Evaluating membrane bioreactor treatment for the elimination of emerging contaminants using different analytical methods.

Since wastewater treatment plants (WWTPs) were not originally designed to eliminate contaminants of emerging concern (CECs), alternative strategies like membrane bioreactor (MBR) technology are gaining importance in achieving effective CEC removal and minimising their environmental impact. In this study, composite wastewater samples were collected from the biggest WWTP in the Basque Country (Galindo, Biscay) and the performance of two secondary treatments (i.e. conventional activated sludge treatment, CAS, and MBR) was assessed. The combination of a suspect screening approach using liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) and multitarget analysis by gas chromatography-mass spectrometry (GC-MS) allowed the detection of approximately 200 compounds in the WWTP effluents. The estimated removal efficiencies (REs) revealed that only 16 micropollutants exhibited enhanced removal by MBR treatment (RE > 70% or 40 - 60%). The environmental risk posed by the non-eliminated compounds after both treatments remained similar, being anthracene, clarithromycin, bis(2-ethylhexyl) phthalate (DEHP) and dilantin the most concerning pollutants (RQ > 1). The Microtox® bioassay confirmed the MBR's efficiency in removing baseline toxicity, while suggesting a similar performance of CAS treatment. These minimal differences between treatments call into question the worthiness of MBR treatment and emphasise the need to seek more efficient alternative treatment methods.

Sample preparation for suspect and non-target screening of xenobiotics in human biofluids by liquid chromatography-High resolution tandem mass spectrometry.

The concept of exposome covers all the exposures an individual suffers from conception to death, which can be partially assessed through the monitoring of human biofluids. In there, target analytical approaches tend to focus on a limited set of xenobiotics, whereas exposomic studies need broad scopes in search of a full understanding. Given the issue, suspect and non-target screening are feasible alternatives. However, adequate sample preparation procedures should minimize interferences without significantly reducing the number of xenobiotics. Within this context, the present article aims to describe comprehensive sample preparation procedures for suspect or non-target screening of organic xenobiotics in several human biofluids, all coupled to unified separation and detection conditions based on ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). The referred biofluids consist of human urine, breast milk, saliva and ovarian follicular fluid.•Analytical methods for untargeted analysis of a wide range of xenobiotics in human biofluids are fully described in order to ensure reproducibility.•The sample preparation procedures balance selectivity and sensitivity.•Unified analysis conditions allow simultaneous analysis of diverse biofluids.

Exploratory optimisation of a LC-HRMS based analytical method for untargeted metabolomic screening of Cannabis Sativa L. through Data Mining.

BACKGROUND: Recent increase in public acceptance of cannabis as a natural medical alternative for certain neurological pathologies has led to its approval in different regions of the world. However, due to its previous illegal background, little research has been conducted around its biochemical insights. Therefore, in the current framework, metabolomics may be a suitable approach for deepening the knowledge around this plant species. Nevertheless, experimental methods in metabolomics must be carefully handled, as slight modifications can lead to metabolomic coverage loss. Hence, the main objective of this work was to optimise an analytical method for appropriate untargeted metabolomic screening of cannabis. RESULTS: We present an empirically optimised experimental procedure through which the broadest metabolomic coverage was obtained, in which extraction solvents for metabolite isolation, chromatographic columns for LC-qOrbitrap analysis and plant-representative biological tissues were compared. By exploratory means, it was determined that the solvent combination composed of CHCl3:H2O:CH3OH (2:1:1, v/v) provided the highest number of features from diverse chemical classes, as it was a two-phase extractant. In addition, a reverse phase 2.6 µm C18 100 Å (150 × 3 mm) chromatographic column was determined as the appropriate choice for adequate separation and further detection of the diverse metabolite classes. Apart from that, overall chromatographic peak quality provided by each column was observed and the need for batch correction methods through quality control (QC) samples was confirmed. At last, leaf and flower tissues resulted to provide complementary metabolic information of the plant, to the detriment of stem tissue, which resulted to be negligible. SIGNIFICANCE: It was concluded that the optimised experimental procedure could significantly ease the path for future research works related to cannabis metabolomics by LC-HRMS means, as the work was based on previous plant metabolomics literature. Furthermore, it is crucial to highlight that an optimal analytical method can vary depending on the main objective of the research, as changes in the experimental factors can lead to different outcomes, regardless of whether the results are better or worse.

Characterization of the contamination fingerprint of wastewater treatment plant effluents in the Henares River Basin (central Spain) based on target and suspect screening analysis.

The interest in contaminants of emerging concern (CECs) has increased lately due to their continued emission and potential ecotoxicological hazards. Wastewater treatment plants (WWTPs) are generally not capable of eliminating them and are considered the main pathway for CECs to the aquatic environment. The number of CECs in WWTPs effluents is often so large that complementary approaches to the conventional target analysis need to be implemented. Within this context, multitarget quantitative analysis (162 compounds) and a suspect screening (>40,000 suspects) approaches were applied to characterize the CEC fingerprint in effluents of five WWTPs in the Henares River basin (central Spain) during two sampling campaigns (summer and autumn). The results indicated that 76% of the compounds quantified corresponded to pharmaceuticals, 21% to pesticides and 3% to industrial chemicals. Apart from the 82 compounds quantified, suspect screening increased the list to 297 annotated compounds. Significant differences in the CEC fingerprint were observed between summer and autumn campaigns and between the WWTPs, being those serving the city of Alcalá de Henares the ones with the largest number of compounds and concentrations. Finally, a risk prioritization approach was applied based on risk quotients (RQs) for algae, invertebrates, and fish. Azithromycin, diuron, chlortoluron, clarithromycin, sertraline and sulfamethoxazole were identified as having the largest risks to algae. As for invertebrates, the compounds having the largest RQs were carbendazim, fenoxycarb and eprosartan, and for fish acetaminophen, DEET, carbendazim, caffeine, fluconazole, and azithromycin. The two WWTPs showing higher calculated Risk Indexes had tertiary treatments, which points towards the need of increasing the removal efficiency in urban WWTPs. Furthermore, considering the complex mixtures emitted into the environment and the low dilution capacity of Mediterranean rivers, we recommend the development of detailed monitoring plans and stricter regulations to control the chemical burden created to freshwater ecosystems.

Suspect screening workflow comparison for the analysis of organic xenobiotics in environmental water samples.

Suspect screening techniques are able to determine a broader range of compounds than traditional target analysis. However, the performance of the suspect techniques relies on the procedures implemented for peak annotation and for this, the list of potential candidates is clearly a limiting factor. In order to study this effect on the number of compounds annotated in environmental water samples, a method was validated in terms of absolute recoveries, limits of quantification and identification, as well as the peak picking capability of the software (Compound Discoverer 2.1) using a target list of 178 xenobiotics. Four suspect screening workflows using different suspect lists were compared: (i) the Stoffident list, (ii) all the NORMAN lists, (iii) suspects containing C, H, O, N, S, P, F or Cl in their molecular formula with more than 10 references in Chemspider and (iv) the mzCloud library. The results were compared in terms of the number of annotated compounds at each confidence level. The same 8 compounds (atenolol, caffeine, caprolactam, carbendazim, cotinine, diclofenac, propyphenazone and trimetoprim) were annotated at the highest confidence level using the four workflows. Remarkable differences were observed for lower confidence levels but only 4 features were annotated at different levels by the four workflows. While the third approach provided the highest number of annotated features, the workflow based on the mzCloud library rendered satisfactory results with a simpler approach. Finally, this latter approach was extended to the analysis of organic xenobiotics in different environmental water samples.

Suspect and non-target screening: the last frontier in environmental analysis.

Suspect and non-target screening (SNTS) techniques are arising as new analytical strategies useful to disentangle the environmental occurrence of the thousands of exogenous chemicals present in our ecosystems. The unbiased discovery of the wide number of substances present over environmental analysis needs to find a consensus with powerful technical and computational requirements, as well as with the time-consuming unequivocal identification of discovered analytes. Within these boundaries, the potential applications of SNTS include the studies of environmental pollution in aquatic, atmospheric, solid and biological samples, the assessment of new compounds, transformation products and metabolites, contaminant prioritization, bioremediation or soil/water treatment evaluation, and retrospective data analysis, among many others. In this review, we evaluate the state of the art of SNTS techniques going over the normalized workflow from sampling and sample treatment to instrumental analysis, data processing and a brief review of the more recent applications of SNTS in environmental occurrence and exposure to xenobiotics. The main issues related to harmonization and knowledge gaps are critically evaluated and the challenges of their implementation are assessed in order to ensure a proper use of these promising techniques in the near future.

Focused ultrasound-based extraction for target analysis and suspect screening of organic xenobiotics in fish muscle.

The development of multitarget and/or suspect screening methods for the analysis of xenobiotics in fish samples is compulsory due to the lack of works in the literature where a deep evaluation of the variables affecting extraction and clean-up steps is performed. The aim of the present work was to optimize and validate a multitarget (180 compounds) method for the analysis of priority and emerging xenobiotics in fish muscle using focused ultrasound-assisted solid-liquid extraction. From the different extraction solvents studied, a single extraction in cold acetonitrile rendered the best consensus results in terms of absolute recoveries and the number of target compounds extracted. Matrix effect was minimized using commercially available Captiva ND-Lipid filters, which provided clean extracts and satisfactory repeatability compared to other approaches. Absolute recoveries were corrected using matrix-matched calibration and apparent recoveries in the 43%-105%, 73%-131% and 78%-128% ranges were obtained at low (20 ng g-1), medium (100 ng g-1), and high (200 ng g-1) spiking levels, respectively. A 60% of the xenobiotics showed limits of identification lower than 20 ng g-1. The developed method was successfully applied to the quantification and suspect screening of samples bought in a local market (hake, gilt-head bream, sea bass and prawn) and fished (thicklip grey mullet) at the Urdaibai estuary (north of Spain). Food additives, antiparasitic drugs and PFOS were quantified at ng g-1 level. Moreover, the targeted method was extended to the suspect screening, revealing the presence of plastic related products (caprolactam, phthalates, polyethylenglycols), pharmaceutical products (albendazole, mebendazole, valpromide) and pesticides or insect repellents (icaridin, myristyl sulfate, nootkatone). Therefore, FUSLE in cold acetonitrile combined with Captiva ND-Lipid filters and liquid chromatography tandem high-resolution mass spectrometry (LC-q-Orbitrap) were successfully applied to both multitarget quantitative analysis and suspect screening of approx. 17,800 compounds.

Toxicology tailored low density oligonucleotide microarray for the thicklip grey mullets (Chelon labrosus): Biomarker gene transcription profile after caging in a polluted harbour.

In aquatic organisms inhabiting polluted waters genes are activated to build an adaptive/compensatory defence against the possible effects of pollutants. Such responses can be used as biomarkers of exposure to chemical compounds, outlining the molecular mechanisms activated under specific pollution scenarios. With the aim of exploiting such approach in environmental health assessment, toxicologically relevant gene fragments were sequenced in the thicklip grey mullet (Chelon labrosus) and a toxicologically tailored low-density (160 genes) oligonucleotide microarray was customised. The tool was validated comparing organ/sex specific gene expression profiles and characterising responses under laboratory exposure to model chemicals. Finally, juvenile mullets were caged in a polluted harbour and hepatic gene expression profiles analysed after 5 and 21 days of deployment. Cages were deployed in the inner (IH) and outer (OH) Pasaia harbour, Bay of Biscay. Mussels (Mytilus galloprovincialis) were also caged as biological matrix for chemical bioaccumulation analysis and stress biomarkers measurements. Slightly higher concentrations of chemicals (metals, tributyltin, PAHs, phthalates) were quantified in IH than in OH, fish bile metabolites also revealing higher availability of PAHs in IH. Lysosome membrane stability in mussels was reduced, indicating stress condition in both sites. The developed microarray discriminated mullets showing distinctive expression profiles depending on site and deployment time. Genes related to immune and hypoxia responses were regulated comparing IH and OH at day 5. Phase I and II biotransformation genes, such as cyp2, cyp3 and ugt, were up-regulated in IH, together with the aryl hydrocarbon receptor 2 (ahr2) and the ahr repressor. Similarly, TBT-binding proteins and genes involved in lipid metabolism (pparγ, cyp7) were up-regulated with deployment time. Even if nowadays higher throughput approaches for gene expression analyses are available, the developed mullet tool constitutes a comprehensive tool to assess molecular responses of mullets exposed to pollutants, although it remains to be explored whether it can be applied to assess pollutant exposure in active pollution monitorings and in environmental health assessment.

Tetraphasic polar organic chemical integrative sampler for the determination of a wide polarity range organic pollutants in water. The use of performance reference compounds and in-situ calibration.

In the present work, a homemade polar organic chemical integrative sampler (POCIS) was studied for the determination of 16 target analytes. The suitability of the combination of triphasic mixture (used in so called pesticides-POCIS) and octadecyl-functionalized silica gel (C18) as sorbent for POCIS was evaluated for the determination of alkylphenols (APs), several hormones, bisphenol-A (BPA), synthetic musk fragrances and herbicides such as trifluralin (Tri) and alachlor (Ala). With this purpose, POCIS laboratory calibration study, using a continuous-flow calibration system, was carried out in order to determine the uptake behavior and sampling rate (Rs) values for each target analyte. While the most hydrophobic compounds, (synthetic musk fragrances, some APs and Tri), showed poor linearity and low accumulation, a linear accumulation was observed for compounds whose logarithmic octanol-water partition coefficient (log Kow) ranged from 5.3 (4-tert-octylphenol, 4t-OP) to 3.1 (cis-androsterone, ADT). The Rs values obtained ranged from 0.190Lday-1 (4t-OP) to 0.042Lday-1 (BPA and equilin, EQ). The addition of C18 to the commonly used triphasic mixture increased the applicability of the POCIS sampler to compounds slightly more non-polar, such as 4t-OP. As far as we know, this is the first time that a combination of tetraphasic sorbent composed by the commercially available triphasic sorbent (Isolute ENV+polystyrene divinylbenzene and Ambersorb 1500 carbon dispersed on S-X3 Biobeads) and C18 was evaluated for passive sampling of the target analytes. The developed POCIS samplers were applied in field experiments from Halle (Germany) wastewater treatment plant (WWTP) effluent. Concerning the calculation of Rs values, the time weighted average (TWA) water concentration CWTWA values were determined considering three different approaches: (i) Rs from laboratory calibration (ii) Rs from laboratory calibration corrected with the use of performance reference compounds (PRCs) and (iii) Rs derived from field experiments or in-situ calibration. Several deuterated compounds such as, [2H3]-17ß-estradiol ([2H3]-E2), [2H4]-nonylphenol ([2H4]-NP), [2H4]-equilin ([2H4]-EQ), [2H3]-tonalide ([2H3]-AHTN) and [2H15]-musk xylene ([2H15]-MX) were also studied for their applicability as PRCs. Finally, a reasonable agreement between grab sampling and CWTWA was found when results from in-situ calibration were considered, but not when PRCs were used for correction.

Fast and simple determination of perfluorinated compounds and their potential precursors in different packaging materials.

A simple and fast analytical method for the determination of fourteen perfluorinated compounds (PFCs), including three perfluoroalkylsulfonates (PFSAs), seven perfluorocarboxylic acids (PFCAs), three perfluorophosphonic acids (PFPAs) and perfluorooctanesulfonamide (PFOSA) and ten potential precursors, including four polyfluoroalkyl phosphates (PAPs), four fluorotelomer saturated acids (FTCAs) and two fluorotelomer unsaturated acids (FTUCAs) in different packaging materials was developed in the present work. In order to achieve this objective the optimization of an ultrasonic probe-assisted extraction (UPAE) method was carried out before the analysis of the target compounds by liquid-chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). 7 mL of 1 % acetic acid in methanol and a 2.5-min single extraction cycle were sufficient for the extraction of all the target analytes. The optimized analytical method was validated in terms of recovery, precision and method detection limits (MDLs). Apparent recovery values after correction with the corresponding labeled standard were in the 69-103 % and 62-98 % range for samples fortified at 25 ng/g and 50 ng/g concentration levels, respectively and MDL values in the 0.6-2.2 ng/g range were obtained. The developed method was applied to the analysis of plastic (milk bottle, muffin cup, pre-cooked food wrapper and cup of coffee) and cardboard materials (microwave popcorn bag, greaseproof paper for French fries, cardboard box for pizza and cinema cardboard box for popcorn). To the best of our knowledge, this is the first method that describes the determination of fourteen PFCs and ten potential precursors in packaging materials. Moreover, 6:2 FTCA, 6:2 FTUCA and 5:3 FTCA analytes were detected for the first time in microwave popcorn bags.

Multiscreening determination of organic pollutants in molluscs using matrix solid phase dispersion.

This work describes the optimisation, validation and application of matrix solid-phase dispersion (MSPD) coupled to gas chromatography mass spectrometry, both single quadrupole (GC-MS) and tandem (GC-MS/MS), for the quantification in molluscs of up to 40 different analytes belonging to several families of priority and emerging organic contaminants, including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), organophosphorus pesticides (OPPs) and musk fragrances. The MSPD procedure was fully optimised with a special focus on the clean-up strategy. The best recoveries were obtained using glass syringes, 0.30 g of freeze-dried sample, 0.30 g of Florisil as solid support, 4.00 g of activated silica and 25 mL of dichloromethane as elution solvent. Using GC-MS/MS the method afforded good linearities (r(2), between 0.980 and 0.9996), adequate repeatability and reproducibility (RSD<17% and 33%, respectively) and low instrumental limits of detection (between 0.010 and 2.74 ng mL(-1)). The accuracy of the method was evaluated using different approaches, i.e. assessment of spiked fish hatchery samples, laboratory reference material and standard reference material (SRM 2977). Satisfactory apparent recoveries were obtained for all the target analytes after correction with the corresponding labelled surrogate, except for PAHs in the case of SRM 2977, which required the use of the standard addition method. Finally, MSPD was applied to mollusc species collected in Colombia and Nicaragua, where PAHs, PCBs, musks and pesticides were detected at low ng g(-1) levels.

Microextraction with polyethersulfone for bisphenol-A, alkylphenols and hormones determination in water samples by means of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry analysis.

In the present work, the suitability of polyethersulfone (PES) tube was assessed for the simultaneous sorptive microextraction of commonly found endocrine disrupting compounds in natural waters such as bisphenol-A (BPA), nonylphenol technical mixture (NP mix), 4-tert-octylphenol (4tOP), 4-n-octylphenol (4-nOP), 17ß-estradiol (E2) and 17α-ethynilestradiol (EE2). After the concentration of target compounds in the PES polymer, the analytes were recovered soaking the polymer with a suitable solvent (ethyl acetate or methanol), derivatized using N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane (BSTFA+1% TMCS) and determined by gas chromatography-mass spectrometry (GC-MS). The analysis was also performed without derivatization step by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction parameters (addition of MeOH, ionic strength, extraction speed and time and desorption time) were evaluated and the optimum conditions were fixed as follows: 150 mL water samples containing a 10% (w/v) of sodium chloride and using 5 tubular PES sorbent fibers (1.5 cm length×0.7 mm o.d.). Equilibrium conditions were achieved after 9 h, with absolute extraction efficiencies ranging from 27 to 56%. On the whole, good apparent recoveries were achieved (68-103% and 81-122% for GC-MS and LC-MS/MS, respectively) using deuterated analogues as surrogates. Achieved quantification limits (LOQs) varied between 2-154 ng/L and 2-63 ng/L for all the compounds using GC-MS and LC-MS/MS, respectively. The effect of organic matter was evaluated previous to apply the final method to the analysis of estuarine and wastewater real samples. The comparison of both methods showed that overall, PES-LC-MS/MS provided shorter sample preparation time and better LODs, but PES-silylation-GC-MS allowed the simultaneous determination of all the studied compounds with adequate repeatability and accuracy.

Matrix effect during the membrane-assisted solvent extraction coupled to liquid chromatography tandem mass spectrometry for the determination of a variety of endocrine disrupting compounds in wastewater.

Membrane-assisted solvent extraction (MASE) coupled to liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) was studied for the determination of a variety of emerging and priority compounds in wastewater. Among the target analytes studied certain hormones (estrone (E1), 17ß-estradiol (E2), androsterone (ADT), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES), equilin (EQ), testosterone (TT), mestranol (MeEE2), 19-norethisterone (NT), progesterone (PG) and equilenin (EQN)), alkylphenols (APs) (4-tert-octylphenol (4tOP), nonylphenol technical mixture (NPs) and 4n-octylphenol (4nOP)) and BPA were included. The work was primarily focused in the LC-MS/MS detection step, both in terms of variable optimization and with respect to the matrix effect study. Both, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were assessed both in the negative and positive mode, including the optimization of MS/MS operating conditions. The best results were obtained, in most of the cases, for ESI using 0.05% ammonium hydroxide as buffer solution in the mobile phase, composed with methanol and water. Under optimum detection conditions, matrix effect during the detection step was thoroughly studied. Dilution, correction with deuterated analogues and clean-up of the extracts were evaluated for matrix effect correction. Clean-up with Florisil together with correction with deuterated analogues provided the most satisfactory results, with apparent recoveries in the 57-136% range and method detection limits in the low ngL(-1) level for most of the analytes. For further validation of the method, two separated extraction procedures, the above mentioned MASE, and conventional solid phase extraction (SPE) were compared during the analysis of real samples and comparable results were successfully obtained for E1, E2, EE2, DES, NT, TT, EQ, PG, BPA, ADT, 4nOP, 4tOP, NPs and EQN.

Health status assessment through an integrative biomarker approach in mussels of different ages with a different history of exposure to the Prestige oil spill.

A battery of cell and tissue-level biomarkers was applied in mussels of 6 size-classes collected from Galicia and the Basque coast in summer 2007 in an attempt to examine the health status of individuals affected as adults (mature before 2003), affected during their developmental or juvenile stages (2003-2004 offspring), or not directly affected by the Prestige oil spill (POS) exposure (presumably 2005-2006 offspring). This battery of biomarkers was akin to those formerly applied on mussels of 3.5-4.5 cm shell length for which there exist biomarker reference values in the studied geographical areas. The cause-effect relationship between biological responses and the different history of exposure to POS fuel oil was intricate for different reasons: (a) growth rate was dissimilar in mussels of the two studied localities and much lower than expected, (b) a chronological basis could not be directly associated to POS events (all mussels except the smallest from Galicia had been subjected to the direct POS impact at one or another stage of their life-cycle); and (c) some biomarkers and histopathology seemingly depended on size/age irrespectively of the locality and the POS chronology. As a whole, the present study gives a very useful set of reference values of biomarkers obtained for Mytilus galloprovincialis of different size-classes. Finally, it is recommended that Mussel Watch programmes should be designed by standardising the age of the sentinel mussels rather than their size, especially if the programme covers large or diverse geographical areas, if long-term trends are relevant or if significant pollution effects on growth are expected.

Intersex condition and molecular markers of endocrine disruption in relation with burdens of emerging pollutants in thicklip grey mullets (Chelon labrosus) from Basque estuaries (South-East Bay of Biscay).

Endocrine disrupting chemicals (EDCs) interfere with the functioning of the endocrine system, causing reproductive and developmental disturbances in aquatic wildlife. Appearance of intersex gonads and elevated plasma levels of vitellogenin in male fish are well known biomarkers of exposure to xenoestrogenic EDCs. In the present study, intersex condition and transcription levels of vtg and cyp19a1b were assessed in five thicklip grey mullet populations from the Basque coast (Bay of Biscay). Levels of EDCs (estrogenic hormones, polycyclic musks, bisphenol-A, phthalates, alkylphenols and pesticides) were determined in water and fish bile. Intersex gonads were observed in three out of five mullet populations. Vtg and cyp19a1b were up-regulated in mullet populations with relatively higher EDCs load. Phthalates and pesticides were the most abundant EDCs in bile, followed by alkylphenols, musks, bisphenol-A and estrogenic hormones. Statistically significant correlations were found between concentrations of individual and total EDCs in bile and water samples and transcription levels of vtg and cyp19a1b.

Glutathione S-transferase, glutathione peroxidase and acetylcholinesterase activities in mussels transplanted to harbour areas.

As part of an integrative monitoring campaign involving water and sediment chemistry, in situ bioassays, and mussel bioaccumulation and biomarkers, Mytilus galloprovincialis mussels of standard size were transplanted from a clean location to five sites in two important harbours from the Atlantic coast of Spain (Vigo and Pasaia). After a 30-day field exposure, the concentrations of major contaminants (trace metals, polychlorinated biphenyls and polycyclic aromatic hydrocarbons) accumulated in mussel tissues were measured at each site, and a mussel bioaccumulation index (MBI) was calculated. The enzymatic activity levels of glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE) were quantified in the gills of transplanted mussels (n=12). Mussels from the most polluted sites consistently exhibited significantly higher GST and GPx activities compared to the control site, whereas AChE activity was significantly inhibited. The responses of the GST and GPx activities were related to MBI, trace metals and PAH concentrations in mussels, whereas AChE activity was related to the trace metals concentrations in mussels. The above results suggest that GST and AChE activities can be used as potential biomarkers for active monitoring in marine coastal ecosystems. However, at this moment, GPx activity is not robust enough to be applicable to harbour areas.

Determination of polycyclic and nitro musks in environmental water samples by means of microextraction by packed sorbents coupled to large volume injection-gas chromatography-mass spectrometry analysis.

In this work the development and validation of a new procedure for the simultaneous determination of 9 nitro and polycyclic musk compounds: musk ambrette (MA), musk ketone (MK), musk mosken (MM), celestolide (ADBI), phantolide (AHMI), tonalide (AHTN), traseolide (ATII), cashmeran (DPMI) and galaxolide (HHCB) in environmental water samples (estuarine and wastewater) using microextraction by packed sorbent (MEPS) followed by large volume injection-gas chromatography-mass spectrometry (LVI-GC-MS) was carried out. Apart from the optimization of the different variables affecting MEPS (i.e., nature of the sorbent, nature of the solvent elution, sample load, and elution/injection volume) extraction recovery was also evaluated, not only for water samples but also for environmental water matrices such as estuarine and waste water. The use of two deuterated analogs ([(2)H3]-AHTN and [(2)H15]-MX) was successfully evaluated in order to correct matrix effect in complex environmental matrices such as influent samples from wastewater treatment plants. Method detection limits (MDLs) ranged from 5 to 25 ng L(-1), 7 to 39 ng L(-1) and 8 to 84 ng L(-1) for influent, effluent and estuarine samples, respectively. Apparent recoveries were higher than 75% for all target compounds in all the matrices studied (estuarine water and wastewater) and the precision of the method, calculated as relative standard deviation (RSD), was below 13.2% at 200 ng L(-1) concentration level and below 14.9% at low level (20 ng L(-1) for all the target analytes, except for AHTN which was set at 40 ng L(-1) and HHCB at 90 ng L(-1), due to the higher MDL values presented by those target compounds). Finally, this MEPS procedure was applied to the determination of the target analytes in water samples, including estuarine and wastewater, from two estuaries, Urdaibai (Spain) and Adour (France) and an established stir-bar sorptive extraction-liquid desorption/large volume injection-gas chromatography-mass spectrometry (SBSE-LD/LVI-GC-MS) method was performed in parallel for comparison. Results were in good agreement for all the analytes determined, except for DPMI.

Application of Toxicity Identification Evaluation (TIE) procedures for the characterization and management of dredged harbor sediments.

This study refers to the performance of Phase I Toxicity Identification Evaluation (TIE) procedures to identify the contaminants (i.e. organic compounds, metals and ammonia) exerting toxicity in marine sediments from the Pasaia harbor (Oiartzun estuary, northern Spain). The effectiveness of the manipulations to reduce toxicity was proved with the marine amphipod survival test (whole-sediment) and the sea urchin embryo-larval assay (elutriates). By means of TIEs it was concluded that organic compounds were the major contaminants exerting toxicity, although toxic effects by metals was also demonstrated. Additionally, the combination of Phase I treatments allowed to investigate the toxicity changes associated to the mobility of contaminants during dredging activities. Therefore, the performance of TIE procedures as another line of evidence in the decision-making process is recommended. They show a great potential to be implemented at different steps of the characterization and management of dredged harbor sediments.

Calibration and field test of the Polar Organic Chemical Integrative Samplers for the determination of 15 endocrine disrupting compounds in wastewater and river water with special focus on performance reference compounds (PRC).

In this work, home-made Polar Organic Chemical Integrative Samplers (POCIS) were studied for passive sampling of 15 endocrine disrupting compounds (4 alkylphenols and steroid hormones) in influent and effluent samples of wastewater treatment plants (WWTPs) as well as up- and downstream of the receiving river water. POCIS calibration at laboratory conditions was carried out using a continuous-flow calibration system. The influence of the exposure position of the POCIS within the calibration device, horizontal or vertical, to the water flow direction was evaluated. While the sampling rates of most of the target substances were not affected by the sampler position, for cis-ADT, E1, E2 and E3, the vertical position provided the highest analyte accumulation. Hence, the POCIS samplers were preferably exposed vertical to the water flow in overall experiments. Using the continuous-flow calibration device, lab-based sampling rates were determined for all the target compounds (RSBPA = 0.0326 L/d; RScisADT = 0.0800 L/d, RSE1 = 0.0398 L/d, RSEQ = 0.0516 L/d, RSTT = 0.0745 L/d, RSE2 = 0.0585 L/d, RSEE2 = 0.0406 L/d, RSNT = 0.0846 L/d, RSPG = 0.0478 L/d and RSE3 = 0.1468 L/d), except for DES, MeEE2, 4tOP, 4OP, 4NPs, where the uptake after 14 days POCIS exposure was found to be insignificant or indicated a no linear behaviour. Recoveries from POCIS extractions were in the range between 71 and 152% for most of the target analytes except for DES and E3 with around 59%. Good precision of the sampling procedure up till 20% was observed and limits of detection were at ng/L level. Two deuterated compounds ([(2)H3]-E2 and [(2)H4]-EQ) were successfully tested as performance reference compounds (PRC, [Formula: see text] = 0.0507 L/d and [Formula: see text] = 0.0543 L/d)). Finally, the POCIS samplers were tested for monitoring EDCs at two wastewater treatment plants, in Halle and Leipzig (Germany). BPA, E1, EQ, E2, MeEE2, NT, EE2, PG and E3 were quantified and their time-weighted average concentrations calculated on the basis of the lab-derived sampling rates were compared with the results based on conventional grab samples. While the influent concentration of BPA, cisADT, E1, TT, PG, EE2 reached the µg/L level, the rest of the target analytes were determined at ng/L. The analyte concentrations in the effluent never exceed ng/L level except for BPA. The concentration determined by spot sampling was partially lower (BPA, E1, TT) or comparable (EQ, E2, EE2, PG, E3) to the concentration obtained by POCIS using performance reference compounds (PRC).

Integrative sediment assessment at Atlantic Spanish harbours by means of chemical and ecotoxicological tools.

This study refers to the integrative assessment of sediment quality in three harbour areas at the Spanish Atlantic Coast: Vigo (Northwestern Spain), Bilbao and Pasajes (Northern Spain). At each site, two lines of evidence have been considered: chemical analyses (metal, PAH and PCB concentrations in sediments and ammonia concentration in bioassays) and toxicity tests (Microtox®, Corophium sp. marine amphipod and Paracentrotus lividus sea urchin larvae). Chemical and ecotoxicological results have been integrated by means of a tabular matrix and a multivariate factorial analysis (FA). Highly toxic samples have been characterised in Vigo and Pasajes harbours while Bilbao samples present toxicity levels ranging from non-toxic to moderately toxic. High toxicity is associated with high levels of contaminants whereas confounding factors (ammonia, organic matter and mud) have been identified to be the main cause of low to moderate toxicity. Based on the obtained results, it can be concluded that deriving potential toxicity of sediments based on comparison with Sediment Quality Guidelines (SQGs) is in agreement to toxicity results in areas presenting high levels of contaminants. However, at lower levels of toxicity (low to moderate), the mismatch between the potential toxicity (SQG approach) and the toxicity measured by bioassays is greater, as the former only accounts for chemical concentrations, without considering the interaction between contaminants and the effect of confounding factors. Contrarily, the multivariate analysis seems to be a robust tool for the integration and interpretation of different lines of evidence in areas affected by different sources of contamination.