Village Settlements in Mountainous Tropical Areas, Hotspots of Fecal Contamination as Evidenced by Escherichia coli and Stanol Concentrations in Stormwater Pulses.

Fecal bacteria in surface water may indicate threats to human health. Our hypothesis is that village settlements in tropical rural areas are major hotspots of fecal contamination because of the number of domestic animals usually roaming in the alleys and the lack of fecal matter treatment before entering the river network. By jointly monitoring the dynamics of Escherichia coli and of seven stanol compounds during four flood events (July-August 2016) at the outlet of a ditch draining sewage and surface runoff out of a village of Northern Lao PDR, our objectives were (1) to assess the range of E. coli concentration in the surface runoff washing off from a village settlement and (2) to identify the major contributory sources of fecal contamination using stanol compounds during flood events. E. coli pulses ranged from 4.7 × 104 to 3.2 × 106 most probable number (MPN) 100 mL-1, with particle-attached E. coli ranging from 83 to 100%. Major contributory feces sources were chickens and humans (about 66 and 29%, respectively), with the highest percentage switching from the human pole to the chicken pole during flood events. Concentrations indicate a severe fecal contamination of surface water during flood events and suggest that villages may be considered as major hotspots of fecal contamination pulses into the river network and thus as point sources in hydrological models.

Experimental and modelling evidence of splash effects on manure borne Escherichia coli washoff.

In tropical montane South-East Asia, recent changes in land use have induced increased runoff, soil erosion and in-stream suspended sediment loads. Land use change is also contributing to increased microbial pathogen dissemination and contamination of stream waters. Escherichia coli (E. coli) is frequently used as an indicator of faecal contamination. Field rain simulations were conducted to examine how E. coli is exported from the surface of upland, agricultural soils during runoff events. The objectives were to characterize the loss dynamics of this indicator from agricultural soils contaminated with livestock waste, and to identify the effect of splash on washoff. Experiments were performed on nine 1 m2 plots, amended or not with pig or poultry manure. Each plot was divided into two 0.5 m2 sub-plots. One of the two sub-plots was protected with a mosquito net for limiting the raindrop impact effects. Runoff, soil detachment by raindrop impact and its entrainment by runoff, and E. coli loads and discharge were measured for each sub-plot. The results show that raindrop impact strongly enhances runoff generation, soil detachment and entrainment and E. coli export. When the impact of raindrops was reduced with a mosquito net, total runoff was reduced by more than 50%, soil erosion was on average reduced by 90% and E. coli export from the amended soil surface was on average 3 to 8 times lower. A coupled physics-based approach was performed using the Cast3M platform for modelling the time evolutions of runoff, solid particles detachment and transfer and bacteria transport that were measured for one of the nine plots. After estimation of the saturated hydraulic conductivity, soil erodibility and attachment rate of bacteria, model outputs were consistent with measured runoff coefficients, suspended sediment and E. coli loads. This work therefore underlines the need to maintain adequate vegetation at the soil surface to avoid the erosion and export of soil borne potential pathogens towards downstream aquatic systems.

Do early diagenetic processes affect the applicability of commonly-used organic matter source tracking tools? An assessment through controlled degradation end-member mixing experiments.

In the development of organic matter (OM) source tracking tools, it is critical to validate if (1) the tracers are conservative with source mixing, and (2) they can be conservative under diagenetic processes (e.g., microbial degradation). In this study, these two critical points were rigorously tested for three commonly-used source tracking tools (i.e., absorbance and fluorescence proxies, stable carbon isotopes and lipid biomarkers) via a controlled experiment at laboratory scale. To this end, two end-members (e.g., soil and algae), which represent the most common and contrasted sources of OM to sediments in an aquatic environment, were mixed in different ratios and then incubated under different oxygen conditions (oxic versus anoxic) in the dark at 25 °C for 60 days. The initial and final signals of the source tracking tools were analyzed and compared for each mixing ratio. Based on three evaluation criteria concerning the linearity of the relationships, discrimination sensitivity, and conservative mixing behavior, we evaluated the applicability of the tools to trace the sediment organic matter in the aquatic environment. Although most of the source tracking proxies evaluated in this study showed a conservative nature after incubation, there are only a few that demonstrated both conservative behaviors with the sources mixing and under early diagenetic processes. The fluorescence proxies such as the relative distribution of a humic-like component associated with refractory source material (Ex/Em: 220/430 nm), modified fluorescence index (YFI), humification index (HIX), and carbon stable isotope ratios were identified to be the most reliable tracers for tracking sedimentary OM sources under early diagenetic processes. This study provides strong insights into the validation of common OM source tracking tools for sediment and a reasonable guideline to select the optimum indices for source discrimination via end-member mixing analysis.

Veterinary pharmaceutical residues in water resources and tap water in an intensive husbandry area in France.

In intensive livestock areas, veterinary pharmaceutical residues (VPRs) can occur in water resources, but also in tap water because treatment processes are not designed to remove these contaminants. The main objective of this study is to assess the occurrence of VPRs in water resources and tap waters in Brittany. As several identical compounds are used in both veterinary and human medicine, a toolbox (stanols and pharmaceuticals) is used to help determine the origin of contamination in the case of mixed-use molecules. Water resources samples were collected from 25 sites (23 surface waters and two groundwaters) used for tap water production and located in watersheds considered as sensitive due to intensive husbandry activities. Samples were also taken at 23 corresponding tap water sites. A list of 38 VPRs of interest was analyzed. In water resources, at least one VPR was quantified in 32% of the samples. 17 different VPRs were quantified, including antibiotics, antiparasitic drugs and anti-inflammatory drugs. Concentration levels ranged between 5 ng/L and 2946 ng/L. Mixed-use pharmaceuticals were quantified in twelve samples of water resources and among these samples nine had a mixed overall fecal contamination. In the context of this large-scale study, it appeared difficult to determine precisely the factors impacting the occurrence of VPRs. VPRs were quantified in 20% of the tap water samples. Twelve VPRs were quantified, including ten compounds exclusively used in veterinary medicine and two mixed-use compounds. Concentration levels are inferior to 40 ng/L for all compounds, with the exception of the antibiotic florfenicol which was quantified at 159 ng/L and 211 ng/L. The population of Brittany may therefore be exposed to these contaminants through tap water. These observations should be put into perspective with the detection frequencies per compound which are all below 10% in both water resources and tap water.

Faecal biomarkers can distinguish specific mammalian species in modern and past environments.

Identifying the presence of animals based on faecal deposits in modern and ancient environments is of primary importance to archaeologists, ecologists, forensic scientists, and watershed managers, but it has proven difficult to distinguish faecal material to the species level. Until now, four 5ß-stanols have been deployed as faecal biomarkers to distinguish between omnivores and herbivores, but they cannot distinguish between species. Here we present a database of faecal signatures from ten omnivore and herbivore species based on eleven 5ß-stanol compounds, which enables us to distinguish for the first time the faecal signatures of a wide range of animals. We validated this fingerprinting method by testing it on modern and ancient soil samples containing known faecal inputs and successfully distinguished the signatures of different omnivores and herbivores.

Veterinary pharmaceutical residues from natural water to tap water: Sales, occurrence and fate.

Veterinary pharmaceuticals (VPs) increasingly used in animal husbandry have led to their presence in aquatic environments -surface water (SW) or groundwater (GW) - and even in tap water. This review focuses on studies from 2007 to 2017. Sixty-eight different veterinary pharmaceutical residues (VPRs) have been quantified worldwide in natural waters at concentrations ranging from nanograms per liter (ng L-1) to several micrograms per liter (µg L-1). An extensive up-to-date on sales and tonnages of VPs worldwide has been performed. Tetracyclines (TCs) antibiotics are the most sold veterinary pharmaceuticals worldwide. An overview of VPRs degradation pathways in natural waters is provided. VPRs can be degraded or transformed by biodegradation, hydrolysis or photolysis. Photo-degradation appears to be the major degradation pathway in SW. This review then reports occurrences of VPRs found in tap water, and presents data on VPRs removal in drinking water treatment plants (DWTPs) at each step of the process. VPRs have been quantified in tap water at ng L-1 concentration levels in four studies of the eleven studies dealing with VPRs occurrence in tap water. Overall removals of VPRs in DWTPs generally exceed 90% and advanced treatment processes (oxidation processes, adsorption on activated carbon, membrane filtration) greatly contribute to these removals. However, studies performed on full-scale DWTPs are scarce. A large majority of fate studies in DWTPs have been conducted under laboratory at environmentally irrelevant conditions (high concentration of VPRs (mg L-1), use of deionized water instead of natural water, high concentration of oxidant, high contact time etc.). Also, studies on VPRs occurrence and fate in tap water focus on antibiotics. There is a scientific gap on the occurrence and fate of antiparatic drugs in tap waters.

Experimental evidence of REE size fraction redistribution during redox variation in wetland soil.

The evolution of rare earth element (REE) speciation between reducing and oxidizing conditions in a riparian wetland soil was studied relative to the size fractionation of the solution. In all size fractions obtained from the reduced and oxidized soil solutions, the following analyses were carried out: organic matter (OM) characterization, transmission electron microscopy (TEM) observations as well as major and trace element analyses. Significant REE redistribution and speciation evolution between the various size fractions were observed. Under reducing conditions, the REEs were bound to colloidal and dissolved OM (<2µm size fractions). By contrast, under oxidizing conditions, they were distributed in particulate (>2µm size fraction), colloidal (<2µm size fraction), organic and Fe-enriched fractions. In the particulate size fraction, the REEs were bound to humic and bacterial OM embedding Fe nano-oxides. The resulting REE pattern showed a strong enrichment in heavy REEs (HREEs) in response to REE binding to specific bacterial OM functional groups. In the largest colloidal size fraction (0.2µm-30kDa), the REEs were bound to humic substances (HS). The lowest colloidal size fraction (<30kDa) is poorly concentrated in the REEs and the REE pattern showed an increase in the middle REEs (MREEs) and heavy REEs (HREEs) corresponding to a low REE loading on HS. A comparison of the REE patterns in the present experimental and field measurements demonstrated that, in riparian wetlands, under a high-water level, reducing conditions are insufficient to allow for the dissolution of the entire Fe nano-oxide pool formed during the oxidative period. Therefore, even under reducing conditions, Fe(III) seems to remain a potential scavenger of REEs.

Application of a microbial source tracking based on bacterial and chemical markers in headwater and coastal catchments.

This study identified sources of fecal contamination in three different French headwater and coastal catchments (the Justiçou, Pen an Traon, and La Fresnaye) using a combination of microbial source tracking tools. The tools included bacterial markers (three host-associated Bacteroidales) and chemical markers (six fecal stanols), which were monitored monthly over one or two years in addition to fecal indicator bacteria. 168 of the 240 freshwater and marine water samples had Escherichia coli (E. coli) or enterococci concentrations higher than "excellent" European water quality threshold. In the three catchments, the results suggested that the fecal contamination appeared to be primarily from an animal origin and particularly from a bovine origin in 52% (Rum2Bac) and 46% (Bstanol) of the samples and to a lesser extent from a porcine origin in 19% (Pig2Bac) and 21% (Pstanol) of the samples. Our results suggested a human fecal contamination in 56% (HF183) and 32% (Hstanol) of the samples. Rainfall also impacted the source identification of microbial contamination. In general, these findings could inform effective implementation of microbial source tracking strategies, specifically that the location of sampling points must include variability at the landscape scale.

Nanoplastic in the North Atlantic Subtropical Gyre.

Plastics can be found in all ecosystems across the globe. This type of environmental pollution is important, even if its impact is not fully understood. The presence of small plastic particles at the micro- and nanoscales is of growing concern, but nanoplastic has not yet been observed in natural samples. In this study, we examined four size fractions (meso-, large micro-, small micro-, and nanoplastics) of debris collected in the North Atlantic subtropical gyre. To obtain the nanoplastic portion, we isolated the colloidal fraction of seawater. After ultrafiltration, the occurrence of nanoscale particles was demonstrated using dynamic light scattering experiments. The chemical fingerprint of the colloids was obtained by pyrolysis coupled with gas chromatography-mass spectrometry. We demonstrated that the signal was anthropogenic and attributed to a combination of plastics. The polymer composition varied among the size classes. At the micro- and nanoscales, polyvinyl chloride, polyethylene terephthalate, polystyrene and polyethylene were observed. We also observed changes in the pyrolytic signals of polyethylene with decreasing debris size, which could be related to the structural modification of this plastic as a consequence of weathering.

Are fecal stanols suitable to record and identify a pulse of human fecal contamination in short-term exposed shellfish? A microcosm study.

In this study, the capacity of oysters to bioaccumulate fecal stanols and to record a source-specific fingerprint was investigated by the short-term contamination of seawater microcosms containing oysters with a human effluent. Contaminated oysters bioaccumulated the typical fecal stanols coprostanol and 24-ethylcoprostanol and their bioaccumulation kinetics were similar to that of the Fecal Indicator Bacteria Escherichia coli used in European legislation. Although stanol fingerprints of contaminated water allowed the identification of the human specific fingerprint, this was not the case for oysters. This discrepancy is attributed to (i) high concentrations of endogenous cholestanol and sitostanol, responsible for "unbalanced" stanol fingerprints, (ii) different accumulation/depuration kinetics of fecal coprostanol and 24-ethylcoprostanol and (iii) the limits of the analytical pathway used. These results show that fecal stanols bioaccumulated by oysters are useful to record fecal contamination but the usefulness of stanol fingerprints to identify specific sources of contamination in shellfish currently seems limited.

Development of the analysis of fecal stanols in the oyster Crassostrea gigas and identification of fecal contamination in shellfish harvesting areas.

The objective of this work was to study the effects of washing and purification steps on qualitative and quantitative analysis of fecal stanols in the oyster Crassostrea gigas using either single or a combination of lipid purification steps on silica gel or aminopropyl bonded silica gel (NH2) or a washing step. Among the three analytical pathways compared, the two including water extraction or NH2 purification did not lead to higher recoveries and decreased repeatabilities of extractions compared to the single purification on silica gel. This latter led to similar recoveries (ca. 80%) and repeatabilities (ca. 10%) for both spiked standards (coprostanol and sitostanol). This analytical pathway has been applied to oysters collected in a harvesting area in Brittany (France) where fecal contaminations are important and allowed to quantify eight stanols in oysters. The relative proportions of fecal stanols of these oysters were combined with principal component analysis in order to investigate the usefulness of their stanol fingerprints to record a fecal contamination and to distinguish its source between human, porcine and bovine contaminations. Oysters non-fecally contaminated by Escherichia coli did not present specific stanol fingerprints while oysters fecally contaminated had a bovine fingerprint, suggesting a contamination of these samples by bovine sources. As a consequence, the method developed here allows the use of stanol fingerprints of oysters as a microbial source tracking tool that can be applied to shellfish harvesting areas subjected to fecal contaminations in order to identify the different sources of contamination and improve watershed management.

Extreme variability of steroid profiles in cow feces and pig slurries at the regional scale: implications for the use of steroids to specify fecal pollution sources in waters.

Thirty-five samples of cow feces (cowpat and cow manure) and pig slurries subjected to different treatment processes and different storage times before land spreading were extracted and analyzed by gas chromatography-mass spectrometry to determine their fecal stanol profiles. The fresh pig slurry data presented here increase considerably the classical range of values obtained for steroid ratios, resulting in an overlap with the range for cow feces. These results lead to the inability to distinguish species source of feces on the basis of steroid ratios alone. The cause of these differences is not known, although it appears likely to be related to differences in the metabolism of animals in relation to their age and/or variations in diet, rather than to secondary mechanisms of steroid degradation during storage or/and treatment of the feces. Nevertheless, the specificity of steroids to serve as a tool to differentiate cow feces from pig slurries is restored by considering the fecal stanol profile, notably, the six most diagnostic stanol compounds, which are 5ß-cholestan-3ß-ol (coprostanol), 5ß-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3ß-ol (campestanol), 24-ethyl-5α-cholestan-3ß-ol (sitostanol), 24-ethyl-5ß-cholestan-3ß-ol (24-ethylcoprostanol), and 24-ethyl-5ß-cholestan-3α-ol (24-ethylepicoprostanol). In this study, chemometric analysis of the fingerprint of these six stanols using principal components analysis (PCA) distinguished pig slurries from cow feces. The application of PCA to the stanol profiles, as developed in this study, could be a promising tool for identifying the animal source in fecal contamination of waters.

Microbial and chemical markers: runoff transfer in animal manure-amended soils.

Fecal contamination of water resources is evaluated by the enumeration of the fecal coliforms and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and ), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure.

Development of a combined isotopic and mass-balance approach to determine dissolved organic carbon sources in eutrophic reservoirs.

A combined mass-balance and stable isotope approach was set up to identify and quantify dissolved organic carbon (DOC) sources in a DOC-rich (9mgL(-1)) eutrophic reservoir located in Western France and used for drinking water supply (so-called Rophemel reservoir). The mass-balance approach consisted in measuring the flux of allochthonous DOC on a daily basis, and in comparing it with the effective (measured) DOC concentration of the reservoir. The isotopic approach consisted, for its part, in measuring the carbon isotope ratios (δ(13)C values) of both allochthonous and autochthonous DOC sources, and comparing these values with the δ(13)C values of the reservoir DOC. Results from both approaches were consistent pointing out for a DOC of 100% allochthonous origin. In particular, the δ(13)C values of the DOC recovered in the reservoir (-28.5±0.2‰; n=22) during the algal bloom season (May-September) showed no trace of an autochthonous contribution (δ(13)C in algae=-30.1±0.3‰; n=2) being indistinguishable from the δ(13)C values of allochthonous DOC from inflowing rivers (-28.6±0.1‰; n=8). These results demonstrate that eutrophication is not responsible for the high DOC concentrations observed in the Rophemel reservoir and that limiting eutrophication of this reservoir will not reduce the potential formation of disinfection by-products during water treatment. The methodology developed in this study based on a complementary isotopic and mass-balance approach provides a powerful tool, suitable to identify and quantify DOC sources in eutrophic, DOC-contaminated reservoirs.

Tracing and quantifying sources of fatty acids and steroids in amended cultivated soils.

Soluble organic fractions from soils of two agricultural sites from Brittany (France) have been analyzed to (i) identify the source of polar compounds in soils and (ii) evaluate the impact of organic fertilization and crop type on the distribution and concentration of polar compounds in soils. The main sources of polar compounds in soils are higher plants; they represent >70% of the polar compounds from the experimental sites and mainly originate from crop residues and animal manure. Crop type and animal manure application significantly increase the polar compound concentrations in soils. Among polar compounds, fatty acids cannot be used as specific markers because their distributions in soils whatever the crop type or organic fertilization type are the same. On the other hand, analysis of steroids provides interesting information. Cow and poultry manure applications increase only the concentration of steroids. Pig slurry fertilization modifies both the concentration and distribution of steroids. The identified pig slurry steroid fingerprint can persist in the soil for 9 years after the slurry application has been stopped. Those compounds are then robust markers to detect pig slurry contribution in soils.

Organic markers in the lipidic fraction of sewage sludges.

The lipidic organic fraction of 48 sewage sludges that originated from food-processing, paper-mill and domestic (urban, small urban, and rural) wastewater-treatment plants of the Lorraine region (Northeast of France) was characterised by gas chromatography coupled with mass spectrometer (GC-MS). This study enables us to define an average organic composition typical of each group of sewage sludges. Linear alkyl benzenes (LABs) are only present in domestic sludges, sterols in food-processing and domestic sludges. Paper-mill sludges are characterised by the specific distribution of n-alkanes. Besides, all the domestic sludges, whatever the size of the wastewater catchment, are characterised by the same distribution of polar compounds. Differences can be evidenced in the distribution of the aliphatic compounds of some domestic sewage sludges and are attributed to the important contribution of petroleum products in their sewer system. Moreover, this study highlights the correlation between abundance of polycyclic aromatic hydrocarbons (PAHs) in the aromatic fraction of some sewage sludges and a distribution of n-alkanes characteristic of heavy petroleum products into the corresponding aliphatic fraction. This might be a clue for the determination of punctual sources of PAHs.