Assessing the Potential Ecotoxicological Risk of Different Organic Amendments Used in Agriculture: Approach Using Acute Toxicity Tests on Plants and Earthworms.

In Europe, spreading organic wastes to fertilize soils is an alternative commonly used instead of chemical fertilizers. Through their contributions of nutrients and organic matter, these wastes promote plant growth and thus agricultural production. However, these organic amendments can also contain mineral and organic pollutants requiring chemical and ecotoxicological analyses to guarantee their harmlessness on soil and its organisms during spreading. The purpose of this study was to assess the potential toxicity of three organic amendments from different sources (sewage sludge, dairy cow manure, dairy cow slurry) by performing chemical analyses and acute toxicity tests on three types of organism: earthworms, plants, soil microbial communities. Chemical analysis revealed a higher content of certain pharmaceuticals, polycyclic aromatic hydrocarbons and metals in sewage sludge in comparison with the two other types of organic wastes. The ecotoxicological assessment showed a dose-dependent effect on soil organisms for the three organic amendments with higher toxic effects during the exposure tests with a soil amended with dairy cow slurry. However, at realistic spreading doses (10 and 20 g kg-1 dry weight of organic amendments) on a representative exposed soil, organic amendment did not show any toxicity in the three organisms studied and had positive effects such as increased earthworm biomass, increased plant root growth and earthworm behavior showing attraction for organic amendment. On the contrary, exposure assays carried out on a limited substrate like sandy soil showed increased toxicity of organic amendments on plant germination and root growth. Overall, the ecotoxicological analysis revealed greater toxicity for soil organisms during the amendment of cow slurry, contrary to the chemical analysis which showed the potential high risk of spreading sewage sludge due to the presence of a higher quantity of pollutants. The analysis of the chemical composition and use of acute toxicity tests is the first essential step for assessing the ecotoxicological risk of spreading organic amendments on soil organisms. In addition to standard tests, the study suggests using a representative soil in acute toxicity tests to avoid overestimating the toxic effects of these organic amendments.

Monitoring the extraction of additives and additive degradation products from polymer packaging into solutions by multi-residue method including solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis.

The use of polymer materials in industry for product packaging is increasing. The presence of additives in the polymer matrix enables the modification or improvement of the properties and performance of the polymer, but these industries are concerned regarding the extractability of these additives. The quantification of these additives is particularly challenging because of the presence of these substances as contaminants in all the analytical equipment and the diversity of their physicochemical properties. In this context, a multi-residue analytical method was developed for the trace analysis of the twenty main additives (and their degradation products) authorized in plastic products such as pharmaceutical packaging (e.g., antioxidants, release agents, and light absorbers). This analytical method consisted of a solid phase extraction (SPE) followed by an analysis using ultra-high performance liquid chromatography coupled to a tandem mass spectrometer (UHPLC-MS/MS). A comparison of two ionization interfaces and the optimization of the extraction procedure were discussed. The influence of the quality of the solvent type (distilled versus not distilled) and the nature of the SPE cartridges (Polypropylene versus Teflon(®)) were demonstrated. The optimized method exhibited a quantification limit lower than 20 ng mL(-1) and recoveries between 70 % and 120 % for all compounds. Finally, the method was validated according to the ICH directive and was subsequently applied to the extraction of polymers under different pH conditions and storage temperatures. To the best of our knowledge, this study presents the first methodology allowing the simultaneous quantification of 24 additives at low ng mL(-1).

Multi-residue analysis of emerging pollutants in sediment using QuEChERS-based extraction followed by LC-MS/MS analysis.

Emerging contaminants are suspected to cause adverse effects in humans and wildlife. Aquatic ecosystems are continuously contaminated by agricultural and industrial sources. To establish a causality relationship between the occurrence of contaminants in the environment and disease, experiments including all environmental matrices must be performed. Consequently, the current analytical tools must be improved. A new multi-residue method for analysing 15 emerging pollutants in sediments based on the Quick, Easy, Cheap, Effective, Rugged and Safe approach is reported. The development of such a multirisque, inter-family method for sediment including pharmaceuticals, pesticides, personal care products and plasticizers is reported for the first time. The procedure involves salting-out liquid-liquid extraction using acetonitrile and clean-up with dispersive solid phase extraction, followed by liquid chromatography coupled with tandem mass spectrometry. The validated analytical procedure exhibited recoveries between 40 and 98% for every target compound. This methodology facilitated the determination of pollutant contents at nanogram-per-gram concentrations.

Single injection LC-MS/MS analytical method for the quantification of diverse families of micropollutants, including PFAS and organotins, in Gammarus fossarum.

Since the last decades, light has been shed on the pollution of aquatic ecosystems. Considering apolar compounds in water, analysis of the dissolved phase is not sufficient due to their possible bioaccumulation. Additional analysis of sediments, biota, or sentinel species is necessary. Among sentinel species, Gammarus fossarum is a small shrimp of 30 mg that lives naturally in the river. Its ability to bioaccumulate makes it a good bioindicator of river pollution. Nevertheless, micropollutants are difficult to extract from gammarids due to their small size and their high level of lipids. Extracted interferences can lead to analytical difficulties. Targeted micropollutants in this work were organotins, personal and pharmaceutical care products (PPCPs), pesticides, flame retardants, and perfluorinated compounds. A sample preparation based on QuEChERS followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed: different salts (acetate and citrate) and purification (addition of hexane, dispersive SPE) were tested and validated. Targeted molecules used to be analyzed by LC-MS/MS, except organotins which are principally analyzed in gas chromatography. One of the main challenges was to quantify them also in LC-MS/MS to implement an original multi-residue method. The analysis of 40 compounds was finally validated according to ICH guidelines, with LOQs ranging from 0.04 ng.g-1 to 313.5 ng.g-1. Regarding Perfluorooctanesulfonic acid (PFOS) and the sum of hexabromocyclododecane, LOQs reached the environmental quality standards in biota which are respectively of 9.1 and 167 ng.g-1. Finally, the method was applied to 15 real samples. Many compounds were quantified: perfluorinated compounds, drugs such as ketoprofen, and even cosmetics products such as octocrylene. This is the first study to quantify as many emerging compounds, especially organotins, in a sentinel species such as G. fossarum.

A potential biomarker of androgen exposure in European bullhead (Cottus sp.) kidney.

The aim of this study was to identify a signal that could be used as an androgen exposure indicator in the European bullhead (Cottus sp.). For this purpose, the ultra-structure of the kidney was characterized to identify normal structure of this organ, and histological changes previously described in the kidney of breeding male bullheads were quantified using the kidney epithelium height (KEH) assay previously developed and validated for the stickleback. In the next step, the effect of trenbolone acetate (TbA), a model androgen, was assessed to identify potential androgenic regulation of bullhead kidney hypertrophy. Measurement of KEH performed on adult non-breeding male and female bullheads exposed for 14 and 21 days to 0, 1.26 and 6.50 µg/L showed that kidney hypertrophy is induced in a dose-dependent manner, confirming the hypothesis that the European bullhead possesses a potential biomarker of androgen exposure. Combined with the wide distribution of the European bullhead in European countries and the potential of this fish species for environmental toxicology studies in field and laboratory conditions, the hypothesis of a potential biomarker of androgen exposure offers interesting perspectives for the use of the bullhead as a relevant sentinel fish species in monitoring studies. Inducibility was observed with high exposure concentrations of TbA. Further studies are needed to identify molecular signals that could be more sensitive than KEH.

Determination of groundwater origins and vulnerability based on multi-tracer investigations: New contributions from passive sampling and suspect screening approach.

Knowledge about groundwater origins and their interactions with surface water is fundamental to assess their vulnerability. In this context, hydrochemical and isotopic tracers are useful tools to investigate water origins and mixing. More recent studies examined the relevance of contaminants of emerging concern (CECs) as co-tracers to distinguish sources contributing to groundwater bodies. However, these studies focused on known and targeted CECs a priori selected regarding their origin and/or concentrations. This study aimed to improve these multi-tracer approaches using passive sampling and qualitative suspect screening by exploring a larger variety of historical and emerging concern contaminants in combination with hydrochemistry and water molecule isotopes. With this objective, an in-situ study was conducted in a drinking water catchment area located in an alluvial aquifer recharged by several water sources (both surface and groundwater sources). CECs determined by passive sampling and suspect screening allowed to provide in-depth chemical fingerprints of groundwater bodies by enabling the investigation of >2500 compounds with an increased analytical sensitivity. Obtained cocktails of CECs were discriminating enough to be used as chemical tracer in combination with hydrochemical and isotopic tracers. In addition, the occurrence and type of CECs contributed to a better understanding of groundwater-surface water interactions and highlighted short-time hydrological processes. Furthermore, the use of passive sampling with suspect screening analysis of CECs lead to a more realistic assessment and mapping of groundwater vulnerability.

Multi-residue analysis of free and conjugated hormones and endocrine disruptors in rat testis by QuEChERS-based extraction and LC-MS/MS.

Endocrine disrupting compounds (EDCs) are suspected to be responsible for many disorders of the human reproductive system. To establish a causality relationship between exposure to endocrine disruptors and disease, experiments on animals must be performed with improved or new analytical tools. Therefore, a simple, rapid, and effective multi-residue method was developed for the determination of four steroid hormones (i.e., testosterone, androstenedione, estrone, and estradiol), glucuronide and sulfate conjugates of estrone and estradiol and four endocrine disruptors in rat testis (i.e., bisphenol A, atrazine, and active metabolites of methoxychlor and vinclozolin). The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was then developed to quantify these compounds at ultra-trace levels by liquid chromatography coupled to tandem mass spectrometry. The QuEChERS extraction was optimized with regard to the acetonitrile/water ratio used in the extraction step, the choice of the cleanup method and the acetonitrile/hexane ratio used in the cleanup step. The optimized extraction method exhibited recoveries between 89% and 108% for all tested compounds except the conjugates (31% to 58%). The detection limits of all compounds were below 20 ng g(-1) of wet weight of testis. The method was subsequently applied to determine the levels of hormones and EDCs in seven rat testis samples.

Advantages of MS/MS/MS (MRM3) vs classic MRM quantification for complex environmental matrices: Analysis of beta-lactams in WWTP sludge.

When dealing with complex matrices such as wastewater treatment plant (WWTP) sludge or animal manure, usual MRM quantification may lack enough sensitivity or accuracy due to the presence of numerous interfering compounds co-extracted from the matrix. To circumvent the sensitivity and specificity loss, the method development can be focused on sample extraction, purification or/and optimisation of the detection. In this study, we propose an enhancement of a method for the analysis of five beta-lactams (Amoxicillin, Ampicillin, Cefapirin, Ceftiofur, and Cloxacillin) in WWTP, with the use of a hybrid triple quadrupole-Linear Ion Trap (LIT) spectrometer, enabling triple stage MS acquisition, namely MRM3, in place of the usual MS/MS detection. The adaptation of various parameters such as the secondary fragmentation energy, excitation, and accumulation times of the secondly generated ion are described. The method was then validated and enabled quantification limits between 0.8 and 14.7 ng g-1 associated with accurate quantification (between 98% and 113%). This method is the first to report the use of MRM3 acquisition in an environmental matrix as complex as sludge.

Improvement of the QuEChERS extraction step by matrix-dispersion effect and application on beta-lactams analysis in wastewater sludge by LC-MS/MS.

In the last decade, beta-lactams use in veterinary and human medicine increased to represent today about 15% of the overall consumption. Beta-lactams tend to degrade and metabolize in the environment. Therefore, analytical methods must be sensitive enough to quantify low concentrations of the parent molecules and also allow detection of metabolites. This study presents the development of a modified QuEChERS method for the extraction of seven beta-lactams and one degradation product (Amoxicillin, Ampicillin, Cefapirin, Cefoperazone, Cefquinome, Ceftiofur, Cloxacillin, and Amoxicillin-Diketopiperazine) from sewage treatment plant sludge and their analysis by liquid chromatography coupled with tandem mass spectrometry. Before the QuEChERS extraction, a dispersion step of the sample with EDTA-treated sand was optimized and added, allowing to facilitate the exchanges between the matrix and the extraction solvent. Then, to decrease the interferences present in the extract, a fast and efficient pass-through SPE was implemented. The optimized method was validated and showed satisfactory performances, in adequacy with the analysis of beta-lactams in solid environmental matrices. Limits of quantification lower than 20 ng.g-1 for all analytes, high accuracy (96%-114% quantification on spiked samples nominal concentration) and interday precision (2%-12% RSD) were obtained. This method was then applied to eight sludge samples. Cefapirin and amoxicillin-diketopiperazine were detected in four samples each, at concentrations of 10.2-53.3 ng.g-1 and 3.0-9.5 ng.g-1 respectively. Thus, the developed method is very effective for the extraction of beta-lactams from environmental solid matrices.

Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment.

Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems.

Ultrasound-assisted sample preparation for simultaneous extraction of anionic, cationic and non-ionic surfactants in sediment.

Despite their very wide use in various fields, knowledge concerning surfactants in environmental solid matrices is generally poor. One of the difficulties encountered in the analysis of surfactants is their very diverse physicochemical properties which require different extraction techniques. The objective of this work was therefore to develop an extraction method in sediments that allows the simultaneous analysis of anionic, cationic and non-ionic surfactants. Different extraction techniques (salting-out, ultrasound), solvents and additives were compared. The optimized method, followed by analysis by coupling liquid chromatography with tandem mass spectrometry, was then validated and applied to real samples in which the analytes were quantified by matrix matched calibration. Optimization of the extraction parameters showed different trends depending on the surfactant family. However, ultrasound assisted extraction with a 90/10 acetonitrile/water mixture at 1% acetic acid and 0.1 M EDTA showed the best results overall. The quantification limits obtained, between 6.4 µg/kg for linear alkylbenzene sulfonate (LAS) C10 and 158 µg/kg for 1-laureth sulfate, allow the analysis of traces in sediments. Eighteen of the 27 targeted surfactants were thus detected. The highest concentrations were found for LAS and quaternary ammoniums. Strong correlations between concentrations of different homologues of the same families of surfactants were observed.

Distribution of pesticides and some of their transformation products in a small lentic waterbody: Fish, water, and sediment contamination in an agricultural watershed.

More than 20 years after the Water Framework Directive was adopted, there are still major gaps in the sanitary status of small rivers and waterbodies at the head of basins. These small streams supply water to a large number of wetlands that support a rich biodiversity. Many of these waterbodies are fishponds whose production is destined for human consumption or for the restocking of other aquatic environments. However, these ecosystems are exposed to contaminants, including pesticides and their transformation products. This work aims to provide information on the distribution, diversity, and concentrations of agricultural contaminants in abiotic and biotic compartments from a fishpond located at the head of watersheds. A total of 20 pesticides and 20 transformation products were analyzed by HPLC-ESI-MS/MS in water and sediment sampled monthly throughout a fish production cycle, and in three fish species at the beginning and end of the cycle. The highest mean concentrations were found for metazachlor-OXA (519.48 ± 56.52 ng.L-1) in water and benzamide (4.23 ± 0.17 ng g-1 dry wt.) in sediment. Up to 20 contaminants were detected per water sample and 26 per sediment sample. The transformation products of atrazine (banned in Europe since 2003 but still widely used in other parts of the world), flufenacet, imidacloprid (banned in France since 2018), metazachlor, and metolachlor were more concentrated than their parent compounds. Fewer contaminants were detected in fish and principally prosulfocarb accumulated in organisms during the cycle. Our work brings innovative data on the contamination of small waterbodies located at the head of a basin. The transformation products with the highest frequency of occurrence and concentrations should be prioritized for further environmental monitoring studies, and specific toxicity thresholds should be defined. Few contaminants were found in fish, but the results challenge the widely use of prosulfocarb.

SIPIBEL observatory: Data on usual pollutants (solids, organic matter, nutrients, ions) and micropollutants (pharmaceuticals, surfactants, metals), biological and ecotoxicity indicators in hospital and urban wastewater, in treated effluent and sludge from wastewater treatment plant, and in surface and groundwater.

The Bellecombe pilot site - SIPIBEL - was created in 2010 in order to study the characterisation, treatability and impacts of hospital effluents in an urban wastewater treatment plant. This pilot site is composed of: i) the Alpes Léman hospital (CHAL), opened in February 2012, ii) the Bellecombe wastewater treatment plant, with two separate treatment lines allowing to fully separate the hospital wastewater and the urban wastewater, and iii) the Arve River as the receiving water body and a tributary of the Rhône River and the Geneva aquifer. The database includes in total 48 439 values measured on 961 samples (raw and treated hospital and urban wastewater, activated sludge in aeration tanks, dried sludge after dewatering, river and groundwater, and a few additional campaigns in aerobic and anaerobic sewers) with 44 455 physico-chemistry values (including 15 pharmaceuticals and 14 related transformation products, biocides compounds, metals, organic micropollutants), 2 193 bioassay values (ecotoxicity), 1 679 microbiology values (including microorganisms and antibioresistance indicators) and 112 hydrobiology values.

Multiresidue method to quantify pesticides in fish muscle by QuEChERS-based extraction and LC-MS/MS.

Pesticide residues in fish muscle are an environmental and a health safety concern which requires analytical methods presenting high sensitivity and low limits of quantification. In this study, adapted QuEChERS method, coupled to liquid chromatography tandem mass spectrometry (Scheduled MRM-5500 QTRAP), was developed to quantify 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in muscle of fish. Quantification limits were below 1 ng g(-1) except for clomazone (1.8 ng g(-1)) and quizalofop (7.4 ng g(-1)). Best recoveries were observed for perch (>80%) and roach (>68%), except for thifensulfuron-methyl. Lower recoveries had been observed for carp (6% to 86%). Relative standard deviation was lower than 28% for intra-day and 29% for inter-day analysis, respectively. This method was successfully tested on three fish species, naturally or orally exposed: roach (Rutilus rutilus), perch (Perca fluviatilis) and carp (Cyprinus carpio). Few levels were observed in fish naturally exposed, but carp and perch orally contaminated showed measurable levels in their muscles.

A multi-family offline SPE LC-MS/MS analytical method for anionic, cationic and non-ionic surfactants quantification in surface water.

Of the large number of emerging pollutants discharged from wastewaters into surface waters, surfactants are among those with the highest concentrations. However, few monitoring in river waters of these substances have already been performed and only on a few families, mostly anionic. This work aimed to develop a multi-family analytical strategy suitable for the quantification of low concentrations of surfactant in surface waters. Twelve families of surfactants, anionic, cationic and non-ionic were selected. Their quantification by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and their extraction by SPE were optimized by comparing different retention mechanisms. The best performances were obtained with a C18 grafted silica LC column and a hydrophilic-lipophilic balanced (HLB) polymeric SPE cartridge. The final analytical method was validated and applied for the quantification of surfactants in 36 river water samples. Method limits of quantification (LQ), intra and inter days precision and trueness were evaluated. With LQ between 15 and 485 ng/L, and trueness over 80%, this method was suitable for monitoring surfactants in surface water. Application on French river water samples revealed the presence of anionic, cationic and non-ionic surfactants with median concentrations from 24 ng/L for octylphenol ethoxylates (OPEO), up to 4.6 µg/L regarding linear alkylbenzene sulfonates (LAS).

Aminoglycosides analysis optimization using ion pairing liquid chromatography coupled to tandem mass spectrometry and application on wastewater samples.

Aminoglycosides are mostly used as veterinary antibiotics. In France, their consumption accounts for about 10% of all prescribed animal medicine. Due to their high polarity nature (log Kow < -3), they require chromatographic separation by hydrophilic interaction liquid chromatography or ion-pairing chromatography. This study presents the development of an ion pairing liquid chromatography with alkanesulfonates coupled to tandem mass spectrometry for the analysis of 10 aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin, apramycin, gentamicin, neomycin and sisomicin) in wastewater samples. The novelty of this method lies in the addition of the ion paring salt directly and only into the sample vial and not in the mobile phase, lowering the amount of salt added and consequently reducing signal inhibition. The optimized method was validated and showed satisfactory resolution, performances suitable with the analysis of aminoglycosides in wastewater samples, with limits of quantifications less than 10 ng/mL for most of the compounds, low matrix effects, high accuracy (85%-115% recoveries) and reproducibility (2%-12%RSD). It was then applied successfully to raw and treated wastewater samples.

Gentamicin Adsorption onto Soil Particles Prevents Overall Short-Term Effects on the Soil Microbiome and Resistome.

Antibiotics used in agriculture may reach the environment and stimulate the development and dissemination of antibiotic resistance in the soil microbiome. However, the scope of this phenomenon and the link to soil properties needs to be elucidated. This study compared the short-term effects of a range of gentamicin concentrations on the microbiome and resistome of bacterial enrichments and microcosms of an agricultural soil using a metagenomic approach. Gentamicin impact on bacterial biomass was roughly estimated by the number of 16SrRNA gene copies. In addition, the soil microbiome and resistome response to gentamicin pollution was evaluated by 16SrRNA gene and metagenomic sequencing, respectively. Finally, gentamicin bioavailability in soil was determined. While gentamicin pollution at the scale of µg/g strongly influenced the bacterial communities in soil enrichments, concentrations up to 1 mg/g were strongly adsorbed onto soil particles and did not cause significant changes in the microbiome and resistome of soil microcosms. This study demonstrates the differences between the response of bacterial communities to antibiotic pollution in enriched media and in their environmental matrix, and exposes the limitations of culture-based studies in antibiotic-resistance surveillance. Furthermore, establishing links between the effects of antibiotic pollution and soil properties is needed.

Ecotoxicological risk assessment of contaminants of emerging concern identified by "suspect screening" from urban wastewater treatment plant effluents at a territorial scale.

Urban wastewater treatment plants (WWTP) are a major vector of highly ecotoxic contaminants of emerging concern (CECs) for urban and sub-urban streams. Ecotoxicological risk assessments (ERAs) provide essential information to public environmental authorities. Nevertheless, ERAs are mainly performed at very local scale (one or few WWTPs) and on pre-selected list of CECs. To cope with these limits, the present study aims to develop a territorial-scale ERA on CECs previously identified by a "suspect screening" analytical approach (LC-QToF-MS) and quantified in the effluents of 10 WWTPs of a highly urbanized territory during three periods of the year. Among CECs, this work focused on pharmaceutical residue and pesticides. ERA was conducted following two complementary methods: (1) a single substance approach, based on the calculation for each CEC of risk quotients (RQs) by the ratio of Predicted Environmental Concentration (PEC) and Predicted No Effect Concentration (PNEC), and (2) mixture risk assessment ("cocktail effect") based on a concentration addition model (CA), summing individual RQs. Chemical results led to an ERA for 41 CEC (37 pharmaceuticals and 4 pesticides) detected in treated effluents. Single substance ERA identified 19 CECs implicated in at least one significant risk for streams, with significant risks for DEET, diclofenac, lidocaine, atenolol, terbutryn, atorvastatin, methocarbamol, and venlafaxine (RQs reaching 39.84, 62.10, 125.58, 179.11, 348.24, 509.27, 1509.71 and 3097.37, respectively). Mixture ERA allowed the identification of a risk (RQmix > 1) for 9 of the 10 WWTPs studied. It was also remarked that CECs leading individually to a negligible risk could imply a significant risk in a mixture. Finally, the territorial ERA showed a diversity of risk situations, with the highest concerns for 3 WWTPs: the 2 biggest of the territory discharging into a large French river, the Rhône, and for the smallest WWTP that releases into a small intermittent stream.

Miniaturization of an extraction protocol for the monitoring of pesticides and polar transformation products in biotic matrices.

Monitoring pesticides in the environment requires the use of sensitive analytical methods. However, existing methods are generally not suitable for analyzing small organisms, as they require large matrix masses. This study explores the development of a miniaturized extraction protocol for the monitoring of small organisms, based on only 30 mg of matrix. The miniaturized sample preparation was developed using fish and macroinvertebrate matrices. It allowed the characterization of 41 pesticides and transformation products (log P from -1.9 to 4.8) in small samples with LC-MS/MS, based on European guidelines (European Commission DG-SANTE, 2019). Quantification limits ranged from 3 to 460 ng g-1 dry weight (dw) for fish and from 0.1 to 356 ng g-1 dw for invertebrates, with most below 60 ng g-1 dw. Extraction rates ranged from 70% to 120% for 35 molecules in fish. Recoveries ranged from 70% to 120% for 37 molecules in macroinvertebrates. Inter-day precision was below 30% for 32 molecules at quantification limits. The method was successfully applied to 17 fish and 19 macroinvertebrates collected from two ponds of the French region of Dombes in November and May 2018, respectively. Both sample matrices were nearly always contaminated with benzamide, imidacloprid-desnitro, and prosulfocarb at respective concentrations of 42-237, 3, and 30-165 ng g-1 dw in fish, and 62-438, 2-6, and 15-29 ng g-1 dw in macroinvertebrates. Results show that this method is an effective tool for characterizing polar pesticides in small biotic samples.

Spatio-temporal variations in chemical pollutants found among urban deposits match changes in thiopurine S-methyltransferase-harboring bacteria tracked by the tpm metabarcoding approach.

The bTPMT (bacterial thiopurine S-methyltransferase), encoded by the tpm gene, can detoxify metalloid-containing oxyanions and xenobiotics. The hypothesis of significant relationships between tpm distribution patterns and chemical pollutants found in urban deposits was investigated. The tpm gene was found conserved among eight bacterial phyla with no sign of horizontal gene transfers but a predominance among gammaproteobacteria. A DNA metabarcoding approach was designed for tracking tpm-harboring bacteria among polluted urban deposits and sediments recovered for more than six years in a detention basin (DB). This DB recovers runoff waters and sediments from a zone of high commercial activities. The PCR products from DB samples led to more than 540,000 tpm reads after DADA2 or MOTHUR bio-informatic manipulations that were allocated to more than 88 and less than 634 sequence variants per sample. The tpm community patterns were significantly different between the recent urban deposits and those that had accumulated for more than 2 years in the DB, and between those of the DB surface and the DB settling pit. These groups of samples had distinct mixture of priority pollutants. Significant relationships between tpm ordination patterns, sediment accumulation time periods and location, and concentrations in PAH, chlorpyrifos, and 4-nonylphenols (NP) were observed. These correlations matched the higher occurrences of, among others, Aeromonas, Pseudomonas, and Xanthomonas tpm-harboring bacteria in recent urban DB deposits more contaminated with chrysene and alkylphenol ethoxylates. Highly significant drops in tpm reads allocated to Aeromonas species were recorded in the oldest DB sediments accumulating naphthalene and metallic pollutants. Degraders of urban pollutants such as P. aeruginosa and P. putida showed conserved distribution patterns over time but P. syringae phytopathogens were more abundant in the oldest sediments. TPMT-harboring bacteria can be used to assess the incidence of high risk priority pollutants on environmental systems.