A multibiomarker approach to assess toxic effects of wastewater treatment plant effluents and activated defence mechanisms in marine (Ruditapes philippinarum) and fresh water (Corbicula fluminea) bivalve species.

Since it has been demonstrated that urban effluents can have adverse effects on aquatic organisms, a multibiomarker study was used to evaluate the effects of wastewater treatment plant (WWTP) effluents discharged into the marine and freshwater environments on clams in Cádiz, Spain. One bioassay was performed in the Bay of Cádiz, exposing Ruditapes philippinarum (marine) to a reference site as well as two sites close to WWTP discharges for 14 days. A second bioassay was performed in the Guadalete River, exposing Corbicula fluminea (fresh water) to three sites for 21 days. The biomarkers analysed included defence mechanisms and various toxic effects. Results indicated that WWTP effluents activated defence mechanisms and induced toxic effects in clams exposed to both environments, thus indicating bioavailability of contaminants present in water. Elevated enzymatic activity was found in clams deployed in La Puntilla and El Trocadero compared to control clams and those exposed to the reference site, and 96% of clams deployed at G2 in the Guadalete River died before day 7. Clams exposed to G1 and G3 indicated significant differences in all biomarkers analysed with respect to control clams (p < 0.05). Both species were sensitive to contaminants present in studied sites. This is the first time that these species were used in cages to assess the environmental risk of wastewater effluent discharges in freshwater and marine column environments. The multibiomarker approach provided important ecotoxicological information and is useful for the assessment of the bioavailability and effect of contaminants from WWTP effluents on marine and fresh water invertebrates.

Health status alterations in Ruditapes philippinarum after continuous secondary effluent exposure before and after additional tertiary treatment application.

A mobile pilot plant was set up in a wastewater treatment plant (WWTP) in southwest Spain to address potential adverse effects of effluents as a whole contaminant, which are discharging into marine environments. Ruditapes philippinarum specimens were exposed to different effluent concentrations (50%, 25%, 12.5%, 6.25%, and 3.15%) during seven days. After effluent exposure, lysosomal membrane stability alterations (LMS), changes in the energy status storage (total lipids content (TLP) and in the mitochondrial electron transport (MET), inhibition of inflammatory mechanisms (cyclooxygenase activity (COX)), and neurotoxic effects (acetylcholinesterase (AChE) were determined in exposed organisms. Furthermore, potential toxic reduction in the effluent was analysed by the application of an additional microalgae tertiary treatment called photobiotreatment (PhtBio). Results after PhtBio confirmed the toxic effect reduction in exposed organisms. Neuroendocrine effects, alterations in energy budget and in lipid storage revealed alterations in clam's health status causing stress conditions after effluent exposure.

Biochemical responses of Solea senegalensis after continuous flow exposure to urban effluents.

Urban effluent potential toxicity was assessed by a battery of biomarkers aimed at determining sub-lethal effects after continuous exposure on the marine organism Solea senegalensis. Specimens were exposed to five effluent concentrations (1/2, 1/4, 1/8, 1/16, 1/32) during 7-days, simulating the dispersion plume at the discharge point. Three different groups of biomarkers were selected in the present study: biomarkers of exposure (Phase I: EROD and DBF; Phase II: GST), biomarkers with antioxidant responses (GR and GPX) and biomarkers of effects (DNA damage and LPO). Additionally, a biological depuration treatment (photobiotreatment (PhtBio)) was tested in order to reduce the adverse effects on aquatic organisms. Effluent exposure caused sub-lethal responses in juvenile fish suggesting oxidative stress. After PhtBio application, concentrations of the major part of measured contaminants were reduced, as well as their bioavailability and adverse effects.

Environmental risk assessment of effluents as a whole emerging contaminant: Efficiency of alternative tertiary treatments for wastewater depuration.

Emerging contaminants (ECs) and regulated compounds (RCs) from three different WWTP effluents were measured in the current study. The efficiency of two tertiary treatments, Photobiotreatment (PhtBio) and Multi-Barrier Treatment (MBT), for removing contaminants was determined. Results indicated different percentages of removal depending on the treatment and the origin of the effluent. Risk Quotients (RQs) were determined for different species of algae, Daphnia, and fish. RQ results revealed diverse risk values depending on the bioindicator species. Tonalide, galaxolide (fragrances), and ofloxacin (antibiotic) were the most persistent and harmful substances in tested effluents. "Negligible risk" category was reached since a wide diversity of ECs were removed by MBT with high removal percentages. Contrarily, PhtBio was effective only in the depuration of certain chemical compounds, and its efficiency depended on the composition of the raw effluent.

Are combined AOPs effective for toxicity reduction in receiving marine environment? Suitability of battery of bioassays for wastewater treatment plant (WWTP) effluent as an ecotoxicological assessment.

Ecotoxicological assessment of three different wastewater treatment plant (WWTP) effluents D1, D2 and D3 was performed before and after tertiary treatment using combination of advanced oxidation processes (AOPs). A multibarrier treatment (MBT) consisting of microfiltration (MF), hydrogen peroxide photolysis (H2O2/UVC) and catalytic wet peroxide oxidation (CWPO) was applied for all effluents. Sparus aurata, Paracentrotus lividus, Isochrysis galbana and Vibrio fischeri, representing different trophic levels, constituted the battery of bioassays. Different acute toxicity effects were observed in each WWTP effluents tested. The percentage of sea urchin larval development and mortality fish larvae were the most sensitive endpoints. Significant reduction (p < 0.05) of effluent's toxicity was observed using a classification pT-method after MBT process. Base on obtained results, tested battery of bioassays in pT-method framework can be recommended for acute toxicity preliminary evaluation of WWTP effluents for the marine environment.

Is the step-wise tiered approach for ERA of pharmaceuticals useful for the assessment of cancer therapeutic drugs present in marine environment?

Methotrexate (MTX) and tamoxifen (TMX) cancer therapeutic drugs have been detected within the aquatic environment. Nevertheless, MTX and TMX research is essentially bio-medically orientated, with few studies addressing the question of its toxicity in fresh water organisms, and none to its' effect in the marine environment. To the authors' knowledge, Environmental Risk Assessments (ERA) for pharmaceuticals has mainly been designed for freshwater and terrestrial environments (European Medicines Agency-EMEA guideline, 2006). Therefore, the purpose of this research was (1) to assess effect of MTX and TMX in marine organism using the EMEA guideline, (2) to develop an ERA methodology for marine environment, and (3) to evaluate the suitability of including a biomarker approach in Phase III. To reach these aims, a risk assessment of MTX and TMX was performed following EMEA guideline, including a 2-tier approach during Phase III, applying lysosomal membrane stability (LMS) as a screening biomarker in tier-1 and a battery of biochemical biomarkers in tier-2. Results from Phase II indicated that MTX was not toxic for bacteria, microalgae and sea urchin at the concentrations tested, thus no further assessment was required, while TMX indicated a possible risk. Therefore, Phase III was performed for only TMX. Ruditapes philippinarum were exposed during 14 days to TMX (0.1, 1, 10, 50 µg L(-1)). At the end of the experiment, clams exposed to environmental concentration indicated significant changes in LMS compared to the control (p<0.01); thus a second tier was applied. A significant induction of biomarkers (activity of Ethoxyresorufin O-deethylase [EROD], glutathione S-transferase [GST], glutathione peroxidase [GPX], and lipid peroxidation [LPO] levels) was observed in digestive gland tissues of clams compared with control (p<0.01). Finally, this study indicated that MTX was not toxic at an environmental concentration, whilst TMX was potentially toxic for marine biota. This study has shown the necessity to create specific guidelines in order to evaluate effects of pharmaceuticals in marine environment which includes sensitive endpoints. The inadequacy of current EMEA guideline to predict chemotherapy agents toxicity in Phase II was displayed whilst the usefulness of other tests were demonstrated. The 2-tier approach, applied in Phase III, appears to be suitable for an ERA of cancer therapeutic drugs in the marine environment.

In situ evaluation of wastewater discharges and the bioavailability of contaminants to marine biota.

Marine sediment quality of wastewater discharges areas was determined by using in situ caged clams Ruditapes philippinarum taking into account the seasonality. Clams were caged in sediment directly affected by wastewater discharges at four sites (P1, P2, P3, P4) at the Bay of Cádiz (SW, Spain), and one reference site (P6). Exposure to contaminated sediments was confirmed by measurement of metals and As, PAH, pharmaceutical products and surfactants (SAS) in bottom sediments. Biological effects were determined by following biomarkers of exposure (activities of 7-ethoxyresorufin O-deethylase - EROD, dibenzylfluorescein dealkylase - DBF, glutathione S-transferase - GST, glutathione peroxidase - GPX, glutathione reductase - GR and acetylcholinesterase - AChE), effects (lysosomal membrane stability - LMS, DNA damage and lipid peroxidation - LPO), energy status (total lipids - TLP and mitochondrial electron transport - MET), and involved in the mode of action of pharmaceutical products (monoamine oxidase activity - MAO, alkali-labile phosphates - ALP levels and cyclooxygenase activity - COX). In winter, urban effluents were detoxified by phase I biotransformation (CYP3A-like activity), phase II (GST), and the activation of antioxidant defence enzymes (GR). Urban effluents lead to the detoxification metabolism (CYP1A-like), oxidative effects (LPO and DNA damage), neurotoxicity (AChE) and neuroendocrine disruption (COX and ALP levels) involved in inflammation (P1 and P2) and changes in reproduction as spawning delay (P3 and P4) in clams exposed in summer. Adverse effects on biota exposed to sediment directly affected by wastewater discharges depend on the chemical contamination level and also on the reproductive cycle according to seasonality.

Toxicological evaluation of sediment samples spiked with human pharmaceutical products: Energy status and neuroendocrine effects in marine polychaetes Hediste diversicolor.

There is a lack of studies about the ecotoxicology of pharmaceutical products on marine environment. To predict possible adverse effects of pharmaceutical products on benthic biota, polychaetes Hediste diversicolor were exposed for 14-days to pharmaceutical-spiked sediments under laboratory conditions. Carbamazepine (CBZ), ibuprofen (IBP) and propranolol (PRO) at concentrations of 500ngg(-1), 50ngg(-1), 5ngg(-1), 0.5ngg(-1) and 0.05ngg(-1), fluoxetine (FX) and 17α-ethynylestradiol (EE2) at concentrations of 100ngg(-1), 10ngg(-1), 1ngg(-1), 0.1ngg(-1) and 0.01ngg(-1), including environmental concentrations (underlined), were spiked in marine sediment samples. After the exposure, cellular energy status (total lipids content - TLP; and mitochondrial electron transport activity - MET), metabolism of monoamines (monoamine oxidase activity - MAO) and inflammation properties (cyclooxygenase activity - COX) were observed in polychaetes. CBZ increased TLP content and MET activity, and decreased MAO activity in polychaetes. IBP did not interfere on the TLP level, but on the MET and MAO activities (environmental concentrations). FX did not cause changes in the energy status. Therefore, environmental concentration diminished MAO activity. EE2 did not affect the energy status, however, MAO activity was significantly lower in polychaetes exposed to environmental concentration. PRO increased TLP level in polychaetes, but not MET activity. MAO activity was significantly lower for polychaetes exposed to environmental concentration. Except FX, all pharmaceuticals showed anti-inflammatory properties confirmed by the decrease of COX activity. Pharmaceutical products affected H. diversicolor physiology and health. As a benthic top predator, adverse effects on sea-worms can potentially culminate in ecosystem perturbations.

Assessing potential risks of wastewater discharges to benthic biota: an integrated approach to biomarker responses in clams (Ruditapes philippinarum) exposed under controlled conditions.

Marine clams Ruditapes philippinarum were exposed under laboratory conditions to sediments sampled at five sites affected by wastewater effluents at the Bay of Cádiz (SW, Spain). Contamination and early biological stress were determined. Metabolism and antioxidant system differed according to seasons. Health status diminished in summer. Metabolism of detoxification, and oxidative effect were related to concentration of metals, PAH, secondary alkane sulfonates (SAS) and antibiotics in winter. Antioxidant system and DNA damage were linked to metals and pharmaceutical products. Phase I and antioxidant system were associated to PAH and SAS in summer. Oxidative stress and effects were related to pharmaceuticals. Phase II was linked to metals and pharmaceuticals. Seasonality of sediment contamination by organic compounds and biological responses was determined. Clams were useful bioindicators, since the set of biomarkers applied was validated as potential tools for sediment quality assessment of wastewater discharges areas.

Are WWTPs effluents responsible for acute toxicity? Seasonal variations of sediment quality at the Bay of Cádiz (SW, Spain).

Adverse effects of wastewater treatment plants (WWTPs) on sediment quality at the Bay of Cádiz (SW, Spain) were evaluated by a battery of acute bioassays and chemical contamination. Five sites directly affected by WWTPs effluents and one control site were chosen. Results evidenced clear deterioration of ecological sediment quality parameters and possible effects on aquatic communities towards WWTPs areas. Acute toxicity and chemical contamination varied significantly across the studied sites and differed between winter and summer seasons. The Bay of Cádiz is contaminated by PAHs, metals, detergents (SAS) and pharmaceutical products. Principal Component Analyses indicated metals, SAS and pharmaceutical products as the major environmental stresses. Sea-urchin embryo-larval and microalgae growth rate were the most sensitive bioassays to evaluate resuspension of contaminants (elutriate) from bulk sediment. Amphipods mortality and Microtox(®) solid phase test bioassays were recommended to evaluate bulk sediment quality. Therefore, the use of multiple-bioassays, sensitive to sediment pollution, may provide complementary information to diagnose environmental factors that can impair aquatic communities. The battery of bioassays is recommended to assess and monitor marine sediments directly affected by a mixture of contaminants released from WWTPs.

A candidate short-term toxicity test using Ampelisca brevicornis to assess sublethal responses to pharmaceuticals bound to marine sediments.

Lethal and sublethal responses related to different phases of metabolism (phases I and II enzymatic activities), neurotoxicity (acetylcholinesterase activity), oxidative stress (lipid peroxidation and antioxidant enzyme activities), and genetic damage (DNA strand breaks) were analysed to assess the possible adverse effects of pharmaceuticals bound to marine sediments. The crustacean amphipod Ampelisca brevicornis was chosen as the bioindicator species. Organisms were exposed for 10 days to sediment spiked with pharmaceutical compounds frequently used and previously detected in the environment: carbamazepine (CBZ), ibuprofen (IBP), fluoxetine (FX), 17α-ethynylestradiol (EE2), propranolol (PRO), and caffeine (CAF). Short-term bioassay to evaluate amphipod mortality was recommended to assess pollution by CBZ, FX, and PRO. IBP and PRO were metabolized by phases I and II detoxification enzymatic activities. Oxidative stress was caused by PRO and CAF. Contrary to expected results, DNA damage (strand breaks) decreased after the exposure of amphipods to sediment spiked with IBP, FX, EE2, PRO, and CAF (including environmental concentrations). FX was neurotoxic to amphipods. The battery of biomarkers tested allowed the assessment of bioavailability, oxidative stress, genotoxicity, and neurotoxicity of the pharmaceuticals analysed. The results of this study suggested that pharmaceutical products at concentrations currently found in the environment might cause a wide variety of adverse effects (based on laboratory studies). The results obtained here are useful for environmental risk assessment of marine sediments contaminated by pharmaceuticals. Nevertheless, more research is needed using field-based marine sediments.

Are standard tests sensitive enough to evaluate effects of human pharmaceuticals in aquatic biota? Facing changes in research approaches when performing risk assessment of drugs.

Nowadays, the presence of pharmaceutical products in aquatic environments is not only common, but is also of significant concern regarding the adverse effect they may produce to aquatic biota. In order to determine the adverse effects of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ) and novobiocin (NOV), at environmental occurring concentrations, standardized endpoints applied in current guidelines were evaluated in four organisms including bioluminescence response in Vibrio fischeri, growth inhibition in Isochrysis galbana (marine water) and Pseudokirchneriella subcapitata (fresh water) and fertilization and embryo-larval development in Paracentrotus lividus. To reach this aim bioassays were implemented by exposing organisms to water spiked with drugs dissolved in DMSO (0.001% v/v). Risk characterization was performed, calculating the environmental impact of drugs by calculating environmental concentration and predicted no effect concentration ratio (MEC/PNEC). Results indicate that acute toxicity was found above environmental concentrations in the order of mg L(-1) for bacteria bioluminescence, microalgae growth inhibition and sea urchin fertilization. However, teratogenicity was observed on sea urchin after exposure to environmental concentrations of drugs at 0.00001 mg L(-1); at this concentration CBZ and IBU were found to reduce significantly the embryo-larval development compared to controls (p<0.01). The risk calculated for selected drugs suggested they are harmless for aquatic environment except when applying the embryo-larval development endpoint. Endpoints applied in this study showed the necessity of using more sensitive responses, when assessing risk of pharmaceuticals in aquatic environments, since endpoints applied in current guidelines may not be suitable.

Bioavailability, oxidative stress, neurotoxicity and genotoxicity of pharmaceuticals bound to marine sediments. The use of the polychaete Hediste diversicolor as bioindicator species.

A set of "early warning responses", measured as biomarkers of exposure and effect, was applied in the marine bioindicator Hediste diversicolor, in a way to assess the environmental quality of sediment affected by pharmaceutical contamination. Sublethal responses were determined in the sea-worms after 14-days of exposure to sediment spiked with some of the most representative pharmaceutical products found in the environment: carbamazepine (CBZ), ibuprofen (IBP), fluoxetine (FX), 17α-ethynylestradiol (EE2) and propranolol (PRO), including the environmental concentrations. Phases I (ethoxyresorufin O-deethylase - EROD and dibenzylfluorescein dealkylase - DBF) and II (glutathione S-transferase - GST) of the metabolism, antioxidant system (glutathione peroxidase - GPX and glutathione reductase - GR), neurotoxicity (acetylcholinesterase - AChE) and oxidative effects (lipid peroxidation - LPO and DNA damage strand breaks) were selected to evaluate the sublethal responses in the sea-worms. FX, EE2 and PRO were detoxified by the phase I of the metabolism (EROD activity). On the other hand, phase II (GST-activity) did not respond in sea-worms exposed to pharmaceutical products, except for the environmental concentrations of CBZ (activation) and PRO (deactivation). Neurotoxicity was induced in sea-worms exposed to EE2 (only the environmental concentrations), FX, IBP and CBZ. Oxidative effect determined as LPO increased in sea-worms exposed to environmental concentrations of IBP, EE2 and PRO. Genetic damage increased in sea-worms exposed to IBP and diminished for FX, EE2 and PRO. Our results indicated the toxicity of pharmaceutical products and recommended the battery of biomarkers and the bioindicator specie H. diversicolor for the environmental quality assessment of sediment affected by pharmaceutical contamination.

Early responses measured in the brachyuran crab Carcinus maenas exposed to carbamazepine and novobiocin: application of a 2-tier approach.

One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50µgL(-1). Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50µgL(-1)). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50µgL(-1)). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50µgL(-1) (p<0.05). Crabs exposed to NOV (50µgL(-1)) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment.

Integrated ecotoxicological assessment of marine sediments affected by land-based marine fish farm effluents: physicochemical, acute toxicity and benthic community analyses.

An integrated ecotoxicological assessment of marine sediments affected by land-based marine fish farm effluents was developed using physicochemical and benthic community structure analyses and standardised laboratory bioassays with bacteria (Vibrio fischeri), amphipods (Ampelisca brevicornis) and sea urchin larvae (Paracentrotus lividus). Intertidal sediment samples were collected at five sites of the Rio San Pedro (RSP) creek, from the aquaculture effluent to a clean site. The effective concentration (EC50) from bacterial bioluminescence and A. brevicornis survival on whole sediments and P. lividus larval developmental success on sediment elutriates were assessed. Numbers of species, abundance and Shannon diversity were the biodiversity indicators measured in benthic fauna of sediment samples. In parallel, redox potential, pH, organic matter and metal levels (Cd, Cu, Ni, Pb and Zn) in the sediment and dissolved oxygen in the interstitial water were measured in situ. Water and sediment physicochemical analysis revealed the exhibition of a spatial gradient in the RSP, evidenced by hypoxia/anoxia, reduced and acidic conditions, high organic enrichment and metal concentrations at the most contaminated sites. Whereas, the benthic fauna biodiversity decreased the bioassays depicted decreases in EC50, A. brevicornis survival, P. lividus larval success at sampling sites closer to the studied fish farms. This study demonstrates that the sediments polluted by fish farm effluents may lead to alterations of the biodiversity of the exposed organisms.

Identification of biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of Carcinus maenas.

A 28-day bioassay was performed with Carcinus maenas to evaluate chronic effects caused by exposure to caffeine and ibuprofen (0.1-50 µg L(-1)) in sea water. Lysosomal membrane stability (LMS) was evaluated in hemolymph applying the neutral red retention assay (NRRA); several biomarkers including ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPX), lipid peroxidation (LPO) and DNA damage were studied in gill, hepatopancreas, muscle and gonad tissues. In crabs exposed to environmental concentrations of the drugs, retention time was reduced by 50%. EROD and DBFOD activities were induced by caffeine in muscle and hepatopancreas tissues (p < 0.05); GST activity was activated by ibuprofen in gill, hepatopancreas and muscle at the highest concentrations tested (p < 0.05). All tissues showed GPX activity and LPO induction (p < 0.05). Crabs exposed to caffeine and ibuprofen showed evidence of DNA damage mainly in hepatopancreas tissues (p < 0.05). Environmental concentrations of pharmaceuticals induce LMS and the biochemical responses studied in this crab. This methodology is a suitable technique for assessing pharmaceutical toxicity in the marine environment.

Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals.

The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked every 2 days with various concentrations (from 0.1 to 50 µg · L(-1)) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p < 0.05) after exposure to environmental concentrations of each pharmaceutical (caffeine = 15 µg · L(-1); carbamazepine = 1 µg · L(-1); ibuprofen = 5 µg · L(-1); and novobiocin = 0.1 µg · L(-1)) when compared with control organisms. The predicted "no environmental effect" concentration/measured environmental concentration results showed that the selected pharmaceuticals are toxic at environmental concentrations and need further assessment. LMS monitoring in crabs is a sensitive tool for evaluating exposure to concentrations of selected drugs under laboratory conditions and provides a robust tier 1 testing approach (screening biomarker) for rapid assessment of marine pollution and environmental impact assessments for analyzing pharmaceutical contamination in aquatic environments.

Designing an integrated environmental monitoring plan for land-based marine fish farms located at exposed and hard bottom coastal areas.

The increase in aquaculture activities in the last few decades has not been accompanied by a corresponding increase in environmental controls and regulations. In this context, the application of environmental monitoring plans (EMPs) has become necessary to assess the environmental impact associated with fish farming wastes. The objective of this review paper is to evaluate the suitability of experimental and analytical procedures as monitoring tools for inclusion in EMPs for intensive land-based marine fish farms (LBMFFs). The strong hydrodynamics and, in particular, the lack of sediment on the rocky coasts where LBMFFs are usually located, greatly limit the monitoring tools that can be used. We propose EMPs that employ a weight-of-evidence approach to evaluate: contamination, trophic and toxic effects, and ecological integrity. Laboratory tests, in situ bioassays and field surveys of local species are presented as key tools for assessing the impact of LBMFFs on ecosystems. The δ(15)N signal along a spatial gradient is proposed for evaluating exposure to contaminants. Trophic effects can be determined by growth of transplanted macro- and microalgae. Toxic effects can be evaluated by responses at different levels of biological organization, including biochemical and histological changes, physiological alterations and survival, in species from different trophic levels. Fouling tests and analysis of community structures are recommended for assessing ecological integrity. This review contributes to the development of environmental controls for intensive LBMFFs, and for other activities that discharge wastewater to rocky shores.

Assessing a bioremediation strategy in a shallow coastal system affected by a fish farm culture--application of GIS and shellfish dynamic models in the Rio San Pedro, SW Spain.

An integrated multi-trophic aquaculture assessment for Pacific oyster (Crassostrea gigas) aquaculture as a bioremediation strategy in areas impacted by fish farm effluents in Rio San Pedro was assessed by combining geographic information system with carrying capacity models. Sites of 0.44 km(2) were evaluated considering constraints; physical factors, growth and survival factors, environmental quality factors, water and sediment quality criteria, factor suitability ranges, and Multi-Criteria Evaluation. Isleta and Flamenco are promising sites for oyster production, and Dorada is of marginal interest. Carbon and nitrogen removal from the water by algae and through detritus filtration was estimated. The biodeposition of organic material from longline leases was found to have little negative impact on sediment. The eutrophication results indicate that phytoplankton removal had a positive impact on water quality at the Dorada. This case study quantified the direct profitability and bioremediative environmental service advantages that fish-shellfish farms can have relative to fish monocultures.

Chronic contamination assessment integrating biomarkers' responses in transplanted mussels--a seasonal monitoring.

This study aimed to provide the first biomonitoring integrating biomarkers and bioaccumulation data in São Paulo coast, Brazil and, for this purpose, a battery of biomarkers of defense mechanisms was analyzed and linked to contaminants' body burden in a weigh-of-evidence approach. The brown mussel Perna perna was selected to be transplanted from a farming area (Caraguatatuba) to four possibly polluted sites: Engenho D'Água, DTCS (Dutos e Terminais do Centro-Oeste de São Paulo) oil terminal (Sao Sebastiao zone), Palmas Island, and Itaipu (It; Santos Bay zone). After 3 months of exposure in each season, mussels were recollected and the cytochrome P4501A (CYP1A)- and CYP3A-like activities, glutathione-S-transferase and antioxidants enzymes (catalase, glutathione peroxidase, and glutathione reductase) were analyzed in gills. The concentrations of polycyclic aromatic hydrocarbons, linear alkylbenzenes, and nonessential metals (Cr, Cd, Pb, and Hg) in whole tissue were also analyzed and data were linked to biomarkers' responses by multivariate analysis (principal component analysis-factor analysis). A representation of estimated factor scores was performed to confirm the factor descriptions and to characterize the studied stations. Biomarkers exhibited most significant alterations all year long in mussels transplanted to It, located at Santos Bay zone, where bioaccumulation of organic and inorganic compounds was detected. This integrated approach using transplanted mussels showed satisfactory results, pointing out differences between sites, seasons, and critical areas, which could be related to land-based contaminants' sources. The influence of natural factors and other contaminants (e.g., pharmaceuticals) on biomarkers' responses are also discussed.