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Medial patellar instability, though infrequently recognized, can be a disabling complication of a lateral retinacular release. Patients with persistent anterior knee pain and instability after lateral release should be evaluated closely. If evidence of increased medial patellar translation is identified on physical examination, a trial of reverse McConnell taping should be prescribed. If there is noted improvement in symptoms after this taping trial, reconstruction of the lateral patellotibial ligament should be considered. This article details our technique for lateral patellotibial ligament reconstruction using iliotibial band and patellar tendon autografts.

There is growing evidence that rising global temperatures resulting from climate change may exacerbate the toxic effect of pollutants and heterotherms, including fish, in which homestatic mechanisms are directly influenced by environmental temperature will be most affected. Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many of their drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity but this has received little consideration in the context of effects in wildlife. Further, these mechanisms are relatively poorly understood, particularly regarding multiple stressor interactions. We used transgenic TG(EpRE:mCherry) zebrafish, developed in our laboratory for detecting OS, as our experimental model. We show that the oxidative effects of high concentrations of pharmaceuticals from three different therapeutic classes (paracetamol, diclofenac and doxorubicin) are increased at temperatures elevated by 2-5 °C above those for zebrafish standard husbandry and relevant to their current natural environment (and predicted under the IPCC 2023 scenarios for intermediate to very high greenhouse gas emissions). These OS responses were primarily seen in the pronephros, liver, and gastrointestinal tract. The increase in OS at the increased water temperature may have resulted from the elevated temperature acting as a direct additive physiological stressor to the OS imposed by the drugs and/or via the temperature increasing the chemicals oxidative effect. For paracetamol, it appeared that the elevated responses at the higher temperature of 33 °C were in part due to an increase in uptake of the drug. Our data illustrate that risk assessments for chemicals inducing OS in fish (and likely other heterotherms) should consider the influence of temperature to ensure environmental protection in future environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0-4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild., Competing Interests: Declaration of competing interest CH was in receipt of a scholarship (awarded to CRT, MJW and LM-C) co-funded by AstraZeneca. SFO is an employee and share holder of AstraZeneca, a biopharmaceutical company with an interest in the discovery, development and commercialisation of prescription medicines including glucocorticoids., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Environmental DNA (eDNA) is a technique increasingly used for monitoring organisms in the natural environment including riverine macroinvertebrates. However, the effectiveness of eDNA for monitoring riverine macroinvertebrates compared with the more traditional method of sampling the organisms directly and identifying them via morphological analysis, has not been well established. Furthermore, the ability of the various gene markers and PCR primer sets to detect the full range of riverine invertebrate taxa has not been quantified. Here we conducted a meta-analysis of the available literature, to assess the effectiveness of eDNA sampling for detecting riverine macroinvertebrates compared with sampling for the organisms directly and applying morphological analysis. We found, on average, eDNA sampling, irrespective of the gene marker used, detected fewer riverine invertebrates than morphological sampling. The most effective PCR primer set for identifying taxa was mlCOIintF/jgHCO2198, (mlCOIintF- forward primer, jgHCO2198, - reverse primer). Regardless of the gene marker or primer sets used, however, many taxa were not detected by eDNA metabarcoding that were detected by sampling directly for these invertebrates, including over 100 members of Arthropoda. eDNA sampling failed to detect any species belonging to Nematoda, Platyhelminthes, Cnidaria or Nematomorpha and these markers applied for eDNA sampling in terrestrial systems also do not detect members of Nematoda. In addition to these issues, uncertainties relating to false positives from upstream DNA sources, the stability of DNA from different species, differences in the propensity for DNA release into the environment for different organisms, and lack of available sequence information for numerous taxa illustrates the use of eDNA is not yet applicable as a robust stand-alone method for the monitoring of riverine invertebrates. As a primary consideration, further methodological developments are needed to ensure eDNA captures some of the key freshwater taxa, notably taxa belonging to the phyla Arthropoda, Nematoda, Platyhelminthes, Cnidaria and Nematomorpha., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Imogen Poyntz-Wright reports financial support was provided by Natural Environment Research Council. Imogen Poyntz-Wright reports financial support was provided by United Kingdom Department for Environment Food and Rural Affairs., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Globally, riverine system biodiversity is threatened by a range of stressors, spanning pollution, sedimentation, alterations to water flow, and climate change. Pesticides have been associated with population level impacts on freshwater invertebrates for acute high-level exposures, but far less is known about the chronic impact of episodic exposure to specific classes of pesticides or their mixtures. Here we employed the use of the UK Environment Agency's monitoring datasets over 40 years (covering years 1980 to 2019) to assess the impacts of AChE (acetylcholinesterase) and GABA (gamma-aminobutyric acid) receptor targeting pesticides on invertebrate family richness at English river sites. Concentrations of AChE and GABA pesticides toxic to freshwater invertebrates occurred (measured) across 18 of the 66 river sites assessed. For one of the three river sites (all found in the Midlands region of England) where data recorded over the past 40 years were sufficient for robust modelling studies, both AChE and GABA pesticides associated with invertebrate family richness. Here, where AChE total pesticide concentrations were classified as high, 46 of 64 invertebrate families were absent, and where GABA total pesticide concentration were classified as high, 16 of 64 invertebrate families were absent. Using a combination of field evidence and laboratory toxicity thresholds for population relevant endpoints we identify families of invertebrates most at risk in the selected English rivers to AChE and GABA pesticides. We, furthermore, provide strong evidence that the absence of the invertebrate family Polycentropodidae (caddisfly) from one field site is due to exposure effects to AChE pesticides., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Imogen Poyntz-Wright reports financial support was provided by Natural Environment Research Council. Imogen Poyntz-Wright reports financial support was provided by United Kingdom Department for Environment Food and Rural Affairs. Susan Zappala reports a relationship with JNCC Support Co that includes: employment., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Globally freshwater biodiversity has experienced major decline and chemical pollutants are believed to have played a significant role in this decline, but this has not been well quantified for most riverine invertebrate populations. Here we applied a biogeographically independent trait-based bioindicator, SPEAR pesticides across sites across five regions (Northern, Midlands and Western, Anglian, Southeast, and Southwest) in England to investigate for associations specifically between pesticide use/pollution and riverine invertebrate communities over a 55-year period (1965-2019). Both spatially and temporally post-1990, the Anglian and Thames regions consistently showed the lowest SPEAR pesticides scores, illustrating the presence of fewer pesticide sensitive species. The Anglian region had the highest pesticide use compared to all other regions from 1990 to 2018 and there were negative relationships between the level of pesticide/insecticide use and the regional SPEAR pesticides score. Biochemical Oxygen Demand and ammonia, as measures of general water quality, were also negatively correlated with the SPEAR pesticides scores across the regions, but these factors were not the driver for the lower SPEAR pesticides scores seen in the Anglian region. Based on SPEAR pesticides scores, riverine invertebrate communities in England have been most impacted in the Anglian region and we evidence chronic insecticide exposure is likely a significant factor in shaping the status of those invertebrate communities., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Imogen Poyntz-Wright reports financial support was provided by Natural Environment Research Council. Imogen Poyntz-Wright reports financial support was provided by United Kingdom Department for Environment Food and Rural Affairs. Susan Zappala reports a relationship with JNCC Support Co that includes: employment., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures., Competing Interests: Declaration of Competing Interest SFO is an employee and shareholder of AstraZeneca PLC. All other authors declare no potentially competing interests., (Copyright © 2023. Published by Elsevier Inc.)

In anuran amphibians (frogs and toads), evidence linking pollution to population declines is limited, in particular through impaired reproduction. Here we review the evidence for pollutant-induced alterations on reproductive endpoints in wild anurans with a particular focus on the application of non-destructive endpoints including on sex ratios, male reproductive phenotypes (data are too scarce for females) and reproductive outputs (reflective of mating success). Data evidencing alterations in sex ratio in wild anurans are scarce, however, both feminisation and masculinisation in response to pollution have been reported (seven studies). Male nuptial pad morphology and calling behaviour display high sensitivity to pollutant-exposure and are important features determining male breeding success, however there is considerable variation in these endpoints and inconsistencies in the responses of them to pollution are reported in wild anurans. Data for clutch size are insufficient to assess sensitivity to pollutants (five studies only). However, hatch success and offspring fitness (tadpole survival/development) are sensitive to pollution, with clear linkages to population stability. In conclusion, there are a wide range of non destructive measures with good potential for application to assess/monitor reproductive health in wild anurans, however, a greater understanding of pollutant effects on these endpoints is needed. There measures deserve wider application as they are relatively simple and inexpensive to implement, and as they can be applied non-destructively are widely applicable to our declining anuran populations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frances Orton reports financial support was provided by Department of Environment, Food and Rural Affairs., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Harmful algal blooms (HABs) can have severe ecological, societal and economic impacts upon marine ecosystems, human health and the seafood industry. We evaluated changes in marine plankton communities with prevailing physico-chemical conditions throughout an exceptionally warm summer (2018), to elucidate key factors governing HABs and their impacts on shellfish mariculture in the western English Channel. Despite warm, stable weather conditions and widespread seasonal stratification throughout the summer, divergent plankton community compositions were observed at two rope-grown mussel (Mytilus edulis) farms (St Austell Bay and Lyme Bay) and a long-term ecological research LTER site (Plymouth L4). There were significant differences between sites in the abundances of HAB species, including Dinophysis spp. and Karenia mikimotoi, whose cell counts bloomed in excess of UK Food Standards Agency (FSA) advisory 'trigger' levels at Plymouth L4 and St Austell Bay, but not at the Lyme Bay site. The K. mikimotoi bloom occurred over two weeks in August and comprised up to 88% of the standing phytoplankton biomass in St Austell Bay. Dinophysis spp. also bloomed here from May to September, constituting up to 28% of phytoplankton biomass. This protracted bloom resulted in concentrations of Dinophysis toxins 1 & 2 and pectenotoxins and okadaic acid in shellfish, which closed shellfish harvesting operations on farms located in St Austell Bay, and other shellfish sites in the west of the western English Channel (but not in the east of the region). Inter-site differences in the abundances of these and other HAB species were associated with variations in water circulation and co-occurring phytoplankton and zooplankton communities. Furthermore, plankton monitoring data obtained from the L4 site over the past 3 decades showed HAB species (including Dinophysis spp.) with abundances commonly occurring above advisory trigger levels during warmer periods, such as that coinciding with our study. Under projected climate warming these blooms are likely to continue to be governed by regionally distinct patterns of water circulation, which need to be taken into account in marine spatial planning, when assessing the suitability of new shellfish mariculture sites., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Legacy organic pollutants persist in freshwater environments, but there is limited understanding of how their trophic transfer and effects vary across riverine ecosystems with different land use, biological communities and food webs. Here, we investigated the trophic magnification of polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and a suite of organochlorines (OCs) across nine riverine food webs in contrasting hydrological catchments across South Wales (United Kingdom). Pollutants biomagnified through the food webs in all catchments studied, in some cases reaching levels sufficient for biological effects on invertebrates, fish and river birds such as the Dipper (Cinclus cinclus). Trophic magnification differed across food webs depending on pollutant characteristics (e.g. octanol-water partitioning coefficient) and site-specific environmental conditions (e.g. land use, water chemistry and basal resource composition). The trophic magnification of PBDEs, PCBs and OCs also reflected food-web structure, with greater accumulation in more connected food webs with more generalist taxa. These data highlight interactions between pollutant properties, environmental conditions and biological network structure in the transfer and biomagnification of POPs in river ecosystems. We advocate the need for further investigations of system-specific transfers of contaminants through aquatic food webs as these factors appear to have important implications for risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Once released into the environment antibiotics can kill or inhibit the growth of bacteria, and in turn potentially have effects on bacterial community structure and ecosystem function. Environmental risk assessment (ERA) seeks to establish protection limits to minimise chemical impacts on the environment, but recent evidence suggests that the current regulatory approaches for ERA for antibiotics may not be adequate for protecting bacteria that have fundamental roles in ecosystem function. In this study we assess the differences in interspecies sensitivity of eight species of cyanobacteria to seven antibiotics (cefazolin, cefotaxime, ampicillin, sufamethazine, sulfadiazine, azithromycin and erythromycin) with three different modes of action. We found that variability in the sensitivity to these antibiotics between species was dependent on the mode of action and varied by up to 70 times for β-lactams. Probabilistic analysis using species sensitivity distributions suggest that the current predicted no effect concentration PNEC for the antibiotics may be either over or under protective of cyanobacteria dependent on the species on which it is based and the mode of action of the antibiotic; the PNECs derived for the macrolide antibiotics were over protective but PNECs for β-lactams were generally under protective. For some geographical locations we identify a significant risk to cyanobacteria populations based upon measured environmental concentrations of selected antibiotics. We conclude that protection limits, as determined according to current regulatory guidance, may not always be protective and might be better derived using SSDs and that including toxicity data for a wider range of (cyano-) bacteria would improve confidence for the ERA of antibiotics., (Copyright © 2019. Published by Elsevier B.V.)

Although microplastics are a recognised pollutant in marine environments, less attention has been directed towards freshwater ecosystems despite their greater proximity to possible plastic sources. Here, we quantify the presence of microplastic particles (MPs) in river organisms upstream and downstream of five UK Wastewater Treatment Works (WwTWs). MPs were identified in approximately 50% of macroinvertebrate samples collected (Baetidae, Heptageniidae and Hydropsychidae) at concentrations up to 0.14 MP mg tissue -1 and they occurred at all sites. MP abundance was associated with macroinvertebrate biomass and taxonomic family, but MPs occurred independently of feeding guild and biological traits such as habitat affinity and ecological niche. There was no increase in plastic ingestion downstream of WwTW discharges averaged across sites, but MP abundance in macroinvertebrates marginally increased where effluent discharges contributed more to total runoff and declined with increasing river discharge. The ubiquity of microplastics within macroinvertebrates in this case study reveals a potential risk from MPs entering riverine food webs through at least two pathways, involving detritivory and filter-feeding, and we recommend closer attention to freshwater ecosystems in future research., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Fish can be exposed to a variety of neuroactive pharmaceuticals via the effluent discharges from wastewater treatment plants and concerns have arisen regarding their potential impacts on fish behaviour and ecology. In this study, we investigated the uptake of 14 neuroactive pharmaceuticals from a treated wastewater effluent into blood plasma and brain regions of roach (Rutilus rutilus) after exposure for 15days. We show that a complex mixture of pharmaceuticals including, 6 selective serotonin reuptake inhibitors, a serotonin-noradrenaline reuptake inhibitor, 3 atypical antipsychotics, 2 tricyclic antidepressants and a benzodiazepine, concentrate in different regions of the brain including the telencephalon, hypothalamus, optic tectum and hindbrain of effluent-exposed fish. Pharmaceuticals, with the exception of nordiazepam, were between 3-40 fold higher in brain compared with blood plasma, showing these neuroactive drugs are readily uptaken, into brain tissues in fish. To assess for the potential for any adverse ecotoxicological effects, the effect ratio was calculated from human therapeutic plasma concentrations (HtPCs) and the measured or predicted fish plasma concentrations of pharmaceuticals. After accounting for a safety factor of 1000, the effect ratios indicated that fluoxetine, norfluoxetine, sertraline, and amitriptyline warrant prioritisation for risk assessment studies. Furthermore, although plasma concentrations of all the pharmaceuticals were between 33 and 5714-fold below HtPCs, alterations in serotonin, glutamate, acetylcholine and tryptophan concentrations were observed in different brain regions of effluent-exposed fish. This study highlights the importance of determining the potential health effects arising from the concentration of complex environmental mixtures in risk assessment studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There is currently limited guidance available for regulators and risk assessors on how to use data from non-guideline methods when assessing the bioaccumulation potential of a chemical. Furthermore, bioaccumulation assessments can be more subjective than they need to be due to the lack of a guidance framework on how to use/include the range of information that may be available for a substance. Under some circumstances, in silico, in vitro and/or in vivo non-test guideline data may be sufficient to classify whether a substance is bioaccumulative without the need for further animal testing. Classifying the bioaccumulative potential of a substance is especially difficult when the bioconcentration factor (BCF) is close to the threshold for defining it as bioaccumulative/very bioaccumulative (B/vB), and a more structured process is required to reduce uncertainty in the BCF estimates. In these situations, in silico and in vitro data can, and should, be used to provide greater confidence in classifying these substances. To aid future evaluations of bioaccumulation data, a proposed tiered assessment strategy is presented incorporating all available data on the bioaccumulative properties of a substance. In addition, a revised scheme is recommended for improving the classification of the bioaccumulative potential of a substance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Current metabolite profiling methods based on liquid chromatography-mass spectrometry (LC-MS) platforms do not detect many of the components present at trace concentrations in extracts of plasma due to their low ionisation efficiency or to interference from highly abundant compounds. Nanoflow LC-nanospray MS platforms, which are commonly used in proteomics, could overcome these limitations and significantly increase analytical sensitivity and coverage of the plasma (xeno)metabolome (i.e., metabolites and xenobiotics), but require small injection volumes (<0.5μL). In this study, we developed sample preparation methods to remove ion suppressive phospholipids and concentrate remaining components of the plasma (xeno)metabolome in order to analyse sub-microliter volumes of plasma extracts for nanoflow ultra-high-performance liquid chromatography-nanoelectrospray ionisation-time-of-flight mass spectrometry (nUHPLC-nESI-TOFMS). These methods use phospholipid filtration plates in combination with polymeric or mixed mode exchange solid-phase extraction (SPE). The phospholipid filtration plates removed >94% of the predominant phospholipid/lysophospholipid species from plasma, whilst absolute recoveries of 63 selected (xeno)metabolites from spiked plasma were generally between 60 and 104%. After a further SPE step, recoveries of test compounds were between 50 and 81%. Studies revealed that both the sample preparation methodology and nUHPLC-nESI-TOFMS analyses gave acceptable repeatability. A qualitative comparison of SPE methods revealed that sample concentration by either polymer or mixed mode ion-exchange SPE gave comprehensive metabolite coverage of plasma extracts, but the use of cation exchange SPE significantly increased detection of many cationic compounds in the sample extracts. Method detection limits for steroid, eicosanoid and bile metabolites were <1.0ng/mL plasma and for pharmaceutical contaminants were between 0.01 and 30ng/mL plasma. Comparison of the phospholipid removal/cation exchange SPE and the classical protein precipitation (PPT) sample preparation methodologies revealed that both methods detected the same range of (xeno)metabolites. However, unlike PPT extracts, the SPE preparations allowed direct injection of more concentrated plasma extracts onto the nUHPLC-nESI-TOFMS platform without blockage of the nanocolumn or nanospray, thus resulting in a wider coverage of the (xeno)metabolome. This is the first work to demonstrate the significantly enhanced sensitivity arising from the use of concentrated SPE sample preparations and direct nUHPLC-nESI-TOFMS analysis for untargeted profiling of plasma samples and constitutes a step forward for identifying mixtures of chemical stressors accumulated in blood as well as the disruption of key metabolite pathways in the same sample., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright 2009 Elsevier B.V. All rights reserved.)

Effluents from wastewater treatment works (WwTWs) contain estrogenic contaminants that can cause feminised responses in fish. In order to assess the identity of estrogenic contaminants taken up by fish exposed to effluents, an analytical method was developed to detect estrogenic substances in fish bile, where many xenobiotics are excreted and concentrated. Estrogenic metabolites in bile were deconjugated using enzymatic hydrolysis and the estrogenic activity was determined using a yeast estrogen receptor transcription screen (YES). Hydrolysed samples were concentrated by solid-phase extraction (SPE) prior to fractionation by reversed-phase high-performance liquid chromatography (HPLC). Active HPLC fractions were detected by YES assay and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The method was validated using bile samples from immature female rainbow trout, which had been exposed to either tap water or an undiluted estrogenic effluent for 10 days. Hydrolysis of bile from effluent-exposed fish was complete within 16 h add most of the estrogenic activity in the bile was released by 3-glucuronidase rather than sulfatase or 3-glucosidase treatment. The estrogenic activity of hydrolysed bile from effluent-exposed fish ranged between 530 and 1440 ng E2eq/mL and was 17-48-fold greater than the activity of bile from reference fish exposed to tap water. The estrogenic activity of bile samples decreased with time in storage (at-70 degrees C by 7% per month). The recovery of estrogenic activity from SPE was 96 +/- 7% (mean +/- SD), from HPLC fractionation 87 +/- 7% and for the whole method 81 +/- 7% (n = 7). 17beta-Estradiol, estrone, 17alpha-ethinylestradiol, nonylphenol and short-chain nonylphenol polyethoxylates were all identified from GC-MS analysis of active HPLC fractions of bile from effluent-exposed trout, whereas only 17beta-estradiol was detected in bile from fish exposed to tap water. There were also several other minor estrogenic components, at present unidentified, in bile of effluent-exposed fish. The work shows that fractionation of fish bile is a useful approach to identifying mixtures of estrogenic contaminants taken up by fish from WwTW effluents and has the potential for application in the detection of other endocrine disrupting chemicals in fish tissues.

To contribute to the development and evaluation of a practical and cost-effective in vivo testing system for endocrine disruption (specifically environmental estrogens), the effects of 17alpha-ethinylestradiol (EE2) were assessed in a gonadal recrudescence assay with the fathead minnow (Pimephales promelas). Mature male and female fathead minnows were kept first at 15 degrees C on a 8 h light/16 h dark regime and then transferred to 25 degrees C and a 16 h light/8 h dark regime to induce gonadal recrudescence. They were then exposed to various nominal concentrations of the synthetic estrogen EE2 (0, 0.1, 1, 3, 10, 100 ng/L). After 3 weeks of chemical exposure, effects on plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI), and condition factor were assessed. Additionally, the effects on liver and gonad tissue morphology were investigated by means of light (LM) and electron microscopy (EM). Reproductive output (egg production) and fertilization rate were measured during a subsequent 3-week period in breeding adults maintained in clean water. Exposure to EE2 resulted in a significant decrease in GSI, condition factor, and number of batches of eggs and their fertilization rate at EE2 exposure concentrations between 10 and 100 ng/L. A reduction in the extent of parenchymatic areas in ovaries and ultrastructural changes in the livers of females could be detected at EE2 concentrations > or =3 ng/L. The lowest observed effective concentration (LOEC) of EE2 for plasma VTG induction in both sexes and for ultrastructural changes in the testes and livers was 1 ng/L. A significant increase in the mean number of eggs spawned per pair occurred at EE2 exposure doses of 0.1 and 1 ng/L. However, at higher EE2 concentrations, a dose-dependent decrease in the mean number of eggs per pair was apparent. Therefore, the LOEC for a biological effect of EE2 in the fathead minnow using the selected endpoints in the recrudescence assay was 1 ng/L for biomarkers such as plasma VTG and number of tubercles, and 0.1 ng/L for an increased number of eggs spawned per pair.