Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.

The growing use of recycled water in large urban centres requires comprehensive public health risk assessment and management, an important aspect of which is the assessment and management of residual trace chemical substances. Bioanalytical methods such as in vitro bioassays may be ideal screening tools that can detect a wide range of contaminants based on their biological effect. In this study, we applied thirteen in vitro assays selected explicitly for their ability to detect molecular and cellular effects relevant to potential chemical exposure via drinking water as a means of screening for chemical contaminants from recycled water at 9 Australian water reclamation plants, in parallel to more targeted direct chemical analysis of 39 priority compounds. The selected assays provided measures of primary non-specific (cytotoxicity to various cell types), specific (inhibition of acetylcholinesterase and endocrine receptor-mediated effects) and reactive toxicity (mutagenicity and genotoxicity), as well as markers of adaptive stress response (modulation of cytokine production) and xenobiotic metabolism (liver enzyme induction). Chemical and bioassay analyses were in agreement and complementary to each other: the results show that source water (treated wastewater) contained high levels of biologically active compounds, with positive results in almost all bioassays. The quality of the product water (reclaimed water) was only marginally better after ultrafiltration or dissolved air floatation/filtration, but greatly improved after reverse osmosis often reducing biological activity to below detection limit. The bioassays were able to detect activity at concentrations below current chemical method detection limits and provided a sum measure of all biologically active compounds for that bioassay, thus providing an additional degree of confidence in water quality.

Assessment of wastewater and recycled water quality: a comparison of lines of evidence from in vitro, in vivo and chemical analyses.

We investigated water quality at an advanced water reclamation plant and three conventional wastewater treatment plants using an "ecotoxicity toolbox" consisting of three complementary analyses (chemical analysis, in vitro bioanalysis and in situ biological monitoring), with a focus on endocrine disruption. The in vitro bioassays were chosen to provide an appropriately wide coverage of biological effects relevant to managed aquifer recharge and environmental discharge of treated wastewater, and included bioassays for bacterial toxicity (Microtox), genotoxicity (umuC), photosynthesis inhibition (Max-I-PAM) and endocrine effects (E-SCREEN and AR-CALUX). Chemical analysis of hormones and pesticides using LCMSMS was performed in parallel to correlate standard analytical methods with the in vitro assessment. For two plants with surface water discharge into open drains, further field work was carried out to examine in situ effects using mosquitofish (Gambusia holbrooki) as a bioindicator species for possible endocrine effects. The results show considerable cytotoxicity, phytotoxicity, estrogenicity and androgenicity in raw sewage, all of which were significantly reduced by conventional wastewater treatment. No biological response was detected to RO water, suggesting that reverse osmosis is a significant barrier to biologically active compounds. Chemical analysis and in situ monitoring revealed trends consistent with the in vitro results: chemical analysis confirmed the removal trends observed by the bioanalytical tools, and in situ sampling did not reveal any evidence of endocrine disruption specifically due to discharge of treated wastewater (although other sources may be present). Biomarkers of exposure (in vitro) and effect (in vivo or in situ) are complementary and together provide information with a high level of ecological relevance. This study illustrates the utility of combining multiple lines of evidence in the assessment of water quality.

Comparison of five in vitro bioassays to measure estrogenic activity in environmental waters.

Bioassays are well established in the pharmaceutical industry and single compound analysis, but there is still uncertainty about their usefulness in environmental monitoring. We compared the responses of five bioassays designed to measure estrogenic activity (the yeast estrogen screen, ER-CALUX, MELN, T47D-KBluc, and E-SCREEN assays) and chemical analysis on extracts from four different water sources (groundwater, raw sewage, treated sewage, and river water). All five bioassays displayed similar trends and there was good agreement with analytical chemistry results. The data from the ER-CALUX and E-SCREEN bioassays were robust and predictable, and well-correlated with predictions from chemical analysis. The T47D-KBluc appeared likewise promising, but with a more limited sample size it was less compelling. The YES assay was less sensitive than the other assays by an order of magnitude, which resulted in a larger number of nondetects. The MELN assay was less predictable, although the possibility that this was due to laboratory-specific difficulties cannot be discounted. With standardized bioassay data analysis and consistency of operating protocols, bioanalytical tools are a promising advance in the development of a tiered approach to environmental water quality monitoring.

Balancing the budget of environmental estrogen exposure: the contribution of recycled water.

Estrogenic endocrine disrupting compounds (e-EDCs) are present in treated sewage and there is concern about estrogenicity of potable recycled water. However e-EDCs are also present in other environmental media and intake from water needs to be considered in relation to these other sources. The concentrations of 13 e-EDCs in foodstuffs and drinking water are reviewed, their predicted concentrations in recycled water are estimated, and the daily estrogenic intake as 17beta-estradiol equivalent (EEq) based on both in vitro and in vivo potencies is calculated as 1.39 and 9.65 microg EEq/d, respectively. Dietary intake accounts for more than 99.8% of that total, and more than 84.2% is due to phytosterols. Drinking 2 L of recycled water per day is expected to add 0.001 to 0.016 microg EEq/d based on in vitro and in vivo potencies, respectively. Exposure to e-EDCs in recycled water is therefore likely to be insignificant compared to current dietary intakes.

Ecotoxicities of polyquaterniums and their associated polyelectrolyte-surfactant aggregates (PSA) to Gambusia holbrooki.

The toxicity of 11 polyquaterniums used in cosmetic applications, and polydimethyldiallylammonium chloride (poly(DADMAC)) were studied for toxicity of the polyquaternium alone, and of a polyquaternium/anionic surfactant complex as occurs in some cosmetic formulations. The surfactant used in the study was sodium dodecyl sulfate (SDS), which is used in cosmetic formulations under its International Nomenclature of Cosmetic Ingredients (INCI) name Sodium Laurel Sulfate. In fish immobilization studies with Gambusia holbrooki, the EC(50) of the polyquaternium/surfactant complex was found to be the same as or similar to the EC(50) for the polyquaternium alone. The toxicity of the polyquaterniums investigated was similar to the published values for other cationic polyelectrolytes and cationic surfactants, in the range from < 1.0 to 10 mg/L, with the exception of low charge density cellulosic polyquaterniums. The anionic surfactant alone was not toxic to fish in the concentration range tested. Results thus showed the toxicity of the polyquaternium was not mitigated by the presence of the anionic surfactant.

Stir bar sorptive extraction and trace analysis of selected endocrine disruptors in water, biosolids and sludge samples by thermal desorption with gas chromatography-mass spectrometry.

Stir bar sorptive extraction (SBSE) in combination with thermal desorption coupled to gas chromatography-mass spectrometry (GC-MS) was successfully applied to analyze a range of endocrine disrupting compounds (EDCs) in wastewater, solids and sludge. The targeted EDCs include sex steroid hormones, phthalates, alkylphenols and tamoxifen. Recovery for the EDCs using this analytical technique ranged from 44% to 128%. Limit of detection (LOD) of the compounds was 2.0 ng/L for water samples and 0.02 ng/g for solid samples, whereas the limit of quantitation (LOQ) was 5.0 ng/L for water samples and 0.06 ng/g for solid samples. When this analytical technique was applied to measure EDC concentration in a biological nutrient removal (BNR) wastewater treatment plant located in South East Queensland, Australia, the results showed that there were high amounts of phthalates, alkylphenols and female hormones present in the raw influent wastewater and solids. These concentrations were dramatically reduced after passing through the various treatment zones of the bioreactor (anaerobic, aerobic and anoxic).

Modelling of the fate of selected endocrine disruptors in a municipal wastewater treatment plant in South East Queensland, Australia.

The aim of this study was to develop a fugacity-based analysis of the fate of selected industrial compounds (alkylphenols and phthalates) with endocrine disrupting properties in a conventional activated sludge wastewater treatment plant (WWTP A) in South East Queensland, Australia. Using mass balance principles, a fugacity model was developed for correlating and predicting the steady-state-phase concentrations, the process stream fluxes, and the fate of four phthalates and four alkylphenols in WWTP A. Input data are the compound's physicochemical properties, measured concentrations and the plant's operating design and parameters. The relative amounts of chemicals that are likely to be volatilized, sorbed to sludge, biotransformed, and discharge in the effluent water was determined. Since it was difficult to predict biotransformation, measured concentrations were used to calibrate the model in terms of biotransformation rate constant. Results obtained by applying the model for the eight compounds showed <40% differences between most of the estimated and measured data from WWTP A. All eight compounds that were modelled in this study had high removal efficacy from WWTP A. Apart from benzyl butyl phthalate and bisphenol A, the majority is removed via biotransformation followed by a lesser proportion removed with the primary sludge. Fugacity analysis provides useful insight into compound fate in a WWTP and with further calibration and validation the model should be useful for correlative and predictive purposes.

Comprehensive study of endocrine disrupting compounds using grab and passive sampling at selected wastewater treatment plants in South East Queensland, Australia.

Chemical (gas chromatography-mass spectrometry, GC-MS) and biological (E-Screen assay) analyses were used to determine the concentrations of 15 endocrine disrupting compounds (EDCs) and estrogen equivalent (EEq) in grab and passive samples from five municipal wastewater treatment plants (WWTPs) in South East Queensland, Australia. EEq concentrations derived by E-Screen assays for the grab samples were between 108-356 ng/L for the influents and < 1-14.8 ng/L for the effluents with the exception of one effluent sample which was at 67.8 ng/L EEq. The EDC concentrations and EEq values for the passive samples were several times lower than those of the grab samples: a decrease probably caused by, but not limited to biofouling, low flow rate, biodegradation and temperature which can progressively reduce the uptake of compounds into the sampler. At this stage, grab sampling is the most reliable method for field monitoring; nevertheless, passive sampler is a useful sampling tool but the method requires more research to ensure that the information obtained can be interpreted appropriately. Although alkylphenols and phthalates were detected at higher concentrations in the wastewater samples as compared to natural hormones, the environmental risk may be negligible as their estrogenic potencies are several orders of magnitude lower than that of the natural estrogens. In most wastewater samples, the natural estrogens contributed to 60% or more of the EEq value. Removal efficacy of most estrogenic and xenoestrogenic compounds from the conventional activated sludge or biological nutrient removal (BNR) WWTPs monitored in this study was in the range of 80-> 99%. The efficiency of the WWTPs in removing estrogenic activity was > 95%. The EEqs of the E-Screen and those calculated from the results of extensive chemical analyses using the estradiol equivalency factors were comparable for most of the WWTPs samples.

Endocrine-disrupting compounds: a review of their challenge to sustainable and safe water supply and water reuse.

The relevance of endocrine-disrupting compounds as potential contaminants of drinking water is reviewed, particularly in the reuse of wastewater. Growing populations and increasing intensification of land and water use for industry and agriculture have increased the need to reclaim wastewater for reuse, including to supplement the drinking water supply. The variety of anthropogenic chemicals that have been identified as potential endocrine disruptors in the environment and the problems arising from their use as human and livestock pharmaceuticals, as agricultural chemicals and in industry are discussed. The potentially adverse impact of these chemicals on human health and the ecology of the natural environment are reviewed. Data for the removal of estrogenic compounds from wastewater treatment are presented, together with the comparative potencies of estrogenic compounds. The relative exposure to estrogens of women on oral contraceptives, hormone replacement therapy, and through food consumption is estimated. A brief overview of some methods available or under development for the assessment of estrogenic activity in environmental samples is provided. The review concludes with a discussion of the directions for further investigation, which include human epidemiology, methodology development, and wastewater monitoring.

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples.

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep (Ovis aries) uteri and rainbow trout (Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51-73 ng/L estradiol equivalents (EEq).

Bioassay-derived androgenic and estrogenic activity in municipal sewage in Australia and New Zealand.

Raw sewage and sewage at various stages of treatment were sampled from 15 municipal sewage treatment plants in south Queensland (Australia) and Canterbury (New Zealand). Estrogenic and androgenic activities were determined with sheep estrogen receptor and rainbow trout androgen receptor binding assays, respectively. Selected estrogenic chemicals were also analyzed by gas chromatography-mass spectrometry. The raw sewage influents contained significant levels of both estrogenic (<4-185 ng/L estradiol equivalents) and androgenic (1920-9330 ng/L testosterone equivalents) activity. Subsequent treatment of raw sewage successfully removed most of the activity so that the estrogenicity and androgenicity associated with the final effluents were very low (<1-4.2 ng/L estradiol equivalents and <6.5-736 ng/L testosterone equivalents, respectively). Secondary treatment was the most effective treatment step to remove estrogenic and androgenic activity from sewage water. Activated sludge treatment in particular removed 92% to >99% of the estrogenic activity and 82% to >99% of the androgenic activity in sewage.

Efficacy of an advanced sewage treatment plant in southeast Queensland, Australia, to remove estrogenic chemicals.

The estrogenicity profile of domestic sewage during treatment at a medium-sized (3800 EP) advanced biological nutrient removal plant in Queensland, Australia, was characterized using a sheep estrogen receptor binding assay (ERBA) and the MCF-7 breast cancer cell proliferation assay (E-Screen). The raw influent was highly estrogenic (20-54 ng/L EEq), and primary treatment resulted in a slight increase in estrogenicity that was detected in one of the assays (6-80 ng/L). Concurrent chemical analysis suggested that most of the estrogenicity in the influent was due to natural hormones (>48%). Secondary activated sludge treatment followed by nitrification/denitrification effectively removed > 95% of the estrogenic activity (to <0.75-2.6 ng/L), and estrogenicity of the final tertiary-treated effluent was below the detection limit of both assays (<0.75 ng/L).