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.

Liquid chromatography-tandem mass spectrometric analysis and regulatory issues of polar pesticides in natural and treated waters.

Pesticides are among the most detected contaminants in the aquatic environment. This is mainly due to their use in agriculture and their physico-chemical properties that enable transportation and a persistent or pseudo-persistent existence in the water media. Several directives and guidelines set maximum levels of pesticides in water in order to protect the human and environmental health. A brief discussion of the existing directives and guidelines concerning pesticides in water is presented, e.g., the new regulatory framework for the Registration, Evaluation and Authorisation of Chemicals (REACH), and the Directive 91/414/EEC concerning the placing of plant protection products on the market. Up-to-date analytical tools to support the REACH program are of prime importance to ensure its complete implementation. Since liquid chromatography (LC) coupled to mass spectrometry (MS) is considered the most appropriate technique for determination of most modern pesticides in environmental waters, the most recent developments and applications in this field are discussed in detail in this review.

Chemical and biological analysis of endocrine-disrupting hormones and estrogenic activity in an advanced sewage treatment plant.

The steroid hormones estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)), 17alpha-ethinylestradiol (EE(2)), and their conjugated forms were surveyed throughout an advanced sewage treatment plant (STP). The estrogen concentrations in water and sludge samples, collected in October 2004 and April 2005, were determined by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Simultaneously, the estrogenic activity was quantified using estrogen-responsive reporter cell lines (MELN) to investigate the behavior of overall estrogenic compounds. The estrogen concentrations in the inlet ranged from 200 to 500 ng/L, with the contribution of conjugated forms being higher than 50%. The major estrogens in influent were E(1) and E(3). The estrogenic activity was between 25 and 130 ng/L of E(2) equivalents (EEQs). Estrogen concentrations and estrogenicity measured in the inlet and in primary treated sewage were similar, showing a weak impact of primary treatment on hormone removal. In contrast, both estrogen concentration and estrogenicity decreased during biological treatment, with high removal efficiencies (>90%). Estrone, E(2), and EE(2) persisted in the treated water below 10 ng/L, whereas the estrogenicity was lower than 5 ng/L of EEQs. Estrogen mass flux in the effluent and sludge represented less than 2 and 4%, respectively, of the inlet. Consequently, the fraction of estrogens sorbed into the sludge was very small, and biodegradation was the main vehicle for estrogen elimination. This dual approach, comparing chemical and biological analysis, allowed us to confirm that most of the estrogenic activity occurring in this STP, which receives mainly domestic sewage, resulted from sex hormones.

Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples.

Estrogens have been often identified as the major contributors to the endocrine-disrupting activity observed in environmental waters. However, their analysis in these, sometimes very complex, matrices is still challenging due to the very low detection limits and the selectivity required for their reliable determination at the very low concentrations at which they are physiologically active. In this work, a polyclonal enzyme-linked immunosorbent assay (ELISA) kit for 17-beta-estradiol analysis, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based on triple-quadrupole analyzer (QqQ), and a newly developed method based on ultra performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS) have been evaluated in terms of performance for the rapid screening, quantitative analysis, and unequivocal identification of some selected, environmentally relevant estrogens in different water matrices, including urban wastewater, river water, and ground water, after solid phase extraction. Compounds quantified and/or identified included the estrogens 17-beta-estradiol, estrone, 17-alpha-ethynyl estradiol and estriol, and the isoflavones daidzein, genistein, and biochanin A. Except for a moderate overestimation using the ELISA kit, especially in the analysis of complex wastewater samples, results obtained by all the investigated techniques were in very good, general agreement. The instrumental sensitivity achieved increased in the order: UPLC-Q-TOF-MS < polyclonal ELISA kit < HPLC-MS/MS (QqQ). Direct analysis of water samples by using the ELISA kit permitted to reach a limit of detection of 2.5 ng L(-1). However, using an appropriated sample pretreatment method detection limits at nanogram to picogram per liter levels can be obtained with all techniques and the risk for matrix effects is minimized. In terms of selectivity, both HPLC-MS/MS (QqQ) and UPLC-Q-TOF-MS show outstanding performance, but the latter allows, in addition, shorter analysis times (16 min vs. 45 min) and the identification of non-target, unknown compounds. The identification of unknown compounds is based on the accurate mass measurements for the precursor and product ions, that permit the elemental compositions calculation and the chemical structures to be identified searching against different databases.

Combined use of biomarkers and in situ bioassays in Daphnia magna to monitor environmental hazards of pesticides in the field.

The aim of this investigation was to evaluate toxicity effects of pesticides in aquatic invertebrates using in situ bioassays with the local species, Daphnia magna. Investigations were carried out in the Delta del Ebro (northeast Spain) during the main growing season of rice (May-August). Measures of energy consumption (i.e., algal grazing) and of specific biochemical responses (biomarkers) were conducted in individuals transplanted at four stations, including a clean site upstream of the affected area and the three main channels that collect and drain the water from the rice fields into the sea. Seventeen pesticides were analyzed in water by on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry. The results obtained indicated high levels of pesticides in water, with peak values of 487 microg/L for bentazone, 8 microg/L for methyl-4-chlorophenoxyacetic acid, 5 microg/L for propanil, 0.8 microg/L for molinate, and 0.7 microg/L for fenitrothion. Measured biological responses denoted severe effects on grazing rates; a strong inhibition of cholinesterases and carboxylesterases, which are specific biomarkers of organophosphorous and carbamate pesticides; and altered patterns of the antioxidant enzyme catalase and the phase II metabolizing enzyme glutathione S-transferase. Correlation analysis with pesticide residue levels converted to toxic units relative to its acute 48-h median lethal concentration of D. magna indicated significant and negative coefficients between the dominant pesticide residues and the observed biological response, thus denoting a clear cause-and-effect relationship. The results emphasize the importance of considering specific (biomarkers) as well as more generalized and ecologically related (grazing) in situ responses to identify and evaluate biological effects of environmental contaminants in the field.

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.

Fate of selected pesticides, estrogens, progestogens and volatile organic compounds during artificial aquifer recharge using surface waters.

The artificial recharge of aquifers has become a valuable tool to increase water resources for drinking water production in many countries. In this work a total of 41 organic pollutants belonging to the classes of pesticides, estrogens, progestogens and volatile organic compounds (VOCs) have been monitored in the water from two artificial recharge plants located in Sweden and Denmark. The results from two sampling campaigns performed in each plant indicate good chemical status of the source water, as the contaminants detected were present at very low levels, far from those established in the legislation as maximum admissible concentrations (when existing) and far from those considered as a risk. Thus, of the 17 pesticides investigated, BAM (2,6-dichlorobenzamide), desethylatrazine, simazine, atrazine, terbuthylazine, diuron, metolachlor, and diazinon were the only compounds detected, and total pesticides levels were below 25ng L(-1), respectively. Estrone-3-sulfate was the only estrogen detected, at concentrations lower than 0.5ng L(-1). Progestogens were not found in any sample. Detected VOCs (benzene, toluene, ethylbenzene, and trichloroethylene) were below 0.04microg L(-1). The efficiency of elimination of these organic contaminants was poor as no significant decrease in their concentrations was observed through the recharge process.

An integrated study of endocrine disruptors in sediments and reproduction-related parameters in bivalve molluscs from the Biosphere's Reserve of Urdaibai (Bay of Biscay).

Urdaibai was declared a Biosphere's Reserve by UNESCO in 1984. Because we observed a high prevalence of hermaphroditism in mussels sampled in Urdaibai in March 2004, we started the present research work in order to determine the presence of endocrine disruptors (EDs) in sediments and to study possible EDs effects on mussels and oysters using gonad index and vitellogenin (VTG)-like protein levels as biomarkers. Samples were collected at five localities in April 2007 and 2008, and in October 2007. Estrogenic hormones were not detected in sediments and levels of bisphenol A and organotin compounds were very low. Alkylphenols were found at moderate levels and showed a time-dependent decrease. Phthalates were found at levels up to 8000 ng/g. High prevalence of oocyte atresia and necrosis occurred in mussels sampled in April. Retarded gametogenesis was observed in an oyster population from a shipyard. Hermaphrodites or alterations in VTG-like protein levels were not found but the high prevalence of histopathological alterations in oocytes is of concern.

Analysis of 17 polar to semi-polar pesticides in the Ebro river delta during the main growing season of rice by automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry.

This work describes the optimization of a fully automated method based on on-line solid-phase extraction-liquid chromatography-electrospray-tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the simultaneous determination of 17 medium to polar pesticides in water. The list of target analytes included organophosphates, triazines, phenylureas, anilides, chloroacetanilides, acidic herbicides and thiocarbamates. Detection limits achieved were below 5 ng/L for all compounds except metolachlor (12 ng/L), alachlor (17 ng/L), malathion (36 ng/L) and fenitrothion (50 ng/L). The method developed was used to investigate the occurrence of the target pesticides in a total of 52 water samples collected monthly (from May to August 2005) at 14 selected locations in the rice cultivation area of the Ebro river delta. The study showed high levels, in the microg/L range, of bentazone, MCPA, propanil, molinate and atrazine, in basically all the samples investigated. The remaining pesticides were present at lower levels (<0.1 microg/L) or only detected sporadically (e.g. fenitrothion and malathion). The sampling campaign performed in July showed comparatively higher levels than the other three campaigns.

Use of chemometric and geostatistical methods to evaluate pesticide pollution in the irrigation and drainage channels of the Ebro river delta during the rice-growing season.

Data sets obtained from quantitative analysis of seventeen pesticides in water samples from a network of irrigation and drainage channels in the Ebro river delta (Catalonia, NE Spain) have been analysed by chemometric and geostatistical methods. Samples were taken at fourteen locations during the main rice-growing season, from May to August 2005. Principal-component analysis enabled investigation of the spatial and temporal distribution of the main pollution patterns caused by application of pesticides in the region under study. A first pesticide-contamination pattern from the Ebro river was differentiated from a second more specific pattern from the water-drainage channels of the delta, collected from the rice fields. The seasonal peak in this more specific rice pesticide source was observed in July. Coupling the results from chemometric data analysis with use of geostatistical methods was shown to be a useful procedure for discovery of the most significant spatial and monthly variations of the main pesticide-contamination patterns, taking into account the particular geographical structure of the area under study.

Analysis of pesticides in water by liquid chromatography-tandem mass spectrometric techniques.

Pesticide residues continue to be the focus of many environmental studies, and the number of articles describing the development of more advanced, multiresidue analytical methodologies does not decline. The use of liquid chromatography-mass spectrometry based on single quadrupole or ion trap analyzers is consolidated for this purpose. The implementation, in the near future, of more sophisticated mass analyzers, such as triple quadrupole and hybrid quadrupole-time-of-flight is anticipated for routine analysis. This article reviews the various works published so far in the literature for the determination of pesticides and transformation products (TPs) in water by means of liquid chromatography (LC) coupled to tandem mass spectrometry. It discusses the various ionization sources and analyzers used for this purpose, as well as the extraction procedures employed for previous sample preconcentration. Because of the widespread use of triple quadrupole analyzers for the generation of pesticides levels in water using tandem mass spectrometry, a table compiling the transitions monitored for ca. 70 compounds is also included.

Evaluation of commercial immunoassays for the detection of estrogens in water by comparison with high-performance liquid chromatography tandem mass spectrometry HPLC-MS/MS (QqQ).

In this work four different commercially available enzyme-linked immunosorbent assays (ELISA) (from Japan EnviroChemicals, Ltd., Tokyo, Japan) were evaluated in terms of performance for the rapid screening of estrogens in different water matrices, including natural and spiked samples from urban wastewater, river water and ground water. All four test kits are based on monoclonal antibodies. The compounds detected by these immunoassays are (1) 17-beta-estradiol, (2) estrone, (3) 17-alpha-ethynyl estradiol and (4) estrogens in general, with high recognition properties for 17-beta-estradiol, estrone and estriol. Standards were prepared in water containing 10% (v/v) methanol. The IC50 (corresponding to the 50% of the effective concentration) values, the dynamic ranges, and the limits of detection of the ELISA kits were 0.060-0.304 microg/L, 0.05-5 microg/L and 0.05 microg/L, respectively. All samples were extracted by solid-phase extraction (SPE) beforehand, and the evaluation was carried out by comparing the results obtained by ELISA with those obtained by HPLC-MS/MS using a triple quadrupole (QqQ) instrument. In addition, two different solid-phase extraction procedures were carried out and compared. Except for moderate overestimation in the results observed with the ELISA kits in the analysis of complex wastewater samples, the results obtained using all of the tested techniques were generally in very good agreement.