Veterinary pharmaceutical contamination in mixed land use watersheds: from agricultural headwater to water monitoring watershed.

Veterinary pharmaceuticals, widely used in intensive livestock production, may contaminate surface waters. Identifying their sources and pathways in watersheds is difficult because i) most veterinary pharmaceuticals are used in human medicine as well and ii) septic or sewer wastewater treatment plants (WWTP) can release pharmaceuticals into surface water, even in agricultural headwater watersheds. This study aimed to analyze the spatiotemporal variability of animal-specific, mixed-use, and human-specific pharmaceuticals, from agricultural headwaters with intensive livestock production and a WWTP to a watershed used for Water Framework Directive monitoring. Grab sampling was performed during one hydrological year upstream and downstream from a WWTP and at three dates in seven nested watersheds with areas of 1.9-84.1km2. Twenty pharmaceuticals were analyzed. Animal-specific pharmaceuticals were detected at all sampling dates upstream and downstream from the WWTP and at concentrations higher than those of human-specific pharmaceuticals. The predominance of animal-specific and mixed-use pharmaceuticals vs. human-specific pharmaceuticals observed at these sampling points was confirmed at the other sampling points. Animal-specific pharmaceuticals were detected mainly during runoff events and periods of manure spreading. A large percentage of mixed-use pharmaceuticals could come from animal sources, but it was difficult to determine. Mixed-use and human-specific pharmaceuticals predominated in the largest watersheds when runoff decreased. In areas of intensive livestock production, mitigation actions should focus on agricultural headwater watersheds to decrease the number of pathways and the transfer volume of veterinary pharmaceuticals, which can be the main contaminants.

Storage of natural water samples and preservation techniques for pharmaceutical quantification.

In order to perform a human and ecological risk assessment of pharmaceutical products (PPs) in natural waters, it is necessary to accurately quantify a broad variety of PPs at low concentrations. Although numerous currently implemented analytical methodologies, less is known about the preservation of PPs in natural water samples within the period before analysis (holding time, storage conditions). This paper is the first literature review about the stability of PPs in natural waters (surface and groundwaters) during sample storage. The current work focuses on a comparison of the performances of the available preservation techniques (filtration, container materials, storage temperature, preservative agents, etc.) for PPs in samples. All 58 reviewed PPs may be successfully stabilized during 7 days in surface waters by at least one appropriate methodology regarding temperature, acidic and non-acidic preservatives. When temperature is not a sufficient preservation parameter for some PPs (hormones and fluoxetine) its combination with the addition of chemical agents into the samples may prolong the integrity of the PPs during storage in surface water. There is a strong need to use standard protocols to assess and compare the stability of PPs in environmental water matrices during storage as well as during analytical preparation or analysis (European criteria 2002/657/EC). Since the stability of PPs during sample storage is a critical parameter that could call into question the quality of the data provided for the concentrations, the design of stability studies should rigorously take into account all critical parameters that could impact the concentrations of the PPs with time.

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers.

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 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)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)" methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.

Relative decay of fecal indicator bacteria and human-associated markers: a microcosm study simulating wastewater input into seawater and freshwater.

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations.

Persistence of microbial and chemical pig manure markers as compared to faecal indicator bacteria survival in freshwater and seawater microcosms.

Natural seawater and freshwater microcosms inoculated with pig manure were set up to determine the persistence of pig faecal microbial and chemical markers in these two types of surface water. The concentrations of Lactobacillus amylovorus, the Bacteroidales Pig-2-Bac 16S rRNA genetic marker, five stanols and the evolution of two ratios of stanols, R1 (coprostanol to the sum of coprostanol and 24-ethylcoprostanol) and R2 (sitostanol to coprostanol) were analyzed during two months along with the concentration of Faecal Indicator Bacteria (FIB). Pig manure was inoculated to unfiltered water microcosms incubated aerobically at 18 °C in the dark. The faecal contamination load represented by the concentrations of culturable Escherichia coli and/or enterococci remained for two months in the freshwater and seawater microcosms water column. These concentrations followed a biphasic decay pattern with a 97% reduction of the initial amount during a first rapid phase (<6 days) and a remaining proportion undergoing a slower or null second decline. The L. amylovorus marker and five stanols persisted as long as the indicators in both treatments. The Pig-2-Bac marker persisted 20 and 27 days in seawater and freshwater, respectively. The ratios R1 and R2 were in the range specific to pig manure until day 6 in both types of water. These results indicate that Pig-2-Bac, L. amylovorus and stanol ratios might be used in combination to complement FIB testing to determine the pig source of fecal pollution. However, stanol ratios are to be used when the time point of the discharge is known.

Influence of salinity and natural organic matter on the solid phase extraction of sterols and stanols: application to the determination of the human sterol fingerprint in aqueous matrices.

Faecal sterols have been proposed as direct chemical markers for the determination of faecal contamination in inland and coastal waters. In this study, we assess the impact of (a) the concentration of dissolved organic carbon (DOC), (b) the nature of DOC, (c) the salinity and (d) the concentration of sterols and stanols on their solid phase extraction. When natural organic matter (NOM) is modelled by humic acid, increasing DOC concentration from 2.7 to 15.4 mg/L has no significant impact on the recovery of sterols and stanols. The modelling of NOM by a mixture of humic acid and succinoglycan induces a significant (24%) decrease in the recovery of sterols and stanols. For all concentrations of target compounds, no significant increase in recovery is associated with increasing the salinity. Moreover, an increase in the recovery of target compounds is induced by an increase in their concentration. The nine target compounds and the recovery standard (RS) exhibit the same behaviour during the extraction step. Thus, we propose that (a) the concentration of target compounds can be corrected by the RS to calculate more realistic concentrations without modifying their profile and (b) the sterol fingerprint can be investigated in the colloidal fraction of aqueous samples without altering the information it could provide about the source. The application of this analytical method to waste water treatment plant influent and effluents yields results in agreement with previous studies concerning the use of those compounds to differentiate between sources of faecal contamination. We conclude that this analytical method is fully applicable to the determination of sterol fingerprints in the dissolved phase (<0.7 µm) of natural aqueous samples.

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France.

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.

Influence of natural organic matter on the solid-phase extraction of organic micropollutants. Application to the water-extract from highly contaminated river sediment.

In freshwater systems, organic micropollutants are bound to natural organic matter (NOM), which is responsible for a decrease in their recoveries by solid-phase extraction (SPE). This "negative effect" has been investigated for the SPE of polycyclic aromatic hydrocarbons (PAHs), oxygenated PAHs, nitrated PAHs and n-alkanes from salt water using Aldrich humic acid as a model of NOM. The effect has been partially obviated by the addition of isopropanol as a surfactant. The SPE protocol, developed with isopropanol, has been applied to the water-extract of a highly contaminated sediment. The water-extract has been size fractionated by cross-flow ultrafiltration into particulate (PM), colloidal (CM) and truly dissolved matter (tDM). Organic extracts from SPE experiments have been analyzed by gas chromatography-mass spectrometry. The major classes of molecules are heteroaromatic PAHs and PAHs. Those molecules are mainly bound to the tDM, which highlights: (1) the competition between organic micropollutants and natural organic molecules for available sorption sites and (2) the toxicological hazard linked to the mobilization of sediments highly contaminated by both industrial and urban activities.