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SEDIMENT GENOTOXICITY AND ITS RELATIONSHIP TO THE FREQUENCY OF CHIRONOMID LABIAL PLATE DEFORMITIES by QUE LAN, B.Sc. (Hons.), China, A Thesis submitted to the Department of Biological Sciences in partial fulfilment of the requirements for the degree of Master of Science June 1988 Brock University St. Catharines, Ontario C Que Lan, 1988 26438001 BFG50945 I hereby declare that. I am the sole author of this thesis. I authorize Brock University to lend this thesis to other institutions or individuals for the purpose of scholarly research and further authorize Brock University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Signature 26433002 BFG50946 3. ACKNOWLEDGEMENTS I am greatly indebted to Dr. M.D. Dickman, my thesis supervisor, for suggesting the study of the relationship between sediment genotoxicity and the frequency of benthic invertebrate abnormalities. This thesis benefited from his encouragement, critical comments and support. I am grateful to Dr. A.W. Bown, Dr. A. Castle, and Dr. C. Lewis, member of my committee, for their helpful advice and encouragement. I thank Dr. B.J. Barclay for his helpful discussion and advice, especially during the genotoxicity tests which were done' in his laboratory at Brock University. I wish to thank Dr. B. Dutka of the Canada Centre for Inland Waters for his help in guiding me through the genotoxicity tests and for kindly providing the test strains which I used in my SOS chromotest study. I am also grateful to Dr. K. Coates of the Royal Ontario Museum for her help in identifying oligochaete species, and Mr. B. Morton of Guelph, Ontario, for the long hours he spent helping me identify my invertebrate species. I wish to thank Miss K. Veltri, T. Huang, N.K. Ondrusak, B. Shannon, M.D. Agbeti, B. Solymar, and M.M. Saavedra Alvarez for their encouragement and support. I express my deep gratitude to Mr. H. Xu for his encouragment thoughout my thesis research. Finally, I thank my family for their strong support thoughout my academic career. BFG50947 26439003 4. page Abstract 1. Acknowledgements Table captions 3. 6. Figure captions 6. Introduction 8. Literature Review Genotoxicity of environmental samples 12. Deformities in benthic invertebrates 17. Mechanisms of deformity induction by genotoxins Toxicity vs genotoxicity of environmental samples Measures of generic richness of benthic invertebrates 19. 20. 22. Materials and Methods Study locations 24. Sample preparation for the SOS chromotest 29. The SOS chromotest 30. Calculation of results 34. Sampling benthic invertebrates 36. Preservation of invertebrate specimens 36. Classification of the invertebrates 37. Statistical analyses 38. Results Genotoxicity of sediment samples Frequency of deformities in chironomids Correlation between genotoxicity and frequency of deformities Benthic invertebrate community BFG50948 40. 55. 63. 66. 26433004 Generic richness and diversity 79. Discussion Sediment genotoxicity Frequency of deformities in chironomids Correlation between genotoxicity and the frequency of chironomid labial plate deformities Toxicity vs genotoxicity of the sediments The Benthic invertebrate community 83. 87. 89. 92. 95. Conclusions 101. References 103. Appendices 1. Industrial discharges in the Welland River (MOE, 1987) 110. 2. The percent alkaline phosphatase activity in the SOS chromotest. 112. 3. The percentage frequency of deformities in chironomid labial plates 114. BFG50949 26433005 Tables page 1. The SOS chromotest induction factor of sediment extracts from seven locations in the Welland River. 43. 3. Relative abundance of benthic invertebrate taxa at seven locations in the Welland River. 70. 3. Benthic invertebrate taxa at seven locations in the Welland River. 72. 4. Abundance of benthic invertebrates at seven locations in the Welland River. 74. 5. Generic diversity of benthic invertebrates at seven locations in the Welland River. 81. Figures 1. Map of the study area (Modified from Dickman et al. 1983). . 27. 2. SOS chromotest induction factor and colour development time. 33. 3. The relationship of dose-response in the SOS chromotest. 45. 4. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts with S9. 47 5. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts without S9. 49 6. The percent alkaline phosphatase activity in the SOS chromotest of distilled water extracts with S9. 51 7. The percent alkaline phosphatase activity in the SOS BFG50950 264: 7. chromotest of distilled water extracts without S9. 8. Normal structure of chironomid labialplate. 9. Deformed chironomid labial plate. 10. The frequency (%) of deformities in chironomid labial plates. 11. Correlation between genotoxicity and frequency of deformities in chironomid labialplates. 12. Generic richness of benthic invertebrates at seven locations in the Welland River. 53. 57, 59. 61. 64. 79. 26439007 BFG50951 REPORT ON THE 1986 INDUSTRIAL DIRECT DISCHARGES IN ONTARIO October, 1987 b?G50952 26433008 INTRODUCTION Pollution from carcinogenic materials has become a serious problem as large amounts of contaminated municipal and industrial wastes annually enter natural waterbodies. For example, over the years, the government of Canada has poured tens of millions of dollars into the Sydney Steel Corporation in Nova Scotia (McMillan, 1988). The technology which was used to produce steel, in this case created an enormous lagoon of highly carcinogenic materials that constitutes the largest chemical carcinogenic industrial landfill in eastern Canada. It is now costing the Canadian and Nova Scotian tax payers 49 million dollars to excavate and incinerate the Sydney Steel Corporation landfill wastes. This represents the second biggest site clean-up in the history of North America (Mcmillan, 1988). Since aquatic communities are sensitive to mutagens, many ecologists have attempted to. use invertebrates to quantify the level of carcinogens in aquatic sediments (Warwick, 1985). To the best of my knowledge, my thesis research is the first study of the relationship between the frequency of benthic invertebrate abnormalities and sediment SOS chromotoxicity. The correlation between these two independent tests of genotoxicity constitutes the main focus of my thesis research. Although many studies have been done on the genotoxicity of environmental samples and the frequency of deformities in chironomids, this is the first study to use the SOS chromotest in conjunction with the study of the frequency of chironomid labial plate deformities. The genotoxicity of environmental samples and BFG50953 26439009 the frequency of deformities of chironomids were both studied in terms of their relationship to industrial and agricultural discharge sources. The Welland River was chosen for this study because of its status as a provincially significant wetland area. The Welland River flows east through the City of Welland to the Niagara River. Upstream of the City of Welland, the river is mainly influenced by agricultural runoff. Within and downstream of the City of Welland, the major discharges to the river are from industrial and domestic sources (Steele, 1981; Dickman et al., 1983). The Welland river has been studied since the 1960's (e.g.' Johnson, 1964). Serious shock loading of industrial waste materials in the Welland River has resulted in aquatic plant dead zones below some industrial discharge sites in the Welland River (Dickman et al., 1983, 1986 and 1986). Several industrial discharge effluents contained detectable genotoxic organic compounds, such as chloroform, carbon tetrachloride and dibromomethane (Kaiser and Coraba, 1983). These organic compounds have been described as carcinogens (McCann and Ames, 1976). Discharges of heavy metals were also found at industrial sites, those, metals included Cr, Pb, Ni (Dickman et al., 1986) some of which are both toxic and genotoxic (Flessel, 1979). Previous studies on the Welland River indicated that the frequency of gonadal neoplasms in wild carp-goldfish hybrids was higher in the industrial influenced portion of the Welland River than in the upstream agricultural portion (Dickman and Steele, BFG50954 26430010 10. 1986). This report prompted me to attempt to determine whether there were any genotoxic materials being discharged into the Welland River. The seven locations selected for this study were divided into two groups: 1). A control location (site A) which was located in an agricultural area upstream of the city of Welland, and 2). Six "treatment'* locations (sites B, C, D-l, D-2, E, and F) which were all located in an industrial area downstream from the city of Welland with the single exception of station B which received insecticides and herbicides from an agricultural area near station A (Fig. 1). It should be noted that similar herbicides and insecticides only reached station A after they had passed through an extensive aquatic plant lined streamcourse. These aquatic plants serve to remove pesticides from the water (Bingham, 1973). The goals of this study were: 1. To determine the genotoxicity of sediment samples from the Welland River, using the SOS chromotest and coded samples to avoid unconscious biases. 2. To sample benthic invertebrates from the Welland River, at the identical locations that the SOS chromotest sediment mutagenicity test samples had been taken. 3. To observe the frequency of chironomid labial plate deformities at these locations, after first again coding the samples to prevent unconscious bias. 4. To determine the correlation between the level of SOS 26433011 11. chromotest genotoxicity and the frequency of chironomid labial plate deformities. 5. To determine the diversity of benthic invertebrates (at the generic level) at each of the seven study sites in the Welland River. BFG50956 26433012 Literature Review 12. Aquatic toxicological studies of freshwater include two major areas of study: 1.) field studies of species pattern, species diversity, species abundance, etc. (Guafin and Tarzwell, 1952). These field studies have been carried out with bacteria, aquatic plants and invertebrates, etc. (Cairns et al., 1972; Buikema and Herricks, 1978; Dickman et al., 1983; Pettibone and Cooney, 1986). 2.) the determination of pollutants in environmental samples by using some sensitive, inexpensive, convenient and short-term methods to estimate genotoxic pollutant loading (de Serres and Matsushima, 1987). Both areas have developed rapidly, as genotoxic pollution problems have become more and more evident (Sato et al., 1983; Samolloff et al., 1983; Xu and Dutka, 1986). In fact, contaminated drinking water was considered at one time as a potential cause of cancer in humans (Velema, 1987). Genotoxicity of environmental samples Genotoxins include mutagens, clastogens, carcinogens, and teratogens (Quillardet et al., 1985). Mutagens cause permanent modifications in DNA structure in both prokaryotes and eukaryotes (Starr and Taggart, 1987). Clastogens result in chromosomal changes in eukaryotes (Adler, 1984). Carcinogens cause gene mutations leading to cancer in eukaryotes (Starr and Taggart, 1987). The differences between these genotoxins are not absolute. BFG50957 26433013 For example, 90% of the carcinogens were also known mutagens tested in the Ames test of McCann and Ames (1976). Theoretically, mutagens cause gene mutations, and consequently they may result in chromosomal changes, and finally they may result in the appearance of abnormalities. Because there is little difference between genotoxic groups, especially when one considers eukaryotes and prokaryotes, the grouping of genotoxins is quite confused. Some authors limit mutagens to DNA damaging agents in bacteria (McCann et al., 1975), while others expand their definition of mutagens to include carcinogenic potentiality in higher eukaryotes (Quillardet et al., 1985; Metcalfe et al., 1985). I preferred to use the term "genotoxicity", in a restricted sense (e.g. DNA damaging agents) as a large range of organisms from bacteria to insect larvae were analyzed in my study. For the purpose of evaluating the genotoxic potentiality of waste materials in the environment, genotoxicologists have been looking for a widely available and uncomplicated method of detecting potential genotoxic compounds. Different methods for detecting potential genotoxic materials have been developed (Venitt and Parry, 1984; de Serres and Matsushima, 1987). The yeast "Saccharomyces cerevisiae" has been used as a simple eukaryotic system for detecting genotoxicity of chemicals (Parry and Parry, 1984). Cultured mammalian cells have also been used in estimating genotoxicity of chemicals and environmental samples (Dean and Danford, 1984). However, bacteria are the most widely used test organisms in genotoxic studies (Hoilstein and McCann, u. 1979). The Ames test The Ames test is a well-known mutagenicity test which has been widely used since 1975 to detect drug genotoxicity as well as chemical and environmental genotoxicity (Ames et al., 1975; Walker, 1983; Dutka et al. , 1981; Samolloff et al., 1983; Sato et al., 1983). The Ames test employs Salmonella bacteria which carry a mutation at an easily detected locus. An increase in the frequency of revertants is related to increasing dosage levels of genotoxic compounds. Fly ash collected from an oil-burning power plant was demonstrated using the Ames test to contain mutagens (Wei et al., 1984). The acid, base and neutral fractions of samples from petrochemical plant wastes were also found to be mutagenic using the Ames test (Brown and Donnelly, 1984). Metcalfe et al. found that the genotoxic materials associated with the discharge of particulate material in oil refinery effluents were present in the most polar silica gel fraction (Metcalfe et al. , 1985). Some extensively used pesticides such as organphosphorous insecticides (Usha Ranni et al. , 1980) were also demonstrated to be genotoxic using the Ames test. Many freshwater bodies have been used as dumpsites for waste materials. Metcalfe et al. (1985) observed a positive relationship between the genotoxicity of particulate materials in petroleum refinery effluents and the concentration of oil and grease samples by using the Ames test and the carcinogenicity test. Samolloff et al. (1984) reported that neutral compounds 3^G50959 26433015 15. extracted from sediments of Tabin Lake (Canada) which was contaminated with agricultural runoff, mining, petrochemical industries, and paper mills and municipalities showed mutagenic activity when tested with the Ames test. Sato et al. (1983) using the Ames test found that some sediments which they removed from the Niagara River and its feeder streams were mutagenic. The SOS Chromotest The SOS Chromotest is both more sensitive and more specific than the Ames test (Quillardet et al., 1982), and was therefore adopted in this study as the principle genotoxicity test. The first report on the SOS Chromotest was made by Quillardet et al. in 1982. The SOS chromotest is based on the "SOS" response to DNA-damaging agents in JE_. coli (Walker, 1984; Little and Mont, 1982). The test strain (E_. coli PQ 37) carries a genfe fusion of the SOS repair operon (lacZ gene). The test strain has a deletion for the normal lac region so that beta-galactosidase (coded for by lacZ) activity is strictly dependent upon sifA expression. Thus, the extent of any DNA damage is measured by quantifying the activity of beta-galactosidase in test cells (Quillardet et al., 1982). It has been shown that the results of the SOS chromotest are closely correlated with those of the Ames test (Quillardet et al., 1982; Quillardet and Hofnung, 1985; Dayan et al., 1987). The SOS chromotest was first used to measure genotoxicity of pure chemicals which were mutagenic and/or carcinogenic in other test systems (Quillardet et al., 1982). These studies revealed BFG50960 26439016 16. that the SOS chromotest was reliable in detecting genotoxicity. Genotoxicity studies of 83 compounds were made in order to compare the results of the SOS chromotest and the Ames test with the carcinogenicity test. It was concluded that there were no false positives with the SOS chromotest (e.g. non-carcinogen but SOS inducer) among the 83 tested chemicals. The Ames test on the other hand produced a number of false positives (38%) (Quillardet et al., 1985). Three out of 42 chemicals which displayed mutagenicity in the Ames test did not induce the SOS response in the SOS chromotest (Ohta et al.# 19B4). Although the SOS chromotest has been used in examining pure chemicals (Quillardet et al., 1985; Ohta et al., 1984; Olivier and Marzin, 1987), only a few environmental studies using the SOS chromotest have been carried out (Xu and Dutka, 1987). Six samples taken from contaminated areas along Prince Edward Island were tested with the SOS chromotest, five of the six samples displayed weak genotoxicity (Xu and Dutka, 1987). The SOS chromotest has a major advantage over the Ames test. In the SOS chromotest, the test strain does not need to survive for as long a time (only two hours) as it does in the Ames test (48 hours). Therefore, the SOS chromotest can'be used for detecting toxic mutagens which are undetectable in the Amos test because they kill the bacteria (Quillardet et al. 1982). Since the SOS chromotest has some major advantages over the Ames test (ie. it gives a quantitative response within a few hours and it is more sensitive than the Ames test), I used it as my primary mutagenicity screening tool in my thesis research. BFG50961 26430017 17. Deformities of benthic invertebrates exposed to mutagenic substances Because it is well recognized that some pollutants cause mutations, the studies pf freshwater pollution were expanded to consider the frequency of benthic invertebrate deformities (Hamilton and Saether, 1971). Genotoxic pollution is a concern because such pollutants frequently accumulate without causing the death of organisms. Instead, long term disabilities occur (Baumann, 1984). Studies in France revealed that in drinking water, mutagenic substances could be detected by using a micronuclear test (Jaylet et al., 1987). Milbrink (1983) found that the frequency of chaetal deformities in oligochaetes was significantly correlated with mercury pollution in Lake Vanern, Sweden. Benthic invertebrate chironomid larvae represent some of the most widely distributed gen.era in freshwater (Roback, 1978). In addition, their larvae represent a sensitive stage in their life cycle. Research on the frequency of deformities of chironomid larvae in polluted freshwater (Hamilton and Saether, 1971) led to the use of deformities of mouth parts, antennae and chironomid body wall thickness (Hamilton and Saether, 1971; Wiederholm, 1984; Warwick et al., 1987). The mouthparts of chironomid larvae normally are symmetrical. Deformed specimens are characterized by asymmetrical labial plates and mandibular structures. Unusual thickenings of the chironomid larval body wall were also considered as a deformity by Hamilton and Saether (1971). It was suggested that BFG50962 26433018 18. different, types of deformities may be caused by different types of pollutants (Hamilton and Saether, 1971). The susceptability to these malformations seemed to differ among the various chironomid species (Wiederholm, 1984). Some species were far more sensitive to mutagens than others. In a study of Lake Erie, it was found that deformed chironomid larvae were generally restricted to locations near known sources of industrial pollution (Hamilton and Saether, 1971). Sediment core studies of the Bay of Quinte in Lake Ontario, revealed that the presence of deformed chironomid larvae increased inversely with depth (in most cases, the age of sediment core is determinated by depth, the deeper the older). The increase in deformed chironomid larvae was associated with increased pollution in the bay (Warwick, 1980). The studies of heavy metal (Hg, Zn, Cd, Ni, Pb, Cu and Cr) polluted lakes in Sweden concluded that in these lakes there were much higher occurrences of deformed chironomids than in unpolluted lakes (Wiederholm, 1984). Experimental pond studies showed that pollution by a synthetic, coaltar-derived oil could induce a high frequency of deformities in Chironomus decorus (Cushmam, 1984). It was suggested that antennal deformities in chironomid larvae could also be used as a biological screening marker for the detection and evaluation of mutagenic contaminants in aquatic ecosystems (Warwick, 1985). It was found that the number of deformed antennae in Chironomus sp. was higher in samples from Tobin Lake (contaminated with chemical pollutants and toxic residues of agricultural and industrial waste) than BFG50963 26433019 19. Last, Mountain Lakes which were comparatively uncontaminated. In the study of Chironomid larvae collected from sediments in the harbour at Port Hope, Ontario, it was found that a direct relationship between the degree of sediment radioactivity and the frequency of deformities in Chironomus spp. existed (Warwick et al., 1987). Hamilton and Saether (1971) reported that aberrant mouth parts, thickened body walls and head capsule malformations might be caused by pollutants from agricultural as well as industrial sources. Mechanisms of deformity induction by genotoxins The mechanisms of deformity induction include gene mutations, interference with transcription and translation, disruption of cell division, and metabolic disturbance (Weis and Weis, 1987; Jaylet et al., 1987). Mutagenic or genotoxic compounds cause deformities in animals by disrupting genes (Metcalfe and Sonstegard, 1905). Genotoxic pollutants which are discharged into water systems as waste materials accumulate in the associated sediments which comprise habitats of many benthic invertebrate taxa (Buikeman and Herricks, 1978; Lafont, 1984; Wetzel, 1983), Although the absolute concentration of genotoxic pollutants may be very low in water and sediment, bioconcentration and accumulation will serve to magnify them in the food chain (Buikema and Herricks, 1978; Tarkpea et al., 1985). For example, PCBs in the water were 8.2 ppt, while in Lake Trout (Salvelinus namycush) they were detected at 28.0 ppm (Metcalfe, 1977). BFG50964 26439020 20. To locate the sources which cause the highest occurrence of deformities in chironomids, Hamilton and Saether (1971) tested several pesticides (including some known ganotoxins) on Lab cultures of Chi.ronomus spp. The authors failed to observe the same high frequencies of deformed chironomid larvae as they obtained in their field study. They concluded that synergistic effects occur in the feild which they could not duplicate in the lab. The studies in experimental ponds revealed that a coal oil derived product did increase the frequency of deformities in Chironomus decorus labial plates (Cushman, 1984), while in otherstudies, it was found that petroleum refinery effluents (Metcalfe et al., 1985) and some crude oils (Vandermeulen et al. 1985) were genotoxic. It was suggested that genotoxic sediments in water bodies cause a high frequency of deformities in benthic invertebrates (Warwick et al., 1987). To the best of my knowledge, there are no published studies of sediment genotoxicity using the SOS chromotest and the frequency of benthic invertebrate deformities for the same sample locations. Toxicity vs genotoxicity of environmental samples Some genotxins act alos as general toxins, e.g. KiCr207 (Olivier and Marzin, 1987), and neocarcinostatin (Quillardet et al., 1982). It was noted that some environmental samples might contain these so-caaled toxic mutagens. Vandermeuleu et al. (1985) found that crude oils and oil products were toxic to the BFG50965 26439021 21. Ames "best, bacteria. Chemical fractions of sediments from contaminated Tobin Lake demonstrated in biological assay systems that major toxic constituents of the sediments were neutral compounds eluted from the Florisil columns by 1:1 hexane-dichloromethane (Samolloff et al., 1983). Sediment extracts of samples from the Niagara River and its feeder streams appeared to have high levels of toxicity (Sato et al. , 1983). Therefore, genotoxic pollutants could exert their effects on the benthic invertebrate community in two ways: 1.) DNA-damaging activity. 2.) direct toxicity to the organisms. Underestimation of the genotoxicity of environmental samples can be due to the toxic effects of contaminated samples (Vandermeulen et al. 1985; Metcalfe et al. 1985). The SOS chromotest has another advantage over the Ames test; the SOS chromotest can also detect the toxicity of samples by their suppression of alkaline phosphatase activity (Quillardet et al. 1982). In the SOS test strain, E_. coli K"12 PQ 37, alkaline phosphatase is noninducible by genotoxins. It is constantly synthesized by living cells. Monitoring the alkaline phosphatase activity can screen for the survival of the bacteria. If a tested sample causes a decrease of alkaline phosphatase activity, it can be considered as a protein biosynthesis inhibitor and therefore, the sample is toxic to the bacteria (Quillardet et al., 1982). In my study, the toxicity of samples was estimated since their toxicity could have serious impacts on the aquatic benthic invertebrate community. BFG50966 26433022 22. Measures of generic richness Changes in species pattern in a benthic invertebrate community can be related to the level of toxic materials entering the environment (Hart and Fuller, 1974; Buikema and Herricks, 1978; Wiederholm, 1984). Earlier studies of freshwater toxins in the environment were mainly field studies. Species diversity and species richness are two ways to characterize community structure. In a normal aquatic system, the number of individuals of each species is comparatively low, and the number of species is high. As the pollution levels increase, a reduction in the complexity of the community is observed as a reduction in the the number of species. At the same time the abundance of a few species generally increases (Cairns et al., 1972). Cushman (1984) reported that a coal-derived oil product reduced the species diversity of benthic insects in his experimental study ponds. In Lytle Creek, U.S.A., an aquatic invertebrate community survey was conducted, and it was found that upstream of a sewage outfall, higher species richness occurred than at the discharge site, and with distance downstream the species richness recovered (Gaufin et al., 1952). Thus, species richness generally decreases when toxic pollutants enter an aquatic system (Cairns et al., 1972; Cushman, 1984). In the Welland River, aquatic macrophytes and attached microbiotic species richness decreased downstream of a number of industrial discharge sites, and then recovered with distance (Dickman et al. 1983 and 1988; ; Albanese et al., 1988). Fish populations BFG50967 26433023 23. declined in species richness where waste materials from agricultural and industrial effluents in the Welland River were high (Steele, 1981). By comparing the species pattern of benthic invertebrates in normal (control areas) with that of polluted areas, it has been found that not only do species patterns change, but a shift in the dominant species also occurs (Buikoma and Herricks, 1978). Therefore, it was concluded that those dominant species surviving in polluted environments were tolerant of the sort of pollutants to which they were exposed. It was found that a high relative abundance of tolerant species in a particular location was related to the specific pollutants in each location (Hart and Fuller, 1974). 26438024 L BFG50968 24. MATERIALS AND METHODS The Welland River The Welland River is a slow flowing, shallow river and it is a provicially significant class 1 wetland in Niagara. The river begins in Ancaster, flows east to the Niagara River which brings water into Lake Ontario Upstream of the City of Welland the river passes through agricultural areas while downstream of Welland the river passes portion in an industrialized areas (Fig. 1). Sampling sites Site A: the upstream Welland River control site was located at the confluence of Beaver Creek and the Welland River, in the agricultural area, upstream of the City of Welland. The emergent aquatic vegetation at this site was dominated by Potamogeton spp. , Nuphar spp., Kymphaea, spp. . Typha spp. , Phragmltes spp. . Sagittaria spp. , Po.ntedgria spp. , Polygonum sp. , YallisnaricL spp. and Cladophora spp. Site B: Welland River entensive agricultural site was located at the confluence of the OWMC Swale and the Welland River, in acore and wheat agricultural area, downstream of site A, and upstream of the City of Welland. The aquatic emergent vegetation at this site was similar to those at site A. Site C was located at the confluence of the discharge pipe of the Atlas Specialty Steels Co. and the Welland River, downstream of the City of Welland. The mean discharge from the Atlas-mansfield storm sewer from which their waste stream entered BFG50969 26433025 25. the Welland River was 2458.9 m3/day (Ontario Ministry of the Environemt (MOE), 1987). Their effluent contained high levels of cadmium, nickel, chromium, lead, zinc, copper, cobalt and iron (Appenix 1). The aquatic emergent vegetation at this site was dominated by Sagittaria spp., and Phragmites spp. Site D-l was located below the discharge pipe of the B.F. Goodrich Co. which produces polyvinyl chlorides. The mean discharge from the sewer was 2213.58 m3/day (MOE, 1987). Biological Oxygen Demand (BOD), a measure of the quantity of oxidisable material present in a water sample), values of the effluents were 8 out of 12 months higher than the MOE requirement, and the discharge contained ammonium ion, vinyl chloride and phosphate. The emergent vegetation at this site was dominated by Potamogeton spp., and Typha spp. Site D-2 was located at the confluence of the discharge V stream of the B.F. Goodrich Co. and the Welland River just downstream of site D-l- The emergent vegetation at this site was dominated by Sagittaria spp., Cladophera spp., and Typha spp. Site E was located in Thompson Creek which was used as the main discharge stream of the Cyanamid Chemical Co. The company produces organic and inorganic nitrogen and phosphorus products. The mean discharge to this creek from Cyanamid was 27342 mJ/day (MOE, 1987). The effluents contained detectable concentrations of cyanide, ammonia, urea, nitrate, phosphorus, chromium, nickel, and zinc. There was no aquatic vegetation at this sample site due to the high toxicity of their ammonia waste discharges during frequent shock loading events (Dickman et al., BFG50970 2643C02S 26. discharges daring frequent, shock loading events (Dickman et al., 1983 and 1988). Site F was located at the confluence of the discharge channel of the Ford Glass Co. with the Welland River, downstream of site D-2. The discharge from their sewer was 8,554 m^/day (MOE, 1987). The effluents contained iron, ammonium ion and phosphate. The dominant emergent aquatic plant species at this site were Typha spp., Sagittaria spp., and Pontederia spp. BFG50971 26433027 Figure 1. Map of the study area (modified from Dickman et al. , 1983 ) * BFG50972 26439028 28 .^NIAGARA-ON BFG50973 26433029 29. Sample preparation for genotoxicity tests Seven sediment samples were collected from the Welland River at the same sites as previously described for the benthic invertebrate sampling (Fig.l) The sediments for genotoxicity testing were placed into sterilized plastic bags and within 2 hours they were returned to the laboratory and stored at 4c in a refrigerator. Extraction of samples was made within 24 hours of sample collection. The dimethyl sulfoxide (DMSO) sediment extracts were made by adding 25 g (the wet weight of sediment was weighed out and placed in a clean bottle) to 25 ml of 10% DMSO in distilled water. The bottle was then stoppered and vortexed for 2 minutes to make the contents homogeneous. The mixture was then poured into a sterilized centrifuge tube and centrifuged for 20 minutes at 2000 rpm at 4dC (IEC CENTRA-7R refrigerated centrifuge). The suspension was next filtered through a 0.22um filter. This was done for purposes of sterilization to permit its use in the SOS chromotest. The distilled water extract procedure was similar to the DMSO extraction procedure except that ultra-pure distilled water replaced the 10% DMSO solution. The E.coli K-12 PQ 37 strain was kindly provided by Dr. B.J. Dutka of the Canada Centre for Inland Waters. The genetic markers of this strain are thr, leu, his, pyr D, thi, gal E, gal K, lac U169, srl300: : TnlO, rpo B, rps h, sfiA*. :Mud (Ap lac) cst, rfa, uvrA, trp: :Muc* and Pho' (Quillardet et al. , 1982). Media and Buffers for the SOS chromotest BFG50974 26433030 30. L medium: 1% Bacto tr/ptone, 0.5% Bacto yeast extract, 1% NaCl, pH 7.2. Bacteria were cultured in L medium supplemented with 20 ug/ml ampicillin. B buffer: Na^HPO^ 161. lg, NaH^PQ^ . 2H^o 5.5g, sodium dodecyl sulfate(SDS) lg, beta-mercaptoethanol 2.7ml per 1 double distilled water, adjusted to pH 7.0 with HC1 or NaOH. P buffer: tris(hydroxymethyl)aminomethane 121g, SDS lg per 1 double distilled water, adjusted to pH 8.8 with HC1. ONPG solution (4 mg/ml): 400Mg 0-nitrophenyl-b D-galacto- pyranoside (ONPG) per 100 ml of 0.1 M phosphate buffer pH 7.0. PNPP solution (4 mg/ml): 400 mg p-nitrophenyl phosphate disodium (PNPP) per 100 ml of P buffer. Activation mixture (S9 mixture): per 10 ml of rat liver S9 (purchased from Litron Laboratories Lit.), salt solution (1.65 M KC1 + 0.4 M MgC1.6H*o) 0.2 ml; G6P(1 M) 0.05 ml; NADP( 0.1 M ) 0.15 ml; P buffer 2.5 ml; L medium 6.1 ml; S9 1 ml. The SOS Chromotest * The SOS chromotest test procedure was based on the published methods of Quillardet et al (1985). A fraction of 0.1 ml (overnight culture) was reconstituted in 5 ml of fresh L medium at 37PC for 2 hours, One ml of reconstituted culture (OD 600 = 0.15-0.2) was then diluted in 9 ml of fresh L medium (without metabolic activation) or 9 ml of fresh S9 mixture ( with metabolic activation). Fractions of 0.3 ml were placed into a series of sterilized test tubes (ice bathed) containing 20 ul of sample extract to be tested. The mixture was incubated by shaking for 2 hours at 37" C. BFG50975 26439031 31. After incubation, the fractions were used for beta-galactosidase and alkaline phosphatase assays. Two parallel series of test tubes were set up at the same time and under the same condition for these two enzyme assays. In order to screen the pro-genotoxic materials, an S9 mammalian microsomal fraction was used in the SOS chromotest. Optimal chromotest development time To determine the optimum SOS chromotest time, several tests for induced colour development time were conducted. The results of the optimum SOS chromotest time test showed that induced colour development of 60 minutes was the optimum time (Fig. 2). Therefore, the results of this study were reported as 60 minutes colour development times expcet where otherwise noted. Assays for beta-galactosidase To each test tube 2.7 ml of B buffer was added to the beta-galactosidase series and incubated at 37*C for 15 minutes. The enzyme assay was then initiated by the addition of 0.6 ml of ONPG solution per tube. The assay of color development was made at 30 minutes, 60 minutes, and 90 minutes after addition of the substrate. The absorbance at 420 nm was read against a colorimeter blank consisting of an assay in which the bacterial culture had been replased by either L medium in experiments performed without metabolic activation or activation mixture (S9 mix) in experiments performed with metabolic activation. Assays of alkaline phosphatase BFG50976 26439032 Figure 2. The SOS chromotest induction factor and colour development time (test concentration = 20 mg wet weight /assay). BFG50977 26439033 SOS CHROMOTEST INDUTiON FACTOR O station D-1 O station F station 0-2 + station C A station E X station A SOS CHJROMOTEST INDUCTION FACTOR AND THE COLOUR DEVELOPMENT TIME BFG50978 26439034 The alkaline phosphatase assay was similar "to that of the beta-galactosidase assay except that instead of B buffer, P buffer was used and instead of ONPG solution, 4mg/ml PNPP solution was used. Controls for the SOS chromotest Positive controls in each set of assays were included. 4NQ0 (4-nitroquinoline-N-oxide) was used in the experiments without metabolic activation and 2AA (2-aminoanthrancene) was used in experiments with metabolic activation. The results obtained from the positive controls were compared with previously published values Quillardet et al., 1985; Xu and Dutka, 1987). Negative controls in each set of assays were also included. 10% DMSO solution and double distilled sterilized water were used in all experiments using DMSO extract and ultra-pure distilled water extracts. The induction factor for each sample was compared with the negative control to determine the statistical significance of the test. Calibration of the SOS chromotest The units of enzyme activity were calculated according to Quillardet and Hofnung's (1985): Enzyme units= 1000 x A420 In this formula, A420 is the optical density at 420 nm read from the incubation mixture, and t is the length of time for incubation in the presence of the substrate (ONPG or PNPP) in BFG50979 35. minutes. The ratio of beta-galactosidase units to alkaline phosphatase units reflects the induction of the sfiA gene (Quillardet et al, 1982): A420 BXtP R=------------------------------------- A420 PXtB where A420 B and A420 P represent the optical density at 420 run read from the incubated mixture. Where tB and tP represent the reaction time from beta-galactosidase and alkaline phosphatase. The induction factor 1(c) is R(c) 1(c)-- ----------------- R(0) where R(c) and R(0) represent the ratio (R) at concentrations (C) and (0), respectively. The induction factor of each sample was compared with the negative control in order to evaluate the statistical significance of the test. 26439036 BFG50980 36. Ekman dredge samples Invertebrate samples were collected from the bottom of the Welland River (10 to 40 cm depth) at seven locations during August and September of 1987. An Ekman Dredge (15.3 x 15.3 x 23.0 cm) was used to collect the sediment samples. The dredge was set up and placed on the surface of the sediment and carefully pressed into the mud to a depth of approximately 4 cm. Before I lifted the dredge out of the sediment, I checked to see that the jaws were tightly closed. At least three replicate dredges were randomly collected at each location and then mixed together to form a single composite sample which was then placed into a plastic bag and returned to the laboratory. Three of these composite samples were taken at each location (ie. 9 Ekman samples) and these were returned to the laboratory for sorting. Sample preservation and mounting techniques K.A.A. solution (Pimentel,1967): kerosene 10 ml acetic acid 20 ml 95% ethanol 100 ml 5% formalin solution: 10% formalin 50 ml distilled water 50 ml Ammans Lactophenol (Pimentel, 1967): kerosene 20 ml lactic acid 20 ml glycerol 40 ml 26433037 37. distilled water 20 ml 20% Glycerin solution (Pennak, 1953): glycerin 20 ml 70% ethanol 80 ml 70% ethanol: 95% ethanol 70 ml distilled water 25 ml Separation of the invertebrates Separation of invertebrates from the sediment sample was made within 12 hours of sample collection. The collected sample, a mixture of mud or clay and water, was poured into a large white . enamel plate and gently mixed with clean water so that the movement of invertebrates could be easily observed. The invertebrates were carefully separated from the sample with forceps and an eye dropper. The separated invertebrates were preserved in K.A.A. solution (Pimentel, 1967). After one day, I replaced the K.A.A. solution with 5% formalin, and stored the invertebrates in vials at room temperature. All vials were coded by Dr.M. Dickman (my thesis supervisor) to avoid unconscious bias before classifying and counting the specimens at each station. Classification of the invertebrates Two days before the identification of the specimens, I replaced the 5% formalin solution with a 70% ethanol solution. For Annelida, one day before identification, I placed the specimens in Ammans Lactophenol solution. For Diptera, one day before observation, X placed the specimens into 20% glycerine BFG50982 26433 38. solution. The Annelids were carefully picked from the vials, and placed on clean glass slides, mounted in Ammans Loctophenol solution and covered with a glass cover slip. Next, they were each examined under a Leitz compound microscope at 400 x magnification. Dichotomous keys (Pennak, 1953; Pimental, 1967; Brinkhurst and Cook, 1974) were used for identifying the collected Annelid specimens. The specimens of Diptera were mounted in pure glycerin solution, observed under a Leitz microscope at 400 x magnification. A dichotomous key (Pennak, 1953) was used for classifying the specimens (Wiederholm, 1983). Large specimens of other classes were observed under the dissecting microscope. Smaller ones mounted in 70% ethanol solution were identified by means of Leitz compound microscope. Dichotomous keys to the betjiic invertebrates were in Pennak (1953) . Evaluation of symmetry patterns and abnormalities The symmetry of each invertebrate was observed (ia. labial plates of each chironomid specimen was checked for symmetry). If a deformed structure was found, it was recorded (see results section). The number of chironomids and the number of malformations observed were listed and were later transcribed into tables (see results section). Statistical Analyses The Student's "t" test was used to determine the difference 26433039 BFG50983 39. between controls and tested samples. The Sperman-Kendall rank correlation analysis was used for non-parametric tests and the relationship between the frequency of Chironomid larvae labial plate deformities and the SOS chromotest induction factor level. Correlation coefficients were used for parametric tests. Calculation of Species Diversity Indices Simpson's diversity index: N ( N-1 ) D =---------------------------------------- En ( n - 1 ) where N was the total number of specimens, n was the individual number of different species. Shannon-Wiener's diversity index: k H= -E p 1=1 log p where p was the proportion of individual species, and k was the number of species. BFG50984 26433040 40. RESULTS Genotoxicity of sediment samples Distilled water extracted sediment samples were tested using the SOS chromotest. All samples gave a negative response to the SOS chromotest with or without metabolic activation (Table 1). The 10% DMSO extracts of sediment samples yielded a higher induction factor value with metabolic activation than did the sample without metabolic activation (Table 1). With metabolic activation, the samples from site D-l, collected under the discharge pipe of the B.F.Goodrich Co. showed the highest induction factor (I.F = 2.053 0.361), and the samples from station B, located in the agriculturally influenced portion of the Welland River, showed the lowest induction factor (I.F = 1.062 0,066). The DMSO extract with metabolic activation for station D-l gave a significant positive response (P<0.01), stations D-2 and E gave a weaker but still significant positive response (p<0.05). The genotoxicity of sediment extracts was higher in the industrial influenced portion than it was in the agriculturally influenced portion of the river (Table 1). Without metabolic activation, all DMSO extracts of samples gave negative responses to the SOS chromotest (Table 1). To confirm these results, another set of samples was taken at the same locations in the river four months after the first set of samples was taken. At this time, only the DMSO extract of the sediment samples from station D-l gave a significant positive SOS chromotest response with metabolic activation (P<0.01). BFG50985 26433041 Table 1. The SOS chromotest induction factor of sediment extracts from seven locations in the Welland River (test concentration = 20 mg wet weight/ assay). o * 64330 BFG50986 42. Table 1. Induction Factor of seven samples sample sites A B C D-l D-2 E F 10% DMSO extract +S9 10% DMSO - S9 (December,1986) mean n S.D. (April,1987) mean n S. D. (December,1986) mean n S. D. 1. 173 1.062 1.664 1.638 1.272 1.450 1.049 6 0.123 0.854 4 0.066 0.915 10 0.429 1.072 4 0.135 2.053 13 0.361 1.231 4 0.070 1.537 9 0.427 1.200 4 0. 168 1.209 10 0.126 0.995 4 0.032 1.179 11 0.209 0.970 4 0.119 4 0.098 2 0.007 3 0.140 3 0.240 3 0.140 6 0.058 Tabl e 1. (continuous) Distilled Water Extract+S9 Distilled Water Extract -S9 sample sites (December,1986) mean n S.D. (December,1987) mean n S.D. A B C D-l D-2 E F 1.064 3 0.086 0.993 4 0.019 0.984 4 0.010 1.285 4 0.041 1.059 4 0.084 1.024 4 0.097 0.964 4 0.026 0.873 4 0.033 0.827 4 0.094 0.806 3 0.066 1.062 3 0.088 1.031 3 0.078 0.827 3 0.031 1.200 6 0.441 j. /W ' 3^G5098'7 26439013 43. Of the samples assayed, only samples from st.at.ion D-l were pro-genotoxic in DMSO extract. To construct a dose-dependent response pattern for the samples which gave a positive response to the SOS chromotest, the enzyme activities of beta-galactosidase and alkaline phosphatase were assayed. The dose-response curve for samples from station D-l showed that the induction factor rose to a maximum at a concentration of 2Omg/assay, and then decreased (Fig. 3). The results of the alkaline phosphatase assay for DMSO extracts with S9 for samples from station D-l indicated that the enzyme units decreased with increasing sample concentration (Fig. 4). In DMSO extracts with S9 added, almost all samples except for those from station A had slight inhibition effects on general protein biosynthesis when doses were increased (Fig. 5). According to the alkaline phosphatase activity levels (as percentage of initial level)., DMSO extracts from stations C and D-l showed a significant (p<0.05) inhibition effect on protein biosynthesis (Fig.5). DMSO extracts without S9 in the SOS chromotest did not appear to inhibit protein biosynthesis (Fig. 6), though station F did display a slight inhibition effect (0.05< p <0.10). Among the distilled water extracts, only samples from stations C and D-i showed a significant (p<0.05) inhibition of protein biosynthesis when tested with S9 (Fig. 7). Distilled water extracts of samples from stations F without S9 slightly inhibited protein biosynthesis (0.05<p<0.10) (Fig. 7). These results implied that some water soluble contaminants in samples BFG50988 28439044 from stations C and D-l were toxic activated by metabolic activation. 44. '^133015 BFG50989 Figure 3. The dose-response relationship for the SOS chromotest. BFG50990 26439016 SAMPLE D-1 DOSE-RESPONSE OAP UNITS Dbeta-G UNITS 46. BFG50991 2B433017 Figure 4. The percent, alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts with S9 (vertical bars represent one standard deviation). BFG50992 26433Q48 26433049 BFG50993 AP ACTIVITY (PERCENTAGE) AP ACTIVITY (PERCENTAGE) DMSO +S9 48. Figure 5. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts without S9 (vertical bars represent one standard deviation). BFG50994 26433030 26433051 BFG50995 AP ACTIVITY (PERCENTAGE) AP ACTIVITY (PERCENTAGE) AP ACTIVfTY (PERCENTAGE) DMSO -S9 50. Figure 6. The percent alkaline phosphatase activity in the SOS chromotest of distilled water extracts with S9 (vertical bars represent one standard deviation). BFG50996 28433052 DW +S9 52 OSAMPLEC QSAMPLE D-1 A SAMPLE D*2 BFG50997 2643S053 Figure 7. The percent, alkaline phosphatase activity in the SOS chromotest of distilled water extracts without S9 (vertical bars represent one standard deviation). BFG50998  SAMPLE A OW -S9 t a SAMPLE B 54. OSAMPLEC ^SAMPLE D-1 ^SAMPLE D-2 r , BFG50999 26433055 55. The frequency of labial plate deformities in Chironomids The appearance of normal mouth parts in the chironomid labial plate is shown in Fig. 8. The structure was symmetrical and well defined. The deformed chironomid labial plates included various types of asymmetry, such as mild asymmetry in the numbers of lateral teeth (Fig. 9), fused processes on the median teeth, gaps in the labial plate. Deformities of mandibles and antennae were not analyzed, although some deformed mandibles were occasionally noted. Six out of 15 chironomid genera which were found in the agricultural influenced portion of the Welland River appeared to have deformed individuals, while 6 out of 10 genera in the industrial influenced portion of the river had deformed specimens (Table 3). Labial plate deformities of chironomid larvae showed that the highest frequency of deformed labial plates was associated with the industrial influenced portion of the Welland River (station D-l, 10.90% 3.23%, Fig. 11). The control site A displayed the lowest frequency of deformities (3.64% 1.32%) (Fig. 10). The frequency of deformed chironomid labial plates (6.42% 3.30%) was higher at station D-2 than the agricultural site A. Site E displayed a slightly higher frequency of deformities (4.20% 0.28%) than site A. Site B (6.29% 1.71%), located at the confluence of the Welland river and an old Co ce discharge pipe carring farm field runoff yielded a higher frequency of deformed Chironomid labial plates than the nearby upstream site A (Fig. 10), though the difference was not significant (p> 0.05). Station F displayed a very low abundance BFG51000 56. of chironomids as only 3 specimens were found in three composite samples. Therefore, site F was not included in the chironomid deformity analyses. The difference in the frequency of deformities in Chironomid labial plates was statistically significant between site A and D-l, though there were no significant differences between other stations. c; BFG51001 0 S C t> 9 2 Figure 8. "Normal" structure of chironomid labial plate (modified from Wiederholm, 1983). *0 CJ ov* .M t; 05 BFG51002 58. BFG51003 Figure 9. Deformed chironomid labial plate. BFG51004 cn w o w> 60 BFG51005 cn > CJ c.o o C'j H* Figure 10. The frequency (percentage) of deformities in chironomid labial plates at 5 sample sites in the Welland River (vertical lines represent one standard deviation). BFG51006 cn Q CO o O') % Frequency of deformities in chironomid labia) plates BFG51007 cn & W u o v.`. Cm 63. The correlation between the genotoxicity of sediments and -the frequency of deformities in chironomid labial plates The highest, genotoxicity of sediments and the highest frequency of deformities in chironomid labial plates occurred at industrial site D-l which was at the end of the discharge pipe of a company which produces polyvinyl chlorides (Fig. 1), The correlation between the genotoxicity of the sediment samples and the frequency of deformities of labial plates of chironomid larvae showed a significant positive correlation ( r = 0.88, p < 0.05). However, when tested using a non-parametric test (Sperman-Kendall Rank Test) the correlation was not significant at the p < 0.05 level because a high correlation coefficient of 0.9 is required by the non-parametric test for five or less degrees of freedom when using the less powerful non-parametric test. It is interesting that station B showed a relatively high frequency of deformities in chironomid labial plates, but the sediment samples had the lowest SOS chromotest induction factor (negative responses). Reasons for this are offered in the discussion section. The standard error for the frequency of deformities was very high (Fig.11). As a result, the differences between most sites were not significant. However, the values of sites A and D-l were significantly different (p<0.05). BFG51008 ^ CO tos vf.- Figure 11. Correlation between the genotoxicity and the frequency of deformities in chironomid labial plates (vertical bars represent one standard deviation of % deformity frequency estimates, and horizontal bars represent one standard deviation in SOS induction factor estimates) bFG5^9 rc >* C'V O C/t % frequency of deformities in chironomid labial plates SOS induction factor BFG51010 0? Q?>WZ <7> 0\