To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

Removal of Antibiotics Using an Algae-Algae Consortium (Chlorella protothecoides and Chlorella vulgaris).

The intensive use of antibiotics (for human, veterinary, and agricultural purposes) has steadily increased over the last 30 years. Large amounts of antibiotic residues are released into aquatic systems, mostly due to inefficient wastewater treatment. Conventional wastewater treatments are not designed to remove emerging contaminants (such as antibiotics) from wastewater. Therefore, algae treatment (phycoremediation) has emerged as a promising choice for cost-effective, eco-friendly, and sustainable wastewater treatment. For this reason, we investigated the removal performance of a well-established algal consortia (Chlorella protothecoides and Chlorella vulgaris) used in passive wastewater treatment ponds (Mosselbay, South Africa). Five antibiotics (sulfamethoxazole, amoxicillin, trimethoprim, ofloxacin, and clarithromycin) were selected for their ubiquity and/or low removal efficiency in conventional wastewater treatment plants (WWTPs). For each antibiotic, two concentrations were used: one environmentally relevant (10 ppb) and another 10 times higher (100 ppb), tested in triplicate and collected at two-time points (7 and 10 days). The algae remained viable over the exposure period (which is similar to the retention time within maturation ponds) and exhibited the capacity to remove sulfamethoxazole (77.3% ± 3.0 and 46.5% ± 5.3) and ofloxacin (43.5% ± 18.9 and 55.1% ± 12.0) from samples spiked with 10 and 100 ppb, respectively. This study demonstrates the potential and innovation of algal remediation for contaminants in a developing country context, where minimal infrastructure is available.

Resilience of barley (Hordeum vulgare) plants upon exposure to tramadol: Implication for the root-associated bacterial community and the antioxidative plant defence system.

Insufficiently treated reclaimed water can act as a source of contamination by introducing recalcitrant contaminants (e.g., pharmaceutical compounds) to various water bodies and/or agricultural soils after irrigation. Tramadol (TRD) is one of these pharmaceuticals that can be detected in influents and effluents of wastewater treatment plants, at discharge points as well as in surface waters in Europe. While the uptake of TRD by plants through irrigation water has been shown, plant responses towards this compound are still unclear. Therefore, this study aims to evaluate the effects of TRD on selected plant enzymes as well as on the root bacterial community structure. A hydroponic experiment was conducted to test the effects of TRD (100 µg L-1 TRD) on barley plants, at two harvesting time points after treatment. Accumulation of TRD in root tissues over time was observed reaching concentrations of 111.74 and 138.39 µg g-1 in total root FW after 12 and 24 days of exposure, respectively. Furthermore, noticeable inductions in guaiacol peroxidase (5.47-fold), catalase (1.83-fold) and glutathione S-transferase (3.23- and 2.09-fold) were recorded in roots of TRD-treated plants compared to controls after 24 days. A significant alteration in the beta diversity of root-associated bacteria due to TRD treatment was observed. Three amplicon sequence variants assigned to Hydrogenophaga, U. Xanthobacteraceae and Pseudacidovorax were differentially abundant in TRD-treated compared to control plants at both harvesting time points. This study reveals the resilience of plants through the induction of the antioxidative system and changes in the root-associated bacterial community to cope with the TRD metabolization/detoxification process.

Quantification of insecticides in commercial seafood sold in East Asian markets: risk assessment for consumers.

The extraction of 21 insecticides and 5 metabolites was performed using an optimized and validated QuEChERS protocol that was further used for the quantification (GC-MS/MS) in several seafood matrices (crustaceans, bivalves, and fish-mudskippers). Seven species, acquired from Hong Kong and Macao wet markets (a region so far poorly monitored), were selected based on their commercial importance in the Indo-Pacific region, market abundance, and affordable price. Among them, mussels from Hong Kong, together with mudskippers from Macao, presented the highest insecticide concentrations (median values of 30.33 and 23.90 ng/g WW, respectively). Residual levels of fenobucarb, DDTs, HCHs, and heptachlors were above the established threshold (10 ng/g WW) for human consumption according to the European and Chinese legislations: for example, in fish-mudskippers, DDTs, fenobucarb, and heptachlors (5-, 20- and tenfold, respectively), and in bivalves, HCHs (fourfold) had higher levels than the threshold. Risk assessment revealed potential human health effects (e.g., neurotoxicity), especially through fish and bivalve consumption (non-carcinogenic risk; ΣHQLT > 1), and a potential concern of lifetime cancer risk development through the consumption of fish, bivalves, and crustaceans collected from these markets (carcinogenic risk; ΣTCR > 10-4). Since these results indicate polluted regions, where the seafood is collected/produced, a strict monitoring framework should be implemented in those areas to improve food quality and safety of seafood products.

Exudates from Miscanthus x giganteus change the response of a root-associated Pseudomonas putida strain towards heavy metals.

The composition of root exudates is modulated by several environmental factors, and it remains unclear how that affects beneficial rhizosphere or inoculated microorganisms under heavy metal (HM) contamination. Therefore, we evaluated the transcriptional response of Pseudomonas putida E36 (a Miscanthus x giganteus isolate with plant growth promotion-related properties) to Cd, Pb and Zn in an in vitro study implementing root exudates from M. x giganteus. To collect root exudates and analyse their composition plants were grown in a pot experiment under HM and control conditions. Our results indicated higher exudation rate for plants challenged with HM. Further, out of 29 organic acids identified and quantified in the root exudates, 8 of them were significantly influenced by HM (e.g., salicylic and terephthalic acid). The transcriptional response of P. putida E36 was significantly affected by the HM addition to the growth medium, increasing the expression of several efflux pumps and stress response-related functional units. The additional supplementation of the growth medium with root exudates from HM-challenged plants resulted in a downregulation of 29% of the functional units upregulated in P. putida E36 as a result of HM addition to the growth medium. Surprisingly, root exudates + HM downregulated the expression of P. putida E36 functional units related to plant colonization (e.g., chemotaxis, motility, biofilm formation) but upregulated its antibiotic and biocide resistance compared to the control treatment without HM. Our findings suggest that HM-induced changes in root exudation pattern may attract beneficial bacteria that are in turn awarded with organic nutrients, helping them cope with HM stress. However, it might affect the ability of these bacteria to colonize plants growing in HM polluted areas. Those findings may offer an insight for future in vivo studies contributing to improvements in phytoremediation measures.

How can environmental conditions influence dicofol genotoxicity on the edible Asiatic clam, Meretrix meretrix?

Genotoxic effects of dicofol on the edible clam Meretrix meretrix were investigated through a mesocosm experiment. Individuals of M. meretrix, were exposed to environmental concentration (D1 = 50 ng/L) and supra-environmental concentration (D2 = 500 ng/L) of dicofol for 15 days, followed by the same depuration period. DNA damage (i.e., strand breaks and alkali-labile sites) was evaluated at day 1, 7 and 15, during uptake and depuration, using Comet assay (alkaline version) and nuclear abnormalities (NAs) as genotoxicity biomarkers. The protective effects of dicofol against DNA damage induced by ex vivo hydrogen peroxide (H2O2) exposure were also assessed. Comet assay results revealed no significant DNA damages under dicofol exposure, indicating 1) apparent lack of genotoxicity of dicofol to the tested conditions and/or 2) resistance of the animals due to optimal adaptation to stress conditions. Moreover, ex vivo H2O2 exposure showed an increase in the DNA damage in all the treatments without significant differences between them. However, considering only the DNA damage induced by H2O2 during uptake phase, D1 animals had significantly lower DNA damage than those from other treatments, revealing higher protection against a second stressor. NAs data showed a decrease in the % of cells with polymorphic, kidney shape, notched or lobbed nucleus, along the experiment. The combination of these results supports the idea that the clams used in the experiment were probably collected from a stressful environment (in this case Pearl River Delta region) which could have triggered some degree of adaptation to those environmental conditions, explaining the lack of DNA damages and highlighting the importance of organisms' origin and the conditions that they were exposed during their lives.

Removal of tramadol from water using Typha angustifolia and Hordeum vulgare as biological models: Possible interaction with other pollutants in short-term uptake experiments.

Tramadol (TRD) is widely detected in aquatic ecosystems as a result of massive abuse and insufficient removal from wastewater facilities. As a result, TRD can contaminate groundwater sources and/or agricultural soils. While TRD toxicity has been reported from aquatic biota, data about TRD detection in plants are scarce. Moreover, information regarding plant capability for TRD removal is lacking. To understand the fate of this opioid, we have investigated the uptake, translocation and removal capacity of TRD by plants, addressing short-term and long-term uptake. The uptake rates of TRD, in excised barley and cattail roots, were 5.18 and 5.79 µg g-1 root fresh weight day-1, respectively. However, TRD uptake was strongly inhibited after co-exposing these roots either with the drug venlafaxine (similar molecular structure as TRD) or with quinidine (an inhibitor of cellular organic cation transporters). When barley seedlings were exposed to TRD in a hydroponic experiment a removal efficiency up to 90% (within 15 days) was obtained, with bioconcentration and translocation factors close to 9 and 1, respectively. The combination of results from both plants and the inhibition observed after treatment with quinidine revealed that organic cation transporters may be involved in the uptake of TRD by plants.

The changes in Lemna minor metabolomic profile: A response to diclofenac incubation.

Metabolomics is an emerging approach that investigates the changes in the metabolome profile. In the present study, Lemna minor -considered as an experimental aquatic plant model- was incubated with 10 and 100 µM diclofenac (DCF) for 96 h, respectively. Knowing that DCF is internationally often problematic in wastewater effluents and that it might affect particularly the metabolic profiles in aquatic plants, mainly the oxidoreductase, dehydrogenase, peroxidase, and glutathione reductase activities, here it was hypothesized (H) that in the common duckweed, DCF might increase the phenolic and flavonoids pathways, as an antioxidant response to this stress (H1). Also, it was expected DCF to alternate the physiological characteristics, especially the molecular interaction and biochemical properties, of Lemna (H2). Metabolic changes were investigated with target and untargeted screening analysis using RPLC-HILIC-ESI-TOF-MS. Twelve amino acids were identified in all treatments, together with three organic acids (p-coumaric, cinnamic, and sinapic acids). In untargeted screening, the important metabolites to discriminate between different treatments were assigned to Lemna such as organic acids, lignin, sugars, amino acids, dipeptides, flavonoids, biflavonoids, fatty acids, among others. In resume, Lemna responded to both DCF concentrations, showing different stress patterns. A similar metabolic response had already been identified in other studies in exposing Lemna to other anthropogenic stressors (like pesticides).

Untargeted Analysis of Lemna minor Metabolites: Workflow and Prioritization Strategy Comparing Highly Confident Features between Different Mass Spectrometers.

Metabolomics approaches provide a vast array of analytical datasets, which require a comprehensive analytical, statistical, and biochemical workflow to reveal changes in metabolic profiles. The biological interpretation of mass spectrometric metabolomics results is still obstructed by the reliable identification of the metabolites as well as annotation and/or classification. In this work, the whole Lemna minor (common duckweed) was extracted using various solvents and analyzed utilizing polarity-extended liquid chromatography (reversed-phase liquid chromatography (RPLC)-hydrophilic interaction liquid chromatography (HILIC)) connected to two time-of-flight (TOF) mass spectrometer types, individually. This study (introduces and) discusses three relevant topics for the untargeted workflow: (1) A comparison study of metabolome samples was performed with an untargeted data handling workflow in two different labs with two different mass spectrometers using the same plant material type. (2) A statistical procedure was observed prioritizing significant detected features (dependent and independent of the mass spectrometer using the predictive methodology Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA). (3) Relevant features were transferred to a prioritization tool (the FOR-IDENT platform (FI)) and were compared with the implemented compound database PLANT-IDENT (PI). This compound database is filled with relevant compounds of the Lemnaceae, Poaceae, Brassicaceae, and Nymphaceae families according to analytical criteria such as retention time (polarity and LogD (pH 7)) and accurate mass (empirical formula). Thus, an untargeted analysis was performed using the new tool as a prioritization and identification source for a hidden-target screening strategy. Consequently, forty-two compounds (amino acids, vitamins, flavonoids) could be recognized and subsequently validated in Lemna metabolic profile using reference standards. The class of flavonoids includes free aglycons and their glycosides. Further, according to our knowledge, the validated flavonoids robinetin and norwogonin were for the first time identified in the Lemna minor extracts.

Post-reclamation microbial diversity and functions in hexachlorocyclohexane (HCH) contaminated soil in relation to spontaneous HCH tolerant vegetation.

The toxicity, volatility and persistence of the obsolete organochlorine pesticide hexachlorocyclohexane (HCH), makes reclamation of contaminated areas a priority for the health and welfare of neighboring human communities. Microbial diversity and functions and their relation to spontaneous vegetation in post-excavation situations, are essential indicators to consider in bioaugmentation or microbe-assisted phytoremediation strategies at field scale. Our study aimed to evaluate the effects of long-term HCH contamination on soil and plant-associated microbial communities, and whether contaminated soil has the potential to act as a bacterial inoculum in post-excavation bioremediation strategies. To scrutinize the role of vegetation, the potential nitrogen fixation of free-living and symbiotic diazotrophs of the legume Lotus tenuis was assessed as a measure of nutrient cycling functions in soil under HCH contamination. Potential nitrogen fixation was generally not affected by HCH, with the exception of lower nifH gene counts in excavated contaminated rhizospheres, most probably a short-term HCH effect on early bacterial succession in this compartment. HCH shaped microbial communities in long-term contaminated bulk soil, where we identified possible HCH tolerants such as Sphingomonas and Altererythrobacter. In L. tenuis rhizosphere, microbial community composition was additionally influenced by plant growth stage. Sphingobium and Massilia were the bacterial genera characteristic for HCH contaminated rhizospheres. Long-term HCH contamination negatively affected L. tenuis growth and development. However, root-associated bacterial community composition was driven solely by plant age, with negligible HCH effect. Results showed that L. tenuis acquired possible HCH tolerant bacteria such as the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade, Sphingomonas, Massilia or Pantoea which could simultaneously offer plant growth promoting (PGP) benefits for the host. Finally, we identified an inoculum with possibly HCH tolerant, PGP bacteria transferred from the contaminated bulk soil to L. tenuis roots through the rhizosphere compartment, consisting of Mesorhizobium loti, Neorhizobium galegae, Novosphingobium lindaniclasticum, Pantoea agglomerans and Lysobacter bugurensis.

Uptake and depuration kinetics of dicofol metabolite 4,4'-dichlorobenzophenone, in the edible Asiatic clam Meretrix meretrix.

Uptake and depuration kinetics of 4,4'-dichlorobenzophenone (main metabolite of dicofol) in the edible clam Meretrix meretrix were evaluated through a mesocosm experiment. M. meretrix was exposed to different dicofol concentrations (environmental concentration, D1 = 50 ng/L; supra-environmental concentration, D2 = 500 ng/L) for 15 days, followed by the same depuration period. To accomplish this goal, an analytical method was successfully optimized for 4,4'-DCBP using QuEChERS as extraction method with a range of concentrations 0.3-76.8 ng/g ww quantified by gas chromatography coupled to tandem mass spectrometry. Our results demonstrated different kinetics of accumulation depending on the two dicofol treatments. For D1, the uptake kinetic was best fitted using a plateau followed by one phase association kinetic model, while for D2 a one phase association kinetic model suited better. Similar bioconcentration factors were obtained for both concentrations but only animals exposed to D2, showed 4,4'-DCBP levels above the limits of quantification after 24 h exposure. These animals also showed lower uptake rate (ku) than organisms exposed to D1. During the depuration period, only organisms exposed to D1 successfully depurated after 24 h. On the other hand, although animals exposed to D2 presented higher elimination factor, they did not reach the original levels after depuration. Moreover, values detected in these clams were higher than the Maximum Residue Level (10 ng/g) established by the European legislation. This indicates that longer periods of depuration time than the ones used in this study, may be needed in order to reach safe levels for human consumption. This work also demonstrated that studies on metabolite kinetics during uptake/depuration experiments, could be a new alternative to understand the impact and metabolism of pesticides in the marine environment.

Estimating volumes from common carp hepatocytes using design-based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists.

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5-7 µm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three-dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20-40 µm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single-section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two-dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single-section disector. Strong correlations existed between profile-derived versus 2D-nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D-nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.

Environmental characterization of 4,4'-dichlorobenzophenone in surface waters from Macao and Hong Kong coastal areas (Pearl River Delta) and its toxicity on two biological models: Artemia salina and Daphnia magna.

The Pearl River Delta (PRD) is one of the areas with higher environmental concentration of organochlorine pesticides (OCPs), being DDT one of the most abundant. In this work, 4,4'-dichlorobenzophenone (4,4'-DCBP), a common metabolite of dicofol (DDT related) and DDT, was quantified in surface waters of Hong Kong and Macao, together with the analysis of physicochemical and nutrients parameters. Hong Kong presented higher 4,4'-DCBP mean levels (12.50 ng/L) than Macao (4.05 ng/L), which may be due to the use of dicofol as a pesticide and DDT as antifouling-paint for ships. The region presented a possible eutrophication state due to the high nutrients' concentration. For the first time, toxicity evaluation of this metabolite in Artemia salina and Daphnia magna was done, in order to compute valid EC50s and theoretically evaluate the risk in the PRD. The toxicity results (EC50 = 0.27 mg/L for A. salina; and EC50 = 0.17 mg/L and LC50 = 0.26 mg/L for D. magna), together with the 4,4'-DCBP levels quantified, indicated a low environmental risk.

PAHs in water and surface sediments from Douro River estuary and Porto Atlantic coast (Portugal)-impacts on human health.

This study investigated the presence of 16 priority polycyclic aromatic compounds (PAHs) in waters from the Douro River estuary and nearby Atlantic seacoast, which both bath the Porto metropolis. In the area, there is an oil refinery, an important harbour, an intense maritime traffic, small marinas and highly inhabited cities. For the analysis of PAHs, water samples were taken from four sampling sites, at six different times of the year (2011), and extracted by solid-phase extraction (dissolved fraction) and by ultrasound technique (suspended fraction), before their quantification by gas chromatography-mass spectrometry. Results not only proved the ubiquitous distribution of all analysed PAHs in the present habitat, but also that their global amounts (∑16PAHs) were extremely high at all sampling sites. Their average concentrations attained ≈ 55 ng/L and ≈ 52 µg/g dry weight (dw), respectively, in water and surface sediments. Accordingly, the surveyed area was classified as highly polluted by these organics and so, in view of the concentrations, mutagenic/carcinogenic responses in both humans and aquatic animals are possible to occur. The percentages of carcinogenic PAHs for humans (group 1) dissolved in water and in surface sediments were ca. 5 and 6%, respectively. These results are the first reported in the area and can be used as a baseline for future control of the PAHs levels locally while serving the building of global scenarios of PAHs pollution in Europe. Graphical abstract Percentage of PAHs, from different categories acordingly to WHO (2016), in both surface sediments and surface waters from Douro River estuary and Porto Atlantic seacoast; group 1 - carcinogenic, group 2A - probably carcinogenic, group 2B - possibly carcinogenic, and group 3 - not classifiable as carcinogenic to humans.

Determination of 54 pesticides in waters of the Iberian Douro River estuary and risk assessment of environmentally relevant mixtures using theoretical approaches and Artemia salina and Daphnia magna bioassays.

As a case study, the estuary of the international Douro River (Iberian Peninsula) was sampled over a year (2010) at six sampling sites to determine the presence of 56 pesticides of different categories (insecticides, herbicides, and fungicides). 96% of measured pesticides were detected in 79% of the quantified samples. Individual average pesticide concentrations ranged from 39 to 1 265ng/L, indicating a ubiquitous presence of the selected compounds; moreover, twelve pesticides were above the 2013/39/EU Directive limits. Due to its highly impacted profile, a theoretical hazard assessment was done considering the average and maximum environmental mixtures of all measured pesticides to identify the most sensitive trophic level. For both environmental mixtures, the theoretical approach suggested that invertebrates were the most sensitive group. Therefore, short-time exposure assays using both invertebrates Artemia salina and Daphnia magna, were done using the referred mixtures. Data demonstrated significant toxic effects ─ high mortality rate and abnormal swimming behaviour ─ of the exposed animals. Both approaches (theoretical and experimental) support the analytical results, alerting for an intervention on this estuarine environment and of other comparable.

Multi-matrix quantification and risk assessment of pesticides in the longest river of the Iberian peninsula.

The distribution of pesticides in dissolved aqueous phase (DAP), suspended particulate matter (SPM) and Scrobicularia plana soft tissues from the Tagus River estuary was determined to evaluate the chemicals pollution status and their hazard potential in this area. Samples were collected in 6 campaigns (December 2012-October 2013), from 3 strategical sites, and analysed via different extraction procedures followed by gas chromatography tandem mass spectrometry (GC-MS/MS) determination. The contamination profile among matrices (DAP, SPM, and soft tissue from bivalves (STB)) was marked by average concentrations of 345ng/L, 0.51mg/kg, and 0.02mg/kg, respectively, with several samples above the 2013/39/EU Directive of environmental quality standards (EQS); no differences were observed between sex. A wider range of pesticides was present in STB (n=53) than in SPM (n=36) and DAP (n=19) matrices. Sediment-water partition coefficient, bioaccumulation factor in both DAP and SPM fraction were estimated ranging between 2.5 and 4.4 and 0.008-2799, respectively. The spatial distribution of most pesticides and physicochemical parameters were consistent, indicating a pollution pattern primarily near the Trancão River mouth. Due to the presence of the target compounds, calculated risk quotients pointed out potential hazards for aquatic organisms, mainly to invertebrates. The estimated average daily intake, theoretical maximum daily intake, and hazard quotient of the studied pesticides-via bivalve ingestion-indicated no risk for human health, although it is important to note possible biomagnification processes that may happen along the estuarine food-chain.

Development and application of a QuEChERS-based extraction method for the analysis of 55 pesticides in the bivalve Scrobicularia plana by GC-MS/MS.

A method for quantitative determination of 55 pesticides in a bivalve matrix was established, based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction and using gas chromatography (GC)-ion trap (IT) mass spectrometry (MS/MS). Accomplishing the European SANCO guidelines, this method was validated using 5 g of homogenized soft tissue, allowing the quantification of pesticides at ng/g of wet weight (ww). Quantification limits and recovery rates ranged from 0.33 to 10.3 µg/L and from 78 to 119 %, respectively. As an important mollusc, not only from an ecological perspective but also for food consumption, the peppery furrow shell (Scrobicularia plana) was sampled at three strategical sites (Ria Formosa Lagoon, in the south of Portugal) during 2012-2013, over six campaigns. A total of 2160 animals were pooled by place and sex. No statistical differences were found among sites or between sexes. Forty percent of the sampled pools were above quantification limits, reaching total annual average concentrations of ∑800 ng/g ww. Additionally, 83 % of the selected compounds showed concentrations above the legal limits set by the European Directive 2013/39/EU. In conclusion, the applied method was successful and proved that bivalves were contaminated by the selected pesticides. In future work, this methodology can be used to monitor body burdens and obtain data for predicting impacts in shellfish consumers. Graphical Abstract Resume of pesticides extraction and analyses process from S. plana.

Pollution by oestrogenic endocrine disruptors and ß-sitosterol in a south-western European river (Mira, Portugal).

The Mira River is a Portuguese water body widely known for its wilderness and is advertised as one of the less polluted European rivers. On this presumption, the levels of endocrine-disrupting compounds (EDCs) in Mira waters were never measured. However, because environmentalists have claimed that the Mira could be moderately polluted, a range of 17 EDCs were measured not only at the estuary but also along the river. The targeted EDCs included natural and pharmaceutical oestrogens (17ß-oestradiol, oestrone and 17α-ethynylestradiol), industrial/household pollutants (octylphenols, nonylphenols and their monoethoxylates and diethoxylates and bisphenol A), phytoestrogens (formononetin, biochanin A, daidzein, genistein) and the phytosterol sitosterol (SITO). For this propose, waters from six sampling sites were taken every 2 months, over a 1-year period (2011), and analysed by gas chromatography-mass spectrometry. Unexpectedly high levels of oestrogens and of industrial/household pollutants were measured at all sampling sites, including those located inside natural protected areas. Indeed, the annual average sum of EDCs was ≈57 ng/L for oestrogens and ≈1.3 µg/L for industrial/household chemicals. In contrast, the global average levels of phytoestrogens (≈140 ng/L) and of SITO (≈295 ng/L) were lower than those reported worldwide. The EDC concentrations were normalised for ethynylestradiol equivalents (EE2eq). In view of these, the oestrogenic load of the Mira River attained ≈47 ng/L EE2eq. In addition, phosphates were above legal limits at both spring and summer (>1 mg/L). Overall, data show EDCs at toxicant relevant levels in the Mira and stress the need to monitor rivers that are allegedly less polluted.

A mollusk VDR/PXR/CAR-like (NR1J) nuclear receptor provides insight into ancient detoxification mechanisms.

The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jß. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.

Environmental assessment of pesticides in the Mondego River Estuary (Portugal).

The Mondego River estuary, located on the North Atlantic Ocean Ecoregion, is a basin affected by agricultural run-off with increasing signs of eutrophication. We evaluated the amounts and distribution of 56 priority pesticides belonging to distinct categories (insecticides, herbicides and fungicides). Temporal trends were considered and a total of 42 surface water samples were collected between 2010 and 2011. More than 55% of the GC-MS/MS-quantified pesticides were above the maximum amounts established by the European Directives (98/83/EC and 2013/39/EU). Based on the concentration addition (CA) and independent action (IA) models, we used a two-tiered approach to assess the hazard of the pesticide mixture, at the maximum concentration found, reflecting a potential risk. Short-term exposure using Artemia salina indicated a significant toxic effect where the locomotion of the animals was clearly affected.