Discovery of large molecules as new biomarkers in wastewater using environmental proteomics and suitable polymer probes.

The capability of monitoring large molecules as possible biomarkers in wastewater will be an important contribution to the new field of sewage epidemiology. Here, we explore the use of polymer probes together with untargeted proteomics for large scale protein analysis in sewage and treated water. Polymeric probes were immersed in the influent, anoxic reactor and effluent waters of a Spanish WWTP during 11 days. Proteins sorbed were extracted and identified by mass spectrometry. A total of 690 proteins from bacteria, plants and animals, including human, were identified showing different proteome profiles in the different sites. Bacterial proteins (510) pointed at 175 genera distributed in 22 bacterial classes. The most abundant were EF-Tu, GroEL and ATP synthase which were contributed by a high number of species. Human was the species contributing the greatest number of identified proteins (57), some in high abundance like keratins. Human proteins dominated in the influent water and were efficiently removed at the effluent. Several of the proteins identified (S100A8, uromodulin, defensins) are known disease biomarkers. This study provides the first insight into the proteome profiles present in real wastewater.

Management actions to mitigate the occurrence of pharmaceuticals in river networks in a global change context.

Human consumption of pharmaceuticals leads to high concentrations of pharmaceuticals in wastewater, which is usually not or insufficiently collected and treated before release into freshwater ecosystems. There, pharmaceuticals may pose a threat to aquatic biota. Unfortunately, occurrence data of pharmaceuticals in freshwaters at the global scale is scarce and unevenly distributed, thus preventing the identification of hotspots, the prediction of the impact of Global Change (particularly streamflow and population changes) on their occurrence, and the design of appropriate mitigation actions. Here, we use diclofenac (DCL) as a typical pharmaceutical contaminant, and a global model of DCL chemical fate based on wastewater sanitation, population density and hydrology to estimate current concentrations in the river network, the impact of future changes in runoff and population, and potential mitigation actions in line with the Sustainable Development Goals. Our model is calibrated against measurements available in the literature. We estimate that 2.74 ± 0.63% of global river network length has DCL concentrations exceeding the proposed EU Watch list limit (100 ng L-1). Furthermore, many rivers downstream from highly populated areas show values beyond 1000 ng L-1, particularly those associated to megacities in Asia lacking sufficient wastewater treatment. This situation will worsen with Global Change, as streamflow changes and human population growth will increase the proportion of the river network above 100 ng L-1 up to 3.10 ± 0.72%. Given this background, we assessed feasible source and end-of-pipe mitigation actions, including per capita consumption reduction through eco-directed sustainable prescribing (EDSP), the implementation of the United Nations Sustainable Development Goal (SDG) 6 of halving the proportion of population without access to safely managed sanitation services, and improvement of wastewater treatment plants up to the Swiss standards. Among the considered end-of-pipe mitigation actions, implementation of SDG 6 was the most effective, reducing the proportion of the river network above 100 ng L-1 down to 2.95 ± 0.68%. However, EDSP brought this proportion down to 2.80 ± 0.64%. Overall, our findings indicate that the sole implementation of technological improvements will be insufficient to prevent the expected increase in pharmaceuticals concentration, and that technological solution need to be combined with source mitigation actions.

Reconciling monitoring and modeling: An appraisal of river monitoring networks based on a spatial autocorrelation approach - emerging pollutants in the Danube River as a case study.

Rivers extend in space and time under the influence of their catchment area. Our perception largely relies on discrete spatial and temporal observations carried out at certain sites located throughout the catchment (monitoring networks, MN). However, MNs are constrained by (a) the distribution of sampling sites, (b) the dynamics of the variable considered and (c) the river hydrological conditions. In this study, all three aspects were captured and quantified by applying a spatial autocorrelation modeling approach. We exemplarily studied its application to 235 emerging contaminants (pesticides, pharmaceuticals, and personal care products [PPCP], industrial and miscellaneous) measured at 55 sampling sites in the Danube River. 22 out of the 235 compounds monitored were present at all sites and 125 were found in at least 50%.We first calculated the Moran Index (MI) to characterize the spatial autocorrelation of the compound set. 59 compounds showed MI≤0, which can be interpreted as 'no spatial correlation'. Next, spatial autocorrelation models were set for each compound. From the autocorrelation parameter ρ, catchment average correlation lengths were derived for each compound. MN optimality was examined and compounds were classified into three groups: (a) those with ρ≤0 [25%]; (b) those with ρ>0 and correl. length0 and correl. length>average distance between consecutive sites [73%]. The MN was considered optimal only for the latter class. Networks with the larger average distance between consecutive sites resulted in a decreasing number of optimally monitored compounds. Furthermore, neighbors vs. local relative contributions were quantified based on the spatial autocorrelation model for all the measured compounds. The results of this study show how autocorrelation models can aid water managers to improve the design of river MNs, which are a key aspect of the Water Framework Directive.

Using a polymer probe characterized by MALDI-TOF/MS to assess river ecosystem functioning: From polymer selection to field tests.

Characterization of river ecosystems must take into consideration both structural and functional aspects. For the latter, a convenient and simple approach for routine monitoring is based on the decomposition of organic matter measured in terms of breakdown of natural organic substrates like leaf litter, wood sticks. Here we extended the method to a synthetic organic material using polymer probes characterized by MALDI-TOF/MS. We first characterized several commercial available polymers, and finally selected polycaprolactonediol 1250 (PCP 1250), a polyester oligomer, as the most convenient for further studies. PCP 1250 was first tested at mesocosms scale under conditions simulating those of the river, with and without nutrient addition for up to 4weeks. Differences to the starting material measured in terms of changes in the relative ion peak intensities were clearly observed. Ions exhibited a different pattern evolution along time depending on their mass. Greatest changes were observed at longest exposure time and in the nutrient addition treatment. At shorter times, the effect of nutrients (addition or not) was indistinguishable. Finally, we performed an experiment in 11 tributaries of the Ebro River during 97days of exposure. Principal Component Analysis confirmed the different behavior of ions, which were clustered according to their mass. Exposed samples were clearly different to the standard starting material, but could not be well distinguished among each other. Polymer mass loss rates, as well as some environmental variables such as conductivity, temperature and flow were correlated with some peak intensities. Overall, the interpretation of field results in terms of environmental conditions remains elusive, due to the influence of multiple concurrent factors. Nevertheless, breakdown of synthetic polymers opens an interesting field of research, which can complement more traditional breakdown studies to assess river ecosystem functioning.

Shared effects of organic microcontaminants and environmental stressors on biofilms and invertebrates in impaired rivers.

Land use type, physical and chemical stressors, and organic microcontaminants were investigated for their effects on the biological communities (biofilms and invertebrates) in several Mediterranean rivers. The diversity of invertebrates, and the scores of the first principal component of a PCA performed with the diatom communities were the best descriptors of the distribution patterns of the biological communities against the river stressors. These two metrics decreased according to the progressive site impairment (associated to higher area of agricultural and urban-industrial, high water conductivity, higher dissolved organic carbon and dissolved inorganic nitrogen concentrations, and higher concentration of organic microcontaminants, particularly pharmaceutical and industrial compounds). The variance partition analyses (RDAs) attributed the major share (10%) of the biological communities' response to the environmental stressors (nutrients, altered discharge, dissolved organic matter), followed by the land use occupation (6%) and of the organic microcontaminants (2%). However, the variance shared by the three groups of descriptors was very high (41%), indicating that their simultaneous occurrence determined most of the variation in the biological communities.

Balancing the health benefits and environmental risks of pharmaceuticals: Diclofenac as an example.

Pharmaceuticals are designed to improve human and animal health, but even the most beneficial pharmaceuticals might raise some questions concerning the consequences of exposure to non-target organisms. To illustrate this situation and using diclofenac as a case-study, we analyze global consumption and occurrence data to identify hot spots of consumption without occurrence data, review the scientific literature on the harmful environmental effects to determine whether the observed concentrations in freshwater are of environmental concern, summarize the current pharmaceutical and environmental policies to highlight policy gaps, and suggest a series of research and policy recommendations, which can be summarized as follows: we need to improve the current knowledge on occurrence in freshwaters to properly implement environmental policies (i), diclofenac might pose a risk to non-target organisms in freshwater (ii); the harmful effects that some pharmaceuticals may have on the environment are not always addressed by environmental policies (iii).

White paper on the promotion of an integrated risk assessment concept in European regulatory frameworks for chemicals.

The vision of a sustainable and safe use of chemicals to protect human health, preserve the environment and maintain the ecosystem requires innovative and more holistic approaches to risk assessment (RA) in order to better inform decision making. Integrated risk assessment (IRA) has been proposed as a solution to current scientific, societal and policy needs. It is defined as the mutual exploitation of environmental risk assessment (ERA) for human health risk assessment (HHRA) and vice versa in order to coherently and more efficiently characterize an overall risk to humans and the environment for better informing the risk analysis process. Extrapolating between species which are relevant for HHRA and ERA requires a detailed understanding of pathways of toxicity/modes of action (MoA) for the various toxicological endpoints. Significant scientific advances, changes in chemical legislation, and increasing environmental consciousness have created a favourable scientific and regulatory environment to develop and promote the concept and vision of IRA. An initial proof of concept is needed to foster the incorporation of IRA approaches into different chemical sectorial regulations and demonstrate their reliability for regulatory purposes. More familiarity and confidence with IRA will ultimately contribute to an overall reduction in in vivo toxicity testing requirements. However, significant progress will only be made if long-term support for MoA-related research is secured. In the short term, further exchange and harmonization of RA terminology, models and methodologies across chemical categories and regulatory agencies will support these efforts. Since societal values, public perceptions and cultural factors are of increasing importance for the acceptance of risk analysis and successful implementation of risk mitigation measures, the integration of socio-economic analysis and socio-behavioural considerations into the risk analysis process may help to produce a more effective risk evaluation and consideration of the risks and benefits associated with the use of chemicals.

Dynamics of suspended sediment borne persistent organic pollutants in a large regulated Mediterranean river (Ebro, NE Spain).

Mediterranean rivers are characterized by highly variable hydrological regimes that are strongly dependent on the seasonal rainfall. Sediment transport is closely related to the occurrence of flash-floods capable to deliver enough kinetic energy to mobilize the bed and channel sediments. Contaminants accumulated in the sediments are likely to be mobilized as well during such events. However, whereas there are many studies characterizing contaminants in steady sediments, those devoted to the transport dynamics of suspended-sediment borne pollution are lacking. Here we examined the occurrence and transport of persistent organic microcontaminants present in the circulating suspended sediments during a controlled flushing flow in the low part of the River Ebro (NE Spain) 12 km downstream of a well-known contaminated hot-spot associated to a nearby chloro-alkali industry. Polycyclic aromatic hydrocarbons (PAHs) and semi-volatile organochlorine pollutants (DDT and related compounds, DDX; polychlorinated byphenils, PCBs; and other organochlorine compound, OCs) were measured in the particulate material by GC-MS and GC-MS/MS, using previously developed analytical methods. The concentration levels observed were compared to previously reported values in steady sediments in the same river and discussed on a regulatory perspective. Hydrographs and sedigraphs recorded showed a peak-flow of 1,300 m(3)s(-1) and a corresponding peak of suspended sediments of 315 mg L(-1). Combination of flow discharge, suspended sediments and pollutants' concentrations data allowed for quantifying the mass flows (mass per unit of time) and setting the load budgets (weight amount) of the different pollutants transported by the river during the monitored event. Mean mass-flows and total load values found were 20.2 mg s(-1) (400 g) for PAHs, 38 mg s(-1) (940 g) for DDX, 44 mg s(-1) (1,038 g) for PCBs and 8 mg s(-1) (200 g) for OCs. The dynamic pattern behavior of PAHs differs substantially to that of organochlorine pollutants, thus reflecting different pollution origins.

Organic carbon content effects on bioavailability of pyrethroid insecticides and validation of solid phase extraction with Poly (2,6-diphenyl-p-phenylene oxide) Polymer by Daphnia magna toxicity tests.

Solid phase extraction with Poly (2,6-diphenyl-p-phenylene oxide) Polymer (Tenax) was used for determining the bioavailability of eleven pyrethroids in field collected sediments with different organic carbon content (OC). The bioavailable fraction of pyrethroids decreased with increasing OC in sediments; the percentages of desorption ranged from 10 to 20% for sediment with higher OC content (5.8%) and 15-40% for that with lower OC (2%). Generally pyrethroids showed low bioavailability and cyfluthrin resulted to be the most bioavailable among the studied pyrethroids. Acute toxicity tests with Daphnia magna were carried out on sediment spiked with three selected pyrethroids (λ-cyhalothrin, cypermethrin and deltamethrin) and served to validate the efficiency of Tenax as a method for assessing the bioavailability of pyrethroids. Toxicity test demonstrated that Tenax was able to remove the toxic bio-available fraction of pyrethroids in sediment. Extracts from Tenax beads after the desorption experiments and spiked sediment before desorption had an equivalent toxicity (LC50) to D. magna neonates at 48 and 72 h of exposure. These results indicate that Tenax beds can be used to predict bio-available and toxic fractions of pyrethroids sorbed to sediments to aquatic organisms like D. magna.

Prioritization of chemicals in the aquatic environment based on risk assessment: analytical, modeling and regulatory perspective.

The extensive and intensive use of chemicals in our developed, highly technological society includes more than 100,000 chemical substances. Significant scientific evidence has lead to the recognition that their improper use and release may result in undesirable and harmful side-effects on both the human and ecosystem health. To cope with them, appropriate risk assessment processes and related prioritization schemes have been developed in order to provide the necessary scientific support for regulatory procedures. In the present paper, two of the elements that constitute the core of risk assessment, namely occurrence and hazard effects, have been discussed. Recent advances in analytical chemistry (sample pre-treatment and instrumental equipment, etc.) have allowed for more comprehensive monitoring of environmental pollution reaching limits of detection up to sub ng L(-1). Alternative to analytical measurements, occurrence models can provide risk managers with a very interesting approach for estimating environmental concentrations from real or hypothetical scenarios. The most representative prioritization schemes used for issuing lists of concerning chemicals have also been examined and put in the context of existing environmental policies for protection strategies and regulations. Finally, new challenges in the field of risk-assessment have been outlined, including those posed by new materials (i.e., nanomaterials), transformation products, multi-chemical exposure, or extension of the risk assessment process to the whole ecosystem.

Integrated Risk Assessment for WFD Ecological Status classification applied to Llobregat river basin (Spain). Part II - Evaluation process applied to five environmental Lines of Evidence.

Many indicators and indices related to a variety of biological, physico-chemical, chemical, and hydromorphological water conditions have been recently developed or adapted by scientists in order to support water managers in the Water Framework Directive (WFD) implementation. In this context, the achievement of a comprehensive and reliable Ecological Status classification of water bodies across Europe is hampered by the lack of harmonised procedures for selecting an appropriate set of indicators and integrating heterogeneous information in a flexible way. To this purpose, an Integrated Risk Assessment (IRA)(2) methodology was developed based on the Weight of Evidence approach. This method analyses and combines a set of environmental indicators grouped into five Lines of Evidence (LoE), i.e. Biology, Chemistry, Ecotoxicology, Physico-chemistry and Hydromorphology. The whole IRA methodology has been implemented as a specific module into a freeware GIS (Geographic Information System)-based Decision Support System, named MODELKEY DSS. This paper focuses on the evaluation of the four supporting LoE (i.e. Chemistry, Ecotoxicology, Physico-chemistry and Hydromorphology), and includes a procedure for a comparison of each indicator with proper thresholds and a subsequent integration process to combine the obtained output with the LoE Biology results in order to provide a single score expressing the Ecological Status classification. The approach supports the identification of the most prominent stressors, which are responsible for the observed alterations in the river basin under investigation. The results provided by the preliminary testing of the IRA methodology through application of the MODELKEY DSS to the Llobregat case study are finally reported and discussed.

Integrated risk assessment for WFD ecological status classification applied to Llobregat river basin (Spain). Part I-Fuzzy approach to aggregate biological indicators.

Water Framework Directive (WFD) requirements and recommendations for Ecological Status (ES) classification of surface water bodies do not address all issues that Member States have to face in the implementation process, such as selection of appropriate stressor-specific environmental indicators, definition of class boundaries, aggregation of heterogeneous data and information and uncertainty evaluation. In this context the "One-Out, All-Out" (OOAO) principle is the suggested approach to lead the entire classification procedure and ensure conservative results. In order to support water managers in achieving a more comprehensive and realistic evaluation of ES, an Integrated Risk Assessment (IRA) methodology was developed. It is based on the Weight of Evidence approach and implements a Fuzzy Inference System in order to hierarchically aggregate a set of environmental indicators, which are grouped into five Lines of Evidence (i.e. Biology, Chemistry, Ecotoxicology, Physico-chemistry and Hydromorphology). The whole IRA methodology has been implemented as an individual module into a freeware GIS (Geographic Information System)-based Decision Support System (DSS), named MODELKEY DSS. The paper focuses on the conceptual and mathematical procedure underlying the evaluation of the most complex Line of Evidence, i.e. Biology, which identifies the biological communities that are potentially at risk and the stressors that are most likely responsible for the observed alterations. The results obtained from testing the procedure through application of the MODELKEY DSS to the Llobregat case study are reported and discussed.

Quality assessment of river waters using risk indexes for substances and sites, based on the COMMPS procedure.

The well established risk assessment COMMPS methodology (Fraunhofer Institut, Schmallenberg, Germany) developed at a European basin scale to identify priority pollutants based on their exposure and effects, has been applied at a local scale along a double step process: (a) first, the corresponding adapted list of priority pollutants has been obtained from monitoring data at a regional scale; (b) second, a new site pollution risk index has been developed for the relative comparison of the chemical pollution status of the investigated geographical region. As a case study, representative of the Mediterranean area, the process has been applied to 17 Catalan rivers (NE Spain), using the monitoring data collected between 1997 and 2006 in 207 different sampling sites. After an appropriate selection procedure, 52 parameters were finally used for the calculation of the local priority substance and site risk indexes. From the exposure point of view the most relevant substances at the Catalan scale were volatile organohalogen compounds (VOX) and volatile aromatic hydrocarbons (toluene and xylene), whereas the priority substance risk index points to dibromomethane (volatile organohalogen compound) and benzo-a-anthracene (PAH) as the most concerning compounds. The local and European priority substance indexes have been compared, showing minor differences. Finally, site risk indexes have been calculated and utilized for comparison of the chemical status of different sites. They can be conveniently displayed in quality geographical maps and are considered a valuable tool for the environmental management and risk assessment of the region under study.

Monitoring and toxicity of sulfonated derivatives of benzene and naphthalene in municipal sewage treatment plants.

Monitoring benzenesulfonates (BS) and naphthalenesulfonates (NS) took place in five municipal sewage treatment plants (STP). A previously optimized method based on solid phase extraction with polymeric cartridges followed by ion-pair liquid chromatography-electrospray-mass spectrometry (SPE-IPC-ESI-MS) was used. This work confirmed the little or no effect of primary settlement on total organic carbon (TOC) and monosulfonated compounds removal, whereas the main reduction is obtained at the biological stage. However, the most polar compounds, such as naphthalenedisulfonates (NDS), were not effectively removed using the biological treatment. An aromatic sulfonated compound is suggested to be used as a tracer of the origin of industrial pollutants discharged into STPs. A bioluminescence inhibition test, Microtox assay, allowed toxicity determination of the most relevant aromatic sulfonated compounds detected and toxicity comparison between primary and secondary effluents.

Monitoring of the gasoline oxygenate MTBE and BTEX compounds in groundwater in Catalonia (northeast Spain).

Headspace (HS) gas chromatography with flame ionisation detection (HS-GC-FID) and purge and trap (P) gas chromatography-mass spectrometry (P) were used for the determination of methyl-tert-butyl ether (MTBE) and benzene, toluene, and xylenes (BTEX) in groundwater. In this work, we present the first data on the levels of MTBE and BTEX in different groundwater wells in the area of Catalonia (northeast Spain). This monitoring campaign corresponded to 28 groundwater wells that were located near petrol service stations, oil refinery storage tanks, and/or chemical industry at different locations of Catalonia during the period of 1998/1999. The levels of MTBE detected varied between 4-300 microg/l, but two sites had MTBE levels up to 3 and 13 mg/l. In many cases, the BTEX levels were below 1 microg/l, whereas 7 sites had levels varying from 19 microg/l up to 3 mg/l. Most of them were related to leakage from underground tanks in petrol service stations, while the remaining three corresponded respectively to chemical industrial pollution of undetermined origin and to a leak from high-ground petrol tanks in petrochemical refinery factories. The aquifers involved were constituted by detritus coarse materials, sands, and conglomerates. Piezometric levels were roughly comprised between 3 and 40 m, and permeability (K) and transmissivity (T) values were estimated from field measurements. The MTBE/BTEX ratio was also calculated and reached values up to 250. These values were expected, since if we consider that spilled oxygenated gasoline is the source of well contamination and based on solubility considerations alone, the MTBE source concentrations would be about 200 times higher than any BTEX compounds.

Fate of MTBE and DCPD compounds relative to BTEX in gasoline-contaminated aquifers.

The aim of this communication is to provide preliminary results on MTBE monitoring, and at the same time to propose some new tracers of gasoline pollution in groundwater. An overview is presented on benzene-toluene-ethylbenzene-xylene (BTEX), methyl tertiary-butyl ether (MTBE), and dicyclopentadienes (DCPD) contents in gasoline formulations. Their specific fate in gasoline-contaminated aquifers are consistent with their physical-chemical properties.

Determination of drugs in surface water and wastewater samples by liquid chromatography-mass spectrometry: methods and preliminary results including toxicity studies with Vibrio fischeri.

In the present work a combined analytical method involving toxicity and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was developed for the determination of pharmaceutical compounds in water samples. The drugs investigated were the analgesics: ibuprofen, ketoprofen, naproxen, and diclofenac, the decomposition product of the acetyl salicylic acid: salicylic acid and one lipid lowering agent, gemfibrozil. The selected compounds are acidic substances, very polar and all of them are analgesic compounds that can be purchased without medical prescription. The developed protocol consisted, first of all, on the use Microtox and ToxAlert 100 toxicity tests with Vibriofischeri for the different pharmaceutical drugs. The 50% effective concentration (EC50) values and the toxicity units (TU) were determined for every compound using both systems. Sample enrichment of water samples was achieved by solid-phase extraction procedure (SPE), using the Merck LiChrolut EN cartridges followed by LC-ESI-MS. Average recoveries loading 11 of samples with pH=2 varied from 69 to 91% and the detection limits in the range of 15-56 ng/l. The developed method was applied to real samples from wastewater and surface-river waters of Catalonia (north-east of Spain). One batch of samples was analyzed in parallel also by High Resolution Gas Chromatography coupled with Mass Spectrometry (HRGC-MS) and the results have been compared with the LC-ESI-MS method developed in this work.

Toxic potency assessment of non- and mono-ortho PCBs, PCDDs, PCDFs, and PAHs in northwest Mediterranean sediments (Catalonia, Spain).

Forty-five marine sediments from the Catalonian coast were analyzed for non-ortho and mono-ortho chlorine substituted PCB congeners, PCDDs and PCDFs, and 16 PAHs. Concentrations of total PCBs ranged from 1.1 to 311 ng/g dry weight (d.w.), and the levels of the sum of the 16 PAHs analyzed ranged between 13.4 ng/g d.w. and 16.7 microg/g d.w. The PCB and PAH contamination was greater near the sites of urban and industrial impact. Total toxicity equivalent (TEQ) values were calculated using the toxicity equivalent factors (TEFs) proposed by WHO for dioxin-like PCBs, PCDDs, and PCDFs. These levels ranged between 0.03 and 24.8 pg WHO-TEQ/g d.w. for PCBs and from 0.4 to 39.2 pg WHO-TEQ/g d.w. for PCDDs/PCDFs. Therefore, the suggested sediment quality guideline was sometimes exceeded. Moreover, TEA values were calculated for PAH concentrations, applying different TEFs proposed by the literature. The results obtained were between 0.3 pg TEQ/g d.w. and 18.4 ng TEQ/g d.w. and showed that the TEQ(PCDD/F) and TEQ(PCB) values were several times lower than the TEQ(PAH) values in the marine sediment samples investigated.