Fate and behaviour of Microplastics (> 25µm) within the water distribution network, from water treatment works to service reservoirs and customer taps.

Water treatment works have previously shown high efficiency in removing microplastics > 25 µm from raw source water. However, what is less well known is the extent to which microplastics of this size class are generated or lost within the water distribution network, particularly whether there is a greater presence in the customer tap than in the water treatment works outlet. This study focused on the presence of 21 different types of synthetic polymer particles with sizes larger than 25 µm examined through multiple rounds of sampling at outlets of water treatment works (WTW), service reservoirs (SR), and customer taps (CT) managed by seven different water companies in Britain. Nineteen different types of polymers were detected; their signature and concentration varied based on the round of sampling, the location within the water supply network, and the water company responsible for managing the supply. Among the polymers examined, polyamide (PA), polyethene terephthalate (PET), polypropylene (PP), and polystyrene (PS) were the most commonly found. Apart from PET having its highest concentration of 0.0189 microplastic per litre (MP/L) in the SR, the concentrations of the other three most frequent polymers (PS = 0.017 MP/L, PA = 0.0752 MP/L, PP= 0.1513 MP/L) were highest in the CT. The overall prevalence of this size of microplastics in the network is low, but there was a high variability of polymer types and occurrences. These spatial and temporal variations suggested that the MP in the distribution network may exist as a series of pulses. Given the presence and polymer types, the potential for some of the microplastics to originate from materials used in the water network and domestic plumbing systems cannot be ruled out. As found before, the absolute number of microplastics in the water distribution network remained extremely low.

Diabetic hyperglycemia promotes primary tumor progression through glycation-induced tumor extracellular matrix stiffening.

Diabetes mellitus is a complex metabolic disorder that is associated with an increased risk of breast cancer. Despite this correlation, the interplay between tumor progression and diabetes, particularly with regard to stiffening of the extracellular matrix, is still mechanistically unclear. Here, we established a murine model where hyperglycemia was induced before breast tumor development. Using the murine model, in vitro systems, and patient samples, we show that hyperglycemia increases tumor growth, extracellular matrix stiffness, glycation, and epithelial-mesenchymal transition of tumor cells. Upon inhibition of glycation or mechanotransduction in diabetic mice, these same metrics are reduced to levels comparable with nondiabetic tumors. Together, our study describes a novel biomechanical mechanism by which diabetic hyperglycemia promotes breast tumor progression via glycating the extracellular matrix. In addition, our work provides evidence that glycation inhibition is a potential adjuvant therapy for diabetic cancer patients due to the key role of matrix stiffening in both diseases.

Semi-automated analysis of microplastics in complex wastewater samples.

In order to assess risks to the natural environment from microplastics, it is necessary to have reliable information on all potential inputs and discharges. This relies on stringent quality control measures to ensure accurate reporting. Here we focus on wastewater treatment works (WwTWs) and the complex sample matrices these provide. Composite samples of both influent and effluent were collected over a 24 h period on two separate occasions from eight different WwTWs across the UK. Sludge samples were taken on five occasions from five WwTWs. The WwTW treatments included activated sludge, trickling filter and biological aerated flooded filter with or without tertiary treatment. Using micro-FTIR analysis, microplastics ≥25 µm were identified and quantified. Procedural blanks were used to derive limits of detection (LOD) and limits of quantification (LOQ). Where values were above the LOQ, microplastics in the influent ranged from 955 to 17,214 microplastic particles/L and in the effluent from 2 to 54 microplastic particles/L, giving an average removal rate of 99.8%. Microplastics could be quantified in sludge at concentrations of 301-10,380 microplastics/g dry weight, this analytical method therefore revealing higher concentrations than reported in previous studies. The most common polymers present overall were polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET). We also report on critical considerations for blank corrections and quality control measures to ensure reliable microplastic analysis across different sample types.

Identification and Quantification of Microplastics in Potable Water and Their Sources within Water Treatment Works in England and Wales.

Microplastics were characterized in eight water treatment works (WTWs) in England and Wales (UK). Sources included river water, groundwater, and an upland reservoir. Water treatment varied from disinfection, filtration, sedimentation, and activated carbon techniques. At each WTW, five repeat samples of raw and potable water and two repeat sludge samples were taken over 5 months. Microplastics in water were captured on 10 µm filters and nonplastic materials digested in the laboratory. Microplastics ≥25 µm were analyzed using Fourier-transform infrared microscopy. Blanks revealed consistent polyethylene (PE), poly(ethylene terephthalate) (PET), and polypropylene (PP) contamination. Spike recoveries for 63-90 µm polyamide microplastics demonstrated 101% (standard deviation, SD 27%) and 113% (SD 15%) recovery for raw and potable waters and 52% (SD 13%) for sludge. Only four of the six WTWs sampled for raw water and only two of eight WTWs in their potable water had microplastics above the limit of quantification. Considering only the WTWs with quantifiable microplastics, then on average, 4.9 microplastic particles/L were present in raw water and only 0.00011 microplastic particles/L were present in potable water (99.99% removal). Values in waste sludge were highly variable. PE, PET, and PP were the most common polymers quantified in raw water and sludge, and polystyrene and acrylonitrile butadiene styrene were the most common polymers quantified in potable water.

Engineered microfluidic bioreactor for examining the three-dimensional breast tumor microenvironment.

The interaction of cancer cells with the stromal cells and matrix in the tumor microenvironment plays a key role in progression to metastasis. A better understanding of the mechanisms underlying these interactions would aid in developing new therapeutic approaches to inhibit this progression. Here, we describe the fabrication of a simple microfluidic bioreactor capable of recapitulating the three-dimensional breast tumor microenvironment. Cancer cell spheroids, fibroblasts, and endothelial cells co-cultured in this device create a robust microenvironment suitable for studying in real time the migration of cancer cells along matrix structures laid down by fibroblasts within the 3D tumor microenvironment. This system allows for ready evaluation of response to targeted therapy.

Does exposure to domestic wastewater effluent (including steroid estrogens) harm fish populations in the UK?

Historic fisheries data collected from locations across the UK over several years were compared with predicted estrogen exposure derived from the resident human population. This estrogen exposure could be viewed as a proxy for general sewage (wastewater) exposure. With the assistance of the Environment Agency in the UK, fisheries abundance data for Rutilis rutilis (roach), Alburnus alburnus (bleak), Leuciscus leuciscus (dace) and Perca fluviatilis (perch) from 38 separate sites collected over 7 to 17year periods were retrieved. From these data the average density (fish/m2/year) were compared against average and peak predicted estrogen (wastewater) exposure for these sites. Estrogen concentrations were predicted using the LF2000-WQX model. No correlation between estrogen/wastewater exposure and fish density could be found for any of the species. Year on year temporal changes in roach population abundance at 3 sites on the middle River Thames and 4 sites on the Great Ouse were compared against estrogen exposure over the preceding year. In this case the estrogen prediction was calculated based on the upstream human population providing the estrogen load and the daily flow value allowing concentration to be estimated over time. At none of the sites on these rivers were temporal declines in abundance associated with preceding estrogen (effluent) exposure. The results indicate that, over the past decade, wastewater and estrogen exposure has not led to a catastrophic decline in these four species of cyprinid fish.

PI3K Inhibition Reduces Mammary Tumor Growth and Facilitates Antitumor Immunity and Anti-PD1 Responses.

Purpose: Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,ß in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered antitumor immunity.Experimental Design: The effect of PI3K inhibition on tumor growth, metastasis, and antitumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ-null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor-infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30 mg/kg, every day) or vehicle-treated mice and PI3Kγnull versus PI3KγWT mice. On the basis of the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30 mg/kg, every day) and anti-PD1 (100 µg, twice weekly) was evaluated in PyMT tumor-bearing mice.Results: Our findings show that PI3K activity facilitates tumor growth and surprisingly restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The antitumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+ T-cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared with either agent alone.Conclusions: PI3K inhibition slows tumor growth, enhances antitumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple-negative breast cancer. Clin Cancer Res; 23(13); 3371-84. ©2016 AACR.

Regional multi-compartment ecological risk assessment: Establishing cadmium pollution risk in the northern Bohai Rim, China.

Ecological risk assessment (ERA) has been widely applied in characterizing the risk of chemicals to organisms and ecosystems. The paucity of toxicity data on local biota living in the different compartments of an ecosystem and the absence of a suitable methodology for multi-compartment spatial risk assessment at the regional scale has held back this field. The major objective of this study was to develop a methodology to quantify and distinguish the spatial distribution of risk to ecosystems at a regional scale. A framework for regional multi-compartment probabilistic ecological risk assessment (RMPERA) was constructed and corroborated using a bioassay of a local species. The risks from cadmium (Cd) pollution in river water, river sediment, coastal water, coastal surface sediment and soil in northern Bohai Rim were examined. The results indicated that the local organisms in soil, river, coastal water, and coastal sediment were affected by Cd. The greatest impacts from Cd were identified in the Tianjin and Huludao areas. The overall multi-compartment risk was 31.4% in the region. The methodology provides a new approach for regional multi-compartment ecological risk assessment.

Risk of endocrine disruption to fish in the Yellow River catchment in China assessed using a spatially explicit model.

The global water availability assessment (GWAVA) model, incorporating regional water abstractions and reservoir information, was used to model the human-sourced steroid estrogens estrone (E1) and estradiol (E2) in the Yellow River catchment (China). The river flows in the main stem were calibrated using gauged flows. Following a review of Chinese data on estrogen discharge from a range of sewage treatment plants, low, median, and high discharge rates were identified and used as best, expected, and worst-case scenarios, respectively. For any given location, the temporal variation of modeled estrogen levels was summarized using the mean and upper 90th percentile, which is where the model predicts 90% of values would be below this concentration. The predicted means and 90th percentiles for E1 were comparable to previous E1 measurements reported in the river. For the whole catchment, only 19% (mean value) of the river system by length was predicted to exceed 1 ng/L E2 equivalents (EEQs) using expected estrogen sewage discharge. Only 3% of the network by length was predicted to exceed the dangerously high 10 ng/L EEQ when considering 90th percentile concentrations. The highest exposures were in the Fen and Wei tributaries. Endocrine disruption risk from estrogens was predicted to be minimal in the main stem. Only in the worst-case discharge scenario and 90th percentile predicted concentrations were the most downstream river reaches of the main stem predicted to be at risk. Reservoirs appeared to be helpful in reducing estrogen concentrations thanks to longer water residence facilitating biodegradation.

The challenge presented by progestins in ecotoxicological research: a critical review.

Around 20 progestins (also called gestagens, progestogens, or progestagens) are used today in assisting a range of medical conditions from endometrial cancer to uterine bleeding and as an important component of oral contraception. These progestins can bind to a wide range of receptors including progestin, estrogen, androgen, glucocorticoid, and mineralocorticoid receptor, as well as sex hormone and corticosteroid binding globulins. It appears that only five of these (four synthetic and one natural) progestins have so far been studied in sewage effluent and surface waters. Analysis has reported values as either nondetects or low nanograms per liter in rivers. Seven of the progestins have been examined for their effects on aquatic vertebrates (fish and frogs). The greatest concern is associated with levonorgestrel, norethisterone, and gestodene and their ability to reduce egg production in fish at levels of 0.8-1.0 ng/L. The lack of environmental measurements, and some of the contradictions in existing values, however, hampers our ability to make a risk assessment. Only a few nanograms per liter of ethynodiol diacetate and desogestrel in water would be needed for fish to receive a human therapeutic dose for these progestins according to modeled bioconcentration factors. But for the other synthetic progestins levels would need to reach tens or hundreds of nanograms per liter to achieve a therapeutic dose. Nevertheless, the wide range of compounds, diverse receptor targets, and the effect on fish reproduction at sub-nanogram-per-liter levels should prompt further research. The ability to impair female reproduction at very low concentrations makes the progestins arguably the most important pharmaceutical group of concern after ethinylestradiol.

Elevated risk from estrogens in the Yodo River basin (Japan) in winter and ozonation as a management option.

A simple model was set up to predict estrogen concentrations and endocrine disruption risk in the Yodo River, Japan. This catchment spans the conurbations of Kyoto and Osaka and is the main source of drinking water for Osaka City, Japan. From the river survey data (5 separate occasions between 2005 and 2008), a maximum of 32 g per day estrone (E1) load was observed in the most downstream site of the river. Predicted E1 concentrations were in reasonable agreement with the measurements taken at several points within the basin from a series of sampling campaigns. The predicted concentrations exceeded a net estradiol (E2) equivalent of 1 ng L(-1) on only a few occasions, suggesting that only limited endocrine disruption phenomena in fish along the Yodo River is likely. The model was then used to examine the impact on estrogen concentrations and endocrine disruption of a number of different scenarios. It was found that in-river biodegradation had little effect on predicted concentrations and the outcome of endocrine disruption along the catchment. However, reduced sewage treatment removal, as can be experienced in winter in Japan, led to levels of 3.1 ng L(-1) E2 equivalents being possible. The reduced river flow in winter in Japan exacerbates the situation as it offers less dilution. It was found that the application of the ozonation process as a tertiary sewage treatment in winter could prevent this higher risk endocrine disruption situation.

Do concentrations of ethinylestradiol, estradiol, and diclofenac in European rivers exceed proposed EU environmental quality standards?

This study used a geographic based water model to predict the environmental concentrations of three pharmaceuticals, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), and diclofenac throughout European rivers. The work was prompted by the proposal of the European Community (COM(2011)876) to consider these chemicals as candidates for future control via environmental quality standards (EQS). National drug consumption information, excretion, national water use, and sewage removal rates, were used to derive per capita sewage effluent values for the European countries . For E2, excretion rates of the natural hormone and national demographics were also included. Incorporating this information into the GWAVA model allowed water concentrations throughout Europe's rivers to be predicted. The mean concentration from the expected sewage discharge scenario indicated that 12% by length of Europe's rivers would reach concentrations greater than the proposed 0.035 ng/L EQS for EE2. For several countries, between a quarter and a third of their total river length would fail such an EE2 EQS. For E2, just over 1% by length of rivers would reach concentrations greater than the 0.4 ng/L proposed EQS, while just over 2% by length of rivers would reach concentrations greater than the proposed EQS of 100 ng/L for diclofenac.

Endocrine disruption due to estrogens derived from humans predicted to be low in the majority of U.S. surface waters.

In an effort to assess the combined risk estrone (E1), 17ß-estradiol (E2), 17α-ethinyl estradiol (EE2), and estriol (E3) pose to aquatic wildlife across United States watersheds, two sets of predicted-no-effect concentrations (PNECs) for significant reproductive effects in fish were compared to predicted environmental concentrations (PECs). One set of PNECs was developed for evaluation of effects following long-term exposures. A second set was derived for short-term exposures. Both sets of PNECs are expressed as a 17ß-estradiol equivalent (E2-eq), with 2 and 5 ng/L being considered the most likely levels above which fish reproduction may be harmed following long-term and short-term exposures, respectively. A geographic information system-based water quality model, Pharmaceutical Assessment and Transport Evaluation (PhATE™), was used to compare these PNECs to mean and low flow concentrations of the steroid estrogens across 12 U.S. watersheds. These watersheds represent approximately 19% of the surface area of the 48 North American states, contain 40 million people, and include over 44,000 kilometers of rivers. This analysis determined that only 0.8% of the segments (less than 1.1% of kilometers) of these watersheds would have a mean flow E2-eq concentration exceeding the long-term PNEC of 2.0 ng/L; only 0.5% of the segments (less than 0.8% of kilometers) would have a critical low flow E2-eq exceeding the short-term PNEC of 5 ng/L. Those few river segments where the PNECs were exceeded were effluent dominated, being either headwater streams with a publicly owned treatment works (POTW), or flowing through a highly urbanized environment with one or several POTWs. These results suggest that aquatic species in most U.S. surface waters are not at risk from steroid estrogens that may be present as a result of human releases.

Comparing predicted against measured steroid estrogen concentrations and the associated risk in two United Kingdom river catchments.

Predicted concentrations of estrone, 17ß-estradiol, and 17α-ethinylestradiol generated from a geographical information systems-based model (LF2000-WQX) have previously been used to assess the risk of causing intersex in male fish in the rivers of England and Wales, United Kingdom. Few measured data of sufficient quality and spatial extent have been available to verify this risk assessment. New measured data have been collected from sewage treatment plant effluents and the receiving waters upstream and downstream of these discharges from the Erewash River and the Avon River systems in England. The model results for these rivers were in good agreement with the measured values in terms of estradiol equivalents. Critically, the risk assessment based on the measured data gave a risk assessment nearly identical to that derived from the modeled results. For individual estrogens, 17α-ethinylestradiol was modeled best and estrone worst. Poor simulations reflected poor estimates of the effluent concentrations, which were more variable from day to day and between works of nominally similar type than is assumed in the model. In support of this, model results for the Erewash River, calculated using observed effluent concentrations, were in excellent agreement with the measured data. The model has proved to be adequate in predicting overall estrogenic potency, and therefore risk, along these rivers; however, improvements are possible, particularly in predicting STP removal efficiency and therefore effluent concentrations.

How seasonality affects the flow of estrogens and their conjugates in one of Japan's most populous catchments.

A detailed study of the free and conjugated estrogen load discharged by the eight major sewage treatment plants into the Yodo River basin, Japan was carried out. Sampling campaigns were focused on the winter and autumn seasons from 2005 to 2008 and the free estrogens estrone(E1), 17ß-estradiol(E2), estriol(E3), 17α-ethynylestradiol(EE2) as well as their conjugated (sulfate and glucuronide) forms. For both sewage effluent and river water E2 and E1 concentrations were greatest during the winter period (December-March). This coincides with the period of lowest rainfall and lowest temperatures in Japan. E1 was the dominant estrogenic component in effluent (means of 10-50 ng/L) followed by E2 (means of 0.5-3 ng/L). The estrogen sulfate conjugates were found intermittently in the 0.5-1.7 ng/L concentration range in the sewage effluents. The greatest estrogen exposure was found to be in the Katsura River tributary which exceeded 1 ng/L E2-equivalents during the winter period.

The arrival and discharge of conjugated estrogens from a range of different sewage treatment plants in the UK.

The occurrence of free and conjugated estrogens was examined in a survey of eleven sewage treatment plants (STPs) and their discharge water in the United Kingdom using grab sampling. The STPs included trickling filter with and without tertiary treatment, and activated sludge with tertiary treatment. For three activated sludge plants both influent and effluent samples were compared. For a further 8STPs only the effluent was examined. The estrone-3-sulphate, estradiol-3-sulphate and estriol-3-sulphate concentrations (up to 20 ng L(-1)) were typically 5-fold that of the respective free estrogen concentration in the effluents. This represents a substantial additional 'potential' estrogen load arriving in the receiving waters. Estrone-3-glucuronide was found at 9 ng L(-1), estradiol-3-glucuronide at 7 ng L(-1), and estriol-3-glucuronide at 32 ng L(-1) in sewage influent. Except on one occasion, no glucuronide conjugates could be found in the effluent. The results suggest in most cases glucuronide conjugates will be completely transformed in sewage treatment whilst sulphate conjugates will only be partially removed.

Predicting national exposure to a point source chemical: Japan and endocrine disruption as an example.

The predicted aquatic estrogen concentrations for the whole of England and Japan were determined and compared using population and flow data. The overall value for English surface waters was 0.9 ng/L estradiol equivalents (EEQ) compared to 0.1 ng/L overall for Japan. Available dilution of sewage effluent was considered to be more important than contraceptive pill usage in this relative risk. A national survey of Japanese rivers using the yeast estrogen assay (YES) gave a median value of 0.27 ng/L EEQ which, while higher than that predicted, confirmed an overall low endocrine disruption risk. Using local population and flow data for 27 separate catchments, the predicted EEQ and measured EEQ (YES) values compared well, confirming the national picture that endocrine disruption would not be a widespread phenomena in Japan. Simple predictions based on population and flow can give an appropriate "ball park" value for catchments and even nations for concentrations of polar organic contaminants which have a majority human origin.

Natural variations in flow are critical in determining concentrations of point source contaminants in rivers: an estrogen example.

Daily steroid estrogen concentrations as 17ß-estradiol equivalents (E2 equiv.) were modeled from 1992 to 2008 for single locations on the well populated Thames and Soar rivers in England. The historic daily mean flow values which were the basis of this exercise came from a selected gauging site on each river. The natural variation in flow from winter to summer typically produced a 20- to 30-fold difference in predicted estrogen concentration over the course of a year. Based on all the predicted values from minimum to maximum over the 1992 to 2008 period there was a 98-fold difference in estrogen concentrations on the basis of flow alone for the Thames (0.1-12.7 ng/L E2 equiv.) and 67-fold for the Soar (0.2-13.3 ng/L E2 equiv.). This compares to a predicted 0.5-fold difference that could arise from differences in sewage treatment and 0.1-fold difference due to differences in in-stream biodegradation. The seasonal variation in flow generated a repeating "roller coaster" in predicted estrogen concentrations. Regularly measured phosphate data for the river Avon over the period 1993 to 1996, where point sources also dominate, was compared against flow and predicted estrogen concentrations. The pattern of predicted estrogen and measured total phosphate concentration were very closely related. This dramatic variation in contaminant concentration over the year due to flow poses questions over what we mean by environmental relevance and the representation of the real environment in aquatic ecotoxicity tests.

The use of modelling to predict levels of estrogens in a river catchment: how does modelled data compare with chemical analysis and in vitro yeast assay results?

Effluent discharges at Rodbourne sewage treatment works (STWs) were assessed using chemical and in vitro biological analysis as well as modelling predictions. Results showed that Rodbourne STW discharged less estrone (E1) than expected, but similar 17beta-estradiol (E2) and 17alpha-ethinyl estradiol (EE2) to those predicted by a widely cited effluent prediction model. The Exposure Analysis Modelling System (EXAMS) model was set up using measured effluent concentrations as its starting point to predict estrogen concentrations along a 10 km length of the receiving water of the River Ray. The model adequately simulated estrogen concentrations along the river when compared to July 2007 measured data. The model predicted combined estrogen equivalents in reasonable agreement with estrogenicity as measured by passive sampler (POCIS) extracts using the yeast estrogen screen. Using gauged mean flow values for 2007 the model indicated that the most important determinand for estrogen exposure in the Ray was not season, but proximity to the Rodbourne effluent. Thus, fish in the first 3 km downstream of Rodbourne were typically exposed to two or even three times more estrogens than those living 7-10 km further downstream. The modelling indicated that, assuming the effluent estrogen concentrations measured in February 2008 were typical, throughout the year the whole length of the Ray downstream of Rodbourne would be estrogenic, i.e. exceeding the 1 ng/L E2 equivalent threshold for endocrine disruption.

Gas-liquid chromatography-tandem mass spectrometry methodology for the quantitation of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents and from wild populations.

Fish can be exposed to a complex mixture of chemical contaminants arising from the exposure to wastewater treatment works (WwTWs) effluents. Some of these contaminants are estrogenic and have been associated with feminisation of male fish and the presence of populations containing intersex individuals. However the detection of trace levels (ng/L) of estrogenic chemicals surface waters can be difficult and does not give information on the exposure of aquatic organisms to these contaminants. In this study we assessed whether the analysis of estrogenic substances that bioconcentrate in fish bile can be used to detect the exposure of fish to feminising contaminants in receiving waters and effluents, and thus facilitate their monitoring of these substances in aquatic environments. Estrogenic metabolites in bile were deconjugated using enzymatic hydrolysis and partially purified by solid phase extraction. Steroidal and xenoestrogens were derivatized to their trimethylsilyl ethers and quantified by gas-liquid chromatography-mass spectrometry (GC-MS/MS) using multiple reaction monitoring. The method was validated using spiked bile samples from immature female rainbow trout (Oncorhynchus mykiss) as well as bile from sexually mature roach (Rutilus rutilus) that had been exposed to either tap water or an undiluted estrogenic effluent for 10 days or captured from a river site downstream of a WwTWs effluent discharge. The mean recovery of target analytes from spiked bile was between 86 and 99% and the limit of detection was between 0.1 and 0.7ng/mL bile for bisphenol A (BPA), 17beta-estradiol (E2), estrone (E1) and 17alpha-ethinylestradiol (EE2), and 11, 60 and 327ng/mL bile for branched nonyl chain isomeric mixtures of 4-nonylphenolethoxylate (NP1EO), 4-nonylphenol (NP) and 4-nonylphenoldiethoxylate (NP2EO), respectively. All target analytes were detected in bile from roach exposed directly to a WwTWs effluent, with concentrations between 6-13microg/mL bile for NP, 18-21microg/mL for NP1EO, 75-135microg/mL for NP2EO, 0.7-2.5microg/mL for BPA, E2 and E1 and 17-29ng/mL for EE2. With the exception of NP2EO, all analytes were detected in at least 2 out of the 5 fish sampled from the River Thames. BPA and NP1EO were detected in all three reference fish held in tap water indicating possible contamination from laboratory plastics. The work shows that analysis of 20-100microL quantities of bile could be a useful approach in detecting exposure to mixtures of estrogenic contaminants taken up by fish from WwTW effluents and has the potential for monitoring the efficacy of remediation strategies that may be adopted for reduction of these endocrine disrupting chemicals in the aquatic environment.