2010 SOUTHEASTERN NATURALIST 9(3):497–506 Mutagenicity of Walnut Creek and Troy (Alabama) Wastewater Treatment Plant Influent and Effluent Alicia Whatley1,* and In Ki Cho1 Abstract - Samples from Walnut Creek, upstream of the Troy Wastewater Treatment Plant (TWWTP), and the influents to and effluents from the TWWTP were assayed for mutagenicity using the Salmonella typhimurium fluctuation test. Samples were prepared with metabolic activation (channel catfish S9 and rat S9 enzymes) and without using TA100 and TA98 strains of Salmonella. Results indicated that catfish S9 enzymes (FS9) were more capable of activating base-pair substitution mutagens in upstream samples than rat S9 enzymes (RS9). For influent samples, RS9 activated higher levels of base-pair and frameshift mutagens than FS9. The comparison of changes from influent to effluent samples showed a significant reduction in base-pair and no change in frameshift mutagens with FS9; conversely, no change in basepair and a significant reduction in frameshift mutagens with RS9 were found. For direct-acting compounds (without enzymatic activation), a significant increase in frameshift mutations was found in effluent compared to influent, while no significant change was seen in base-pair substitutions. These results indicate that Walnut Creek contains both mutagenic and promutagenic compounds, and influents to TWWTF exhibit mutagenicity that may be refractory to or created by treatment processes. The generally higher mutagenicity ratios following RS9 activation vs. FS9, suggest that current toxicity studies in fish species and water quality requirements may be inadequate to assess the hazards of water resources that receive municipal wastewater treatment discharges and that may be habitat to both fish and mammalian wildlife and may eventually become sources for human exposures. Introduction Influents to municipal wastewater treatment plants (WWTPs) from various sources, ranging from industries to households, have been shown to contain genotoxic compounds (Claxton et al. 1998, Watanabe et al. 2002, White et al. 1998). Assessments of discharges from textile (Mathur et al. 2007), pulp and paper (Claxton et al. 1998), and dye-processing industries (Umbuzeiro et al. 2005) have found that they contain both mutagenic and promutagenic compounds. Hospitals are often a source of genotoxic chemicals, such as anti-cancer drugs and anti-microbial agents, in discharges to wastewater treatment plants (Jolibois and Guerbet 2006, Jolibois et al. 2003). Although the genotoxicity of influents with industrial origin may be established, some municipal wastewater treatment facilities receive their major loads from households and other domestic sources that have been found to possess genotoxic hazards (Koren and Bisesi 2003, White and Rasmussen 1998). Human sanitary wastes may also include a variety of endocrinedisrupting chemicals that are teratogenic, carcinogenic, and mutagenic 1Department of Biological and Environmental Sciences, Troy University, Troy, AL 36082. *Corresponding author - awhatley@troy.edu. 498 Southeastern Naturalist Vol. 9, No. 3 (Choi et al. 2004). Examination of effluents from numerous WWTPs have shown that many of the genotoxic compounds may either be refractory to treatment processes or converted to active forms (Liney and Hagger 2006). This can even be a problem in an area with minimal industry. Ohe et al. (2002, 2004) have provided considerable evidence that many water bodies are contaminated with potent mutagens as a result of treated and untreated discharges. While few putative mutagens have been identified (Watanabe et al. 2005), the profusion of these compounds introduced through municipal wastewater discharges are an important aspect of the carcinogenic, genotoxic, and mutagenic risks to aquatic species and to humans who may ultimately use these water resources for a variety of reasons (Filipic and Toman 1996, White and Rasmussen 1998). As part of its nationwide reconnaissance of water resources, the US Geological Survey has found that effluent-dominated waters are becoming increasingly prevalent (Barnes et al. 2002). Depending upon the classification of these water resources, effluent discharges may be permitted under the Clean Water Act (US Congress 1977). In the State of Alabama, Walnut Creek, a third-order stream in the Choctawhatchee River basin that flows through Troy (Alabama), has been classified for “fish and wildlife” use (ADEM 2007). Although this classification designates Walnut Creek as suitable for fishing, propagation of fish, aquatic life, wildlife, and other uses (except for swimming, water-contact sports, or as a source of water supply for drinking or food-processing purposes), there are numerous non-point sources of residential, agricultural, construction, and roadway runoff, along with a sewage treatment plant point source. The City of Troy Wastewater Treatment Plant (TWWTP) treats approximately 5 million gallons of wastewater per day up to the secondary level. It receives influents from households, a few industries (including a lead battery recycling company, three post-consumer plastic recycling companies, and a food processing plant), several restaurants, a hospital, and stormwater runoff. The treatment facility discharges its effluent into Walnut Creek, in accordance with permit AL0032310 under the National Pollutant Discharge Elimination System (NPDES) (ADEM 2007). Since secondary treatment is not sufficient to remove 100% of toxic compounds, requirements under the facility’s NPDES permit include whole effluent toxicity (WET) tests using Ceriodaphnia dubia Richard and Pimephales promelas Rafinesque (Fathead Minnow) (ADEM 1997). At present, there are no requirements for measuring mutagenicity of effluents or removal of specific mutagens under the WET policy (US Code of Federal Regulations, Title 40, Part 136.3). The lack of regulatory requirements or established guidelines for mutagenicity and genotoxic effects identifies a need to conduct more detailed investigations to validate methods for assessing mutagenicity as well as for predicting such effects in exposed populations. The objectives of our study were to examine the mutagenicity of Walnut Creek upstream of the TWWTP, as well as TWWTP influent and effluent, using the Salmonella typhimurium fluctuation test. Given the differences that exist in the metabolic processes of species that may be affected by exposures to contaminated water, the study also examined differences in metabolic activation 2010 A. Whatley and I.K. Cho 499 of potential mutagens by rat and fish enzymes (monooxygenases) following exposure to water from Walnut Creek and TWWTP influents and effluents. At this time, the focus of our study is on the effects of exposure to whole samples and does not attempt to characterize individual contaminants or their origins. Materials and Methods During September 2008, two 1-L samples each were collected in glass containers from (1) the continuous-flow composite mixture of influents to TWWTP, (2) TWWTP effluent at the 24-hour retention, pre-discharge tank, and (3) Walnut Creek, approximately 1.6 km upstream from the TWWTP; samples were designated as influent (IN), effluent (EF), and upstream (UP), respectively. Sample containers were capped, transported to the laboratory in an ice chest, and refrigerated at 4 °C until tested the next day. Mutagenicity test kits, including Salmonella bacterial strains (TA98 and TA100), reagents, rat-liver extract S9 (RS9; containing monooxygenases), standard mutagens, ultrapure water, and membrane filters, were purchased from Environmental Biodetection Products Inc. (EBPI, Mississauga, ON, Canada). Falcon 96-well microtiter plates were purchased from Ward’s Natural Science (Rochester, NY). Fish-liver S9 fractions (FS9; containing monooxygenases) were obtained from another study conducted in our laboratory during spring 2008 that involved exposure of Ictalurus punctatus (Rafinesque) (Channel Catfish) to TWWTP effluent to induce increased monooxygenases. Catfish S9 was prepared according to a modification of the methods by Chan (2005), and Burke and Mayer (1974), by homogenizing liver tissue (0.1 g) in 3 mL buffer (0.05 M Tris, 0.15 M KCl; pH 7.8). The homogenate was centrifuged at 9000g for 20 minutes at 4 °C. After decanting the supernatant, the liver pellet was re-suspended in 2 mL buffer (0.1 M potassium phosphate, 0.5 mM DTT, 1 mM EDTA, and 20 % glycerol; pH 7.4), removed to sterile tubes, and stored at -80 °C until needed for the current study. The Salmonella fluctuation tests were performed as outlined in the Environmental Biodetection Products, Inc. instructions (2008) in accordance with the procedure described in Legault et al. (1994). The studies were conducted with rat-liver S9 and fish-liver S9, and without metabolic activation. Reaction mixtures were prepared by mixing 21.62 mL Davis Mingioli concentrate (5.5 times concentrated), 4.75 mL D-glucose (40%, w/v), 2.38 mL bromocresol purple (2 mg/mL), 1.19 mL D-biotin (0.1 mg/mL), and 0.06 mL L-histidine (0.1 mg/mL). Liver S9 mixtures were prepared by mixing 0.4 mL magnesium chloride/potassium chloride (0.4 M, 1.65 M), 0.09 mL glucose-6-phosphate (1.0 M), 0.81 mL nicotine amide di-nucleotide phosphate (0.1 M), 9.98 mL phosphate buffer (0.05 M, pH 7.4), sterile distilled water (6.72 mL), and either rat-liver S9 or fish-liver S9 (2 mL). Non-concentrated water samples (2 replicates each for IN, EF, and UP) were sterilized by vacuum-filtration through 0.22-μm membrane filters immediately prior to testing. Single assay preparations were made for blank and positive controls, and duplicate preparations for background and test samples as shown in Table 1. Sodium azide (5 μg/mL) and 2-nitrofluorine (0.3 mg/mL) were used as 500 Southeastern Naturalist Vol. 9, No. 3 positive controls without metabolic activation for TA100 and TA98 strains, respectively. As a positive control in TA100 and TA98 strains, 2-amino anthracene (0.1 mg/mL) was used with both rat-liver S9 and fish-liver S9 metabolic activation. Sterile, ultrapure water samples without metabolic activation were used as negative controls (or backgrounds) for both TA100 and TA98 strains. Sterile, ultrapure water samples with both rat-liver S9 and fishliver S9 liver metabolic activation were also used as negative controls for both TA100 and TA98 strains. Assay treatments containing bacteria, reagent mixture, S9 mixture, and sample treatments were thoroughly mixed in sterile tubes. Contents were poured into multichannel pipette boats and dispensed in 200-mL aliquots into each well of 96-well microtiter plates. Plates were covered, sealed in plastic bags, and incubated at 37 °C for 5 days. Table 1. Assay preparations for blank and positive controls, duplicate negative controls (Backgrounds), and duplicate study samples (Walnut Creek upstream, Troy Wastewater Treatment Plant influent and effluent) with and without metabolic activation (S9) in Salmonella strains TA100 and TA98. Reaction Sample (mL) H2O (mL) mix (mL) S9 mix (mL) Bacteria (5 μL) Blank 0.0 17.5 2.5 None None Background 1 0.0 17.5 2.5 None TA100 Background 2 0.0 15.5 2.5 2.0 (rat) TA100 Background 3 0.0 15.5 2.5 2.0 (fish) TA100 Background 4 0.0 17.5 2.5 None TA98 Background 5 0.0 15.5 2.5 2.0 (rat) TA98 Background 6 0.0 15.5 2.5 2.0 (fish) TA98 2-AA 0.1 15.4 2.5 2.0 (rat) TA100 2-AA 0.1 15.4 2.5 2.0 (fish) TA100 2-AA 0.1 15.4 2.5 2.0 (rat) TA98 2-AA 0.1 15.4 2.5 2.0 (fish) TA98 NaN3 0.1 17.4 2.5 None TA100 NaN3 0.1 17.4 2.5 None TA98 2-NF 0.1 17.4 2.5 None TA100 2-NF 0.1 17.4 2.5 None TA98 Upstream 15.0 0.5 2.5 2.0 (fish) TA100 15.0 0.5 2.5 2.0 (rat) TA100 15.0 2.5 2.5 None TA100 Effluent 15.0 0.5 2.5 2.0 (fish) TA100 15.0 0.5 2.5 2.0 (rat) TA100 15.0 2.5 2.5 None TA100 Influent 15.0 0.5 2.5 2.0 (fish) TA100 15.0 0.5 2.5 2.0 (rat) TA100 15.0 2.5 2.5 None TA100 Upstream 15.0 0.5 2.5 2.0 (fish) TA98 15.0 0.5 2.5 2.0 (rat) TA98 15.0 2.5 2.5 None TA98 Effluent 15.0 0.5 2.5 2.0 (fish) TA98 15.0 0.5 2.5 2.0 (rat) TA98 15.0 2.5 2.5 None TA98 Influent 15.0 0.5 2.5 2.0 (fish) TA98 15.0 0.5 2.5 2.0 (rat) TA98 15.0 2.5 2.5 None TA98 2010 A. Whatley and I.K. Cho 501 Plates were scored visually with all yellow, partially yellow, or turbid wells considered positive (revertant colonies), and all purple wells scored as negative. The statistical differences between revertant colonies in treatment plate vs negative control or treatment vs treatment plates were determined using the procedure for analysis of results of fluctuation tests developed by Gilbert (1980). Results The mutagenic profiles of upstream, influent, effluent, and negativecontrol samples are shown in Table 2. Revertant colonies in negative-control plates were minimal, with the exception of 21 revertants in the negative control containing rat S9 and TA98 strain of bacteria. Results, expressed as mutagenicity ratios (MR; number of positives wells in test plates/number of positives wells in the appropriate negative-control plate), are an average of two replicates for each treatment. Walnut Creek upstream had signifi- cant levels of mutagenicity using the TA100 strain of Salmonella with fish S9 (P < 0.001); furthermore, the mutagenicity ratio with FS9 was higher than with RS9. Influent to TWWTP had significant levels of mutagenicity in TA100 with both fish S9 (P < 0.001) and rat S9 (P < 0.001), as well as in Table 2. Mutagenic profiles of Walnut Creek upstream (UP) and Troy Wastewater Treatment Plant influent (IN) and effluent (EF) with fish-liver metabolic activation (FS9), with rat-liver metabolic activation (RS9), and without metabolic activation (–S9) using the Salmonella fluctuation test. Bacteria Test plate Negative-control Sample strain positivesA plate positivesA treatment S9 (Salmonella) (SD) (SD) MRB SignificanceC Upstream UP FS9 Fish TA100 12 (5.66) 1 (0.00) 12.00 <0.001 UP RS9 Rat TA100 0 (0.00) 1 (0.00) 1.00 UP –S9 None TA100 3 (1.41) 7 (2.83) 0.43 Influent IN FS9 Fish TA100 24 (5.66) 1 (0.00) 24.00 <0.001 IN RS9 Rat TA100 96 (0.00) 1 (0.00) 96.00 <0.001 IN –S9 None TA100 5 (0.00) 7 (2.83) 0.71 Effluent EF FS9 Fish TA100 0 (0.00) 1 (0.00) 1.00 EF RS9 Rat TA100 96 (0.00) 1 (0.00) 96.00 <0.001 EF –S9 None TA100 3 (2.82) 7 (2.83) 0.43 Upstream UP FS9 Fish TA98 0 (0.00) 1 (0.00) 1.00 UP RS9 Rat TA98 1 (0.00) 21 (4.95) 0.05 UP –S9 None TA98 2 (2.82) 1 (0.00) 2.00 Influent IN FS9 Fish TA98 0 (0.00) 1 (0.00) 1.00 IN RS9 Rat TA98 96 (0.00) 21 (4.95) 4.57 <0.001 IN –S9 None TA98 1 (0.00) 1 (0.00) 1.00 Effluent EF FS9 Fish TA98 2 (1.41) 1 (0.00) 2.00 EF RS9 Rat TA98 6 (5.66) 21 (4.95) 0.29 EF –S9 None TA98 5 (4.24) 1 (0.00) 5.00 0.050 ARevertant colonies on microplates: yellow, partially yellow, or turbid wells (average of two replicates; SD). BMutagenicity ratio (number of positives wells in test plate vs. number of positives wells in the appropriate negative-control plate). CChi-square analysis of fluctuation test results (Gilbert 1980). 502 Southeastern Naturalist Vol. 9, No. 3 TA98 with rat S9 (P < 0.001). Influent mutagenicity ratios were higher with RS9 than FS9 in both TA100 and TA98. Effluent from TWWTP had signifi- cant levels of mutagenicity in TA100 with rat S9 (P < 0.001) and in TA98 without enzymatic activation (P = 0.05) When comparing removal or creation of mutagenicity by TWWTP treatment processes (Table 3), significant reductions of mutagenicity from influent to effluent were found in the TA100 strain of Salmonella with fish S9 (P < 0.001) and in the TA98 strain of bacteria with rat S9 (P < 0.001). However, effluent had significantly higher mutagenicity than influent in TA98 without metabolic activation (P = 0.05) and a slight, but insignificant, increase was shown in TA98 with fish enzymatic activation. Discussion Consistent with previous research (Doerger et al. 1992, Ohe et al. 2004), the positive mutagenic responses of our study suggest that this toxicological hazard is present in Walnut Creek upstream of the TWWTP, that mutagens are present in influents that are either reduced or not removed by TWWTP processes, and in some cases, that promutagens may be transformed to their active forms in effluents. Attempts were not made at this time to isolate the chemical components that are responsible for this activity. As shown in Figure 1, Walnut Creek upstream contains mostly indirectacting base-pair substitution mutagens (as identified by effects seen in TA100). Assays indicated that fish enzymes (UP FS9 in TA100 bacteria) were more capable of metabolizing compounds to these base-pair substitution mutagens than rat enzymes (UP RS9 in TA100). Significant levels of indirect-acting base-pair mutagens were found in influents following both fish (IN FS in TA100) and rat (IN RS9 in TA100) enzyme activation. Significant levels of indirect-acting frameshift mutagens (as identified by effects in TA98) were found in influents with rat liver enzymes (IN RS9 in TA98 bacteria). This result is even more pronounced, given the 21 revertant colonies in the negative control containing rat S9 and TA98 strain of bacteria Table 3. Comparison of removal (or creation) of mutagenicity by treatment processes at Troy Wastewater Treatment Plant for influent vs. effluent in two Salmonella strains (TA100 and TA98) with fish-liver S9 (FS9), with rat-liver S9 (RS9), and without S9 (–S9) metabolic activation using the fluctuation test. Influent Effluent (positive revertants) (positive revertants) Change SignificanceA FS9 TA100 24 0 Reduction <0.001 RS9 TA100 96 96 No change –S9 TA100 5 3 No changeB FS9 TA98 0 2 No changeB RS9 TA98 96 6 Reduction <0.001 –S9 TA98 1 5 Increase 0.05 AChi-square analysis of fluctuation test results (Gilbert 1980). BThe mathematical difference is not statistically significant. 2010 A. Whatley and I.K. Cho 503 (Table 2). The higher-than-expected level of reverse mutations may be due to rat S9 containing potentially carcinogenic compounds or promutagens (Environmental Biodetection Products, Inc. 2008). Rat liver enzymes were more effective than fish liver enzymes in metabolizing both base-pair substitution (strongly significant levels) and frameshift mutagens in influent. Although other studies have shown a predominance of frameshift mutagens in water, wastewater, and sludge samples (Mathur et al. 2007, Ohe et al. 2004, Perez et al. 2003, Waldron and White 1989), indirect-acting base-pair substitution mutagens were much more common in TWWTP influent in our study. Changes in mutagenicity from influent to effluent samples for compounds that require metabolic activation varied depending on the strain of bacteria and on whether rat S9 or fish S9 was used. A significant reduction in base-pair substitution mutagens and slight insignificant increase in frameshift mutagens were observed in effluent when fish S9 was used for assays. On the other hand, no change in base-pair mutagens and a significant reduction in frameshift mutagens were observed in effluent when rat S9 was used. While Filipic and Toman (1996) reported that certain commonly found frameshift mutagens Figure 1. Intensities of genotoxicity response based on mutagenicity ratios (number of positives wells in test plate vs. number of positives wells in the appropriate negativecontrol plate) of water samples from Walnut Creek Upstream (UP), and Troy Wastewater Treatment Plant Influent (IN) and Effluent (EF) with fish-liver metabolic activation (FS9), with rat-liver metabolic activation (RS9), and without metabolic activation (-S9) in Salmonella strains TA100 and TA98. 504 Southeastern Naturalist Vol. 9, No. 3 may not be inactivated by wastewater treatment, our results suggest that the greater risk at Walnut Creek may be from base-pair substitution mutagens. Troy WWTP effluent was found to have some frameshift mutagenicity without metabolic activation and very high levels of base-pair mutagenicity with rat-liver enzyme activation. The result that neither base-pair substitution nor frameshift mutagens were significantly activated by catfish enzymes is noteworthy; given that channel catfish are an important and indigenous species for Walnut Creek. Other studies have suggested that channel catfish are a tolerant species that either does not produce mutagenic metabolites from certain potentially mutagenic compounds or if mutagenic metabolites are produced, they are quickly and efficiently eliminated following Phase II conjugation (Willet et al. 2000). While the TWWTP may be capable of removing some potentially mutagenic compounds it is also possible that others are refractory to or may be created by the treatment processes. Although previous chronic studies (ADEM 1997) have found that TWWTP effluents may be toxic to daphnia and fish, our results suggest that these toxic responses may not be mediated by mutagenic mechanisms in fish exposed to TWWTP effluent. Given the general higher level of mutagenicity ratios following metabolic activation by rat enzymes compared to activation by fish enzymes, fish species may be inadequate to assess all of the hazards of water resources that receive municipal wastewater treatment discharges. This consideration is particularly important when these water resources may be habitat to both fish and mammalian wildlife and may eventually become sources for human exposures. Conclusion Researchers estimate that as much as 75% of the thousands of chemicals that enter the environment have not been studied. And of those remaining chemicals that have been studied, many are resistant to breakdown by the ambient environment, with significant implications for risks to living organisms (Muir and Howard 2006). Following dilution of chemicals in wastewater discharges, some of these contaminants are not detected analytically, while others may form harmful mixtures with other compounds already in the environmental media (Stackelberg et al. 2004). Our pilot study points to a need to study these reactions which may affect the toxicity of Walnut Creek, and TWWTP influents and effluents over time. Given the mutagenicity and genotoxicity potential of discharges from TWWTP and Walnut Creek itself, additional human and aquatic ecological risks associated with designated and subsequent uses of Walnut Creek should be investigated. Further research is also needed to examine relationships among long time exposures to small concentrations below water quality standards limitations and the proliferation of resistant pathogens, undesirable mutants, and other speciation effects. Requirements to remove mutagens to concentrations below no-observedeffect levels should also be considered. Regardless of whether chemical characterization of wastewaters can be made or not, testing of individual chemicals rarely provides adequate assessment of their potential hazards 2010 A. Whatley and I.K. Cho 505 (Claxton et al. 1998). So genotoxic assays of whole effluents at a minimum should be a requirement of all permitting and other regulatory decisions in order to control the discharge of mutagens to water resources. Acknowledgments This study was completed with the support of a Troy University Faculty Development grant and the assistance of the City of Troy Wastewater Treatment Facility staff. The authors are grateful to Paul M. Stewart for his advice in editing the manuscript. Literature Cited Alabama Department of Environmental Management (ADEM). 1997. Water quality assessment, Walnut Creek, Troy, Alabama, Pike County. Environmental Indicators Section, Field Operations Division, Montgomery, AL. ADEM. 2007. Administrative Code 335–6. 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