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The goal of this thesis was to examine the sources and ecological behaviour of genotoxic organics in the St. Lawrence ri

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Canada

• DETECTION, DlSCHARGE AND ECOLOGICAL BEHAVIOUR OF GENOTOXIC ORGANIC CONTAMINANTS IN THE ST. LAWRENCE AND SAGUENAY RIVERS

Paul Andrew White Department of Biology, McGill University

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree Doctor of Philosophy

November, 1995.



© Paul Andrew White

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ISBN 0-612-12510-6

Canada

1





AB5TRACT The goal of this thesis was to examine the sources and ecological behaviour of genotoxic organics in the St. Lawrence river system. A rapid and effective version of the SOS Chromotest was developed to accomplish the task of genotoxicity assessment. The method, validated 'Vith standard reference materials, ;s particularly weil suited to comple~ environmer.tal e~tracts. The endpoint investigated throughout the thesis, SOS genolOxicity, is empirically related 10 more familiar endpoints such as mutageniciLy and carcinogenicity. Analyses of lite.. .ature '---,

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significantly higher tissue levels of BaP equivalents than pike/1arge perch, which in tum are higher than walleye. When available evidence indieates that contaminants partition inte the lipid pools of exposed bio!a, it is conventional that chemical concentrations be cxpressed on a per unit lipid basis (Thoman, 1989). Both models (1) and (2) indicate that the coefficient oflog lipd content is not significantly different from 1.0. Therefore, the lipid effect is strong enough te justify lipid-correction ofgenotoxicity values. Figure 5 compares the mean lipid normalized concentrations ofgenotoxins for the different biota samples investigated. In contrast to the results summarized in Table 2, the results indieate that after eotreetion for differences in lipid content, macroinvertebrate samples are more contaminated than fish. ANOVA comparing only the mean fish value te the mean invertebrate value revealed a



326

Figure 5. Comparison of mean lipid nonnalized tissue concentrations of genoroxic substances. Values are arithmetic means. Error bars are one standard error of the mean. Leners adjacent to bars indicate the results of post-hoc contrasts. Separate ANOVAs were perfonncd for each group of bars. Bars accompanied by the sarne lener are not significantly different at «=0.05.

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significant difference (F Ratio = 6.1. p < 0.02). A separate ANOVA revealed a significant effect of fish-type on mean genotoxic contamination. The results in Figures 3 and 4 do not address the fact that previous industrial effluent analyses (White et al.• 1995d) indicated that genotoxic loadings at the sites examined vary from less than 1 g BaP equivalent per day at La Prairie to over 30 kg per day at Pointe-auxTrembles. We investigated the effeet of total regionalloading on the contamination of biota samples collected in the receiving system. The results obtained are illustrated in Figure 6. Regression analyses failed to demonstrate any significant relationship between biota contamination and regional industrialloading. Although there is no significant trend, the results suggest a negative effeet of genotoxic loading on genotoxic contamination of downstrearn aquatic biola. Despite the lack of relationship berween biota contamination and regional genotoxic loading. the biota results are consistent Sites with contaminated invenebrates tend to have contaminated fish and vice versa. Figure 7 illustrates the relationship between fish contamination and invenebrate contamination acro:;s the 12 sites for which data are available. Linear regression analyses revealed the following model (r2 =0.3'•• F Ratio = 5.1. p Cl. .--Oil

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DISCUSSION

Most publishee! research effons that employee! bioassays to investigate genmoxins and mutagens in aquatic biota have been resoietee! to bivalve molluscs. They are sessile, longlivee! suspension feeders known to accumulate genmoxic carcinogens such as BaP (Rodriguez-Ariza, 1992; Dunn, 1991). For example, Rodriguez-Ariza et al. (1992) deteetee! oxidati'/e mutagens in the tissues of three ~pecies of marine molluscs collectee! from Spanish coastal regions that are contaminated with a variety of toxic metals. Marvin et al. (1994) detected both frameshift and base-pair substitution mutagens in zebra mussels (Dreissina poiymorpha) from Hamilton Harbour (Lake Ontario), an area renownee! for PAH contamination (PAH concentration> 500 ppm dry wt., Marvin et al., 1993). To our knowlee!ge ooly Kinac et al. (1981) investigatee! the accumulation ofmutagens'in fish. Using the Ames/Salmonella mutagenicity assay and a Bacillus subrilus DNA damage assay they deteetee! both genotoxins and frameshift mutagens in the Iivers of spotted sea trout (Nibea mirsukuril) collectee! from areas that receive pulp and paper mill wastes. The SOS Chromotest results obtainee! here reveal that aquatic biota in the St. Lawrence system do acclo..,ulate direct acting genotoxic substances. Although we did not thoroughly investigate the presence ofgenotoxins that require S9 activation, samples tested in the presence of S9 yielded a weaker, often negative response. Our analyses of soft-shell clams from the Saguenay Fjord revealed a similar reduction in potency upon addition of S9 (White et al., 1995b). Similar decreases in activity were observed by Rodriguez-Ariza et al. (1992) andFrezza et al.(1982). However, Marvin et al. (1994), Sparks et al. (1981) and Kira et al. (1989) observed increased mutagenicity in the presence ofatl S9 metabolic activation mixture. Both Marvin et al. (1994) and Kira et al. (1989) indicatee! that the sites examined are heavily contaminated with progenotoxic PAHs such as BaP. The results presentee! in Figure 3 indicate that although both fish and invertebrate extI'aetS contain SOS genotoxins, there is a difference in their relative ability to invoke the SOS response pathway. A lack of mechanistic understanding of SOS response induction make interpretation of these results difficult. However, they likely indicate a difference in the physical-chemical properties of the putative genotoxins accumu1ated in different types of biola. The results obtained indieate that molluscs, panicularly Mya arenaria (White et al., 1995b), accumulate genotoxins that can elicit higher SOS Induction Factors than tho~ accumulatee! by other biola. This difference is likely attributable to differences in the biochemistry of toxicant metabolism and excretion in aquatic biola. For example, metabolism ofPAHs such as BaP in teleost fish is mediated by cyIochrome P-450 monooxygenase. However, cyIochrome P-450 monooxygenase is thought to play ooly a

336





minor role in mollusc:m PAH metabolism (Stegman and Lech. 1991). Analyses of pure compounds also reveaI large differences in the level of SOS induction at which zero-order kinetics is manifested. For example. SOS Chromotest results for beilzo(a)pyrene reveal saturation of the response pathway at induction factors between 3 and 8 (White. unpublished; Quillardet, unpublished). In contras!, compounds such as 4-nitroquinoline-lmode can produce SOS induction factors that easily exceed 25, with zero-order kinc:tics manifested at values above 60 (White, unpublishc:d; Quillarde!, unpublishc:d). Organisms with a high lipid contc:nttended to be more contaminatc:d with gc:notoxic organics. This resu1t (see Figure 4) indicates that SOS genOloxins partition into the lipid pools of the exposed biola. Furthermore, the lipid trend is strong enough to justify lipid normalization. However, lipid content alone was only able to account for 22% of the variation in tissue concentrations of BaP equivalents. For a given lipid content the results reveaIed a wide range in observed tissue contamination. At 1% lipid for c:xample. there is a 3 order of magnitude variation in genotoxic contamination. ANCOVA analyses confirmc:d that part of the variability can be accounted for by biota type :\Jld reveaIed that organisms at lower trophic levels have higher 1evels of cont.'mination. When on1y the fish data were consideree!, ANCOVA reveaIed a similar pattern. In this case, fish occupying lower trophic levels were found to have higher tissue concentrations of genotoxins. While we do nOI cIaim to have detai1ed knowledge about the aquatic food chains of the St. Lawrence river, the results obtained here are supported by the trophic leve1 assignments of Vander Zanden ~ al. (1995). The resuIts presented in Figure 5 indicate that walleye have the lowest tissue 1evels of genotoxins. folloy':.:d by pike and large perch, small percb :::::!. f;!llll1y macroinvertebrates. Using diet studies and nitrogen stable isotope analyses Vander Zanden et ai. (1995) revealed that walleye usually occupy a trophic position that is higher than other piscivores such as pike. Large perch and pilee generally occupy a similar trophic position that is below walleye. Small perch generally occupy a position that is be10w pike and large perch, and slightly above crayfish. The observed effect of trophic position on the tissue concentrations of genotoxins is quite different from that observed for persistent organic contaminants such as PCBs. Efficient traDsfer of PCBs from prey to predator results in a positive effect of trophic position on lipid normalized concentrations (Rasmussen et al., 1990). For genotoxic organics, the results obtained reveaI a negative effect of trophic position on lipid normalized genotoxin concentrations. This bio-diminurion like1y results from the ability of fish 10 metabolize and excrete genotoxins contained in the tissues of their prey. Higher tissue burdens of genotoxins in invertebrates are likeIy the result of a lower trophic position and a decreased capacity for genotoxin metabolism and excretion. This decreased capacity for

337





genotolÔn metabolism is likely related to difference in MFO (mixed function oxidase) activities. Fish MFO levels are usually much higher (= 200 pmol min· t mg'!) than those of invenebrates (= 10-50 pmol min'! mg'!) (Connor. 1984; Stegman and Lech, 1991; Dunn, 1991). Although il is nOl clear how increased MFO levels would effecl SOS genoloxins, we would expecl an increased ability for metabolism and excretion of organics 10 work againsl the biomagnification of genoloxins. Although the observed effecl of trophic position on genololÔn concentration is distinctly differenl from thal observed for persislenl substances, il is similar 10 that observed for more readily metabolized substances such as PAHs. For example, fish to sediment ratios for persislent, chlorinated hydrocarbons such as DDT and PCBs are in the 1()210 1()3 range (Connor, 1984). In contrast, PAHs have little tendency to biomagnify and generally exhibit biota to sediment ratios of 0.01 10 1.0. Eadie et al. (1982) found that oligochaeles from Lake Erie have PAH levels that are approlÔmately 0.2 to 0.5 limes sediment levels. Kauss and Hamdy (1991) found that tissue to sediment ratios for genolOxic PAHs in bivalve molluscs are generally less than 1. Varanasi et al (1985) found that tissue to sediment ratios for PAHs in marine invenebrates ranged from 0.05 to 0.5, with higher values being associated with larger PAHs. Black et al. (1980) found that PAH levels in brown trout and white sucker from the Hershey river (Michigan) ranged from not deteetable to about 1% of the sediment levels. Wide variations in the biota to sediment ratios for PAHs are generally attributed 10 a variable capacity for PAH metabolism and excretion (McElroy et al, 1989; Livingstone, 1994; James, 1989; Stegman, 1981). Comparisons of the tissue genolOxicity levels obtained here, with previously published suspended particulate matter and bottom sediment results (White et al, 1995a) are consistent with PAH-like degradability. Mean concentrations of SOS genolOxins in biota are about one-tenth (0.07 to 0.17)1 the mean suspended particulate matter values (22lJ.g BaP equiv. per g dry wt. - no 59). Ratios to bottom sediment are slightly higher (0.251.25), but generally less than 1. Few researchers have published bioassay results that can he used 10 calculate similar biota to sediment ratios for genotoxic substances. The Ames/Salmonella results for zebra mussels and bottom sediment from Hamilton Harbour (Marvin et al., 1994; Marvin et al, 1993) pi:>vide invertebrate 10 bottom sedimen' "atios for mutagens that are less than 0.1. This low value Cao likely he attributed 10 the extremely high levels ofPAHs in HamillOn Harbour sediments. The surface water (SOS Chromotest) reslùs of Langevin et al. (1~'92) indicate that the St. Lawrence river contains approximately 0.35 ppD BaP equivalents. The results presented 1Ratios ofbiOla (jJ.g BaP per g dry tissue) lO sediment (J.Lg BaP per g dry sediment). Wet weighllO dry weight conversion assumed 85% wau:r content.

338





in Table 2 indicate that BCFs (bioconcentraùon factor) of genotoxins in the St. Lawrence system are = 1()2 - 103. Since Langevin et al. made an effort to sample in open waters not directly effeeted by local indusoial and municipal discharges. this value should he considered a maximum. Several pub1ished regression models relate BeF values in fish and invertebrates tO physical-chemical properùes such as Kow (Mackay, 1982: Neely et al.• 1974; Veith et al., 1979; Bysshe, 1982). However, the ability of aquatic biota to metabolize and excrete genotoxic organics such as PAHs generally results in bioconcentraùon factors that are at least an order of magnitude lower than that of persistent organochlorines with similar Kow's (Pruell et al., 1986: Southworth et al. 1980; Southwonh et al.. 1981). We used a Kow - BCF re1ationship for genotoxic PAHs2 tO calculate the Kow of genotoxins accumulared by St. Lawrence river biota. The results indicate that the accumulated substances have Kow's in the 1()4 range. As a result we might expect a high proporùon of surface water genotoxins to be present in the disso1ved, rather than parocle-bound sute (e.g. at 5 mg per L suspended solids =95% disso1ved). The low rendency for parùcle sorption may result in increased bioavailability of genotoxins (Allan, 1986). It is interesring to note !hat the calculated Kow is similar to that inferred from the sorption parùtion coefficient of genotoxins in domestic wastewaters

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