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The lampricides 3-trifluoromethyl-4-nitrophemol (TFM) and niclosamide have been used for over 60 years to control the invasive sea lamprey (Petromyzon marinus) population in the Laurentian Great Lakes. In this review, we summarize these findings in the context of the mode of action of both lampricides, with a focus on: (1) the physiology of uptake, bodily distribution and mode of action, detoxification, and excretion of lampricides in lamprey and non-target fishes, (2) the development of an Adverse Outcome Pathway for TFM and niclosamide, and (3) the identification of novel avenues for future research that can be further explored to ensure continuous suppression of the sea lamprey population in the Great Lakes. We explored how research on the mode of action of lampricides has provided novel insights into the gill microenvironment and how this impacts lampricide toxicity; described new information on mitochondria and tissue physiology; and discussed how the activity of enzymes that are involved in detoxification pathways impacts the response of fishes to xenobiotics. Considering the information that has been generated over the years on sea lamprey and bony fish physiology from studying the mode of action of lampricides, here we propose an Adverse Outcome Pathway for TFM and niclosamide and identify novel avenues for research on the short and long-term effects of lampricide applications, either alone or in combination. Lastly, we discuss how the differences in physiology between sea lampreys and non-target fishes can be further exploited to ensure continuous suppression of the sea lamprey population in the Great Lakes.

Predator encounters during early life can elicit behavioral and physiological responses that have fitness consequences during subsequent prey life stages. In threatened lake sturgeon (Acipenser fulvescens) and other lithophilic-spawning fishes, newly hatched larvae (free embryos) are exposed to abundant predators including aquatic insect larvae that co-occupy stream substrates. We investigated stress effects on lake sturgeon larvae after encounters with common aquatic insect predators by quantifying mortality, body size, cortisol levels, and swimming behavior. Free embryos were exposed to either Perlidae (stonefly obligate predators) or Isonychiidae (mayfly filterers and facultative predators) or to no predators (controls). Free embryos that encountered perlids experienced high mortality, elevated cortisol levels, and exhibited cortisol reactivity when subsequently exposed to an acute stressor. Free embryos that encountered isonychiids exhibited elevated mortality, and elevated cortisol and cortisol reactivity relative to controls. Findings indicate that lake sturgeon free embryos are stressed by exposure to members of benthic stream communities during early life stages (predation of nearby conspecifics), and that metrics of stress exhibited threat sensitivity. Data are consistent with predictions that individuals modulate antipredator behavior in response to the intensity of perceived predation threat in the environment. We determined that behavioral outcomes associated with encounters with aquatic insects altered future behavioral trajectories, potentially as an adaptive response that can affect predation rates in subsequent life stages. Results contribute to a broader understanding of how interspecies interactions among co-occurring predator and prey communities may impact individual fitness and fish population recruitment.

Introduction Adult sea lamprey (Petromyzon marinus) cease feeding and migrate to spawning streams where males build nests, undergo final sexual maturation, and subsequently produce and release large quantities of bile acid pheromones that attract mature females. These animals are predicted to rearrange their metabolic pathways drastically to support their reproductive strategies, presenting advantageous opportunities to examine how sex and the maturation processes affect metabolism.Objectives The objective is to investigate the metabolic differences between sexes and maturation states in sea lamprey that support changes in physiological functions.Methods We compared plasma metabolomes of spawning and prespawning sea lamprey in both sexes using both non-targeted and targeted metabolomics approaches using UPLC/MS-MS with electrospray ionization in both positive and negative modes. The data were processed using Progenesis QI, Compound Discoverer and XCMS softwares for alignment, peak picking, and deconvolution of the peaks. Principle component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed using SIMCA and Metaboanalyst softwares to identify discriminating features, followed by fragmentation matching with extensive database search and pathway mapping. Results The pheromonal bile acid biosynthesis was upregulated significantly in males compared to females. Spermiating males further upregulated bile acid biosynthesis by altering amino acid metabolisms, upregulating cofactors and nucleotide metabolisms, but downregulating carbohydrate and energy metabolisms. Conclusion Plasma metabolomes are sex- and maturation-dependent and reflect the special metabolic demands at each life stage and reproductive strategy.

Quantifying transgenerational effects of stress is important to predict outcomes of anthropogenic disturbances for wildlife species. Maternal stress can programme physiological and behavioural phenotypes in offspring, which may be maladaptive if maternal and offspring environments are mismatched. We investigated effects of a match and mismatch between egg cortisol and offspring stress levels in lake sturgeon, Acipenser fulvescens, using artificially elevated egg cortisol levels (simulating maternal stress) and a chronic unpredictable stress regime for offspring after hatch. Offspring cortisol levels were quantified at baseline and after an acute stressor. Multiple measures of offspring swimming activity were assessed in behaviour trials. Individuals that experienced elevated egg cortisol and high offspring stress exhibited a diminished cortisol response to an acute stressor, but responses varied among offspring from different families. Results suggest that the interaction between maternal and offspring experience may cue an offspring phenotype that is adaptive in high-stress conditions. Principal components analysis characterizing interindividual variation in offspring behavioural variables showed that treatment significantly affected multivariate offspring response along the PC1 axis (associated with inactivity), and both treatment and family significantly affected response along the PC2 axis (associated with shorter distance moved). The largest differences for PC1 occurred between the ‘mismatch’ treatments (high egg cortisol and low offspring stress exhibiting lower activity; low egg cortisol and high offspring stress exhibiting higher activity), indicating that the combination of egg cortisol and offspring stress is more important in determining offspring behaviour than is egg cortisol or offspring stress alone. Findings suggest that family effects, such as genetic components or maternal experience, may mediate how the interaction of maternal and offspring stress influences offspring physiological and behavioural outcomes, and indicate the need for further research into environmental factors experienced by females that influence how offspring respond to egg cortisol and early life stress.

Use of the first fish pheromone biopesticide, 3-keto petromyzonol sulfate (3kPZS) in sea lamprey (Petromyzon marinus) control requires an understanding of both how the amount 3kPZS applied to a trap relates to catch, and how that relationship varies among stream types. By conducting 3kPZS dose-response experiments over two years and across six varied trapping contexts, we conclude (1) that 3kPZS application is best standardized by how much is emitted from the trap instead of the fully mixed concentration achieved downstream, and (2) that 3kPZS is more effective in wide streams (>30 m). In wide streams, emission of 3kPZS at 50 mg hr.−1 from the trap increased capture rate by 10–15% as sea lamprey were 25–50% more likely to enter the trap after encounter. However, in narrow streams (< 15 m), 50 mg hr.−1 3kPZS generally reduced probabilities of upstream movement, trap encounter, and entrance. While 3kPZS significantly influenced upstream movement, encounter, and capture probabilities, these behaviors were also highly influenced by water temperature, stream width, sea lamprey length, and sex. This study highlights that a pheromone component in a stream environment does not ubiquitously increase trap catch in all contexts, but that where, how, and when the pheromone is applied has major impacts on whether it benefits or hinders trapping efforts.

Reintroduction programs are important tools for wildlife conservation. However, captive rearing environments may lead to maladaptive behavior and physiological alterations that reduce survival probability after release. For captive rearing programs that raise individuals captured from the wild during early ontogeny for later release, there is a lack of information about when during ontogeny the detrimental effects of captive rearing may become evident. In this study we compared cortisol levels, predation rates and swimming behavior between hatchery-produced and wild-caught larval lake sturgeon (Acipenser fulvescens), a threatened fish species, at three times over 9 days. Cortisol levels did not indicate that hatchery-produced individuals were more stressed, but cortisol reactivity to an acute stressor disappeared for both hatchery-produced and wild-caught larvae after 9 days in the hatchery. Swimming activity levels decreased over time for hatchery-produced larvae but increased over time for wild-caught larvae, suggesting that behavioral trajectories may be programmed prior to the larval stage. Neither increasing nor decreasing activity levels was advantageous for survival, as predation rates increased over time in captivity for larvae from both treatments. Results suggest that physiological and behavioral phenotypes may not accurately predict survival for individuals released from reintroduction programs and that the captive environment may inhibit transition to the wild even if cortisol levels do not indicate high stress. Findings emphasize that even a short amount of time in captivity during early ontogeny can affect phenotypes of individuals captured from wild populations, which may impact the success of reintroduction programs.

Per- and polyfluoroalkyl substances (PFAS) are contaminants of concern due to their widespread occurrence in the environment, persistence, and potential to elicit a range of negative health effects. Per- and polyfluoroalkyl substances are regularly detected in surface waters, but their effects on many aquatic organisms are still poorly understood. Species with thyroid-dependent development, like amphibians, can be especially susceptible to PFAS effects on thyroid hormone regulation. We examined sublethal effects of aquatic exposure to four commonly detected PFAS on larval northern leopard frogs (Rana [Lithobates] pipiens), American toads (Anaxyrus americanus), and eastern tiger salamanders (Ambystoma tigrinum). Animals were exposed for 30 days (frogs and salamanders) or until metamorphosis (toads) to 10, 100, or 1000 μg/L of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), or 6:2 fluorotelomer sulfonate (6:2 FTS). We determined that chronic exposure to common PFAS can negatively affect amphibian body condition and development at concentrations as low as 10 µg/L. These effects were highly species dependent, with species having prolonged larval development (frogs and salamanders) being more sensitive to PFAS than more rapidly developing species (toads). Our results demonstrate that some species could experience sublethal effects at sites with surface waters highly affected by PFAS. Our results also indicate that evaluating PFAS toxicity using a single species may not be sufficient for accurate amphibian risk assessment. Future studies are needed to determine whether these differences in susceptibility can be predicted from species' life histories and whether more commonly occurring environmental levels of PFAS could affect amphibians. Environ Toxicol Chem 2022;41:1407-1415. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Water scarcity is increasingly a global cause of concern mainly due to widespread changes in climate conditions and increased consumptive water use driven by the exponential increase in population growth. In addition, increased pollution of fresh water sources due to rising production and consumption of pharmaceuticals and organic chemicals will further exacerbate this concern. Although surface water contamination by individual chemicals is often at very low concentration, pharmaceuticals for instance are designed to be efficacious at low concentrations, creating genuine concern for their presence in freshwater sources. Furthermore, the additive impact of multiple compounds may result in toxic or other biological effects that otherwise will not be induced by individual chemicals. Globally, different legislative frameworks have led to pre-emptive efforts which aim to ensure good water ecological status. Reports detailing the use and types of effect-based measures covering specific bioassay batteries that can identify specific mode of actions of chemical pollutants in the aquatic ecosystem to evaluate the real threat of pollutants to aquatic lives and ultimately human lives have recently emerged from monitoring networks such as the NORMAN network. In this review, we critically evaluate some studies within the last decade that have implemented effect-based monitoring of pharmaceuticals and organic chemicals in aquatic fauna, evaluating the occurrence of different chemical pollutants and the impact of these pollutants on aquatic fauna with special focus on pollutants that are contaminants of emerging concern (CEC) in urban wastewater. A critical discussion on studies that have used effect-based measures to assess biological impact of pharmaceutical/organic compound in the aquatic ecosystem and the endpoints measurements employed is presented. The application of effect-based monitoring of chemicals other than assessment of water quality status is also discussed.

Costs to producing sexual signals can create selective pressures on males to invest signaling effort in particular contexts. When the benefits of signaling vary consistently across time, males can optimize signal investment to specific temporal contexts using biological rhythms. Sea lamprey, Petromyzon marinus, have a semelparous life history, are primarily nocturnal, and rely on pheromone communication for reproduction; however, whether male investment in pheromone transport and release matches increases in spawning activity remains unknown. By measuring (1) 3keto-petromyzonol sulfate (3kPZS, a main pheromone component) and its biosynthetic precursor PZS in holding water and tissue samples at six points over the course of 24 hours and (2) 3kPZS release over the course of several days, we demonstrate that 3kPZS release exhibits a consistent diel pattern across several days with elevated pheromone release just prior to sunset and at night. Trends in hepatic concentrations and circulatory transport of PZS and 3kPZS were relatively consistent with patterns of 3kPZS release and suggest the possibility of direct upregulation in pheromone transport and release rather than observed release patterns being solely a byproduct of increased behavioral activity. Our results suggest males evolved a signaling strategy that synchronizes elevated pheromone release with nocturnal increases in sea lamprey behavior. This may be imperative to ensure that male signaling effort is not wasted in a species having a single, reproductive event.

Wastewater (WW) reuse and biosolid application for vegetable crop culture is a practice applied worldwide. This strategy helps mitigate the pressure on water resources and improve the fertility of soil. Wastewater reuse is currently not included in chemical risk assessment, but its application has risk of potential accumulation of contaminants of emerging concern such as pharmaceutical active compounds (PhACs). In fact, this practice has caused the uptake of PhACs by plant and their subsequent entrance on the food chain. Residual quantities of contaminants may enter in soil, and they can be accumulated or percolated, consequently leading to contamination of groundwater. Herein, we report the main factors that play an important role on the accumulation of PhACs in soil after irrigation with treated wastewater. Limited data is actually available on the fate of PhACs in field studies because several processes are in competition for their dissipation including sorption and formation of non-extractable residues, leaching, as well as biotransformation. Consequently, an approach based on enantiomeric fractionation of chiral PhACs has been suggested to discriminate between biotic and abiotic dissipation processes.

Sexual signals evolve via selective pressures arising from male–male competition and female choice, including those originating from unintended receivers that detect the signal. For example, males can acquire information from other males signaling to females and alter their own signal. Relative to visual and acoustic signals, less is known about how such communication networks influence chemical signaling among animals. In sea lamprey (Petromyzon marinus), the chemical communication system is essential for reproduction, offering a useful system to study a pheromone communication network that includes signalers and both intended and unintended receivers. Male sea lamprey aggregate on spawning grounds, where individuals build nests and signal to females using sex pheromones. We examined how exposure to a major component of the male pheromone, 3keto-petromyzonol sulfate (3kPZS), influenced male pheromone signaling, and whether females had a preference for males that altered their signal. Exposure to 3kPZS, at a concentration of 5×10−10 mol l−1, simulated the presence of other male(s) and led to increased 3kPZS release rates within 10 min, followed by a return to baseline levels within 30 min. Exposure also led to increases in hepatic synthesis and circulatory transport of pheromone components. In behavioral assays, females preferred the odor of males that had been exposed to 3kPZS; therefore, males likely benefit from upregulating 3kPZS release after detecting competition for mates. Here, we define how a specific pheromone component influences chemical signaling during intrasexual competition, and show a rare example of how changes in chemical signaling strategies resulting from male competition may influence mate choice.

The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.

Biliary atresia (BA) is a rare neonatal disease with unknown causes. Approximately 10% of BA cases develop in utero with other congenital defects that span a large spectrum of disease variations, including degeneration of the gall bladder and bile duct as well as malformation of the liver, intestines, and kidneys. Similar developmental alterations are manifested in a unique animal model, the sea lamprey (Petromyzon marinus), in which BA occurs naturally during metamorphosis. With the likelihood of conserved developmental mechanisms underlying organogenesis and degeneration, lamprey developmental BA may be a useful model to infer mechanisms underlying human embryonic BA. We reasoned that hepatobiliary transcriptomes regulate the transition between landmark stages of BA. Therefore, we examined sea lamprey hepatobiliary transcriptomes at four stages (M0, metamorphic stage 0 or larval stage, no BA; M2, metamorphic stage 2, onset of BA; M5, metamorphic stage 5, BA, and heightened hepatocyte proliferation and reorganization; and JV, juvenile, completion of BA) using messenger RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analyses. We found gene‐expression patterns associated with the transition between these stages. In particular, transforming growth factor β (TGF‐β), hedgehog, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase‐Akt, Wnt, and mitogen‐activated protein kinase pathways were involved during biliary degeneration. Furthermore, disrupting the TGF‐β signaling pathway with antagonist or small interfering RNA treatments at the onset of BA delayed gall bladder and bile duct degeneration. Conclusion: Distinctive gene‐expression patterns are associated with the degeneration of the biliary system during developmental BA. In addition, disrupting TGF‐β signaling pathway at the onset of BA delayed biliary degeneration.

The bioaccumulation of 20 pharmaceuticals in cockle (Cerastodema glaucum), noble pen shell (Pinna nobilis), sea snail (Murex trunculus), golden grey mullet (Liza aurata) and black goby (Gobius niger) was evaluated, considering their distribution throughout the Mar Menor lagoon and their variations in spring and autumn 2010. The analytical procedure was adapted for the different matrices as being sensitive and reproducible. Eighteen out of the 20 compounds analysed were found at low ng g−1 in these species throughout the lagoon. Hydrochlorothiazide and carbamazepine were detected in all species considered. The bioaccumulation of pharmaceuticals was heterogeneous in the lagoon, with a higher number of pharmaceuticals being detected in fish (18) than in wild molluscs (8), particularly in golden grey mullet muscle (16). В-blockers and psychiatric drugs were preferentially bioccumulated in fish and hydrochlorothiazide was also confirmed in caged clams. The higher detection frequency and concentrations found in golden grey mullet suggested that mugilids could be used as an indicator of contamination by pharmaceuticals in coastal areas. To the best of our knowledge, this is the first study that shows data about hydrochlorothiazide, levamisole and codeine in wild marine biota., SI

Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.

There is concern that psychoactive drugs present in the aquatic environment could affect the behaviour of fish, and other organisms, adversely. There is considerable experimental support for this concern, although the literature is not consistent. To investigate why, fish were exposed to three concentrations of the synthetic opiate tramadol for 23-24 days, and their anxiolytic behaviour in a novel tank diving test was assessed both before and after exposure. The results were difficult to interpret. The positive control drug, the anti-depressant fluoxetine, produced the expected results: exposed fish explored the novel tank more, and swam more slowly while doing so. An initial statistical analysis of the results provided relatively weak support for the conclusion that both the low and high concentrations of tramadol affected fish behaviour, but no evidence that the intermediate concentration did. To gain further insight, UK and Japanese experts in ecotoxicology were asked for their independent opinions on the data for tramadol. These were highly valuable. For example, about half the experts replied that a low concentration of a chemical can cause effects that higher concentrations do not, although a similar number did not believe this was possible. Based both on the inconclusive effects of tramadol on the behaviour of the fish and the very varied opinions of experts on the correct interpretation of those inconclusive data, it is obvious that more research on the behavioural effects of tramadol, and probably all other psychoactive drugs, on aquatic organisms is required before any meaningful risk assessments can be conducted. The relevance of these findings may apply much more widely than just the environmental risk assessment of psychoactive drugs. They suggest that much more rigorous training of research scientists and regulators is probably required if consensus decisions are to be reached that adequately protect the environment from chemicals.

This study investigated the occurrence of 135 contaminants of emerging concern (CECs) – pharmaceuticals, pesticides, a set of endocrine disrupting compounds (EDCs) (parabens, bisphenols, hormones, triazoles, organophosphorus flame retardants and triclosan), UV-filters, perfluoroalkyl substances (PFASs) and halogenated flame retardants (HFRs) – in 59 fish samples, collected in 2010 in 4 Spanish Rivers (Guadalquivir, Júcar, Ebro and Llobregat). Of the 135 CECs, 76 including 8 pharmaceuticals, 25 pesticides, 10 EDCs, 5 UV-filters, 15 PFASs and 13 HFRs were detected. Pharmaceuticals were the less frequently found and at lower concentrations. Pesticides, EDCs, UV-filters, PFASs and HFRs were detected more frequently (>50% of the samples). The maximum concentrations were 15 ng/g dry weight (dw) for pharmaceuticals (diclofenac), 840 ng/g dw for pesticides (chlorpyrifos), 224 ng/g dw for EDCs (bisphenol A), 242 ng/g dw for UV-filters (EHMC), 1738 ng/g dw for PFASs (PFHxA) and 64 ng/g dw for HFRs (Dec 602). The contaminants detected in fish are commonly detected also in sediments. In light of current knowledge, the risk assessment revealed that there was no risk for humans related to the exposure to CECs via freshwater fish consumption. However, results provide detailed information on the mixtures of CECs accumulated that would be very useful to identify their effects on aquatic biota., This research has been supported by the European Union 7th Framework Programme funding under Grant Agreement No. 603629-ENV-2013-6.2.1-Globaqua, by the Generalitat de Catalunya (Consolidated Research Groups 2017 SGR 1404 - Water and Soil Quality Unit) and by the Generalitat Valenciana (ANTROPOCEN@, PROMETEO/2018/155).

Aquatic organisms from freshwater ecosystems impacted by waste water treatment plant (WWTP) effluents are constantly exposed to constant concentrations of pharmaceuticals, endocrine disruptors and related compounds, among other anthropogenic contaminants. Macroinvertebrates inhabiting freshwater ecosystems might be useful bioindicators of exposure to contaminants, since their lives are long enough to bioaccumulate, but at the same time may integrate short-term changes in the environment. However, studies about potential bioaccumulation of emerging contaminants in these organisms are very scarce. The objectives of this study were to develop an analytical methodology for the analysis of 41 pharmaceuticals and 21 endocrine disruptors in freshwater invertebrates. In addition, bioaccumulation of these contaminants in three macroinvertebrate taxa inhabiting a waste water treatment plant -impacted river was evaluated. The method for the simultaneous extraction of both families of compounds is based on sonication, purification via removal of phospholipids, and analysis by ultra performance liquid chromatography coupled to a mass spectrometer (UPLC-MS/MS) in tandem. Recoveries for pharmaceuticals were 34-125%, and for endocrine disruptors were 48-117%. Method detection limits (MDLs) for EDCs were in the range of 0.080-2.4 ng g-1, and for pharmaceuticals, 0.060-4.3 ng g-1. These pollutants were detected in water samples taken downstream the waste water treatment plant effluent at concentrations up to 572 ng L-1. Two non-esteroidal anti-inflammatory drugs, diclofenac and ibuprofen, and four endocrine disruptors - estrone, bisphenol A, TBEP, and nonylphenol - were detected in at least one macroinvertebrate taxa in concentrations up to 183 ng g-1 (dry weight). An isobaric interference was identified during the analysis of diclofenac in Hydropsyche samples, which was successfully discriminated via accurate mass determination by TFC-LTQ Orbitrap This study has been financed by Spanish Ministry o fEconomy and Competitiveness through the project SCARCE (Consolider-Ingenio 2010CSD200900065). This work was partly supported by the Generalitat de Catalunya (Consolidated Research Group: Catalan Institute for water Research 2014 SGR 291)

Recent species-extrapolation approaches to the prediction of the potential effects of pharmaceuticals present in the environment on wild fish are based on the assumption that pharmacokinetics and metabolism in humans and fish are comparable. To test this hypothesis, we exposed fathead minnows to the opiate pro-drug tramadol and examined uptake from the water into the blood and brain and the metabolism of the drug into its main metabolites. We found that plasma concentrations could be predicted reasonably accurately based on the lipophilicity of the drug once the pH of the water was taken into account. The concentrations of the drug and its main metabolites were higher in the brain than in the plasma, and the observed brain and plasma concentration ratios were within the range of values reported in mammalian species. This fish species was able to metabolize the pro-drug tramadol into the highly active metabolite O-desmethyl tramadol and the inactive metabolite N-desmethyl tramadol in a similar manner to that of mammals. However, we found that concentration ratios of O-desmethyl tramadol to tramadol were lower in the fish than values in most humans administered the drug. Our pharmacokinetic data of tramadol in fish help bridge the gap between widely available mammalian pharmacological data and potential effects on aquatic organisms and highlight the importance of understanding drug uptake and metabolism in fish to enable the full implementation of predictive toxicology approaches.

In many arid and semi-arid systems, biological communities in river ecosystems are submitted to flow interruption and desiccation, as well as to the impact of urban wastewaters. In this work, we studied (using a LC-LTQ-Orbitrap) the metabolomic response of biofilm communities exposed to both hydrological and chemical stressors. Fluvial biofilms were exposed to a mixture of 9 pharmaceuticals at a total concentration of 5000ng/L (mimicking concentrations and compounds found in polluted aquatic environments) and/or to seven days of desiccation, under laboratory conditions. The biosynthesis of fatty acids was the main metabolic pathway disrupted in biofilms. Endogenous biofilm's metabolites (metabolome) altered due to these stressors were identified. The metabolites that significantly changed only due to one of the stressors could be proposed as potential specific biomarkers. A biomarker of pharmaceutical exposure was the lysophosphatidic acid, which decreased a 160%, while for desiccation stearidonic acid (increased 160%), 16-Oxohexadecanoic acid (increased 340%) and palmitoleic acid (decreased 290%) were the biomarkers proposed. Besides, other metabolites showed different responses depending on the treatment, such as palmitic acid, linolenic acid, behenic acid, lignoceric acid and azelaic acid. The Carbon:Phosphorus (C:P) molar ratio increased due to all stress factors, whereas the algal community composition changed mainly due to desiccation. A possible relationship between those changes observed in structural parameters and the metabolome of biofilms was explored. Overall, our findings support the use of metabolomics to unravel at molecular level the effects from chemical and physical stressors on complex microbial communities, such as biofilms, and pinpoint biomarkers of exposure.

This study was aimed to determine the abundance of four antibiotic resistance genes (blaTEM, ermB, qnrS and sulI), as well as bacterial community composition associated with the intestinal mucus of wild freshwater fish species collected from the Foix and La Llosa del Cavall reservoirs, which represent ecosystems with high and low anthropogenic disturbance, respectively. Water and sediments from these reservoirs were also collected and analyzed to determine the pollution level by antibiotics. The blaTEM gene was only detected in brown trout and Ebro barbel, which were collected from La Llosa del Cavall reservoir. In contrast, the sulI and qnrS genes were only detected in common carp, which were collected from the Foix reservoir. Although the ermB gene was also detected in common carp, the values were below the limit of quantification. Likewise, water and sediment samples from the Foix reservoir had higher concentrations and more classes of antibiotics than those from La Llosa del Cavall. Pyrosequencing analysis of 16S rRNA genes revealed significant differences in bacterial communities associated with the intestinal mucus of fish species. Therefore, these findings suggest that anthropogenic activities are not only increasing the pollution of aquatic environments, but also contributing to the emergence and spread of antibiotic resistance in organisms that inhabit such environments.

Chemometric techniques like Principal Component Analysis (PCA) and Partial Least Squares Regression (PLS) are used to explore, analyze and model relationships among different water quality parameters in wastewater treatment plants (WWTP). Different data sets generated by laboratory analysis and by an automatic multi-parametric monitoring system with a new designed optical device have been investigated for temporal variations on water quality parameters measured in the water influent and effluent of a WWTP over different time scales. The obtained results allowed the discovery of the more important relationships among the monitored parameters and of their cyclic dependence on time (daily, monthly and annual cycles) and on different plant management procedures. This study intended also the modeling and prediction of concentrations of several water components and parameters, especially relevant for water quality assessment, such as Dissolved Organic Matter (DOM), Total Organic Carbon (TOC) nitrate, detergent, and phenol concentrations. PLS models were built to correlate target concentrations of these constituents with UV spectra measured in samples collected at (1) laboratory conditions (in synthetic water mixtures); and at (2) WWTP conditions (in real water samples from the plant). Using synthetic water mixtures, specific wavelengths were selected with the aim to establish simple and reliable prediction models, which gave good relative predictions with errors of around 3-4% for nitrates, detergent and phenols concentrations and of around 15% for the DOM in external validation. In the case of nitrate and TOC concentrations modeling in real water samples from the effluent of the WWTP using the reduced spectral data set, results were also promising with low prediction errors (less than 20%).

A new sensitive method based on pressurized liquid extraction (PLE) and purification by gel permeation chromatography (GPC) prior to ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination in fish homogenate, liver and muscle of twenty pharmaceuticals compounds and metabolites from seven commonly used therapeutic families. An extensive matrix effect evaluation was performed in order to select the best approach when analyzing such complex matrices. Limits of detection (MDLs) for the target compounds were in the range of 0.03-0.50ng/g for fish homogenate, 0.01-0.42ng/g for fish muscle, and 0.08-0.98ng/g for fish liver. The method was applied to fish tissues of eleven fish species from four heavily impacted Mediterranean rivers. Nine compounds from five therapeutic families were measured at concentrations higher than MDLs. Highest levels were found in trout liver, with a maximum concentration of 18ng/g for carbamazepine, whereas the most ubiquitous compound was diclofenac.

There is a growing interest in evaluating the presence of pharmaceutical residues and their metabolites in aquatic biota. In this study, twenty pharmaceuticals, including carbamazepine (CBZ) and two metabolites, were analyzed in homogenates of two fish species (Gambusia affinis and Jenynsia multidentata) captured in polluted areas of the Suquía River (Córdoba, Argentina). The twenty target pharmaceuticals were found in G. affinis, while only fifteen were detected in J. multidentata. We observed a noticeable difference in the accumulation pattern of both fish species, suggesting different pathways for the bioaccumulation of polar pharmaceuticals in each fish. In order to investigate uptake and tissue distribution of pharmaceuticals, a detailed study was performed under controlled laboratory conditions in J. multidentata, exposed to CBZ. CBZ and two of its metabolites (carbamazepine-10,11-epoxide – CBZ-EP and 2-hydroxycarbamazepine – 2-OH-CBZ) were monitored in five organs of fish under laboratory exposure. To our knowledge, this is the first report on the presence of CBZ and its metabolite 2-OH-CBZ in gills, intestine, liver, brain and muscle of fish, while the metabolite carbamazepine-10,11-epoxide (CBZ-EP) was detected in gills and muscle. A ratio CBZ-EP/CBZ close to 0.1 suggests that gills and muscle of J. multidentata could metabolize CBZ through the CBZ-EP pathway. Our results reinforce the need of analyzing multiple species to account for the environmental impact of pollutants, negating the simplification of a single, “representative model” during ecotoxicological biomonitoring. To our knowledge, the biotransformation of CBZ to its metabolites (CBZ-EP, 2-OH-CBZ) in fish, under controlled laboratory in vivo exposures, is reported for the first time. Fil: Valdés, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina Fil: Huerta, B.. Universidad de Girona; España Fil: Wunderlin, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina Fil: Bistoni, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Barceló, D.. Universidad de Girona; España. Consejo Superior de Investigaciones Científicas; España Fil: Rodriguez Mozaz, S.. Universidad de Girona; España

The Adverse Outcome Pathway (AOP) framework represents a valuable conceptual tool to systematically integrate existing toxicological knowledge from a mechanistic perspective to facilitate predictions of chemical-induced effects across species. However, its application for decision-making requires the transition from qualitative to quantitative AOP (qAOP). Here we used a fish model and the synthetic glucocorticoid beclomethasone dipropionate (BDP) to investigate the role of chemical-specific properties, pharmacokinetics, and internal exposure dynamics in the development of qAOPs. We generated a qAOP network based on drug plasma concentrations and focused on immunodepression, skin androgenisation, disruption of gluconeogenesis and reproductive performance. We showed that internal exposure dynamics and chemical-specific properties influence the development of qAOPs and their predictive power. Comparing the effects of two different glucocorticoids, we highlight how relatively similar in vitro hazard-based indicators can lead to different in vivo risk. This discrepancy can be predicted by their different uptake potential, pharmacokinetic (PK) and pharmacodynamic (PD) profiles. We recommend that the development phase of qAOPs should include the application of species-specific uptake and physiologically-based PK/PD models. This integration will significantly enhance the predictive power, enabling a more accurate assessment of the risk and the reliable transferability of qAOPs across chemicals.

We used a short-term microcosm approach to investigate the influence of two different subinhibitory concentrations of ciprofloxacin (0.01 and 0.1 μg/ml) on both the abundance of a plasmid-mediated quinolone resistance determinant (qnrS) and the structure and composition of bacterial communities from impaired and pristine water supply reservoirs. The results showed that the abundance of the qnrS gene increases in water samples exposed to both subinhibitory concentrations of ciprofloxacin, especially in water samples from La Llosa del Cavall, which represents the pristine system. Subinhibitory ciprofloxacin concentrations also induced changes in bacterial community composition as indicated by the relative abundances of each operational taxonomic unit (OTU) across treatments. Therefore, our findings may be of significant importance because subinhibitory ciprofloxacin concentrations may promote antibiotic resistance and affect bacterial community composition in environmental settings.

The presence of pharmaceuticals in the aquatic environment is an ever-increasing issue of concern as they are specifically designed to target specific metabolic and molecular pathways in organisms, and they may have the potential for unintended effects on nontarget species. Information on the presence of pharmaceuticals in biota is still scarce, but the scientific literature on the subject has established the possibility of bioaccumulation in exposed aquatic organisms through other environmental compartments. However, few studies have correlated both bioaccumulation of pharmaceutical compounds and the consequent effects. Analytical methodology to detect pharmaceuticals at trace quantities in biota has advanced significantly in the last few years. Nonetheless, there are still unresolved analytical challenges associated with the complexity of biological matrices, which require exhaustive extraction and purification steps, and highly sensitive and selective detection techniques. This review presents the trends in the analysis of pharmaceuticals in aquatic organisms in the last decade, recent data about the occurrence of these compounds in natural biota, and the environmental implications that chronic exposure could have on aquatic wildlife.

Psychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggests that they may elicit mode of action-mediated effects in fish as they do in humans, and the key open question is at what exposure concentrations these effects might occur. In the present study, the authors investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 d of waterborne exposure to 0.8 μg L

Increasing evidence exists that emerging pollutants such as pharmaceuticals (PhACs) and endocrine-disrupting compounds (EDCs) can be bioaccumulated by aquatic organisms. However, the relative role of trophic transfers in the acquisition of emerging pollutants by aquatic organisms remains largely unexplored. In freshwater ecosystems, wastewater treatment plants are a major source of PhACs and EDCs. Here we studied the entrance of emerging pollutants and their flow through riverine food webs in an effluent-influenced river. To this end we assembled a data set on the composition and concentrations of a broad spectrum of PhACs (25 compounds) and EDCs (12 compounds) in water, biofilm, and three aquatic macroinvertebrate taxa with different trophic positions and feeding strategies (Ancylus fluviatilis, Hydropsyche sp., Phagocata vitta). We tested for similarities in pollutant levels among these compartments, and we compared observed bioaccumulation factors (BAFs) to those predicted by a previously-developed empirical model based on octanol-water distribution coefficients (Dow). Despite a high variation in composition and levels of emerging pollutants across food web compartments, observed BAFs in Hydropsyche and Phagocata matched, on average, those already predicted. Three compounds (the anti-inflammatory drug diclofenac, the lipid regulator gemfibrozil, and the flame retardant TBEP) were detected in water, biofilm and (at least) one macroinvertebrate taxa. TBEP was the only compound present in all taxa and showed magnification across trophic levels. This suggests that prey consumption may be, in some cases, a significant exposure route. This study advances the notion that both waterborne exposure and trophic interactions need to be taken into account when assessing the potential ecological risks of emerging pollutants in aquatic ecosystems This study has been financially supported by the Spanish Ministry of Economy and Competitiveness through the SCARCE project (Consolider-Ingenio 2010 CSD2009-00065), and by the Economy and Knowledge Department of the Catalan Government (Consolidated Research Group 2014 SGR 291—ICRA

Increasing concentrations of pharmaceutical compounds occur in many rivers, but their environmental risk remains poorly studied in stream biofilms. Flow intermittency shapes the structure and functions of ecosystems, and may enhance their sensitivity to toxicants. This study evaluates the effects of a long-term exposure of biofilm communities to a mixture of pharmaceutical compounds at environmental concentrations on biofilm bioaccumulation capacity, the structure and metabolic processes of algae and bacteria communities, and how their potential effects were enhanced or not by the occurrence of flow intermittency. To assess the interaction between those two stressors, an experiment with artificial streams was performed. Stream biofilms were exposed to a mixture of pharmaceuticals, as well as to a short period of flow intermittency. Results indicate that biofilms were negatively affected by pharmaceuticals. The algal biomass and taxa richness decreased and unicellular green algae relatively increased. The structure of the bacterial (based on denaturing gradient gel electrophoresis of amplified 16S rRNA genes) changed and showed a reduction of the operational taxonomic units (OTUs) richness. Exposed biofilms showed higher rates of metabolic processes, such as primary production and community respiration, attributed to pharmaceuticals stimulated an increase of green algae and heterotrophs, respectively. Flow intermittency modulated the effects of chemicals on natural communities. The algal community became more sensitive to short-term exposure of pharmaceuticals (lower EC50 value) when exposed to water intermittency, indicating cumulative effects between the two assessed stressors. In contrast to algae, the bacterial community became less sensitive to short-term exposure of pharmaceuticals (higher EC50) when exposed to water intermittency, indicating co-tolerance phenomena. According to the observed effects, the environmental risk of pharmaceuticals in nature is high, but different depending on the flow regime, as well as the target organisms (autotrophs vs heterotrophs) This study was funded by the Spanish Ministry of Economy and Competitiveness through the project SCARCE (Consolider-Ingenio 2010 CSD2009-00065) and by the European Union through the European Regional Development Fund (ERDF)

The presence of emerging pollutants such as pharmaceuticals (PhACs) in the aquatic environment is well known, but their bioaccumulation in aquatic organisms has only been studied in the last years. It is, therefore, an issue of emerging concern, particularly in Mediterranean regions, where the WWTP effluents may represent a high percentage of some streams’ flow (especially under water scarcity), and thus bioaccumulation of pollutants can be favored. In this chapter, studies worldwide about bioaccumulation of PhACs in aquatic organisms are first reviewed, and the species and contaminants of concern are tentatively identified. Results from a recent study performed in different river basins in Spain are presented in this work. In our study, biota samples from different trophic levels – periphyton (basal resource), macroinvertebrates (Hydropsyche sp. (collector-filterer insect larvae) and Dreissena polymorpha (zebra mussel)), and different species of fish – were collected from four rivers basins in Spain: Ebro, Llobregat, Jucar, and Guadalquivir. A total of 20 sites were sampled for the analysis of up to 61 multi-class pharmaceuticals. Several aspects are addressed and discussed in this chapter such as trends regarding the most prevalent compounds in different river basins and trophic levels and in relation to their physicochemical characteristics. Levels found in aquatic biota were in general in the low ng/g range but occasionally reached values up to 229.8 ng/g of sertraline in periphyton, 93.7 of diclofenac in macroinvertebrates, or 17.9 ng/g of carbamazepine in fish liver. Diclofenac was overall the most prevalent compound; it was the only compound detected in all types of biological matrix analyzed. Llobregat was identified as the most polluted river according to the number and levels of pharmaceuticals in biota. Finally, periphyton is proposed as a potential sentinel of exposure to pharmaceuticals in the aquatic environment: they are able to retain higher number of compounds and at higher concentrations than other organisms.

A fast, simple and robust method has been developed for the simultaneous determination of 23 pharmaceuticals (including some major metabolites) in bivalve mollusks. The analytes belong to eight different therapeutic groups: antibiotics, psychiatric drugs, analgesics/anti-inflammatories, tranquilizer, calcium channel blockers, diuretic, and prostatic hyperplasia. The method is based on pressurized liquid extraction (PLE) followed by solid phase extraction clean-up (SPE), and ultra performance liquid chromatography–triple quadrupole mass spectrometry (UHPL–MS/MS) for the identification and quantification of the target analytes. It has been developed and validated in three different species of bivalves: Crassostrea gigas (Pacific oyster), Mytilus galloprovincialis (Mediterranean mussel), and Chamelea gallina (striped venus clam). The majority of the compounds were extracted with a recovery between 40 and 115%. The developed analytical method allowed the determination of the compounds in the lower ng/g concentration levels. The relative standard deviation was under 12% for the intra-day and 20% inter-day analyses, respectively. Finally, the method was applied to oyster, clam and mussel samples collected from the Ebro delta, Spain. The most ubiquitous compounds detected were the psychiatric drug venlanfaxine and the antibiotic azithromycin, with the highest concentrations found in mussel (2.7 ng/g dw) and oyster (3.0 ng/g dw), respectively. To the best of our knowledge, this is the first time that azithromycin has been reported in environmental samples of marine biota The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007 2013) under the EC safe SEAFOOD project (grant agreement no.311820). The scientific facilities of ICRA have been cofinanced by the Spanish Ministry of Economy and Competitiveness (MINECO) and by the European Regional Development Fund (ERDF) in the frame of the Catalonia ERDF Operative programme 2007–2013. This work was partly supported by the Generalitat de Catalunya (Consolidated Research Group: Catalan Institute for Water Research 2014 SGR 291)

In this study we characterised the ciprofloxacin-resistant strains isolated in biofilm and sediments from a wastewater treatment plant (WWTP) discharge point and its receiving river. We also examined the prevalence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes in these isolates and determined whether they harbour plasmid-encoded β-lactamases such as TEM, SHV and CTX-M. Moreover, antibiotic concentrations were also measured to evaluate the level of contamination of these pharmaceuticals in the sampling area. Antibiotics were found in the range of ng L(-1) in WWTP effluents, but most of them were no longer found in downstream river. However, some fluoroquinolones were detected in sediment downstream demonstrating their high persistence and their capacity to be retained in the river sediments. Most of the ciprofloxacin-resistant isolates belonged to the Gammaproteobacteria class and 17 of them, 8 (7.6%) from the first sampling and 9 (6.1%) from the second sampling, carried a qnr gene. In particular, 15 isolates carried the qnrS gene and 2 carried the qnrB gene. Among the qnr-positive isolates, 12 harboured the aac(6')-lb-cr gene and 2 of them also carried a β-lactamase on the same plasmid, indicating that they may be transferred simultaneously. It is also noteworthy that all qnr-positive isolates identified as Aeromonas species harboured the same qnrS allele, namely the qnrS2. This study reinforces the importance of environmental bacteria as vehicles for dissemination of antibiotic resistance genes.

We assessed the tolerance acquired by stream biofilms to two non-steroidal anti-inflammatory-drugs (NSAIDs), ibuprofen and diclofenac. Biofilms came from a stream system receiving the effluent of a wastewater treatment plant (WWTP). The response of biofilms from a non-polluted site (upstream the WWTP) was compared to that of others downstream with relevant and decreasing levels of NSAIDs. Experiments performed in the laboratory following the pollution-induced community tolerance (PICT) approach determined that both algae and microbial communities from biofilms of the sites exposed at the highest concentrations of ibuprofen and diclofenac acquired tolerance to the mixture of these NSAIDs occurring at the sites. It was also observed that the chronic pollution by the WWTP effluent affected the microbial metabolic profile, as well as the structure of the algal community. The low (at ngL-1 level) but chronic inputs of pharmaceuticals to the river ecosystem result in tolerant communities of lower diversity and altered microbial metabolism This research was financed by the Spanish Ministry of Economy and Competitiveness through the Project SCARCE (Consolider- Ingenio 2010 CSD2009-00065) and by the European Union through the European Regional Development Fund (FEDER), and by a Marie Curie European Reintegration Grant (PERG07-GA-2010-259219) (V. Acuña). This work was partly supported by the Generalitat de Catalunya (Consolidated Research Group: Water and Soil Quality Unit 2009-SGR-965)

A new, sensitive and rapid method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach followed by ultra high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination of nineteen endocrine disruptors (EDCs) and related compounds belonging to different classes in various fish species. Matrix effect on the analytical performance was evaluated, and thus, internal sample calibration was chosen as the most appropriate approach when analyzing such complex matrices as biota. The procedure provided adequate recoveries in the range from 40% to 103% for most of the compounds, low method detection limits (MDLs) in the range from 0.002 to 3.09ng/g for fish homogenates and high accuracy

Antibiotic resistance represents a growing global health concern due to the overuse and misuse of antibiotics. There is, however, little information about how the selective pressure of clinical antibiotic usage can affect environmental communities in aquatic ecosystems and which bacterial groups might be responsible for dissemination of antibiotic resistance genes (ARGs) into the environment. In this study, chemical and biological characterization of water and sediments from three water supply reservoirs subjected to a wide pollution gradient allowed to draw an accurate picture of the concentration of antibiotics and prevalence of ARGs, in order to evaluate the potential role of ARGs in shaping bacterial communities, and to identify the bacterial groups most probably carrying and disseminating ARGs. Results showed significant correlation between the presence of ARG conferring resistance to macrolides and the composition of bacterial communities, suggesting that antibiotic pollution and the spreading of ARG might play a role in the conformation of bacterial communities in reservoirs. Results also pointed out the bacterial groups Actinobacteria and Firmicutes as the ones probably carrying and disseminating ARGs. The potential effect of antibiotic pollution and the presence of ARGs on the composition of bacterial communities in lacustrine ecosystems prompt the fundamental question about potential effects on bacterial-related ecosystem services supplied by lakes and reservoirs. © 2013 Elsevier B.V., We thank Carles Borrego, Enrique Navarro, and Veronica Ribé for helpful discussions and comments to the article, and Jaime Ordóñez for help during field work. We also thank George A. Jacoby, Carmen Torres and Joan Jofre for providing us with bacterial strains. This research work was funded by the 1st Interdisciplinary Water Research Projects-ICRA Grants Program 2010 (Project RES2), the Spanish Ministry of Economy and Competitiveness for its financial support through the project SCARCE (Consolider-Ingenio 2010 CSD2009-00065), and by the European Union through the European Regional Development Fund (FEDER). This work was also partly supported by the Generalitat de Catalunya (Consolidated Research Group: Water and Soil Quality Unit 2009-SGR-965). Prof. Barceló acknowledges King Saud University for his visiting professorship.

The presence of pharmaceuticals in the aquatic environment is an ever-increasing issue of concern as they may have the potential for unintended effects on nontarget species. Scientific literature on the subject has established the possibility of bioaccumulation in exposed biota through other environmental compartments. Analytic methodology for the determination of pharmaceutical compounds in complex matrices such as aquatic organisms has advanced greatly over the past few years, particularly the development of highly sensitive analytic tools and instrumentation in combination with selective extraction and purification. Nonetheless, there are still unresolved analytic challenges, such as how to improve sample preparation to resolve analytic problems, and more information on uptake by organisms, absorption, metabolism, and elimination of specific pharmaceuticals. This chapter presents the trends in the analysis of pharmaceuticals in aquatic organisms in the last few years and recent data about the occurrence of these compounds in aquatic biota.

Wastewater treatment plants (WWTPs) are one of the main sources of pharmaceuticals and endocrine disrupting compounds in freshwater ecosystems, and several studies have reported bioaccumulation of these compounds in different organisms in those ecosystems. River biofilms are exceptional indicators of pollution, but very few studies have focused on the accumulation of these emerging contaminants. The objectives of this study were first to develop an efficient analytical methodology for the simultaneous analysis of 44 pharmaceuticals and 13 endocrine disrupting compounds in biofilm, and second, to assess persistence, distribution, and bioaccumulation of these contaminants in natural biofilms inhabiting a WWTP-impacted river. The method is based on pressurized liquid extraction, purification by solid-phase extraction, and analysis by ultra performance liquid chromatography coupled to a mass spectrometer (UPLC-MS/MS) in tandem. Recoveries for pharmaceuticals were 31-137%, and for endocrine disruptors 32-93%. Method detection limits for endocrine disruptors were in the range of 0.2-2.4ng g-1, and for pharmaceuticals, 0.07-6.7ng g-1. A total of five endocrine disruptors and seven pharmaceuticals were detected in field samples at concentrations up to 100ng g-1 This study has been co-financed by the Spanish Ministry of Economy and Competitiveness through the project WATER-Fate (CTM2012-38314-C0201), by the EU through the FP7 project GLOBAQUA (Grant agreement No. 603629) and by the European Union through the European Regional Development Fund (ERDF). This work was partly supported by the Economy and Knowledge Department of the Catalan Government (Consolidated Research Group 2014 SGR 291 - ICRA)