Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.
The induction of adaptive stress response pathways is an early and sensitive indicator of the presence of chemical and non-chemical stressors in cells. An important stress response is the Nrf-2 mediated oxidative stress response pathway where electrophilic chemicals or chemicals that cause the formation of reactive oxygen species initiate the production of antioxidants and metabolic detoxification enzymes. The AREc32 cell line is sensitive to chemicals inducing oxidative stress and has been previously applied for water quality monitoring of organic micropollutants and disinfection byproducts. Here we propose an algorithm for the derivation of effect-based water quality trigger values for this end point that is based on the combined effects of mixtures of regulated chemicals. Mixture experiments agreed with predictions by the mixture toxicity concept of concentration addition. The responses in the AREc32 and the concentrations of 269 individual chemicals were quantified in nine environmental samples, ranging from treated effluent, recycled water, stormwater to drinking water. The effects of the detected chemicals could explain less than 0.1% of the observed induction of the oxidative stress response in the sample, affirming the need to use effect-based trigger values that account for all chemicals present.
The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.
Disinfection of drinking water is the most successful measure to reduce water-borne diseases and protect health. However, disinfection byproducts (DBPs) formed from the reaction of disinfectants such as chlorine and monochloramine with organic matter may cause bladder cancer and other adverse health effects. In this study the formation of DBPs through a full-scale water treatment plant serving a metropolitan area in Australia was assessed using in vitro bioanalytical tools, as well as through quantification of halogen-specific adsorbable organic halogens (AOXs), characterization of organic matter, and analytical quantification of selected regulated and emerging DBPs. The water treatment train consisted of coagulation, sand filtration, chlorination, addition of lime and fluoride, storage, and chloramination. Nonspecific toxicity peaked midway through the treatment train after the chlorination and storage steps. The dissolved organic matter concentration decreased after the coagulation step and then essentially remained constant during the treatment train. Concentrations of AOXs increased upon initial chlorination and continued to increase through the plant, probably due to increased chlorine contact time. Most of the quantified DBPs followed a trend similar to that of AOXs, with maximum concentrations observed in the final treated water after chloramination. The mostly chlorinated and brominated DBPs formed during treatment also caused reactive toxicity to increase after chlorination. Both genotoxicity with and without metabolic activation and the induction of the oxidative stress response pathway showed the same pattern as the nonspecific toxicity, with a maximum activity midway through the treatment train. Although measured effects cannot be directly translated to adverse health outcomes, this study demonstrates the applicability of bioanalytical tools to investigate DBP formation in a drinking water treatment plant, despite bioassays and sample preparation not yet being optimized for volatile DBPs. As such, the bioassays are useful as monitoring tools as they provide sensitive responses even at low DBP levels.
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