Diana Álvarez-Muñoz

Multi-residue method for the analysis of pharmaceuticals and some of their metabolites in bivalves.

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 (2.7ng/g dw) and oyster (3.0ng/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.

Oxidative stress and histopathology damage related to the metabolism of dodecylbenzene sulfonate in Senegalese sole.

Surfactants such as linear alkylbenzene sulfonates (LAS) are widely utilised in the formulation of detergents in commercial products. After use, they pass through waste water treatment plants (WWTP) and are then discharged to aquatic ecosystems, causing risk to aquatic life. The exposure of marine animals to these compounds enhances the production of reactive oxygen species (ROS) with subsequent damage to macromolecules, and produces histological alterations. A flow-through experiment with Senegalese sole (Solea senegalensis) has been devised with the object of correlating the metabolism of LAS including sulfophenylcarboxylic acids (SPCs) by fish with their antioxidant defence system (generation of oxyradicals) and histopathological damage. The generation of intermediate degradation products (SPCs) by the organism, the histopathological responses, the antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione S-transferase (GST)), as well as other kinds of enzyme such as acid and alkaline phosphatases (AcP, ALP), were measured. SPCs from 5ØC(6) to 11ØC(12) were identified and quantified in fish and water; their concentrations differed depending on the sampling moment. In general, the responses found in the enzymes were slight: a decrease in the enzymatic activity in gills and activation in the digestive tract. The evidence of histopathological damage identified was also small; the organisms defensive mechanism against pollutants should enable it to recover easily. A direct relationship was established between biotransformation and the generation of SPCs and ROS. In conclusion, the correct functioning of the antioxidant defence system with absence of large variations, the short-term histopathological damage, and the evidence of SPCs indicate an adequate metabolism of 2-phenyl-C(12)-linear alkylbenzene sulfonates (2ØC(12)LAS) by this specie and non-toxic effects at environmentally realistic levels.

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds

This study analysed the levels of androgen receptor antagonist activity in extracts of coastal sediments sampled from estuaries in southern UK and northern France. Anti-androgenic (AA) activity varied between <0.2 and 224.3±38.4μg flutamide equivalents/g dry weight of sediment and was significantly correlated with the total organic carbon and silt content of samples. AA activity was detected in tissues extracts of clams, Scrobicularia plana, sampled from a contaminated estuary, some of which was due to uptake of a series of 4 or 5 ring polycyclic aromatic hydrocarbons (PAHs). Initial studies also indicated that fractionated extracts of male, but not female, clams also contained androgen receptor agonist activity due to the presence of dihydrotestosterone in tissues. This study reveals widespread contamination of coastal sediments of the Transmanche region with anti-androgenic compounds and these contaminants should be investigated for their potential to disrupt sexual differentiation in aquatic organisms.

Experimental determination of bioconcentration, biotransformation, and elimination of linear alkylbenzene sulfonates in Solea senegalensis

The bioconcentration, biotransformation, and elimination of anionic surfactants, linear alkylbenzene sulfonates (LAS), specifically 2ØC(10) and 2ØC(12) LAS, which are pure isomers from two different homologues, have been characterized in Solea senegalensis using an experimental flow-through system with environmental exposure levels. The LAS and their carboxylated metabolites, sulfophenylcarboxylic acids (SPCs), were analyzed by pressurized liquid extraction, followed by solid-phase extraction, liquid chromatography with fluorescence detection, and mass spectrometry. The bioconcentration factors obtained in the steady state were 17.2 +/- 1.2 L/kg for 2ØC(10) LAS and 386.5 +/- 31.5 L/kg for 2ØC(12) LAS. A kinetic study also was performed with 2ØC(12) LAS, and the rate constants of uptake and elimination were K(1) = 177.04 L/kg d and K(2) = 0.92/d, respectively. The formation of the intermediate degradation products was monitored during the 2ØC(10) LAS experiment, thus allowing, to our knowledge for the first time, the identification and quantification of SPCs from 5ØC(6) to 9ØC(10) in marine organisms and in the depuration water as a result of biotransformation by the organisms, diffusive elimination, and excretion. The elimination percentage of the surfactants is both time- and concentration-dependent, and this suggests the existence of a concentration limit for the surfactant in the fish below which the elimination processes are slow. Overall, LAS are not persistent bioaccumulative compounds at environmental exposure levels.

Influence of the molecular structure and exposure concentration on the uptake and elimination kinetics, bioconcentration, and biotransformation of anionic and nonionic surfactants

In vivo experiments with fish (sole) were performed for pure homologs of linear alkylbenzene sulfonates (LAS) and alcohol ethoxylates (AEO), the most widely used anionic and nonionic surfactants. The surfactant concentration measured in the organism was higher than in the experimental water, and increased with the exposure concentration for both compounds. At the exposure levels tested the bioconcentration factors (BCF) for AEO were one order of magnitude higher than for LAS. Two linear relationships for hydrophobicity were established, one with BCF and the other with uptake rate. The influence of the exposure concentration on the uptake (k(1)) and elimination (k(2)) velocities was researched. The value obtained for k(1) for AEO was higher than for LAS, while k(2) was very similar for both compounds, indicating differences in the incorporation and but not in the depuration rate. For the first time, internal degradation products of LAS were identified and quantified in fish and water and the glucuronic conjugate of AEO was detected in an organism. The predominant biotransformation process for these compounds may be different: the results suggest a phase I biotransformation for LAS and phase II for AEO, due to their different molecular structures.

Actividades enzimáticas de las fosfatasas ácida y alcalina y la catalasa en Ruditapes philippinarum como biomarcadores del estrés generado por tensioactivos aniónicos (C11-LAS) y no iónicos (NPEO2.8)

A subchronic toxicity study was performed by exposing organisms to two surfactants in a continuous flow-through system for 120 h, followed by a depuration phase of 48 h. The bivalve mollusc Ruditapes philippinarum, widely distributed in intertidal zones and of high commercial value, was used. The main anionic and non-ionic surfactants employed were linear alkylbenzene sulphonate (LAS) and nonylphenol ethoxylate (NPEO2.8), respectively. The organisms were exposed to three environmental levels (μg L–1), and the concentrations of C11-LAS measured in the tissues were 2.4, 6.7 and 12.8 μg g–1 for the low, intermediate and high exposure levels, while those of NPEO2.8 were 13.4, 14.8 and 31.7 μg g–1, respectively. The responses used as “end point” of subcellular toxicity were the enzymatic activity of catalase (CAT), acid phosphatase (AcP) and alkaline phosphatase (ALP). No significant differences were observed in CAT activity at the end of the exposure phase; however, at the end of the depuration phase there was an increase in CAT activity in the organisms exposed to the low concentration of C11-LAS (P < 0.05) and a significant decrease in the individuals exposed to the high concentration of NPEO2.8, which is related to the largest accumulation of the compound inside the organism. The organisms exposed to the intermediate and high concentrations (P < 0.05) of C11-LAS showed inhibition for AcP and activation for ALP at the end of the experiment (day 7). In the organisms exposed to the high concentration of NPEO2.8 there was an increase in the specific activity at the end of the depuration stage.

Fluvial biofilms exposed to desiccation and pharmaceutical pollution: New insights using metabolomics

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 biofilms 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.

Intersex related gene expression profiles in clams Scrobicularia plana : molecular markers and environmental application

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Global metabolite profiling reveals transformation pathways and novel metabolomic responses in Solea senegalensis after exposure to a non-ionic surfactant

Alcohol polyethoxylate (AEO) surfactants are widely used in household and industrial products, but the health effects arising from short-term exposure to sublethal concentrations are unknown. A metabolomic approach was used to investigate the biotransformation and effects of exposure to sublethal concentrations of hexaethylene glycol monododecylether (C12EO6) in juvenile sole, Solea senegalensis. After 5 days, C12EO6 was rapidly metabolized in the sole by oxidation, glucuronidation, and ethoxylate chain shortening. C12EO6 exposure at either 146 or 553 μg L(-1) resulted in significant metabolite disruption in liver and blood samples, including an apparent fold increase of >10(6) in the circulating levels of C24 bile acids and C27 bile alcohols, disturbance of glucocorticoid and lipid metabolism, and a 470-fold decrease in levels of the fatty acid transport molecule palmitoyl carnitine. Depuration resulted in rapid elimination of the surfactant and normalization of metabolites toward pre-exposure levels. Our findings show for the first time the ability of metabolomic analyses to discern effects of this AEO on metabolite homeostasis at exposure levels below its no effect concentrations for survival and reproduction in juvenile fish. The pronounced alteration in levels of liver metabolites, phospholipids, and glucocorticoids in S. senegalensis in response to surfactant exposure may indicate that this contaminant could potentially impact a number of health end points in fish.

Contaminants in the Marine Environment

Abstract This chapter presents an overview of the different groups of chemical substances that pollute the marine environment. Metals, persistent organic contaminants, and emerging organic contaminants such as polycyclic aromatic hydrocarbons (PAHs), surfactants, polychlorinated biphenyls (PCBs), pesticides, dioxins, per- and polyfluorinated alkyl substances (PFASs), brominated flame retardants (BFRs), endocrine-disrupting compounds (EDCs), pharmaceuticals and personal care products (PPCPs), marine biotoxins, nanomaterials, polydimethylsiloxanes (PDMS), and microplastics are included. Their main sources of contamination and physicochemical properties are described, as well as the most used analytical techniques for their detection and quantification in the marine environment. Their usual levels found in seawater, sediments, and marine organisms are reported, providing an exposure scenario to wildlife that will be further studied along the book.