenic potential of chemicals contained in Detroit River sediments. As mentioned above, the
FIG. 2. Examples of dose response curves used for classifying the mutagenic potential of sediments from the Detroit River. A) Non-mutagenic response over the dose range tested. B) Weakly mutagenic response as determined by increases in His+ revertants that were not twice the spontaneous reversion rate. C) Mutagenic response as determined by an increase in His+ revertants that was more than twice the spontaneous rate at one dose (600 p,g). D) Strongly mutagenic as determined by increases in His+ revertants that were more than twice the spontaneous at more than one dose (200-1,000 p,g). Organic extracts were tested in strain TA98 with metabolic activation. All points are the mean of three plates per level of residue and the lines through the points are one standard deviation.
mutagenic response can be suppressed in the presence of certain compounds. For example, complex mixtures of aromatic compounds from coalderived oil (Haugen and Peak 1983) and petroleum distillates (Carver et at. 1985) inhibited the mutagenic response to known mutagens. This inhibition was presumably caused by partial inactivation of enzymes in the S9 mix that are needed to activate some mutagens. An additional source of suppression of mutagenic response could come from chemicals in the crude extract that are toxic or inhibitory to the tester strain used. Our results demonstrated that there were substantial amounts
of toxic materials in the crude extracts a evidenced by depleted background lawns of bacteria at high residue concentrations. This toxicity was usually reduced with S9 treatment, suggesting some of the toxic materials were metabolized. However, in several cases, toxicity of extracts appeared to block the mutagenic response. Support for this suggestion comes from preliminary studies in which the mutagenicity data were examined by a computer model that accounts for toxicity (Stead et at. 1981). Analysis of the data using this model demonstrated that, with the exception of stations LM and 83, with metabolic activation, doses at which reversion
MUTAGENICITY OF SEDIMENTS FROM THE DETROIT RIVER TABLE 2. River"
Mutagenicity ofsediments from the Detroit
His+ His+ Linear Correlation Revertants/ge Coefficientd Station b Revertants/mg c 25 30CR 34 41 43 44A 45 47 51 77 82 105 110 111 112 113
160 30 30 120 170 230 100 370 260 160 210 20 150 190 140 350
.81 .75 .67 .82 .91 .92 .60 .93 .83 .76 .72
.54 .87 .75 .88 .95
288 444 531 504 1,139 138 320 629 130 336 84 140 1,275 1,501 2,604 735
aTester strain TA98 of Salmonella typhimurium was used with S9. Solvent control: 26 ± 8 His + revertants. Positive control (Benzo[a]pyrene): 112±30 His+ revertants. bStation numbers are from Figure I. cHis + revertants/mg organic extract was calculated from the linear portion of dose response curves by linear regression analysis of net His+ revertants and p.g of organic extract. dCorreiation coefficients were significant at p > .95 for all stations except 45 and 105 which were significant at p > .90. eHis + revertants/g sediment were determined from the His +/mg organic extract and the 070 extractable material in a gram of sediment.
rates doubled could be calculated for Detroit River sediments (Parkerton, Maccubbin, and DiToro, unpublished). Moreover, similar analysis of results of Ames tests without metabolic activation revealed doses of organic extract that caused doubling of background reversion rates for about 50070 of the stations (Parkerton et al. unpublished). Finally, we only tested the mutagenic potential of the sediment extracts with one tester strain, TA98. TA98 detects frameshift mutagens and is widely used in general mutagenicity testing (Maron and Ames 1983). However, it is possible that some frameshift mutagens can be missed by TA98 and the use of an additional strain, e.g., TA97, would increase detection of this type of mutagen. In addition, it is possible that the other mutagens could be detected by assaying with different tester strains TAlOO (for base-pair mutagens) or TA102 (for oxidative mutagens) (Maron and Ames 1983). Although the results of the Ames tests on Detroit River sediments may underestimate the mutagenic potential, they provide a rapid way to
319
get relative measures of mutagenicity. The exact cause of the mutagenic response warrants further experimentation and results of the Ames test can be used to choose sediments for more extensive analysis. In addition, the Ames test data can be combined with other data from chemical analysis, acute toxicity testing, and so forth to form the basis of a ranking system for sites with contaminated sediments (Kreis and Woodring 1988). This ranking system may prove to be a valuable tool for ecosystem managers in deciding what action to take on contaminated sediments and to determine priorities for remediation of polluted waterways . ACKNOWLEDGMENTS
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Suns, K. Crawford, G., and Russell, D. 1985. Organochlorine and mercury residues in young-ofthe-year spottail shiners from the Detroit River, Lake St. Clair, and Lake Erie. J. Great Lakes Res. 11:347-352. Thornley, S., and Hamdy, Y. 1984. An assessment of the bottom fauna and sediments of the Detroit River. Ontario Ministry of the Environment Report, Toronto, Ontario. _ _ _ _ , and Hamdy, Y. 1985. Macrozoobenthos of the Detroit and St. Clair Rivers with comparisons to neighboring waters. J. Great Lakes Res. 11:290-296. West, W. R., Smith, P. A., Booth, G. M., Wise, S. A., and Lee, M. L. 1986a. Determination of genotoxic polycyclic aromatic hydrocarbons in a sediment from the Black River (Ohio). Arch. Environ. Contam. Toxieo!. 15:241-249. _ _ _ _ , Smith, P. A., Booth, G. M., and Lee, M. L. 1986b. Determination and genotoxicity of nitrogen heterocycles in a sediment from the Black River. Environ. Toxieo!. and Chem. 5:511-519. _ _ _ _ , Smith, P. A., Booth, G. M., and Lee, M. L. 1988. Isolation and detection of genotoxic components in a Black River sediment. Environ. Sci. Teehno/. 22:224-228.