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Experimental design and protocols
The experimental monitoring were carried out between 1st June 2015 and 31 August 2016, with sampling once a month and once every two weeks outside and during the period of irrigation by treated wastewater, respectively. Wastewaters samplings concerned (Figure II.1) raw wastewater just after screening (i.e. before sand and oil removals), treated wastewater at the outlet of the wastewater treatment plant, and treated wastewater at the outlet of the last (eighth) lagoon during the irrigation period. Conventional surface waters were sampled in the Artière River upstream and downstream of the treatment plant discharge and just before the confluence with the Allier River, and in the Allier River upstream and downstream from this confluence. Underground conventional waters were sampled within the Allier alluvial aquifer 7.5 km downstream of the confluence with the Artière River in wells used for agricultural irrigation at 80 and 400 m from the Allier bank. Additional measurements performed simultaneously concerned the temperature and the pH of the waters. Environmental concentrations in noroviruses (genogroups I and II), rotaviruses, enteroviruses, adenoviruses and hepatitis A viruses were monitored by using a procedure of water sampling, virus concentration and purification nearly identical to the procedure of Prevost et al. (2015b) that derived from the method 1615 of the US-Environmental Protection Agency (Cashdollar et al., 2013; Gibson et al., 2012). For each sampling of conventional surface or groundwater, 100 L of water were pumped at a rate of 2 L. minute-1 with a SMART L40 3-roller peristaltic pump (Verderflex®, Castleford, U.K.) using Verderprene® tubes 19 4.8 mm, and filtered in situ on Argonide® filter cartridges NanoCeram® VS2.5-5 (Argonide Corporation, Sanford, FL, U.S.) having a surface area of 12.9 dm2 within H2.5-5C Clear-Housing; NanoCeram® are electropositive filters with alumina fiber of 2 nm as active phase. After having been emptied of water, the filter cartridges within their Clear-Housing were returned under cold conditions to the laboratory. For each wastewater sampling (at the inlet and outlet of the wastewater treatment plant, and within lagoons), 1 L of water was stored in borosilicate bottles and returned in the Lab under cold conditions; it was then filtered on 90 mm non-laminated NanoCeram disks at about 100 mL.minute-1 water flow to ensure approximately the same flux of water with regard to filter surface, by using glass filter holder kit from Millipore.
Viruses were eluted from the filter using an elution buffer (0.05 M glycine, 1% w/v Bacto Beef Extract, 0.1% Na triphosphate, 0.1% Tween, 0.1% Silicon Emulsion Anto-foam) adjusted at pH=9.5 just before its use. 450 and 50 mL of this buffer were poured into each of the Clear-Housing containing their filter cartridge and into 50 mL Falcon tubes already containing the filter discs cut into small pieces, respectively. Clear-Housing and Falcon tubes were immersed during 1 h in an ultrasonic bath XUB 18 (Grant Instruments Ltd., Royston, UK) previously filled with fresh water and ice cubes to ensure a water temperature always below 10°C, at 16 W.L-1. The buffer solutions with eluted viruses were then transferred in 500 mL glass bottles and 50 mL Falcon tubes, and their pH was adjusted to 3.5 with concentrated HCl. They were gently stirred during 1 h at the laboratory temperature using a Reax 2 rotary shaker at minimum speed (Heidolph Instruments GmbH & Co, Walpersdorfer, Germany). The solutions were then centrifuged during 2 h at 4500 g and 4°C, using a centrifuge 6K15 (Sigma, US). After centrifugation, the pellet was resuspended in 8 mL phosphate buffer (0.15 M Na2HPO4 at pH=9.0) and centrifuged during 15 min at 4500 g and 4°C to clarify the viral concentrated suspension. The supernatant was filtered on a lowbinding Millex HV-HPF 0.45 μm, 25 mm in diameter. The viral suspensions were concentrated one last time and their beef extract that inhibit PCR partly removed by ultracentrifugation during 2 h at 143 000 g and 4°C on sucrose cushion (1.5 mL at 40% sucrose) on a L8-70M Ultracentrifuge (Beckman-Coulter Inc., US) using a SW41Ti rotor.
The supernatant was then eliminated with at least 0.5 mL of the sucrose cushion to minimize the amount of remaining beef extract. The pellet was resuspended in the 0.6 ml remaining sucrose cushion.
Water flow data
Additional data concerning the flow of rivers (Crouel on Artière River upstream discharge of wastewater (45.771548 N, 3.1424498 E), Vic-le-Comte on Allie River r upstream the Artière River confluence (45.661528, 3.2020719) and Limons on Allier River downstream the Artière River confluence (45. 968896, 3.448480)) were obtained from the Hydro databank of the French ‘Ministère de l’Ecologie, du Développement Durable et de l’Energie’ (Ministère de l’Ecologie, du Développement Durable et de l’Energie, 2015). Data relating to the wastewater treatment were provided by the wastewater treatment plant with the agreement of Clermont-Auvergne-Métropole. We assumed that the Artière River flow both downstream wastewater discharge and near the Allier River confluence were equal to the Artière River flow upstream the discharge decreased by water withdrawal for filling the lagoons. By doing this, we neglected other contributions to River flows, including small stream contribution (mainly Le Bec stream), surface runoff during rainy periods and exchanges between rivers and alluvial aquifers.
Epidemiologic data
Acute gastroenteritis cases and adapted drug data estimated through the reimbursement of medical expenses by the French Social Security and reported during the viral monitoring period and the previous 6 years were kindly provided by Santé Publique France, the French national public health agency. For each person with gastroenteritis who consulted a doctor, available information includes the age group (either 1-4, 5-65 or >65 years old), the residence place, and the medical prescriptions (antiemetic, antidiarrheal, both antiemetic and antidiarrheal, oral rehydration solutions (ORS) for <1 year or 1-15 years children, and others). Prescriptions entitled « Others » include antispasmodics and intestinal adsorbents. Note that antiemetics are very rarely prescribed, or even contraindicated, for children; for this reason, the proportion of vomiting people among those consulting a physician were estimated without taking into account people having ORS and « Others » prescriptions.
Epidemiologic and drug data were analysed during the period of virus monitoring. Total declared acute gastroenteritis cases with the optional distinction of prescriptions were quantified for each of the three age groups and related to the size of these groups according to the French official census of 2014 (i.e. 12 100, 184 600 and 41 000 inhabitants of 1-4, 5-65 and >65 years old, respectively, for the 19 towns included in the wastewater collection basin arriving at the ‘Trois Rivières’ wastewater treatment plant). After preliminary tests (results not shown), their temporal variations were described using sliding averages over 7 days to avoid insignificant fluctuations (associated with the randomness of infections) while avoiding erasing true temporal trends (Figure II.2).
Acute gastroenteritis and drug consumption; relationship with virus shedding
Over the 1st September 2015 – 31 August 2016 period, the 22 914 declared AGE cases corresponded to probabilities to have been ill and to consult a physician of about 0.096 for all the inhabitants of the wastewater collection basin. This probability depended on the age group in the order « 1-4 years »> »5-65 years »> »>65 years » (. Table II-1). The type of drugs varied with the age group: main differences between them concern young children (1-4 years old) who are first re-hydrated with oral rehydration solutions (ORS). Without taking into account the two ORS groups (26% of the young children prescriptions) and the ‘other’ group, the corrected ratio for vomitive symptoms concern 67, 82 and 70% of the « <5 years », « 5-65 years » and « >65 years » groups suggesting that viral symptoms could have concerned a great proportion of all the age groups. The probabilities of vomiting for each age group in the whole sick population were estimated from the probability of vomiting within the subset of sick people consulting a physician. Values for the <5 years’ group (0.48) and ‘>65 years’ group (0.51) were under the presently used threshold (0.52) to have viral gastroenteritis, whereas its higher value (0.67) for ‘5-65 years’ group lead to an estimate of 100% of viral cases within this group. However, working only on the annual proportion of vomiting symptoms didn’t enable to take into account periods during which the proportion of vomiting symptoms exceeds the 0.52 threshold retained in our study for exclusively non-viral gastroenteritis, and this threshold as well as the 0.66 threshold for exclusively viral gastroenteritis are questionable. In addition, the proportion of viral gastroenteritis cases in the < 5 years age class was surely underestimated, as antiemetics are rarely prescribed for young children (Assathiany et al., 2013); in 2008, antiemetics were still prescribed in France for about 75% of children < 9 years having vomiting symptoms (Pfeil et al., 2008).
Viral contaminations of wastewaters and freshwater bodies; virus flows
Quantified viruses were found almost systematically in raw wastewater with the exception of the hepatitis A virus, which was never detected (Figure II.5a). When above their detection thresholds, their concentrations were comprised between 10+3.1 and 10+7.0, 10+3.8 and 10+5.8, 10+5.1 and 10+7.0, 10+3.8 and 10+5.2, and 10+5.8 and 10+10.0 GC.L-1 for noroviruses GI, noroviruses GII, rotaviruses, enteroviruses and adenoviruses, respectively. When they were above their detection thresholds, their concentrations in treated wastewater at the outlet of the wastewater treatment plant (i.e. without additional tertiary treatment in lagoons) were about one to one thousandth times the corresponding values in raw wastewater (Figure II.5b). Detection was much less sensitive than that for river viruses due to the initial filtration of 100 L of water. Thus, since treated wastewater discharge contributed for 76% (between 39 and 90%) of the flow of Artière River downstream of their point of discharge, the flow of viruses at the outlet of the wastewater treatment plant was better assessed from the increase in the amount of viruses carried by the Artière River between upstream and downstream of the wastewater discharge point, increased by wastewater withdrawal for filling the lagoons and decreased by virus flow resulting from by-pass.
Virus removal in wastewater treatment plant was estimated by the ratio of the daily virus flow at the inlet of the wastewater treatment plant to the daily flow of viruses at the outlet of the treatment plant (see Annexe II). While the Artière River received only treated wastewater from a small treatment plant (120 PE) upstream the discharge by the ‘Trois Rivières’ treatment plant, virus concentrations upstream (results not shown) were sometimes of the same order of magnitude as virus concentrations downstream the discharge by the ‘Trois Rivières’ treatment plant (Figure II.5c), although generally lower. The annual geometric averages of virus removals within the wastewater treatment plant over the annual period 1st September 2015 – 31st August 2016 were 2.4, 1.9, 3.3, 2.6 and 2.5 log10 for the norovirus GI, norovirus GII, rotavirus, enterovirus and adenovirus, respectively. The high variability between estimates led to uncertainties in the seasonal variations (see Figure S4 in Annexe II); nonetheless, the differences of log10 removals between summer 2015 and winter 2016 were 0.9, 2.7, 1.7 and 2.2 for norovirus GI, norovirus GII, enterovirus and adenovirus, respectively. These orders of magnitude could be explained taking into account variations in wastewater temperature (see Figure S5 in Annexe II), and a rate of virus immobilization/destruction varying with temperature according to a Q10 equation with Q10 of about 1.4. However, for simultaneous raw and treated wastewater samplings at about 10 h a.m. U.T., the apparent virus removals exceed the daily averages of about 0.2, 0.3, 0.1 and 0.2 log10 for the considered periods in autumn, winter, spring and summer seasons, respectively (see Figure S6 in Annexe II).
Table of contents :
CHAPITRE I. SYNTHÈSE BIBLIOGRAPHIQUE
1. LES EAUX USÉES ET LEUR RÉUTILISATION
2. LES VIRUS ENTÉRIQUES PATHOGÈNES DE L’HOMME
A. DESCRIPTION GÉNÉRALE DES VIRUS
B. LES VIRUS ENTÉRIQUES
3. DEVENIR ENVIRONNEMENTAL DES VIRUS (ÉTAT DES CONNAISSANCES)
A. DEVENIR DANS LES EAUX USÉES
B. DEVENIR DANS LES EAUX DOUCES ET SOUTERRAINES
C. DEVENIR DANS LE SOL
CHAPITRE II. PROPOSITION ET ÉVALUATION D’UN MODÈLE DE PRODUCTION DE VIRUS ENTÉRIQUES PATHOGÈNES DE L’HOMME ET DE LEUR DEVENIR EN STATION D’ÉPURATION, EN RIVIÈRES ET EN NAPPE D’ACCOMPAGNEMENT POUR SIMULER LES IMPACTS DE DIFFÉRENTS MODES DE GESTION DES EAUX USÉES 31
1. ABSTRACT:
2. INTRODUCTION:
3. MATERIALS AND METHODS
A. EXPERIMENTAL CONTEXT
B. EXPERIMENTAL DESIGN AND PROTOCOLS
C. WATER FLOW DATA
D. EPIDEMIOLOGIC DATA
E. VIRAL SHEDDING, TRANSPORT AND REMOVAL MODELLING
4. RESULTS AND DISCUSSION
A. ACUTE GASTROENTERITIS AND DRUG CONSUMPTION; RELATIONSHIP WITH VIRUS SHEDDING
B. VIRAL CONTAMINATIONS OF WASTEWATERS AND FRESHWATER BODIES; VIRUS FLOWS
C. FLOW OF VIRUSES; FITTING SIMULATIONS BASED ON EPIDEMIOLOGY AND SHEDDING TO ESTIMATES BASED ON MEASUREMENTS.
5. CONCLUSIONS
CHAPITRE III. PROPOSITION ET ÉVALUATION D’UN MODÈLE D’ÉLIMINATION ET D’IMMOBILISATION RÉVERSIBLE DES NOROVIRUS MURINS DANS UN PHAEOZEM CALCIQUE POUR DIVERSES CONDITIONS DE CONTAMINATION ET DE RINÇAGE 63
1. SUMMARY
2. INTRODUCTION
3. MATERIALS AND METHODS
A. VIRUS, SOIL AND ARTIFICIAL SOIL SOLUTION
B. EXPERIMENTAL DESIGN AND PROCEDURE
C. PROCESS MODELING, UNCERTAINTY ANALYSIS AND EXPERIMENTAL DATA ANALYSIS
4. RESULTS AND DISCUSSION
A. IMPACT OF TEMPERATURE, AGGREGATE SIZE, SOIL SATURATION PROCEDURE AND WATER FILTRATION ON VIRUS CONCENTRATIONS
B. REVERSIBLE AND IRREVERSIBLE IMMOBILIZATION IN SATURATED CONDITIONS OVER A WEEK
C. SOIL DRYING IMPACT ON THE REVERSIBILITY OF IMMOBILIZATIONS
D. THE IMPACT OF GEOCHEMISTRY ON IMMOBILIZATION AND SUBSEQUENT DESORPTION
5. CONCLUSIONS AND PERSPECTIVES
CHAPITRE IV. DISCUSSION GÉNÉRALE – CONCLUSIONS
1. RAPPEL DES OBJECTIFS ET FINALITÉS DE LA THÈSE :
2. DEVENIR DES VIRUS DANS UN CONTEXTE DE REJET : ACQUIS ET LIMITES DU TRAVAIL
3. DEVENIR DES VIRUS DANS LE SOL DANS UN CONTEXTE DE RÉUTILISATION D’EAU USÉE : ACQUIS ET LIMITES DUTRAVAIL
4. DÉFIS À ABORDER POUR ATTEINDRE LES FINALITÉS DU TRAVAIL
BIBLIOGRAPHIE
