Occurrence of regulated pollutants in populated Mediterranean basins: Ecotoxicological risk and effects on biological quality.

Chemical stressors co-occur in mixtures into watercourses and this complicates predicting their effects on their ecological status. Our knowledge of river basin specific pollutants (RBSPs) is still limited, but it remains necessary to ensure the good chemical and ecological status. We performed an exercise on Mediterranean river sites exposed to urban and industrial pressures in order to, i) prioritize the occurring chemicals, ii) assessing the site's specific chemical risk (RQsite), and iii) relating the chemical risk to the biological quality, using as evidences invertebrates and diatom indices. Mediterranean rivers suffer from strong pressures which lead to a poor dilution ability, which makes the inhabiting biota highly vulnerable. The most frequent pollutants in the 89 sites surveyed included pharmaceutical products such as the antibiotics azithromycin, clarithromycin, and erythromycin, and the anti-inflammatory diclofenac, and products of industrial origin such as perfluorinated PFOS, nickel, and nonylphenol. Both the diatom index IPS and the macroinvertebrate index IBMWP hold strong negative correlations to RQsite, indicating a significant contribution of chemicals to biological impairment. Chemical contaminants (but not nutrients or dissolved organic carbon) were associated with significant changes to the taxonomic composition of invertebrate communities, but not to that of diatom communities. Our analyses indeed reveal that the impact of co-occurring chemicals translates onto negative effects in the biological quality. Our approach may be of use to evidence impacts on water resources and water quality in rivers under strong human pressure.

Long-term continuous treatment of non-sterile real hospital wastewater by Trametes versicolor.

BACKGROUND: Hospital wastewater is commonly polluted with high loads of pharmaceutically active compounds, which pass through wastewater treatment plants (WWTPs) and end up in water bodies, posing ecological and health risks. White-rot fungal treatments can cope with the elimination of a wide variety of micropollutants while remaining ecologically and economically attractive. Unfortunately, bacterial contamination has impeded so far a successful implementation of fungal treatment for real applications. RESULTS: This work embodied a 91-day long-term robust continuous fungal operation treating real non-sterile hospital wastewater in an air pulsed fluidized bed bioreactor retaining the biomass. The hydraulic retention time was 3 days and the ageing of the biomass was avoided through partial periodic biomass renovation resulting in a cellular retention time of 21 days. Evolution of microbial community and Trametes abundance were evaluated. CONCLUSIONS: The operation was able to maintain an average pharmaceutical load removal of over 70% while keeping the white-rot fungus active and predominant through the operation.

Fungal treatment of metoprolol and its recalcitrant metabolite metoprolol acid in hospital wastewater: Biotransformation, sorption and ecotoxicological impact.

Hospital wastewater (HWW) effluents represent an important source of contaminants such as pharmaceutical compounds and their human metabolites. To better evaluate dedicated treatment of hospital effluents for pollutant mitigation, not only the parent compounds should be considered but also the intermediates generated during treatment. The metabolite metoprolol acid (MTPA) has been found in urban wastewaters at higher concentration than its parent compound metoprolol (MTP), being more recalcitrant to biodegradation. The aim of this study was to investigate degradation, transformation and sorption of the ß-blocker MTP, and its recalcitrant metabolite MTPA, during water treatment based on the fungi Ganoderma lucidum, Trametes versicolor and Pleurotus ostreatus. Fourteen intermediates were identified in MTP biotransformation while five of them also attributed to MTPA biodegradation and two to MTPA only. Their identification allowed their correlation in separate biotransformation pathways suggested. The highest degradation rate of metoprolol (up to 51%) and metoprolol acid (almost 77%) was found after 15-days treatment with Ganoderma lucidum, with an increase in toxicity up to 29% and 4%, respectively. This fungus was further selected for treating real HWW in a batch fluidized bed bioreactor (FBB). Treated wastewater and fungal biomass samples were used to evaluate the distribution of the target compounds and the intermediates identified between solid and liquid phases. While similar elimination capabilities were observed for the removal of metoprolol, and even higher for its persistent metabolite metoprolol acid, the extent on compound transformation diminished considerably compared with the study treating purified water: a high level of the persistent α-HMTP and TP240 were still present in effluent samples (15% and 6%, respectively), being both TPs present at high proportion (up to 28%) in fungal biomass. This is the first time that pharmaceutical TPs have been investigated in the fungal biomass.

Perfluoroalkyl phosphonic acids adsorption behaviour and removal by wastewater organisms.

In this study we have experimentally assessed different physicochemical parameters such as the distribution constant between octanol - water and between water and sludge for three perfluoroakyl substances (PFASs) widely used in waxes and coating materials: perfluorohexane (PFHxPA), perfluorooctane (PFOPA) and perfluorodecane (PFDPA) phosphonic acids. Distribution coefficients were assessed based on the procedures described in the OECD guideline 123 for partition coefficients while the studies of adsorption-desorption in sludge based on the indirect method of the OECD guideline 106. Besides, the removal behaviour of selected compounds has been evaluated using the green algae Desmodesmus subspicatus and microorganisms present in an effluent wastewater. These last experiments were carried out using laboratory scale bioreactors under aerobic conditions according to the OECD guideline 309. The main results of this study showed the resistance to biodegradation of selected compounds by both treatments, <5% was eliminated using D. subspicatus and similar results were obtained by aerobic degradation with wastewater microorganisms. However, it was observed that PFDPA induced changes in algae colour while it was detected to be accumulated in a floccula generated by the microorganisms present in wastewater. According to distribution coefficients the three compounds have values of logDow below 3, indicating their capability to be present in both phases. Finally, the results of the adsorption/desorption experiments showed that PFOPA and PFDPA reach the equilibrium after 10 days of contact with a sorption percentage higher than 40% and 70%, respectively.

Stropharia rugosoannulata and Gymnopilus luteofolius: Promising fungal species for pharmaceutical biodegradation in contaminated water.

Pharmaceuticals are environmental micropollutants that pose an emerging challenge because they are poorly eliminated in conventional wastewater treatment plants. Over the last decade, many attempts have been made to solve this problem, and wastewater fungal treatment is a promising alternative. In this study, six different ligninolytic fungi (Trametes versicolor, Ganoderma lucidum, Irpex lacteus, Stropharia rugosoannulata, Gymnopilus luteofolius and Agrocybe erebia) were studied as bioremediation candidates for the removal and degradation of six recalcitrant pharmaceutical micropollutants: Carbamazepine (CBZ), Venlafaxine (VFX), Iopromide (IPD), Diclofenac (DCF), Cyclophosphamide (CFD) and Ifosfamide (IFD). Self-immobilization in a pellet shape was achieved for all fungal mycelia (which was the first time that this was reported for S. rugosoannulata, G. luteofolius, and A. erebia). Biodegradation achievement was greater than 90% for IPD with G. luteofolius and greater than 70% for CBZ with S. rugosoannulata, which suggests a great potential for this alternative biological treatment. Besides, this was the first report where fungal treatment achieved CFD and IFD removals greater than 20% for the treatment with T. versicolor, G. lucidum and S. rugosoannulata.

Contribution of bacteriophage and plasmid DNA to the mobilization of antibiotic resistance genes in a river receiving treated wastewater discharges.

In this study, we quantified eleven antibiotic compounds and nine antibiotic resistance genes (ARGs) in water samples collected upstream and downstream of the discharge point from a municipal wastewater treatment plant (WWTP) into the Ter River. Antibiotics were analyzed by liquid chromatography coupled to mass spectrometry, whereas the concentration of ARGs in bacterial, phage and plasmid DNA fractions was determined by real-time PCR to explore their contribution to environmental antibiotic resistance. WWTP discharges resulted in higher concentrations of antibiotic residues as well as ARGs in water samples collected downstream the impact point. Specifically, genes conferring resistance to macrolides (ermB), fluoroquinolones (qnrS) and tetracyclines (tetW) showed significant differences (p<0.05) between upstream and downstream sites in the three DNA fractions (i.e. bacteria, plasmids and phages). Interestingly, genes conferring resistance to ß-lactams (blaTEM, blaNDM and blaKPC) and glycopeptides (vanA) only showed significant differences (p<0.05) between upstream and downstream sites in phage and plasmid DNA but not in the bacterial DNA fraction. Our results show for the first time the extent to which phages and plasmids contribute to the mobilization of ARGs in an aquatic environment exposed to chronic antibiotic pollution via WWTP discharges. Accordingly, these mobile genetic elements should be included in further studies to get a global view of the spread of antibiotic resistance.

Occurrence of pharmaceuticals and UV filters in swimming pools and spas.

The occurrence of 32 pharmaceuticals and 14 UV filters in swimming pools and spas was studied. Fifty-one water samples were collected from 17 pools located in sport centres and hotels in Catalonia, Spain. The samples were analysed by liquid chromatography-tandem mass spectrometry. The pharmaceuticals atenolol, carbamazepine, hydrochlorothiazide, metronidazole, ofloxacin, sulfamethoxazole, acetaminophen, ibuprofen, ketoprofen and phenazone were measured in water samples at concentrations higher than their limit of quantification (LOQ). The highest concentration of any individual pharmaceutical was measured for the diuretic hydrochlorothiazide (904 ng/L). The most frequently detected pharmaceutical was carbamazepine, as it was observed in more than half of all the water samples measured (53 %, 27/51). The UV filters at concentrations higher than LOQ in water samples were BP1, BP2, BP3, BP8, THB, 4DHB, 4MBC, OD-PABA, 1HBT, MeBT and DMeBT. The highest concentration of UV filter observed was 4MBC (69.3 ng/L) while the most frequent UV filters in the samples were 1HBT (59 %, 30/51). The results also showed that pharmaceuticals and UV filters were most frequently found in spas. Finally, from a water treatment technology perspective, the lowest occurrence of pharmaceuticals was in the pools applying sand filters followed by disinfection by sodium hypochlorite, while the lowest occurrence of UV filters was in the pools applying coagulation, sand filtration, UV and salt electrolysis.

Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact.

Reports on pesticides elimination during wastewater treatment are rare since these substances are typically considered of agricultural rather than of urban origin. In this context, the aim of this work was to evaluate the presence, removal and environmental relevance of 22 selected pesticides in three different wastewater treatment plants (WWTPs), paying attention not only to their occurrence and elimination but also to the toxicity of each pesticide against three aquatic micro organisms (algae, daphnia and fish) through the calculation of the so-named Environmental Relevance of Pesticides from Wastewater treatment plants Index (ERPWI). For this purpose, an analytical method based on isotope dilution on-line solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was optimized, allowing the determination of the 22 target pesticides in wastewater with satisfactory sensitivity (limits of detection below 30 ng/L), accuracy and precision. Concerning the results, total pesticide levels were in most instances below 1 µg/L but removal in the WWTPs was variable and often poor, with concentrations in the effluent sometimes higher than in the corresponding influent. Possible explanations for these poor or negative removal rates are, among many others considered (e.g. sampling, sample preservation, method biases, atmospheric deposition), deconjugation of metabolites and/or transformation products of the pesticides, hydrolysis, and desorption from particulate matter during wastewater treatment. The most significant pesticides in terms of concentration and frequency of detection were diazinon and diuron. These two pesticides, followed by atrazine, simazine and malathion, were also the most relevant from the environmental point of view, according to the calculated ERPWI.

Endocrine disruption in thicklip grey mullet (Chelon labrosus) from the Urdaibai Biosphere Reserve (Bay of Biscay, Southwestern Europe).

Endocrine disruptors (EDs) interfere with the development and functioning of the endocrine system, causing reproductive disturbance in aquatic wildlife. The aim of the present work was to determine the presence of EDs in sediments and to investigate possible exposure and effects of EDs in the estuary of the Urdaibai Biosphere Reserve (Gernika) in comparison with the Arriluze marina. For this, gonad histology, plasma vitellogenin (VTG) protein levels and mRNA levels of vitellogenin (vtg), cyp19 aromatases, estrogen receptor (er) and retinoid X receptor (rxr) were studied in Chelon labrosus. The presence of alkylphenols (APs) in fish bile was also assessed. In sediments, estrogenic hormones were below the detection limit and levels of bisphenol A were very low. In Gernika organotin compounds were low but in Arriluze levels of up to 12 µg/g were found. Moderate levels of APs and phthalate levels of up to 8 µg/g were found in sediments. In fish, a high prevalence up to 33% of intersex gonads was found in Gernika, whereas only one intersex was found in Arriluze. Accordingly, mullets from Gernika showed higher concentrations of APs in bile. VTG protein levels were detected not only in females but also in some undifferentiated, male and intersex fish. mRNA of vtg was detected in one male from Gernika. mRNA of er and rxr showed significant differences between seasons. In conclusion, the present study demonstrated that C. labrosus from the Urdaibai estuary were exposed to EDs and showed clear signs of endocrine disruption.

Chemometric evaluation of different experimental conditions on wheat (Triticum aestivum L.) development using liquid chromatography mass spectrometry (LC-MS) profiles of benzoxazinone derivatives.

Different chemometric techniques have been used to evaluate the effect of distinct experimental conditions and factors on Triticum aestivum L. plant development. The study was conducted using three wheat varieties, Astron, Ritmo and Stakado. These varieties were grown under organic and conventional cultivation systems. Samples were collected at five growth stages. Shoots and roots of each plant at these stages were analysed. Three replicates of each analysed sample were performed to improve representativeness and to allow for the evaluation of natural variability and interaction effects. All samples were analysed using Liquid Chromatography Mass-Spectrometry (LC-MS), and the Total Ion Current (TIC) profiles of benzoxazinone derivatives obtained for each sample were investigated. Qualitative and quantitative assessments of these TIC profiles and of their changes in the analysed samples were carried out using different chemometric techniques. Estimation of main effects, and of their possible interaction, was performed by means of Analysis of Variance combined to Principal Component Analysis (ANOVA-PCA) and of Analysis of Variance combined to Simultaneous Component Analysis (ASCA).

LC-QqLIT MS analysis of nine sulfonamides and one of their acetylated metabolites in the Llobregat River basin. Quantitative determination and qualitative evaluation by IDA experiments.

Robust instrumental analytical chemistry and the subsequent development of improved analytical methodologies and extraction procedures have enabled the detection at environmental levels of new emerging contaminants, for example pharmaceuticals. The objective of this study was to explore the potential of liquid chromatography-tandem quadrupole-linear ion trap (LC-QqLIT MS) for quantitative determination of nine sulfonamides (SAs) and one acetylated metabolite in surface water from the Llobregat River and one of its main tributaries, the Anoia River, in Catalonia (Spain). A total of 21 samples were taken in three sampling campaigns. Recoveries ranging from 26% to 123% were calculated for the SAs studied, and method limits of detection (MLODs) achieved were in the range 0.05-0.2 ng L(-1). Through the different campaigns, concentrations ranged from 0.74 ng L(-1) (sulfamethizole) to 2,482 ng L(-1) (sulfamethazine) in the Llobregat River, and from 0.27 ng L(-1) (sulfamethizole) to 168 ng L(-1) (sulfamethoxazole) in the Anoia River. Sulfamethoxazole and sulfapyridine were the two SAs most frequently detected (80% and 71% respectively). N(4)-acetylsulfamethazine was detected in both rivers, but with different frequencies (4% of the samples from the Anoia River and 43% of those from the Llobregat River). Information-dependent acquisition (IDA) experiments were also developed in order to obtain enhanced product-ion spectra in surface water samples.

Analysis and occurrence of alkylphenolic compounds and estrogens in a European river basin and an evaluation of their importance as priority pollutants.

As a part of a project aiming to assess the potential toxicological effects of contaminants in aquatic ecosystems, the objective of this work was to determine the occurrence of several selected endocrine-disrupting compounds in water and sediment and to estimate the estrogenicity of the water. The study consisted of four sampling campaigns at seven sampling points in the lower Llobregat catchment area (NE Spain). Water and sediment samples underwent chemical target analysis for 19 steroid estrogens and alkylphenols, which are known to be endocrine-disrupting compounds. In this study, the only estrogens detected in the water samples were estrone and estrone-sulfate, which were found at low levels (2-5 ng l(-1)). The alkylphenolic compound showing the highest concentrations was nonylphenol di-ether carboxylate (NP2EC), which was found at levels up to 30.62 microg l(-1) in water samples and 535 ng g(-1) in sediment samples. K (d) was determined for several alkylphenolic compounds and showed the expected trend of decreasing K (d) with increasing polarity. The concentrations of nonylphenol and octylphenol only exceeded the annual average of the European Union's environmental quality standards (EQS) in one sampling point. However, the calculated estrogenic potential surpassed the expected effect concentration in several sampling points, indicating a potential risk. Therefore, we recommend that future EQS include short-chain alkylphenol ethoxylates and carboxylates.

Bridging levels of pharmaceuticals in river water with biological community structure in the Llobregat River basin (northeast Spain).

A wide range of human pharmaceuticals are present at low concentrations in freshwater systems, particularly in sections of polluted river. These compounds show high biological activity, often associated with a high stability. These characteristics imply a potential impact of these substances on aquatic biota even when present at low environmental concentrations. Low flow conditions in Mediterranean rivers, most of which flow through densely populated areas and are subjected to intensive water use, increase the environmental risk of these emergent compounds. Here, we studied whether pharmaceuticals in river water affect the local benthic community structure (diatoms and invertebrates). For this purpose, we analyzed the occurrence of pharmaceuticals along the Llobregat River and examined the benthic community structure (diatoms and invertebrates) of this system. Some pharmaceutical products in the Llobregat River registered concentrations greater than those cited in the literature. Multivariate analyses revealed a potential causal association between the concentrations of some anti-inflammatories and beta-blockers and the abundance and biomass of several benthic invertebrates (Chironomus spp. and Tubifex tubifex). Further interpretation in terms of cause-and-effect relationships is discussed; however, it must be always taken with caution because other pollutants also may have significant contributions. Combined with further community experiments in the laboratory, our approach could be a desirable way to proceed in future risk management decisions.

Effects of low concentrations of the phenylurea herbicide diuron on biofilm algae and bacteria.

A system of recirculating channels was used in this study to examine the long-term effects (29d) of environmentally realistic concentrations of the herbicide diuron (from 0.07 to 7 microg L(-1)) on biofilm communities. The autotrophic activity of biofilms was affected by this herbicide, as reflected by a marked decrease in the photosynthetic efficiency. Diuron exposure also increased chlorophyll-a content and reduced the biovolume of diatom taxa at low concentrations. The effects on bacteria were also remarkable. Bacterial abundance was reduced after a week of exposure to the herbicide at a range of concentrations. Effects were on the number of live bacteria and on the increase in the leucine-aminopeptidase activity. It is suggested that inputs of herbicides to the river ecosystem at low concentrations may cause a chain of effects in the biofilm, which include inhibitory effects on algae but also indirect effects on the relationships between biofilm components.

Toward an integrated assessment of the ecological and chemical status of European river basins.

Here, recommendations to improve ecological and chemical status assessments in accordance with the European Water Framework Directive (WFD) are made on the basis of experience gained from the MODELKEY project database, linking existing biological and chemical monitoring data of 3 case study river basins (Elbe, Scheldt, and Llobregat). The data analysis within and across river basins revealed major obstacles to be tackled, including scarcity of matching ecological and chemical monitoring sites for cause-effect relationships as well as a general lack of stressor-specific metrics for single biological quality elements (BQE) to enable a comprehensive risk assessment of all predominant stressors, including toxicity. An example of such a metric, which is recommended for the BQE of benthic macroinvertebrates, is the trait-based species-at-risk index (SPEAR) that correlated well with a respective measure for toxic stress, referred to as toxic units, based on simple mixture toxicity concepts. Surprisingly, the assessment of chemical status of a total of 695 monitoring sites for 2000 to 2004 showed that environmental quality standards (EQSs) were exceeded for at least 1 of the currently 41 priority pollutants (PPs) in 92% to 98% of the cases in all 3 of the river basins, which, according to definition, indicates potential effects on ecological status. A comparison of compliance with EQSs for 41 PPs with a respective effect threshold (derived for benthic macroinvertebrates) revealed that the rather conservative concept of chemical status is most likely not protective in all cases. Furthermore, to account for the many other compounds that are detected frequently in European surface waters and that may also have ecotoxicological effects, we introduced a provisional predicted no-effect concentration that is in accordance with the EQS methodology and is suggested to identify potential emerging compounds for which no or insufficient toxicity data exist. In conclusion, this study aims to support the implementation of the WFD by drawing conclusions from the analysis of heterogeneous data sets of various member states and by introducing new tools to move toward an integrated European assessment of ecological and chemical status.

Environmental analysis of fluorinated alkyl substances by liquid chromatography-(tandem) mass spectrometry: a review.

Fluorinated alkyl substances (FASs) are widely distributed contaminants that have been found in many environmental, human and biological samples throughout the world. Perfluorochemicals are used in many industry and consumer products, such as polymers and surfactants, because they have unique and useful properties (they are stable, chemically inert and generally unreactive). However, these compounds have also been found to be toxic, persistent and bioaccumulative. In recent years various analytical methods have been developed for the analysis of FASs in environmental samples. Most of these methods are based on liquid chromatography coupled to mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS), since this is considered to be the technique of choice. This article reviews the various LC-(tandem)MS methods described so far for the analysis of FASs in water, sediment, sludge and biota samples. It discusses the main experimental conditions used for sample pretreatment and for analysis as well as the most relevant problems encountered and the limits of detection achieved.

Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry.

A new analytical method based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction with LiChrolut RP C18 cartridges was evaluated for the sample preparation, extraction, and cleanup of eight naturally occurring benzoxazinone derivatives, 2-beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one, 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one, benzoxazolin-2-one, and 6-methoxybenzoxazolin-2-one in plant samples. Afterward, liquid chromatography-electrospray mass spectrometry, using the selected ion monitoring mode and internal standard (2-MeO-DIBOA, indoxyl-beta-D-glucoside, and quercetin-3-O-rutinoside) quantification method was performed. This paper demonstrates the effectiveness of the PLE method, in conjunction with sensitive and specific mass spectrometric detection, for the quantitative recovery of compounds of the benzoxazinone class from plants. The recoveries of the analytes ranged from 66 to 110% with coefficients of variation ranging from 1 to 14%. This method gave detection limits between 1 and 27 microg/g. The method was applied to foliage and roots of three different wheat cultivars, and the analytes were detected in the range of 11-3261 microg/g of dry weight.

Benzoxazinoid allelochemicals in wheat: distribution among foliage, roots, and seeds.

In this study, the distribution of eight allelochemicals among the foliage, roots, and seeds of different wheat varieties is reported for two different sampling campaigns, corresponding to two consecutive years. The determination of benzoxazinoid derivatives was performed by combining pressurized liquid extraction--solid-phase extraction followed by liquid chromatography--electrospray mass spectrometry. To the authors' knowledge, there are no previous works about the content of allelochemicals in seed tissue of germinated wheat seedlings. Allelochemicals found in seeds were detected at levels similar to those found in foliage and roots. The results showed that the type of metabolites detected depends strongly on the working up procedure of the plant material, as well as of plant growth stage. A general decrease of the total amount of allelochemical content in the plants was observed with plant age. There was a significant difference in the total amount of benzoxazinoid derivatives in the different wheat varieties analyzed.