Anne-Sophie Le Gal
email: alegal@univ-perp.fr

Pauline Priol

Jean-Yves Georges

Olivier Verneau

Population structure and dynamics of the Mediterranean Pond turtle Mauremys leprosa (Schweigger, 1812) in contrasted polluted aquatic environments

Keywords: Testudines, Wastewater treatment plant, Water pollution, Capture-mark-recapture, Robust design model, Multi-state models Akaike information criterion AMPA: Aminomethylphosphonic acid BM: Body mass OSR: Operational sex ratio SCL: Straight carapace length SMI: Scaled mass index TSD: Temperature-dependent sex determination WWTP: Wastewater treatment plant

  L'archive ouverte pluridisciplinaire

Although inland waters cover less than 1% of the world's surface area, they are critical habitats for a highly diverse assemblage of animal and plant species [START_REF] Balian | The freshwater animal diversity assessment: an overview of the results[END_REF][START_REF] Dudgeon | Freshwater biodiversity : importance, threats, status and conservation challenges[END_REF]. Unfortunately, these ecosystems are being impacted by climate change leading to increased water temperature, and by human activities leading to physical degradation, biological invasions, and chemical pollution [START_REF] Rahel | Homogenization of freshwater faunas[END_REF][START_REF] Malmqvist | Threats to the running water ecosystems of the world[END_REF].

Urban wastewater, which is usually treated by wastewater treatment plants (WWTP) before being discharged into waterways, is one of the main sources of water pollution worldwide [START_REF] Kosmala | Evaluation écotoxicologique de l'impact des effluents de stations d'épuration sur Ies cours d'eau : intérêt d'une approche intégrée[END_REF][START_REF] Kümmerer | Drugs in the environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sourcesa review[END_REF]. Wastewater is composed of dissolved organic matter, inorganic nutrients, and microcontaminants [START_REF] Marti | Nutrient retention efficiency in streams receiving inputs from wastewater treatment plants[END_REF][START_REF] Mandaric | Impact of urban chemical pollution on water quality in small, rural and effluent-dominated Mediterranean streams and rivers[END_REF].

Inorganic nutrient inputs promote algal growth and primary production [START_REF] Carey | Contribution of wastewater treatment plant effluents to nutrient dynamics in aquatic systems: a review[END_REF]) that provide abundant trophic resources for herbivorous and detritivorous consumers [START_REF] Debruyn | The role of sewage in a large river food web[END_REF][START_REF] Singer | Anthropogenic subsidies alter stream consumer-resource stoichiometry, biodiversity, and food chains[END_REF]. However, excessive nitrate and phosphate discharges can produce ammonia, which can have sublethal or lethal effects on aquatic organisms [START_REF] Manahan | Environmental chemistry[END_REF]. Microcontaminants such as drug residues can bioaccumulate through the food web [START_REF] Vinodhini | Bioaccumulation of heavy metals in organs of freshwater fish Cyprinus carpio (Common Carp)[END_REF] and modify the sex ratio of some species [START_REF] Vajda | Demasculinization of male fish exposed to estrogenic wastewater effluent[END_REF]. As a whole, wastewater discharges into aquatic environments have been reported to reduce community specific richness and diversity [START_REF] Mor | Invertebrate community responses to urban wastewater effluent pollution under different hydro-morphological conditions[END_REF], negatively impact pollution-sensitive predators [START_REF] Mor | Energy limitation or sensitive predators? Trophic and non-trophic impacts of wastewater pollution on stream food webs[END_REF], and ultimately disturb stream food webs (Emmanuel, 2014;[START_REF] Sabater | Effects of human-driven water stress on river ecosystems: a meta-analysis[END_REF].

Some invertebrates and vertebrates can survive in highly polluted environments [START_REF] Buikema | Effects of pollution on freshwater invertebrates[END_REF][START_REF] Kreutz | Acute toxicity test of agricultural pesticides on Silver Catfish (Rhamdia quelen) fingerlings[END_REF] through genetic mechanisms [START_REF] Posthuma | Heavy-metal adaptation in terrestrial invertebrates: A review of occurrence, genetics, physiology and ecological consequences[END_REF][START_REF] Reid | The genomic landscape of rapid repeated evolutionary adaptation to toxic pollution in wild fish[END_REF]. Such species are useful proxies for measuring watershed pollutions and their effects on wildlife. By exploiting both terrestrial and aquatic environments, J o u r n a l P r e -p r o o f aquatic (marine and freshwater) turtles are exposed to diverse pollutions throughout their life cycle [START_REF] Gibbons | Why do turtles live so long?[END_REF][START_REF] Vargo | Study of the effects of oil on marine turtles[END_REF]. Agriculture pesticides can be absorbed by turtle eggs laid in the ground [START_REF] De Solla | Organochlorine pesticides, PCBs, dibenzodioxin, and furan concentrations in common Snapping Turtle eggs (Chelydra serpentina serpentina) in Akwesasne, Mohawk Territory, Ontario, Canada[END_REF][START_REF] De Solla | Absorption of current use pesticides by Snapping Turtle (Chelydra serpentina) eggs in treated soil[END_REF] and reduce embryo survivorship [START_REF] Nagle | Accumulation and selective maternal transfer of contaminants in the turtle Trachemys scripta associated with coal ash deposition[END_REF]. Pollutants can also be transferred from female turtles to the yolks of their eggs, which can interfere with hatchling sex determination [START_REF] Thompson | Major global changes interact to cause male biased sex ratios in a reptile with temperature-dependent sex determination[END_REF] and early development [START_REF] Bishop | Temporal and geographic variation of organochlorine residues in eggs of the common Snapping Turtle (Chelydra serpentina serpentina) and comparisons to trends in the Herring Gull (Larus argentatus) in the Great Lakes basin in Ontario, Canada[END_REF][START_REF] De Solla | Toxicity of pesticides associated with potato production, including soil fumigants, to Snapping Turtle eggs (Chelydra serpentina)[END_REF], depending on the pollutant and its concentration. Similarly, as a result of the exposure to pollutants in aquatic environments, genomic damages, physiological changes, and reduced fitness can occur in adult turtles [START_REF] Adel | Mercury, lead, and cadmium in tissues of the Caspian Pond Turtle (Mauremys caspica) from the southern basin of Caspian Sea[END_REF][START_REF] Hutchinson | A review of the effects of pollution on marine turtles[END_REF][START_REF] Meyers-Schöne | Comparison of two freshwater turtle species as monitors of radionuclide and chemical contamination: DNA damage and residue analysis[END_REF]. These impacts threaten the long-term survival of both marine and freshwater turtles [START_REF] Hopkins | Mercury exposure is associated with negative effects on turtle reproduction[END_REF][START_REF] Iverson | Patterns of survivorship in turtles (Testudines)[END_REF][START_REF] Vargo | Study of the effects of oil on marine turtles[END_REF][START_REF] Wilbur | Life history evolution in turtles[END_REF]. Because freshwater turtles can be easily captured and exhibit measurable responses at the individual and population levels when exposed to pollutants, they are considered suitable bioindicators for monitoring long-term exposures to contaminants in continental hydrosystems [START_REF] Greenfield | Introduction to wetland monitoring[END_REF][START_REF] Meyers-Schöne | Comparison of two freshwater turtle species as monitors of radionuclide and chemical contamination: DNA damage and residue analysis[END_REF].

The Mediterranean Pond Turtle Mauremys leprosa (Schweigger, 1812) is a freshwater turtle endemic to the western Mediterranean basin, distributed from North Africa (Libya, Tunisia, Algeria, and Morocco) to southern Europe (Spain, Portugal, and southern France) [START_REF] Bertolero | Mauremys leprosa (Schoepff in Schweigger 1812) -Mediterranean Pond Turtle, Spanish Terrapin, Mediterranean Stripe-necked Terrapin[END_REF]. This wetland-dwelling species inhabits mainly lentic environments of Mediterranean rivers, which are mostly characterized by very low flows, except during exceptional massive floods in winter. These extreme weather events have major effects, both in terms of water flow and anthropogenic pollution discharge [START_REF] Mandaric | Impact of urban chemical pollution on water quality in small, rural and effluent-dominated Mediterranean streams and rivers[END_REF][START_REF] Reoyo-Prats | Multicontamination phenomena occur more often than expected in Mediterranean coastal watercourses: Study case of the Têt River (France)[END_REF]. The densest recorded populations are mostly located in heavily degraded environments, where turtles appear to benefit from water, organic matter, and macroinvertebrate supplies provided by the pollution sources [START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF]. Because J o u r n a l P r e -p r o o f the body size and reproductive output of M. leprosa are enhanced in environments degraded by organic waste and pollutants coming from industrial, agricultural, and urban sources, this species appears to be well adapted to pollution [START_REF] Da Silva | Notes on clutch size and egg size of Mauremys leprosa from Spain[END_REF][START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF][START_REF] Gahmous | Reproductive traits demonstrate how well the Mediterranean Stripe-necked Turtle Mauremys leprosa can flourish under highly degraded-polluted conditions[END_REF][START_REF] Naimi | Clutch and egg allometry of the turtle Mauremys leprosa (Chelonia: Geoemydidae) from a polluted peri-urban river in west-central Morocco[END_REF]. As a result of its tolerance to high levels of pollution and eutrophication of its habitat, M. leprosa has been proposed as a biological indicator of pollution [START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF]. It has been shown that it could bioaccumulate pollutants, affecting its body condition [START_REF] Slimani | Large-scale geographic patterns of mercury contamination in Morocco revealed by freshwater turtles[END_REF]. Additionally, an increased oxidative stress has been reported, suggesting that pollutants can have adverse effects on its physiology in polluted environments [START_REF] Héritier | Oxidative stress biomarkers in the Mediterranean Pond Turtle (Mauremys leprosa) reveal contrasted aquatic environments in Southern France[END_REF]. These deleterious effects could ultimately affect population status, as it has been reported in other freshwater turtles [START_REF] Bergeron | PCBs as environmental estrogens: turtle sex determination as a biomarker of environmental contamination[END_REF][START_REF] Polo-Cavia | Body condition does not predict immunocompetence of western pond turtles in altered versus natural habitats[END_REF]. Considered together these results suggest potential trade-offs between the benefits derived from water and food supplies provided by the pollution sources on the one hand, and deleterious effects of the contaminants on the other.

This study explored the medium-term trends of a population of M. leprosa inhabiting the Fosseille River, a Mediterranean river located in the peri-urban area of Cabestany City, in southern France, where a WWTP is settled. To investigate the extent effluents of this WWTP impact the quality of the Fosseille River, a set of pesticides commonly found in the surface waters of the Pyrénées-Orientales county were examined in the upstream section of the river, in effluents coming down from the WWTP, and in the section located downstream of the WWTP. Although WWTPs are set up for cleaning urban waters, not all pollutants can be completely degraded [START_REF] Saleh | Removal of pesticides from water and wastewater: Chemical, physical and biological treatment approaches[END_REF], so we expected an increasing pollution gradient from upstream to downstream of the WWTP. To highlight the potential effects of pollution on the population, morphology, body condition, survival and transition were studied among turtles sampled upstream and downstream of the WWTP. Ultimately, we sought to determine whether the primary driver of population functioning is WWTP-induced resources or WWTP-induced J o u r n a l P r e -p r o o f pollutions by testing two hypotheses : H1 -WWTP-induced resources are the primary driver of population functioning (as suggested by El [START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF], and H2 -WWTP-induced pollutions are the primary driver of population functioning. If H1 is correct, body condition, as well as density and survival of turtles downstream of the WWTP should increase, leading turtles to seek beneficial effluents by transiting from upstream to downstream of the WWTP.

Alternatively, if H2 is correct, body condition, density, and survival of turtles downstream of the WWTP should decrease, leading turtles to evade the polluted area by transiting from downstream to upstream of the WWTP.

Material and methods

Study area

The field work was carried out in the Fosseille River, nearby the city of Cabestany (42°40'N 2°57'E), which is located on the Mediterranean basin, in Southern France (Fig. 1). The river benefits from a Mediterranean climate: the average minimum and maximum temperatures were 12.7 ± 0. WWTP outfall into the Fosseille River, two river sections were defined: upstream of the WWTP and downstream of the WWTP (Fig. 1). Following a bed of common reed Phragmites australis that is dry except during rainy events, the upstream section flows over 0.6 kilometer. The second river section permanently receives wastewater from the WWTP and is 1.5 kilometers long. It J o u r n a l P r e -p r o o f continues to flow in brackish water that provides an unsuitable environment for M. leprosa, based on field observations. Between the two river sections there is a 0.5 kilometer stretch completely filled with P. australis, which is dry most of the year except during rainy episodes in winter, spring, and fall, thus constituting a temporary obstacle to aquatic migrations. While the hydrological characteristics of the two river sections differ from each other because of the WWTP, the morphological characteristics of the river are homogeneous along its entire length (personal observations), with a width of one to four meters, a maximum depth of two meters, and a sandy-muddy mixed substrate. Agricultural fields with crops and grapevines are located along the entire length of the river, and the edge vegetation consists of sparse vegetation, dominated by P. australis, the lakeshore bulrush Schoenoplectus lacustris, and the yellow flag Iris pseudacorus.

Levels of water pollution

Wastewater treatment plants aim to treat industrial and domestic wastewater before it is discharged into waterways, but some incoming contaminants cannot be completely eliminated.

Additionally, non-point source pollution (e.g., pesticides, fertilizers) can be washed into waterways by rain and drainage water from cultivated soils. To determine if the Cabestany WWTP effluents contribute to the chemical degradation of the Fosseille River, water samples were collected from the two river sections as well as from the outfall of the WWTP (Fig. 1). preference to avoid biasing capture probabilities. The annual capture effort consisted of two to three capture-mark-recapture sessions, lasting two to three days, which were conducted every one to four weeks depending on the year. When a turtle was captured, it was individually identified by notching the marginal scutes of the carapace [START_REF] Cagle | A system of marking turtles for future identification[END_REF] and its sex was determined (MAL for males, and FEM for females) by examining the secondary sexual characteristics such as plastron concavity, and the distance from the plastron notch to the cloaca opening [START_REF] Pérez | Crecimiento de Mauremys caspica leprosa (Schweigger, 1812) (Reptilia, Testudines) en la Reserva Biológica de Doñana[END_REF][START_REF] Meek | Aspects of the population ecology of Mauremys capsica in north west Africa[END_REF][START_REF] Busack | Variation in Mediterranean populations of Mauremys Gray 1869 (Reptilia, Testudines, Emydidae)[END_REF][START_REF] Fritz | Die Europäische Sumpfschildkröte (Emys orbicularis)[END_REF]. Because secondary sexual characters are first apparent from 80 mm straight carapace length in the study population, individuals smaller than 80 mm were considered as immatures (IMM), without reference to sex. The body mass (BM) was measured using a balance (precision = 0.1 g), and the straight carapace length (SCL) with a hand caliper (precision = 1 mm). After measurements, turtles were released at the place of capture.

Data analysis

2.4.1. Levels of pesticides

J o u r n a l P r e -p r o o f

The concentrations of pesticides were classified into four magnitude classes: 0.00-0.01, 0.01-0.10, 0.10-1.00, 1.00-10.00 µg.L -1 (Appendix A). Following point-sampling on river sections and outfall (2018/08, 2021/05, and 2021/09), the pesticides detected in the water were identified and quantified (Fig. 2). Finally, to test the difference in pesticide concentrations between the three water sections, non-parametric Wilcoxon rank sum tests were performed for each of the three sampling periods because of the non-normality of the data. the turtle group and river section set as fixed effects, and the turtle identity set as random effect to account for pseudo replication [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. To determine the adult sex ratio, hereafter referred as the operational sex ratio (OSR), of the turtles' groups located in upstream and downstream WWTP sections (Ns = 2) across the study period, a weight relative to the number of individuals caught was associated with each annual sex ratio (Ny = 7). The number of distinct males and females caught every year (y) on each river section (s) was used to calculate the OSR as below. The OSR difference between the upstream and downstream sections was tested using a Wilcoxon rank-sum test.

J o u r n a l P r e -p r o o f OSR y,s = MAL y,s MAL y,s + FEM y,s

; OSR total,s = ∑ (N y,s * OSR y,s ) y y=1:7

Body condition

The individual body condition is a relevant parameter on animal relative body reserves, health, quality, and vigor [START_REF] Peig | New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method[END_REF] readily available to monitor populations [START_REF] Lecq | Body condition and habitat use by Hermann's tortoises in burnt and intact habitats[END_REF]. To investigate the body condition, the scaled mass index (SMI) was calculated following the procedure developed by [START_REF] Peig | New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method[END_REF]. The SMI is a hypothetical mass that allows for consideration of the relationship between body mass and body length within the group of interest, and the impact of individual variability and measurement errors on BM and SCL variables. Because of age and sex-specific body composition, immatures, males, and females were analyzed separately. To avoid bias in the measurements related to the reproductive status of the females, the morphometric measurements were acquired at the beginning of the activity season of each year, i.e., outside the breeding season. To assess the effect of the two river sections on SMI in each group of turtles, ANOVA were used on linear mixed-effects models, with the individual identity set as a random effect to account for pseudo replication [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF].

Statistical analyses were performed in R environment (RStudio Team, 2020), using the lme4 [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF], car [START_REF] Fox | An R companion to applied regression[END_REF], and lmodel2 (Legendre, 2018) R packages, and data visualization was done using the ggplot2 R package [START_REF] Wickham | Ggplot2: Elegant graphics for data analysis[END_REF]. All estimates were provided with standard errors. Significance levels were set as 5%.

Population dynamics

J o u r n a l P r e -p r o o f

Validation of models' application conditions

Because turtle population stability depends on adult survival [START_REF] Blamires | Influence of habitat and predation on population dynamics of the freshwater turtle Myuchelys georgesi[END_REF][START_REF] Crouse | A stage-based model for loggerhead sea turtles and implications for conservation[END_REF][START_REF] Escoriza | Demographics and survivorship in the European Pond Turtle (Emys orbicularis) : a 31-years study[END_REF][START_REF] Frazer | Sea turtle conservation and halfway technology[END_REF][START_REF] Galbraith | Survivorship of adult females in a northern population of common snapping turtles, Chelydra serpentina[END_REF][START_REF] Heppell | Application of life-history theory and population model analysis to turtle conservation[END_REF], only adult population dynamics were considered for modelling. For turtles that were captured first as immature and later as adult, only the capture history of adults was considered in the models. Because transience and trap-dependence can bias estimates of population dynamics models, a goodness-of-fit test [START_REF] Pradel | Multievent: an extension of multistate capture-recapture models to uncertain states[END_REF] was performed using U-Care 2.3.2 [START_REF] Pradel | Estimating population growth rate from capture-recapture data in presence of capture heterogeneity[END_REF], and the occurrence of trap-dependence was tested with the CAPTURE program, implemented in MARK 6.2 [START_REF] White | Program MARK: Survival estimation from populations of marked animals[END_REF]. To perform robust-design models, intra-annual sessions were assumed to be closed, with no additions (recruitment, births)

or losses (permanent emigrations, deaths) in the population. To ensure the condition of application of this model, closure tests were performed using CloseTest [START_REF] Stanley | A closure test for time-specific capture-recapture data[END_REF].

Robust design

A robust design Pradel survival and seniority model [START_REF] Pradel | Utilization of capture-mark-recapture for the study of recruitment and population growth rate[END_REF][START_REF] Pradel | Estimating population growth rate from capture-recapture data in presence of capture heterogeneity[END_REF]) was used to estimate population trends over the study period (2013-2018 and 2021). It considered the primary and secondary sessions' data to estimate the probability of capture (p) and recapture (c), the survival (φ), the seniority, and the population size (N). The probability of recruitment (γ) is estimated from the seniority (γ = 1 -seniority). To permit the models to converge and provide outputs, the capture and recapture probabilities were considered equivalent. Both the additive and interactive effects of the following variables were tested to build the robust design models: season (spring and summer), year (2013-2018 and 2021), catching effort per intraannual session (< 100 and 100-200 traps.days), and sex. Because no capture was done in 2019

J o u r n a l P r e -p r o o f and 2020, these two years were excluded by setting specific time intervals in the specifications for the MARK analyses. In a first step, the covariates of recapture probability were tested, with both survival and seniority probabilities depending on the interaction between sex and year. In a second step, the covariates of seniority were tested on the best model of recapture probability, with the survival depending on the interaction between sex and year. In a third step, the covariates of survival probability were tested on the best models of recapture and seniority probabilities. A set of 26 models was obtained and then fitted using MARK 6.2. Model selection was based on AIC [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF], and the least number of parameters (Appendix B).

Multi-state

Multi-state for live recaptures models [START_REF] Nichols | Estimating transition probabilities for stage-based population projection matrices using capture-recapture data[END_REF][START_REF] Nichols | Estimating breeding proportions and testing hypotheses about costs of reproduction with capture-recapture data[END_REF], with the states being the WWTP upstream and downstream river sections, were used to evaluate dynamic parameters upstream and downstream of the WWTP. These models estimated the inter-annual probabilities of survival (φ), recapture (c), and inter-section transition (Ψ), using all intra-year captures merged to build a yearly dataset. The effect of cumulate intra-annual catching efforts (< 250, 250-350, and > 350 traps.days), sex, year, and river sections, were tested on the probabilities of recapture, survival, and transition. The years of analyses ranged from 2013 to 2021, with 2019 and 2020 excluded as explained above. In a first step, the recapture probability covariates were tested, holding the survival and transition probabilities dependent on the interaction between sex, year, and river section. In a second step, the different assumptions on transition probabilities were tested on the best recapture probability model, holding the survival probability dependent on the interaction between sex, year, and river section. In a third step, the different assumptions on survival probabilities were tested on the best recapture and transition probability models. A set of 44 models was obtained and fitted using MARK 6.2. Model J o u r n a l P r e -p r o o f selection was based on AIC and the least number of parameters (Appendix C). Because three models were supported, AIC weighting and a model averaging procedure were used to get the average estimate.

Results

Levels of pesticides

The daily minimal temperature, maximal temperature and cumulative rainfall were 24°C, 33°C pollution levels at the outfall and downstream of the WWTP were similar to each other and both higher than upstream of the WWTP (Fig. 2). Because pollution levels at the outfall did not J o u r n a l P r e -p r o o f differ significantly from the pollution levels assessed from the WWTP downstream river section, only WWTP upstream and downstream sections were considered further for the population structure and dynamics study, the WWTP downstream section then referring to both outfall and second river section.

Population dynamics

Raw captures of turtles

From 2013 to 2018 and in 2021, a total of 786 captures contained 358 distinct turtles. A total of 90 distinct immature turtles were captured among 123 captures (i.e., 15.6% of the total captures), with 57 turtles being recaptured as immatures only, and 33 being recaptured as adults over the study period. A total of 301 distinct adult turtles were captured among 663 captures (i.e., 84.4% of the total captures), including 213 males and 88 females. Of the adults, 58 (30 males and 28 females) and 243 (183 males and 60 females) turtles were captured upstream and downstream of the WWTP, respectively. The population density was 97 adults.km -1 and 122 adults.km -1 upstream and downstream of the WWTP, respectively.

Models' application conditions

Transience and trap dependence phenomena, as well as population closure, were tested prior to modeling to avoid bias in population dynamics estimates. Throughout the study period, 132 individuals (87 males and 45 females) were captured only once. Transience was not detected in either males (GOF test, z = -0.98; p = 0.84) or females (z = 0.58; p = 0.28). Both closure and trap-dependence tests could be performed for years with a minimum of three capture sessions

Medium-term demographics

Robust design Pradel survival and seniority modelling was used to determine population dynamics and trends over the study period. Two models had most of the support, with a combined AIC weight of 0.97 (Appendix B). Both models considered that the probability of capture depended on the interaction between year and catching effort, and that the seniority depended on the year. However, while the first model considered that survival was stable over time, the second model considered that survival depended on sex. Because these models were separated by a ΔAICc of 2.19, the most parsimonious model was selected to assess population trends. The retained robust design model, with an AIC of 666.53 and an AIC weight of 0.73, was p(effort x year) φ( .) γ(year). This model indicated that throughout the 18 capture sessions performed over the study period, (i) the probability of capture depended on the interaction between year and catching effort (p = 0.17 ± 0.02; min = 0.06 ± 0.01 (2017); max = 0.32 ± 0.04 (2013); Fig. 3), (ii) the survival was stable (φ = 0.88 ± 0.02) regardless the sex, and (iii) the recruitment depended on the year (γ = 0.14 ± 0.07; min = 0; max = 0.42 ± 0.07; Fig. 3). The adult population size remained stable over the study period and was estimated to Kruskal-Wallis' H = 0.8, p < 0.01; Fig. 4), resulting in a male biased OSR of 0.67.

Transition probabilities

The multi-state models estimated the population dynamic parameters for the upstream and downstream river sections. Three models had most of the support, with a combined AIC weight of 0.87 (Appendix C). They diverged in their estimates of survival by considering survival as stable (model 1), sex-dependent (model 2), or river section-dependent (model 3). For all three models, the probability of recapture varied across the years with bidirectional transition.

Because these three models were separated by low ΔAICc of 0.92 and 1.83, a model averaging was performed to estimate the population dynamics parameters. The model averaging procedure indicated that (i) the probability of recapture (c) varied across years (0.29 ± 0.05), ranging from 0.05 ± 0.02 in 2017 to 0.41 ± 0.09 in 2021 (Appendix D), (ii) the survival estimate was 0.90 ± 0.03 for males and 0.91 ± 0.03 for females, being similar between upstream and downstream of the WWTP (φ = 0.91 ± 0.03), and (iii) the transition occurred preferentially in one direction, with 17% of the individuals transiting from upstream to downstream of the WWTP annually (ψ up-down = 0.17 ± 0.04; ψ down-up = 0.05 ± 0.01; Fig. 5). Raw captures indicated that only four individuals (two males and two females) transited in both directions during their capture history. belonged to the predominant size class 80-120 mm (Fig. 6). In contrast, the distribution of turtles in the size classes was more balanced in 2021, with 92% of the turtles (n = 95 of 103 turtles captured) being distributed in three predominant size classes: 80-120, 120-160, and 160-200 mm (Fig. 6).

Population

OSR, morphology, and body condition

To determine how population structure could be impacted by WWTP-induced pollutions, sex ratio and body condition were examined over the entire study period, upstream and downstream of the WWTP. Concerning sex ratio: OSR was balanced upstream (OSRtotal,up = 0.48 ± 0.01) and male biased downstream (OSRtotal,down = 0.76 ± 0.00), and this difference was significant over the study period (n = 7 years; Wilcoxon's W = 28, p = 0.02). Concerning body condition:

J o u r n a l P r e -p r o o f firstly, SCL and BM values of immatures were significantly higher downstream than upstream of the WWTP (SCL: ANOVA's F(1,54) = 8.86, p < 0.01; BM: F(1,54) = 7.34, p = 0.01), but SMI values of immatures (SMIimm = BMi x (62/SCLi) 2.4 ) did not differ between upstream and downstream sections (ANOVA's F(1,52) = 1.20, p = 0.28) (Appendix E). Secondly, neither SCL nor BM values of males varied between the two sections (SCL: ANOVA's F(1,310) = 0.14, p = 0.71; BM: F(1,261) = 0.07, p = 0.80), whereas males SMI (SMIM = BMi x (119/SCLi) 2.8 ) did not vary (ANOVA's F(1,350) = 0.06, p = 0.80) (Appendix E). Thirdly, SCL values of females were higher downstream than upstream of the WWTP (SCL: ANOVA's F(1,109) = 8.59, p < 0.01), BM values of females tended to be higher downstream than upstream of the WWTP (ANOVA's F(1,94) = 3.10, p = 0.08), and SMI values of females (SMIF = BMi

x (141/SCLi) 2.9 ) tended to be significantly higher downstream that upstream of the WWTP (ANOVA's F(1,129) = 3.89, p = 0.05) (Appendix E).

Discussion

Origin and fate of pesticides

Our study on pesticides in 2018 and 2021 indicated that the Fosseille River is heavily polluted.

Importantly, whether both the diversity and concentration of pesticides were higher downstream than upstream of the WWTP, it showed similar pesticides levels between outfall and downstream of the WWTP, indicating that the WWTP did not fully removing the pesticides that were likely originating in the urban area (Fig. 2). Although runoff and leaching from cultivated surfaces during rain events represent another source of river contamination [START_REF] Reoyo-Prats | Multicontamination phenomena occur more often than expected in Mediterranean coastal watercourses: Study case of the Têt River (France)[END_REF], the low rainfalls by end of summer 2021 (see above section 2.2) could not explain the occurrence of pesticides. The wide range of contaminants detected in September 2021, upstream and downstream of the WWTP, could be related to the timing of agricultural J o u r n a l P r e -p r o o f pest control in the region, with vineyard treatments running from January to September (Chambre d'Agriculture du Tarn, 2022). The four recurring parent compounds recorded over the three sampling periods were glyphosate, diuron, imidacloprid, and terbutryn, all four of which are classified as very toxic to aquatic life with long-lasting effects under the Globally Harmonized System of Classification and Labeling of Chemicals (National Center for Biotechnology Information, 2022). The AMPA was also detected during all three sampling periods. This by-product can result from the degradation of glyphosate, but also from aminomethylene-polyphosphonate, which are components of detergents, washing machines, and cooling systems. Because the half-life of the above-described five compounds is less than one year, their presence in surface waters indicated their recent use following agricultural practices, which was unexpected for terbutryn, diuron, and imidacloprid that are restricted for use in the European Union (European Chemicals Agency, http://echa.europa.eu/, accessed 2023/04/13).

Their high toxicity and persistence in the water column makes them likely to impact ecosystems in the long-term. As far as freshwater turtles are concerned, some pesticides are endocrine disruptors that can interfere with hatchling behavior and survival (Neuman-Lee and Janzen, 2011), as well as in early development [START_REF] Bishop | Temporal and geographic variation of organochlorine residues in eggs of the common Snapping Turtle (Chelydra serpentina serpentina) and comparisons to trends in the Herring Gull (Larus argentatus) in the Great Lakes basin in Ontario, Canada[END_REF].

Population trends

Our study revealed that the M. leprosa population over the two sections of the Fosseille River and outfall was composed of about 200 adults and has remained stable over the nine years of the study period. The composition of the captures showed that the population was mainly composed of adults (84%) of which 2/3 were males. M. leprosa exhibited a high survival rate (91%) similar to that reported in other studies [START_REF] Bakhouche | Phenology and population structure of the Mediterranean Stripe-necked Terrapin Mauremys leprosa (Schweigger, 1812) in the Reghaia Lake (Northern Algeria)[END_REF][START_REF] Keller | Ecología de poblaciones de Mauremys leprosa y Emys orbicularis en el Parque Nacional de Doñana[END_REF], indicating that this species can tolerate WWTP-induced pollution and can survive in a degraded environment over the medium-term. In our study, the most parsimonious robust design model J o u r n a l P r e -p r o o f indicated a capture probability consistent yet slightly lower than in former studies on the European Pond Turtle Emys orbicularis (Linnaeus, 1758), another freshwater turtle with a similar ecology [START_REF] Escoriza | Demographics and survivorship in the European Pond Turtle (Emys orbicularis) : a 31-years study[END_REF][START_REF] Olivier | Assessing spatial and temporal population dynamics of cryptic species : an example with the European Pond Turtle[END_REF]Owen-Jones et al., 2015), permitting estimation of demographic parameters. The robust design model also indicated that the detected bias in the OSR neither resulted from a higher mortality in females, nor from a higher recruitment in males, although the low recruitment rates may have prevented the detection of unequal recruitment between males and females. Because M. leprosa is a long-lived and slow maturing animal, a long-term monitoring may detect a sex dependent adult survival in the population, following bimaturism [START_REF] Lovich | Does the timing of attainment of maturity influence sexual size dimorphism and adult sex ratio in turtles?[END_REF]. These results were consistent with previous studies that reported biased adult sex ratio, toward either males [START_REF] Bakhouche | Phenology and population structure of the Mediterranean Stripe-necked Terrapin Mauremys leprosa (Schweigger, 1812) in the Reghaia Lake (Northern Algeria)[END_REF][START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF][START_REF] Palacios | Demographic structure and genetic diversity of Mauremys leprosa in its northern range reveal new populations and a mixed origin[END_REF] or females [START_REF] Domínguez | Biometría sex ratio y tasa de recaptura de una población de Galápago Leproso Mauremys leprosa del Sur de la Península Ibérica[END_REF][START_REF] Keller | Ecología de poblaciones de Mauremys leprosa y Emys orbicularis en el Parque Nacional de Doñana[END_REF].

Influence of the WWTP effluents on the population dynamics

The capture data showed that the OSR was balanced upstream (OSR = 0.46), yet male biased downstream of the WWTP (OSR = 0.76), illustrating a positive covariation between biased sex ratio and pollution levels. The multi-state model revealed that this male bias in the sex ratio did not result from increased mortality of females downstream of the WWTP. Additionally, as the model indicated that transitions occurred primarily from upstream to downstream of the WWTP, with 1/5 of the turtles transiting annually regardless their sex, it demonstrates that the male biased sex-ratio observed downstream of the WWTP did not result from sex dependent transition rates. Therefore, the male biased sex ratio downstream of the WWTP is likely the result of a biased sex ratio in younger age classes, which might result from pollution. Indeed, the biased OSR can originate from a variation in hatchlings or primary sex ratio, influenced by either environment or genetic sexual determination [START_REF] Ewert | Sex determination in turtles: Diverse patterns and some possible adaptive values[END_REF]. In this regard, [START_REF] Thompson | Major global changes interact to cause male biased sex ratios in a reptile with temperature-dependent sex determination[END_REF] considered that environmental pollution could increase the proportion J o u r n a l P r e -p r o o f of males in clutches of the Snapping Turtle Chelydra serpentina (Linnaeus, 1758), via maternal transfer of endocrine disruptors impacting sexual determination, or following modifications in temperature-dependent sexual determination (TSD). Consistent with this, the absence of a sex chromosome in M. leprosa [START_REF] Díaz-Paniagua | Galápago leproso -Mauremys leprosa[END_REF] suggests TSD in that species. Because it has been reported that endocrine disrupting pollutants can interfere with TSD in reptiles [START_REF] Crews | The role of estrogen in turtle sex determination and the effect of PCBs[END_REF][START_REF] Kohno | Endocrine disruption and reptiles: Using the unique attributes of temperature-dependent sex determination to assess impacts[END_REF][START_REF] Thompson | Major global changes interact to cause male biased sex ratios in a reptile with temperature-dependent sex determination[END_REF][START_REF] Warner | Corticosterone exposure during embryonic development affects offspring growth and sex ratios in opposing directions in two lizard species with environmental sex determination[END_REF], a comparative study of the sex ratio of M. leprosa hatchlings under different temperature and pollution conditions would be relevant to conduct. Such experiments should be done by following field and laboratory protocols implemented for the survey of C. serpentina (see [START_REF] Thompson | Major global changes interact to cause male biased sex ratios in a reptile with temperature-dependent sex determination[END_REF].

Influence of the WWTP effluents on the population structure

To test the hypothesis that turtles primarily transit from upstream to downstream of the WWTP to benefit from the water and trophic resources provided by effluents, morphometrics and body condition of turtles were analyzed on both WWTP upstream and downstream sections.

Examination of turtles' size classes at the beginning and at the end of this nine years long study period highlighted the sexual size dimorphism in M. leprosa, with females being larger than males both upstream and downstream of the WWTP. This was consistent with Lovich et al.

(2010), who previously reported sexual size dimorphism in this species. Besides, females tended to have a higher body condition downstream than upstream of the WWTP, as previously reported for that species in another heavily polluted river [START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF]. A higher body condition of females compared to males has been already reported in M. leprosa [START_REF] Escoriza | Habitat overlap and body condition in aquatic turtles: are there additive effects between invasive and native species?[END_REF], and may result from an increased energy storage in females for reproductive allocation. Some studies have suggested that the high nutrient content provided by sewage can benefit aquatic invertebrates and thus turtle nutrition [START_REF] Germano | Ecology of western pond turtles (Actinemys marmorata) at sewagetreatment facilities in the San Joaquin Valley, California[END_REF][START_REF] Lindeman | Comparative life history of painted turtles (Chrysemys picta) in two habitats in the inland Pacific Northwest[END_REF].

J o u r n a l P r e -p r o o f

Similarly, detritus coming from effluents have been observed in the diet of the Caspian Turtle, Mauremys caspica (Gmelin, 1774), in polluted areas [START_REF] Sidis | Food habits of the Caspian Terrapin (Mauremys caspica rivulata) in unpolluted and polluted habitats in Israel[END_REF]. Because M.

leprosa has an opportunistic diet consisting primarily of aquatic arthropods [START_REF] Bakhouche | Phenology and population structure of the Mediterranean Stripe-necked Terrapin Mauremys leprosa (Schweigger, 1812) in the Reghaia Lake (Northern Algeria)[END_REF][START_REF] Díaz-Paniagua | Galápago leproso -Mauremys leprosa[END_REF], effluent resources may enhance its food supply by providing a suitable ecological niche for preys throughout the year, that may be beneficial for turtles' body condition. However, a better body condition in turtles can also be associated with a deterioration of health status [START_REF] Polo-Cavia | Body condition does not predict immunocompetence of western pond turtles in altered versus natural habitats[END_REF][START_REF] Rangel-Mendoza | Health and aquatic environment assessment of captive Central American river turtles, Dermatemys mawii, at two farms in Tabasco, Mexico[END_REF]. Turtles transiting from upstream to downstream of the WWTP may have been attracted by abundant resources, but they can also have passively transited from the upstream to the downstream sections during rain seasonal events, after both sections were hydrologically connected.

Conclusions and prospects

Our study focused on Mauremys leprosa, a long-lived and low-dispersal turtle species that exploits terrestrial environments during the earliest life history stages (i.e., incubation of eggs), and both terrestrial and aquatic environments from hatchling to adult stages. We showed that a turtle population located in a highly degraded river was stable over the medium term, suggesting that it can cope with environmental pollution. Although both diversity and concentration of the investigated pesticides were higher in the river section located downstream of the WWTP hydrologically connected to the Fosseille River, turtle transitions occurred primarily towards the downstream section. The turtle population density was the highest downstream, with both immatures and adult females being larger than those located upstream, whereas no such difference was observed in adult males. In addition, the sex ratio was male-biased downstream of the WWTP. Our results thus supported our first hypothesis, i.e., the primary driver of the population structure and dynamics of the study population is WWTP-induced resources, consistent with previous findings [START_REF] El Hassani | Morphological and physiological assessments reveal that freshwater turtle (Mauremys leprosa) can flourish under extremely degraded-polluted conditions[END_REF][START_REF] Gahmous | Reproductive traits demonstrate how well the Mediterranean Stripe-necked Turtle Mauremys leprosa can flourish under highly degraded-polluted conditions[END_REF][START_REF] Naimi | Clutch and egg allometry of the turtle Mauremys leprosa (Chelonia: Geoemydidae) from a polluted peri-urban river in west-central Morocco[END_REF]

J

  o u r n a l P r e -p r o o f 2.3. Turtle capture and handling Field captures occurred during the peak activity of the turtles, from March to September, in the years 2013 to 2018, and 2021. Capture-mark-recapture protocols consisted in deploying baited collapsible mesh crayfish traps every 100 m along the river borders. The size of the traps used for animal capture depended to the water level (60 x 30 cm or 80 x 35 cm). Each trap contained an empty water bottle to prevent the turtles from drowning. Traps were deployed evenly throughout the two river sections and outfall. No assumptions were made about turtle location

  2.4.2. Population structure 2.4.2.1. Composition of the captures To study the size-class structure of the population and whether it has changed over time, size classes were analyzed for years 2013 and 2021 because they represented the beginning and end of our study period, and because they had the highest annual capture rates. From measurements of SCL, five turtle size classes were defined: < 80, 80-120, 120-160, 160-200, ≥ 200 mm. To assess the effects of WWTP-induced pollution on SCL and BM in turtles captured, analyses of variance (ANOVA) were used on linear mixed-effects models following normality tests, with

  and 4 mm on the 2018/08/07, 15°C, 20°C, and 0 mm on the 2021/05/06, and 20°C, 31°C, and 0 mm on the 2021/09/27 (Historique-Météo.net, http://www.historique-meteo.net/, accessed 2023/03/10). Of a total of 68 pesticides and by-products searched in the water, 16 pesticides were detected: eight were detected at least one time upstream of the WWTP (i.e., 12% occurrence), 14 were detected at least one time at the WWTP outfall (21% occurrence), and 15 were detected at least one time downstream of the WWTP (22% occurrence) (Appendix A; Fig.2). Five residues were systematically detected in the Fosseille River during the three sampling periods, including aminomethylphosphonic acid (AMPA), diuron, glyphosate, imidacloprid, and terbutryn, with AMPA and glyphosate detected at the highest concentrations. The concentrations in pesticides were higher downstream than upstream of the WWTP (Wilcoxon rank-sum tests ; 2018/08/07: W = 86, p = 0.05 ; 2021/05/06: W = 86, p = 0.07 ; 2021/09/27: W = 70, p = 0.02), higher at the outfall than upstream of the WWTP in 2021 (2018/08/07: W = 110, p = 0.33; 2021/05/06: W = 86, p = 0.07; 2021/09/27: W = 59.5, p = 0.01), and not significantly different between the outfall and downstream of the WWTP (2018/08/07: W = 151, p = 0.32; 2021/05/06 : W = 125, p = 0.92; 2021/09/27 : W = 106.5, p = 0.43). In short,

  (i.e., 2013, 2018, and 2021). There was no trap-dependence in 2013 or 2018, where M(t) was J o u r n a l P r e -p r o o f selected as the appropriate model, i.e., the probability of capture varied over time (t) but not among individuals. However, trap-dependence related to the turtle's sex was detected in 2021, as M(h) was selected as the appropriate model, i.e., heterogeneity (h) among individuals impacted the capture probability. The intra-annual population closure test indicated that the population was closed in 2013(Stanley and Burnham test, N hat = 168, χ² = 0.17, p = 0.68), 2018 (N hat = 144, χ² = 0.43, p = 0.51), and 2021 (N hat = 234, χ² = 9.06, p = 0.06). Finally, neither transience nor trap dependence (except in 2021) was detected, and the population was closed intra-annually. Thus, the application conditions were validated to perform population dynamics models.

J

  o u r n a l P r e -p r o o f approximatively 200 adults (min = 162 individuals (2014), max = 238 individuals (2021)) (Fig.4). The rate of population change for each interval varied from 0.88 in 2015-2016 to 1.53 in 2014-2015 (lambda-hat mean = 1.05 ± 0.10), indicating a low population growth. The males (134 ± 9 turtles) outnumbered the females (67 ± 5 turtles) in the population (n = 14 years.sex,

  structure J o u r n a l P r e -p r o o f 3.3.1. Size classes To assess whether transition of turtles from upstream to downstream of the WWTP could be related to a better environment downstream of the WWTP, changes in size classes between the first (2013) and last (2021) years of the study period were investigated. Most of the captured turtles overall were males: 60 of the 105 turtles in 2013 (i.e., 57% ) and 87 of the 145 turtles in 2021 (i.e., 60%) (Fig.6). Most of the captures occurred downstream of the WWTP: 80 captures in 2013 (i.e., 76%) and 103 captures in 2021 (i.e., 71%). Upstream of the WWTP, turtle SCL ranged from 34 to 222 mm in 2013, and from 52 to 203 mm in 2021. In 2013, 88% of the turtles (n = 22, out of 25 turtles captured) belonged to size classes 80-120, 120-160, and 160-200 mm (Fig. 6). In contrast, in 2021, the distribution of turtles in the size classes was unbalanced, with 83% of the turtles (n = 35, out of 42 turtles captured) distributed in size classes < 80 mm and 80-120 mm (Fig. 6). Downstream of the WWTP, turtle SCL ranged from 36 to 206 mm in 2013, and from 61 to 211 mm in 2021. In 2013, 44% of the turtles (n = 35 of 80 turtles captured)

FiguresFigure 1 .

 1 Figures

Figure 2 .Figure 4 .Figure 6 .

 246 Figure 1. a. South of France; b. The Pyrénées-Orientales county, with the study area near

Acknowledgments

We thank our colleagues Bruno Charrière, Christine Sotin, and Charlyne Groussard for their help in the field in 2021, and Laurent Héritier, Léon Meyer, Noémie Régli and Marine Pezin who contributed to the data collection in the field from 2013 to 2018. We also thank the members of the LUTOX project who contributed to the collection of the 2018 water samples, J o u r n a l P r e -p r o o f

Funding sources

Anne-Sophie Le Gal received a fellowship from the MESRI (Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation), and a funding from the CNRS and the University of Perpignan (BQR) was granted to Anne-Sophie Le Gal and Olivier Verneau for the 2021 field activities and chemical analyses.

2012

). However, the masculinization of the population could affect its sustainability over the long term. Therefore, future studies should first focus the sex ratio of clutches laid under different temperature and pollution levels, to determine whether such environmental variables may affect the sexual determination of M. leprosa; and second, on the bioaccumulation of pollutants in the follicles of M. leprosa in polluted environments, to determine whether morphometrics may be impacted by pollutants.

Credit authorship contribution statement

Anne-Sophie Le Gal: Conceptualization, Methodology, Field work, Data curation, Formal analysis, Writingoriginal draft and editing. Pauline Priol: Methodology, Formal analysis, Writingreview and editing. Jean-Yves Georges: Conceptualization, Methodology, Writing review and editing, Supervision. Olivier Verneau: Conceptualization, Methodology, Field work, Writingreview and editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendices

Appendix A. Concentration of the 16 pesticides detected in the river sections sampled on 2021/08/07 (2018_08), 2021/05/06 (2021_05), and 2021/09/27 (2021_09). Non-detected pesticides are shown in white, and each level of gray represents a range of concentrations (in µg.L-1): 0.00-0.01 (gray 1), 0.01-0.10 (gray 2), 0.10-1.00 (gray 3), and 1.00-10.00 (gray 4). J o u r n a l P r e -p r o o f

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.