Jérémy Lemaire
email: jeremy.lemaire@univ-lr.fr

Paco Bustamante

Rosanna Mangione

Olivier Marquis

Carine Churlaud

Maud Brault-Favrou

Charline Parenteau

François Brischoux

Lead, mercury, and selenium alter physiological functions in wild caimans (Caiman crocodilus)

Keywords: Blood chemistry, Trace elements, Crocodilians, Corticosterone, Osmoregulation

Environmental contaminants affect ecosystems worldwide and have deleterious effects on biota. Non-essential mercury (Hg) and lead (Pb) concentrations are well documented in some taxa and are described to cause multiple detrimental effects on human and wildlife.

Additionally, essential selenium (Se) is known to be toxic at high concentration but, at lower concentrations, Se can protect organisms against Hg toxicity. Crocodilians are known to bioaccumulate contaminants. However, effects of these contaminants on physiological processes remain poorly studied. In the present study, we quantified Hg, Pb and Se concentrations in spectacled caimans (Caiman crocodilus) and investigated effects of these contaminants on several physiological processes linked to osmoregulatory, hepatic, endocrine and renal functions measured through blood parameters in 23 individuals. Mercury was related to disruption of osmoregulation (sodium levels), hepatic function (alkaline phosphatase levels) and endocrine processes (corticosterone levels). Lead was related to disruption of hepatic functions (glucose and alanine aminotransferase levels). Selenium was not related to any parameters, but the Se:Hg molar ratio was positively related to the Na + and corticosterone concentrations, suggesting a potential protective effect against Hg toxicity. Overall, our results suggest that Hg and Pb alter physiological mechanisms in wild caimans and highlight the need to thoroughly investigate the consequences of trace element contamination in crocodilians.

INTRODUCTION

Environmental contamination is recognized to be a widespread phenomenon, leading to increasing concerns as to its potential impact on biodiversity [START_REF] Fleeger | Indirect effects of contaminants in aquatic ecosystems[END_REF][START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF][START_REF] Brühl | Biodiversity decline as a consequence of an inappropriate environmental risk assessment of pesticides[END_REF][START_REF] Ferronato | Waste mismanagement in developing countries: a review of global issues[END_REF][START_REF] Pain | Effects of lead from ammunition on birds and other wildlife: a review and update[END_REF][START_REF] Kasonga | Endocrine-disruptive chemicals as contaminants of emerging concern in wastewater and surface water: a review[END_REF]. Environmental contaminants have multiple sources, and their recent increase mainly relates to anthropogenic activities such as fossil fuel combustion, chemical production and use, and mining activities [START_REF] Richardson | Emerging environmental contaminants: Challenges facing our next generation and potential engineering solutions[END_REF]. In addition, global changes have recently been identified as a cause of modification in the distribution and behaviour of environmental contaminants at a global scale [START_REF] Noyes | The toxicology of climate change: Environmental contaminants in a warming world[END_REF][START_REF] Obrist | A review of global environmental mercury processes in response to human and natural pertirbations: Changes of emissions, climate and land use[END_REF]. Providing information on environmental contamination levels and distribution across geographical areas, biomes or species is critical. One of the current challenges is to quantify how environmental contaminants affect physiological functions in various organisms and to understand the potential consequences for wildlife and human health.

Mercury (Hg) and lead (Pb) are two non-essential trace elements which are present in ecosystems worldwide and induce deleterious effects on humans and wildlife [START_REF] Clarkson | The toxicology of mercury and its chemical compounds[END_REF]Wani et al., 2015;[START_REF] Evers | The effects of methylmercury on wildlife; a comprehensive review and approach for interpretation[END_REF][START_REF] Pain | Effects of lead from ammunition on birds and other wildlife: a review and update[END_REF]. They naturally occur in the environment, though human activities increase their levels (e.g., fuel combustion, mining activities and ammunition from hunting, [START_REF] Fisher | A review of lead poisoning from ammunition sources in terrestrial birds[END_REF][START_REF] Pirrone | Global mercury emissions to the anthropogenic and natural sources[END_REF][START_REF] Beckers | Cycling of mercury in the environment: Source, fate, and human health implications: A review[END_REF][START_REF] Fry | Anthropogenic contamination of residential environments from smelter As, Cu and Pb emissions: Implications for human health[END_REF]. In addition, Hg and Pb bioaccumulate in various tissues of organisms (Vizuete et al., 2018;[START_REF] De Almeida Rodrigues | Mercury in aquatic fauna contamination: a systematic review on its dynamics and potential health risks[END_REF][START_REF] Pain | Effects of lead from ammunition on birds and other wildlife: a review and update[END_REF]. They have multiple adverse effects on reproductive, renal and hepatic functions, and are recognized as immune and endocrine disruptors [START_REF] Eisler | Lead hazards to fish, wildlife, and invertebrates: a synoptic review. Contaminant Hazard Reviews[END_REF][START_REF] Fingerman | Impact of heavy metal exposure on the nervous system and endocrine-mediated processes in crustaceans[END_REF][START_REF] Pattee | Lead in the environment[END_REF]Zahir et al., 2005;[START_REF] Scheuhammer | Relationships among mercury, selenium and neurochemical parameters in common loons (Gavia immer) and bald eagles (Haliaeetus leucocephalus)[END_REF][START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF][START_REF] Grillitsch | The ecotoxicology of metals in reptiles[END_REF][START_REF] Bergeron | Interactive effects of maternal and dietary mercury exposure have latent and lethal consequences for amphibian larvae[END_REF][START_REF] Hopkins | Mercury exposure is associated with negative effects on turtle reproduction[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]Whitney and Cristol, 2017;[START_REF] Monclús | Lead contamination in raptors in Europe: A systematic review and meta-analysis[END_REF]. Selenium (Se) is an essential trace element which is involved in functions of various physiological processes, such as immune system and thyroid hormone homeostasis [START_REF] Roy | Supplementation with selenium restores age-related decline in immune cell function[END_REF][START_REF] Ramauge | Evidence that type III iodothyronine deiodinase in rat astrocyte is a selenoprotein[END_REF][START_REF] Köhrle | Selenium, the Thyroid, and the Endocrine System[END_REF][START_REF] Avery | Selenium, Selenoproteins, and Immunity[END_REF][START_REF] Qian | Selenium and selenoproteins in prostanoid metabolism and immunity[END_REF]. Its sources are both natural and anthropogenic with major releases into the environment from coal combustion [START_REF] Rowe | Ecotoxicological implications of aquatic disposal of coal combustion residues in the united states: a review[END_REF][START_REF] Lemly | Aquatic selenium pollution is a global environmental safety issue[END_REF][START_REF] He | Selenium contamination, consequences and remediation techniques in water and soils: A review[END_REF]Ullah et al., 2019). A disruption of the normal Se balance can negatively affect metabolic processes [START_REF] Rayman | The importance of selenium to human health[END_REF][START_REF] Taylor | Recent developments in selenium research[END_REF][START_REF] Finger | Chronic ingestion of Coal Fly-Ash contaminated prey and its effects on health and immune parameters in juvenile American Alligators (Alligator mississippiensis)[END_REF]. Selenium toxicity is known to affect growth, reproduction, and the immune system [START_REF] Heinz | Reproduction in mallards fed selenium[END_REF][START_REF] Hoffman | Interactive effects of boron, selenium and dietary protein on survival, growth, and physiology in mallard duckling[END_REF][START_REF] Hoffman | Role of selenium toxicity and oxidative stress in aquatic birds[END_REF][START_REF] Hopkins | Trophic and maternal transfer of selenium in brown snakes (Lamprophis fuliginosus)[END_REF][START_REF] Naderi | A comprehensive review on the neuropathophysiology of selenium[END_REF]. Nevertheless, due to its high affinity to Hg, Se qualifies as a natural antagonist: Low Se concentrations can protect organisms against toxic effects of Hg by reduction or even elimination of its toxicity [START_REF] Sugiura | Selenium protection against mercury toxicity: high binding affinity of methylmercury by selenium-containing ligands in comparison with sulfur-containing ligands[END_REF][START_REF] Gajdosechova | The role of selenium in mercury toxicity -Current analytical techniques and futures trends in analysis of selenium and mercury interactions in biological matrices[END_REF][START_REF] Rahman | Selenium and zinc protections against metal-(loids)-induced toxicity and disease manifestations: A review[END_REF].

Crocodilians, being apex predators, accumulate high levels of metal contaminants due to their life history characteristics (e.g. aquatic habitat, long lifespan, high trophic level, high tissue conversion rate) and can serve as indicators of ecosystem health (Yanochko et al., 1997, Vieira et al., 2011;[START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF][START_REF] Somaweera | The ecological importance of crocodylians: towards evidence-based justification for their conservation[END_REF]Lemaire et al., 2021a). However, many crocodilians have a concerning conservation status, and populations of several species are currently decreasing [START_REF] Targarona | Crocodylus rhombifer. The IUCN Red List of Threatened Species[END_REF][START_REF] Ferreira | Degradation of the crocodile population in the Olifants River Gorge of Kruger National Parck, South Africa[END_REF][START_REF] Bezuijen | Crocodylus siamensis. The IUCN Red List of Threatened Species[END_REF]Van Weerd et al., 2016;[START_REF] Balaguera-Reina | Crocodylus intermedius. The IUCN Red List of Threatened Species[END_REF][START_REF] Ortiz | Long-term monitoring reveals population decline of Spectacled Caimans (Caiman crocodilus) at a Black-Water Lake in Ecuadorian Amazon[END_REF]. Few studies which focused on a limited number of species have evaluated Pb and Hg concentrations in crocodilians [START_REF] Burger | Metals and metalloids in tissues of American Alligators in three Florida lakes[END_REF][START_REF] Jeffree | Element concentrations in the flesh and osteoderms of Estuarine Crocodiles (Crocodylus porosus) from the Alligator Rivers Region, Northern Australia: biotic and geographic effects[END_REF][START_REF] Correia | Mercury contamination in alligators (Melanosuchus niger) from Mamirauá Reservoir (Brazilian Amazon) and human health risk assessment[END_REF]Trillanes et al., 2014;Nilsen et al., 2017b). Further knowledge on their negative effects is restricted to four crocodilian species (Alligator mississippiensis, Caiman crocodilus, Paleosuchus trigonatus and Crocodylus niloticus) and few markers (morphology, DNA and reproduction; [START_REF] Lance | Chronic incidental lead ingestion in a group of captive-reared alligators (Alligator mississippiensis): possible contribution to reproductive failure[END_REF]Warner et al., 2016;Nilsen et al., 2017a;[START_REF] Marrugo-Negrete | Mercury levels and genotoxic effect in caimans from tropical ecosystems impacted by gold mining[END_REF]Lemaire et al., 2021b).

To our knowledge, no studies have investigated the combined effects of Hg, Pb and Se, and the possible protective effect of Se on physiological parameters of caimans, particularly in French Guiana, where said contaminants are highly abundant in the environment.

To investigate potential effects of such contaminants on physiological functions of humans and wildlife, blood chemistry analysis is a relevant tool. Blood integrates indices of most organisms' physiological processes. Therefore, blood chemistry offers a generalist approach to investigate several physiological functions (e.g., energetic metabolism, reproduction, detoxification, osmoregulation; [START_REF] Aguiree | Blood biochemistry values of Green Turtles, Chelonia mydas, with and without fibropapillomatosis[END_REF][START_REF] Brischoux | Hypernatremia in Dice Snakes (Natrix tessellata) from a coastal population: implications for osmoregulation in marine snake prototypes[END_REF][START_REF] Barão-Nóbrega | The metabolic cost of nesting: body condition and blood parameters of Caiman crocodilus and Melanosuchus niger in Central Amazonia[END_REF][START_REF] Hudson | Steroid hormones, energetic state, and immunocompetence vary across reproductive contexts in a parthenogenetic lizard[END_REF] and can additionally be used for contamination level assessment [START_REF] Bickham | Environmental contaminants in Texas, USA, wetland reptiles: evaluation using blood samples[END_REF]. Because blood sampling is non-lethal and performed relatively easily, the effect of trace elements on biochemical parameters can be monitored over time.

In the present study, we assessed the concentrations of selected trace elements (Hg, Pb, and Se) and 15 physiological parameters indicative of osmoregulatory (e.g., sodium, chlorine), metabolic (e.g., glucose, calcium), hepatic (e.g., aspartate aminotransferase, alanine aminotransferase), endocrine (e.g., corticosterone) and renal (e.g., total protein) functions in the blood of Caiman crocodilus from French Guiana. Additionally, we evaluated relationships between Hg, Pb, and Se with blood chemistry and corticosterone to investigate the effects of these trace elements on crocodilian physiology.

MATERIAL AND METHODS

Sample collection

Between November 2019 and February 2020, we captured and sampled 23 Spectacled

Caimans Caiman crocodilus at "Pripis de Yiyi" (N 05°25'25", W 53°02'50") and the nature reserve "Kaw-Roura" (N 04°29'20", W 52°03'38") in French Guiana. Each individual was measured (Snout-Vent Length, SVL) and weighed. Blood was drawn from the lateral tail vein using a 2.5 mL syringe and a 23 or 21 gauge -50 mm heparinized needle (heparin sodium).

Due to logistical constraints inherent to field procedures and the study species, time between the successful capture of an individual and the end of blood sampling was 14 ± 3 minutes (range 10 -19 minutes). Blood samples were immediately placed in cold temperatures (4°C) until being processed at the laboratory. After biochemistry analysis (see below), half of the whole blood was frozen at -28°C, the second part was centrifuged at 6500 rpm for 5 minutes, then plasma and red blood cells were separately frozen at -28°C.

After sampling, all caimans were released at their capture locations. Captures and sample collection were performed under a permit from the French authorities (Direction Régionale des Territoires et de la Mer) after evaluation by the CSRPN, the regional scientific committee (Permit: R03-2019-01-09-001 & R03-2019-10-24-007).

Blood biochemistry analysis

An average of four hours after sampling, we analyzed blood parameters using VetScan VS2 Chemistry Analyzer (Abaxis, Inc., Union City, California, USA) by applying 100µL of whole blood on "Avian-Reptilian" and/or "Preventive Care Profile Plus" designated rotors to measure total calcium (Ca ++ ), potassium (K + ), sodium (Na + ), chloride (Cl -), phosphorus (P), uric acid (UA), glucose (GLU), total bilirubin (TBIL), total protein (TP), creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), total carbon dioxide (tCO2), blood urea nitrogen (BUN), bile acids (BA), creatinine (CRE) and globulin (GLOB). Five blood biochemistry parameters (ALB, BUN, CRE, BA and GLOB) could not be quantified as their levels were under the detection limit of the VetScan.

Corticosterone analysis

We measured plasma corticosterone levels using radioimmunoassay, as described in [START_REF] Lormée | Sex-specific patterns in baseline corticosterone and body condition changes in breeding Red-footed Boobies[END_REF]. Corticosterone was measured after ethyl ether extraction using a commercial antiserum. After adding dextran-coated charcoal and centrifugation, the free corticosterone was separated. Bound fraction containing corticosterone was measured using a liquid scintillation counter. The minimum detectable corticosterone concentration was 0.28 ng.ml - 1 , and the inter-assay coefficients of variation were 6.24% and 9.63% respectively (samples were assayed in duplicate, in two assays).

Trace element analysis

Whole blood was freeze-dried for 48 hours, ground and homogenized. Total Hg concentrations were determined by direct measurement in the whole blood, using an atomic absorption spectrometer AMA-254 (Advanced Mercury Analyser-254; Altec®). Certified Reference Material (CRM) TORT-3 (lobster hepatopancreas; certified Hg concentration: 0.292 ± 0.022 µg.g -1 dry weight (dw), NRCC) was analyzed at the beginning and at the end of the analytical cycle to validate the method. For each individual, two replicates of blood (~0.6 mg dw) were analyzed, and the reproducibility for replicate samples was approved when the relative standard deviation (RSD) was < 10 %. Measured values for TORT-3 were 0.292 ± 0.006 µg.g -1 dw (n = 20), with a recovery of 100.1 ± 1.9 %. The limit of quantification was 0.05 ng.

Se and Pb were analyzed in freeze-dried whole blood using Inductively Coupled Plasma Mass Spectrometry (ICP-MS II Series Thermo Fisher Scientific), (aliquots mass: 8 -139 mg dw).

Whole blood samples were microwave-digested in a mixture of 6 mL of 70% HNO3 (VWR Quality SUPRAPUR) and 2 mL of 30% HCl (VWR Quality SUPRAPUR); for samples weighing less than 100 mg, the volumes of HNO3 and HCl were divided by half. Samples were further diluted with ultrapure water to 50 mL (25 mL for samples < 100mg). To avoid contamination, all utensils used were soaked in a bath of diluted nitric acid for at least 48 h, rinsed in ultrapure water and dried. Certified Reference Materials (CRM; dogfish liver DOLT-3, NRCC, and lobster hepatopancreas TORT-2, NRCC) were treated and analyzed as samples. Results of Hg, Pb and Se quantification were in agreement with the certified values, and the standard deviations were low, proving good repeatability of the methods. The results for CRMs displayed recoveries of the elements ranging from 92.1 ± 17.5 % (n= 8) for Pb, 110.7 ± 4.7 % for Se (n=8).

Hg, Pb and Se results are further expressed in µg.g -1 dw.

Statistical analyses

All analyses were performed using the Software R v.3.

RESULTS AND DISCUSSION

Non-essential trace elements affected physiological parameters, in particular osmoregulatory, hepatic, endocrine and renal functions. Below we discuss the physiological mechanisms which appear to be potentially affected by these contaminants.

Mercury

Hg concentrations ranged from 0.168 to 1.532 µg.g -1 dw (Table 1) and were negatively correlated with natremia (R 2 = 0.345, p = 0.005, Fig. 1, Table 2) and, to a lesser extent with chloremia (R 2 = 0.437, p = 0.053). This suggests that higher Hg concentrations negatively influenced ionic regulation which is related to renal functions in caimans.

We found a negative relationship between Hg and alkaline phosphatase (ALP) concentrations (R 2 = 0.601, p = 0.024, Fig. 1, Table 2). Elevation of Hg concentrations is linked to a diminution of ALP in the blood of Caiman crocodilus, suggesting an alteration of hepatic function.

Our results show a negative relationship between corticosterone levels and Hg concentrations (R 2 = 0.276, p = 0.021, Table 2), which suggests that elevation of Hg concentrations may disrupt endocrine processes in caimans by a diminution of corticosterone production (HPA axis).

Although Hg toxicity is well documented in several taxa [START_REF] Scheuhammer | Effects of environmental methylmercury on the health of wild birds, mammals, and fish[END_REF][START_REF] Morcillo | Mercury and its toxic effects on fish[END_REF][START_REF] Evers | The effects of methylmercury on wildlife; a comprehensive review and approach for interpretation[END_REF]Zheng et al., 2019), its effects remain poorly studied in reptiles [START_REF] Schneider | Mercury contamination in reptiles: An emerging problem with consequences for wild life and human health[END_REF]. Hg can act as an inhibitor of the Na + /K + -ATPase [START_REF] Kramer | In vitro inhibition of Na-K-ATPase by trace metals: relation to renal and cardiovascular damage[END_REF][START_REF] Magour | Studies on the inhibition of brain synaptosomal Na+/K+-ATPase by mercury chloride and methyl mercury chloride[END_REF][START_REF] Chuu | Differential neurotoxic effects of methylmercury and mercuric sulfide in rats[END_REF], and can lead to a disruption of osmoregulation in taxa such as fishes and crustaceans [START_REF] Lock | Effects of mercuric chloride and methylmercuric chloride on the osmoregulatory function of the gills in Rainbow Trout, Salmo gairdneri Richardson[END_REF][START_REF] Bianchini | Toxicity and accumulation of mercury in three species of crabs with different osmoregulatory capacities[END_REF][START_REF] Handayani | Change of osmoregulatory and hematological parameters in tilapia (Oreochromis niloticus) after exposure to sublethal mercury concentrations[END_REF].

In Alligatorids, osmoregulation principally occurs in the kidneys [START_REF] Mazzotti | Osmoregulation in Crocodilians[END_REF][START_REF] Grigg | Osmoregulation by the broad-snouted caiman, Caiman latirostris in estuarine habitat in southern Brazil[END_REF] and our results suggest that renal function may be negatively affected by Hg. Freshwater species need to maintain osmolality in a hyposmotic environment, as the regulation of the hydromineral balance is crucial for survival [START_REF] Schmidt-Nielsen | Animal physiology: adaptations and environments[END_REF].

Hyponatremia has been shown to cause neurological dysfunction, muscle damage and death [START_REF] Patterson | The impact of hyponatremia[END_REF]Gankam Kengne and Decaux, 2018;Martemyanov and Pddubnaya, 2020[START_REF] Arieff | Hyponatremia, convulsions, respiratory arrest, and permanent brain damages after elective surgery in healthy women[END_REF][START_REF] Arieff | Influence of hypoxia and sex on hyponatremic encephalopathy[END_REF]. Therefore, consequences of chronic hyponatremia in Hg-contaminated freshwater crocodilians needs to be assessed.

The negative relationship between Hg and ALP suggests that some liver functions in Caiman crocodilus are disrupted due to Hg contamination, as previously found in fish [START_REF] Sastry | Mercury induced haematological and biochemical anomalies in Ophiocephalus (Channa) punctatus[END_REF] and rodents [START_REF] El-Shenawy | Comparative evaluation of the protective effect of selenium and garlic against liver and kidney damage induced by mercury chloride in the rats[END_REF]. ALP is a liver cytoplasmic enzyme involved in the hepatocytic functions, and perturbations of its activity occur via mechanisms such as ATPase disruption [START_REF] El-Shenawy | Comparative evaluation of the protective effect of selenium and garlic against liver and kidney damage induced by mercury chloride in the rats[END_REF]. Potential effects of decreased ALP, and/or altered liver functions linked to Hg contamination in caimans deserve further investigation.

Mercury accumulates in the pituitary gland and the thyroid and alters the endocrine system in vertebrates [START_REF] Colborn | Developmental effects of endocrine-disrupting chemicals in wildlife and humans[END_REF][START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF][START_REF] Meyer | Mercury exposure associated with altered plasma thyroid hormones in the declining Western Pond Turtle (Emys marmorata) from California Mountain Streams[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF].

Consistent with these studies, the negative relationship between corticosterone levels and Hg concentration suggests that Hg may disrupt endocrine processes in caimans as already shown in other taxa [START_REF] Moore | Lifelong exposure to methylmercury disrupts stress-induced corticosterone response in Zebra Finches (Taeniopygia guttata)[END_REF][START_REF] Meillère | Corticosterone levels in relation to trace element contamination along an urbanization gradient in the common blackbird (Turdus merula)[END_REF][START_REF] Soto | Chronic exposure to inorganic mercury alters stress responses in male prairie voles (Microtus ochrogaster)[END_REF]. Disruption of corticosterone levels has consequences on metabolism, behaviour and reproduction [START_REF] Denardo | Effects of corticosterone on social behavior of male lizards[END_REF][START_REF] Guillette | Biology of stress: Interactions with reproduction, immunology and intermediary metabolism[END_REF]Scott et al., 2019). Effects of environmental contaminants (e.g., pesticides, trace elements) on the endocrine system of crocodilians have been already reported [START_REF] Guillette | Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile Alligator from contaminated and control lakes in Florida[END_REF][START_REF] Arukwe | Developmental alterations and endocrine-disruptive responses in farmed Nile crocodile (Crocodylus niloticus) exposed to contaminants from the Crocodile River, South Africa[END_REF][START_REF] Finger | Dietary Selenomethionine administration and its effects on the American Alligator (Alligator mississippiensis): Oxidative status and corticosterone levels[END_REF], while endocrine disruption associated to Hg contamination is yet unknown but demands future evaluation.

Selenium and Se:Hg molar ratio

Se concentrations ranged from 0.76 to 1.92 µg.g -1 dw (Table 1) and was not related to other parameters (all p > 0.05, Table 2), suggesting that Se concentrations were not high enough to induce any effects.

Our results show that natremia (Na + : R 2 = 0.378, p = 0.004, Fig. 2, Table 2) and corticosterone levels (R 2 = 0.272, p = 0.026, Table 2) increased with the Se:Hg molar ratio. These results show that Na + and corticosterone levels are positively influenced by an excess of Se against Hg, suggesting a potential positive effect of Se.

Selenium is an essential trace element involved in antioxidant defense and thyroid metabolism which can be toxic in high concentrations [START_REF] Behne | Cellular and subcellular distribution of selenium and selenium-containing proteins in the rat[END_REF][START_REF] Ramauge | Evidence that type III iodothyronine deiodinase in rat astrocyte is a selenoprotein[END_REF][START_REF] Rayman | The importance of selenium to human health[END_REF]. In the American alligator, Alligator mississippiensis, chronic Se exposure affects stress parameters such as corticosterone levels [START_REF] Finger | Examining the effects of chronic selenium exposure on traditionally used stress parameters in juvenile American Alligator (Alligator mississippiensis)[END_REF]. Our results suggest that Se concentrations were not high enough to trigger toxic effects. However, the relationships found between Se:Hg molar ration and natremia and corticosterone levels suggests positive effects of this ratio. As already discussed, Hg concentrations show a negative relationship with Na + and corticosterone concentrations. Because Se reduces Hg toxicity in many taxa (Beijer and Jernolov, 1978;[START_REF] Freidman | Protective effects of freeze-dried swordfish on methylmercury chloride toxicity in rats[END_REF][START_REF] Ohi | The protective potency of marine animal meat against the neurotoxicity of methylmercury: its relationship with the organ distribution of mercury and selenium in the rat[END_REF][START_REF] Culvin-Aralar | Mercury and selenium interaction: a review[END_REF][START_REF] Suzuki | Equimolar Hg-Se complex binds to selenoproteins P[END_REF][START_REF] Ralston | Effects of dietary selenium and mercury on house crickets (Acheta domesticus L.): implications of environmental co-exposures[END_REF][START_REF] Ralston | Dietary selenium's protective effects against methylmercury toxicity[END_REF], we suggest that Se:Hg molar ratio has a role of protection against Hg toxicity in Caiman crocodilus. This hypothesis is reinforced by the marginal relationship detected between Se concentration and Na + values. Our results emphasize the need for future studies on this potential protective effect of the Se:Hg molar ratio in crocodilians.

Lead

Pb concentrations range from 0.04 to 0.28 µg.g -1 dw (Table 1) and were negatively related to glucose levels (R 2 = 0.239, p = 0.025, Fig. 3, Table 2), suggesting that Pb affects mechanisms related to the regulation of glucose. Our results show a positive relationship between Pb concentration and alanine aminotransferase (ALT) (R 2 = 0.740, p = 0.003, Fig. 3, Table 2)

suggesting that the organism increases the production of ALT in response to elevated Pb concentration in the blood.

The detrimental effects of lead contamination are well studied and affect vascular, nervous, renal, hepatic, immune, endocrine and reproductive systems [START_REF] Eisler | Lead hazards to fish, wildlife, and invertebrates: a synoptic review. Contaminant Hazard Reviews[END_REF][START_REF] Pattee | Lead in the environment[END_REF][START_REF] Grillitsch | The ecotoxicology of metals in reptiles[END_REF]. The biokinetics of Pb in the blood of crocodilians is shorter than in other vertebrates, with a half-life of 3 days, in comparison to 13 days in birds and mammals [START_REF] Anders | Morphological, pharmacokinetic, and hematological studies of leadexposed pigeons[END_REF][START_REF] Castellino | Kinetics of the distribution and excretion of lead in the rat[END_REF][START_REF] Hammerton | Experimental study of blood lead kinetics in estuarine crocodiles (Crocodylus porosus) exposed to ingested lead shot[END_REF]. No clinical signs of Pb toxicity were found in crocodilian studies yet, suggesting resistance of the taxon to the contaminant (Cook et al., 1988;[START_REF] Camus | Elevated lead levels in farmed American alligators Alligator mississippiensis consuming nutria Myocastor coypus meat contaminated by lead bullets[END_REF][START_REF] Hammerton | Experimental study of blood lead kinetics in estuarine crocodiles (Crocodylus porosus) exposed to ingested lead shot[END_REF][START_REF] Lance | Chronic incidental lead ingestion in a group of captive-reared alligators (Alligator mississippiensis): possible contribution to reproductive failure[END_REF]Warner et al., 2016). However, our results show a negative relationship between Pb and glucose level suggesting that Pb affects the endocrine systems of Caiman crocodilus and alters its liver function. Glucose is regulated by the liver and complex interactions with the hypothalamus, pituitary and adrenal glands [START_REF] Lin | Hormonal regulation of hepatic glucose production in health and disease[END_REF][START_REF] Cady | Hypothalamic growth hormone receptor (GHR) controls hepatic glucose production in nutrient-sensing leptin receptor (LepRb)[END_REF]. Our results are consistent with studies in marine turtles exposed to Pb [START_REF] Komoroske | Pollutants and the health of green sea turtles resident to an urbanized estuary in San Diego[END_REF]. Our findings are strengthened by the positive relationship we additionally found between Pb and ALT, an indicator of hepatocellular damages [START_REF] Kew | Serum aminotransferase concentration as evidence of hepatocellular damage[END_REF][START_REF] Maheswari | Hepatoprotective activity of Orthosiphon stamineus on liver damage caused by paracetamol in rats[END_REF]. While our initial findings show some toxic effects of Pb on liver functions in crocodilians, it deserves further investigation.

CONCLUSION

The present study provides the first evidence that Hg and Pb affect physiological parameters in Caiman crocodilus. Mercury was related to disruptions of sodium, alkaline phosphatase, and corticosterone levels, which suggests a negative effect on osmoregulation, hepatic functions and endocrine processes. Lead was related to disruption of glucose and alanine aminotransferase levels, suggesting hepatocellular damages. Although the Hg and Pb concentrations of the present study are commonly found in crocodilians, the relationship between contaminant levels and blood parameters are of concern. Interestingly, results that investigate the Se:Hg molar ratio suggest a protective effect of Se against Hg toxicity in caimans. This study is a starting point for further evaluation of trace element consequences on physiological mechanisms in caimans, particularly those more vulnerable to exposure.

Indeed, our sampled individuals were relatively small and thus probably young individuals which suggest that physiological alterations linked to non-essential trace elements can occur early in the life of crocodilians. : in cm; 2 : in g; 3 : in µg.g -1 ; 4 : in mg.L -1 ; 5 : mmol.L -1 ; 6 : in g.L -1 ; 7 : in U.L -1 ; 8 : mosm.L -1 ; 9 : ng.mL -1

  6.1. (R development Core Team). The data was first checked for normality and homogeneity of variances. Analyses on trace elements were performed on log-transformed values. The Selenium:Mercury (Se:Hg) molar ratio was calculated using the Hg and Se concentrations (in µg.g -1 dw) divided by the molecular weight of each element, respectively 200.59 for Hg and 78.96 for Se, following the equation : The relation between trace elements, biometric measurements, and biochemistry parameters were assessed by linear regression. Corticosterone concentrations of Caiman crocodilus were not influenced significantly by the time of handling (F2,18 = 0.274, p = 0.787) and relations between trace elements and corticosterone concentrations were assessed with linear regressions.

Figure 1 .Figure 2 .

 12 Figure 1. Relation between Hg concentration (Log µg.g -1 dw) and (A) sodium (mmol.L -1 , R 2 = 0.345, p = 0.005) and (B) alkaline phosphatase (U.L -

Figure 3 .

 3 Figure 3. Relation between Pb concentration (Log µg.g -1 dw) and (A) Alanine aminotransferase (U.L -1 , R 2 = 0.740, p = 0.003) and (B) Glucose

Table 1 .

 1 Morphometrics, biochemistry parameters and trace element concentrations of the Spectacled Caiman (Caiman crocodilus) from French Guiana.

		Caiman crocodilus
	N	Mean ± SD	Min -Max

Table 2 .

 2 Summary statistics of linear regression between morphometrics, metal concentrations and biochemistry parameters in the Spectacled Caiman (Caiman crocodilus) in French Guiana. Bold denotes significant relationship (p < 0.05) and arrows indicate a positive or negative correlation.

	Parameters		Hg			Se			Se:Hg			Pb
	Caiman crocodilus	n	R 2	p	n	R 2	p	n	R 2	p	n	R 2	p
	Snout-vent length (SVL)	21	0.143	0.091	20	0.091	0.198	20	0.161	0.080	21	0.290	0.012 
	Weight (W)	21	0.163	0.070	20	0.130	0.118	20	0.197	0.050	21	0.129	0.110
	Total calcium (Ca ++)	21	0.112	0.138	20	0.017	0.587	20	0.058	0.307	21	0.122	0.121
	Potassium (K + )	20	0.045	0.370	19	0.074	0.259	19	0.058	0.319	20	0.186	0.057
	Sodium (Na + )	21	0.345	0.005 	20	0.184	0.059	20	0.378	0.004 	21	0.001	0.897
	Chlorine (Cl -)	9	0.437	0.053	9	0.109	0.386	9	0.395	0.070	9	0.041	0.602
	Phosphorus (P)	13	0.091	0.315	12	0.030	0.592	12	0.062	0.436	13	0.099	0.295
	Uric acid (UA)	13	0.009	0.757	12	0.161	0.197	12	0.079	0.376	13	0.032	0.562
	Glucose (GLU)	21	0.014	0.612	20	0.073	0.251	20	0.031	0.455	21	0.239	0.025 
	Total bilirubin (TBIL)	9	0.2085	0.217	9	0.082	0.455	9	0.200	0.228	9	0.096	0.418
	Total protein (TP)	21	0.008	0.700	20	0.024	0.514	20	0.015	0.611	21	0.006	0.740
	Creatine kinase (CK)	13	0.000	0.972	12	0.014	0.716	12	0.005	0.828	13	0.003	0.866
	Aspartate aminotransferase (AST)	21	0.003	0.824	20	0.078	0.232	20	0.013	0.629	21	0.162	0.070
	Alanine aminotransferase (ALT)	9	0.001	0.929	9	0.002	0.910	9	0.002	0.921	9	0.740	0.003 
	Alkaline phosphatase (ALP)	8	0.601	0.024 	8	0.035	0.655	8	0.477	0.058	8	0.003	0.894
	Total carbon dioxide (tCO2)	9	0.142	0.318	9	0.028	0.667	9	0.125	0.351	9	0.175	0.263
	Corticosterone	19	0.276	0.021 	18	0.090	0.228	18	0.272	0.026 	19	0.064	0.295

Acknowledgements

We would like to thank the teams of the nature reserves "Kaw-Roura", the protected area of "Pripris de Yiyi" and the conservatoire du littoral for logistics and assistance in the field. We would also like to thank Philippe Gaucher, Frédéric Beau, Laura Beau, Thibaut Foch, Fanny Veinante, Clarisse Lhez and Jennifer Devillechabrolle for their help in the field. We are grateful to Ashley Pearcy Buitenwerf for the English revision and valuable comment on the manuscript.

This study was supported by the Office de l'Eau de Guyane (OEG), the Office Française pour la Biodiversité (OFB), the Direction Générale des Territoires et de la Mer de Guyane (DGTM), the Parc Zoologique de Paris, the CNRS and the Fondation d'entreprise Hermès. The CPER (Contrat de Projet Etat Région) and the FEDER (European regional Development Fund) are acknowledged for funding the Vetscan, the AMA and the ICP-MS of the Plateforme Analyses Elémentaires of the LIENSs laboratory. The Institut Universitaire de France (IUF) is acknowledged for its support to P. Bustamante as a Senior Member.

Trillanes, C.E., Pérez-Jiménez, J.C., Rosíles-Martínez, R., González-Jáuregui, M. 2014. Metals in the caudal scutes of Morelet's Crocodile (Crocodylus moreletii) from the Southern Gulf of Mexico. Bull. Environ. Contam. Toxicol. 93, 423-428. Ullah, H., Liu, G., Yousaf, B., Ali, M.U., Irshad, S., Abbas, Q., Ahmad, R. 2019 Zheng, N., Wang, S., Dong, W., Hua, X., Li, Y., Song, X., Chu, Q., Hou, S., Li, Y. 2019. The toxicological effects of mercury exposure in marine fish. Bull. Environ. Contam. Toxicol. 102,[714][715][716][717][718][719][720]


Jérémy Lemaire
email: jeremy.lemaire@univ-lr.fr

François Brischoux

Olivier Marquis

Rosanna Mangione

Paco Bustamante

F J Lemaire

J Brischoux

J Lemaire

O Brischoux

R Marquis

P Mangione

Bustamante

Variation of total mercury concentration in different tissues of three neotropical caimans: implications for minimally invasive biomonitoring

Keywords: Trace metals, Ecotoxicology, Keratinized tissues, Crocodilians, Bioaccumulation

Mercury (Hg) is a global environmental contaminant that affects ecosystems. It is known to biomagnify through food webs, and to bioaccumulate especially in the tissues of top predators.

Large scale comparisons between taxa and geographic areas are needed to reveal critical trends related to Hg contamination and its deleterious effects on wildlife. Yet, the large variety of tissues (keratinized tissues, internal organs, blood) as well as the variability in the units used to express Hg concentrations (either in wet-or dry-tissue weight) limits straightforward comparisons between studies. In the present study, we assessed the moisture content that could influence the total Hg (THg) concentrations measured in several tissues (claws, scutes, total blood and red blood cells) of three caiman species. First, we aimed to evaluate the moisture content from the different tissues, and second to provide information on THg concentrations in various matrices. Our results show a difference of THg concentrations between the tissues and intra-and interspecific variations of moisture content, with the highest THg values found in keratinized tissues (scute keratinized layers and claws). For the three species, we found positive relationships between body size and THg concentration in keratinized tissues. In the blood, the relationship between body size and THg concentration was species-dependent. Our results emphasize the need for a standardized evaluation of THg concentration and trace elements quantification based on dry weight analytical procedures. In addition, the use of both blood and keratinized tissues offers the possibility to quantify different time scales of THg exposure by non-lethal sampling.

INTRODUCTION

Mercury (Hg) is one of the major contaminants of concern in ecosystems [START_REF] Ericksen | Accumulation of atmospheric mercury in forest foliage[END_REF][START_REF] Fitzgerald | Marine biogeochemical cycling of mercury[END_REF][START_REF] Selin | Global biogeochemical cycling of mercury: a review[END_REF]. In addition to naturally present geological Hg, human activities such as deforestation, fossil fuel combustion and gold mining activities have been shown to increase the level of Hg in the environment, particularly in aquatic ecosystems [START_REF] Ericksen | Accumulation of atmospheric mercury in forest foliage[END_REF][START_REF] Scheuhammer | Recent advances in the toxicology of methylmercury in wildlife[END_REF][START_REF] Hsu-Kim | Challenges and opportunities for managing aquatic mercury pollution in altered landscapes[END_REF]. In anoxic conditions, aquatic microorganisms can transform inorganic Hg into methylmercury (MeHg), the most bioavailable and toxic form of Hg [START_REF] Jensen | Biological methylation of mercury in aquatic organisms[END_REF][START_REF] Benoit | Geochemical and biological controls over methylmercury production and degradation in aquatic ecosystems[END_REF].

Importantly, MeHg biomagnifies through food webs, and bioaccumulates in the tissues of top predators, which makes them particularly vulnerable to this contaminant (Eagles-Smith et al.

2018).

Crocodilians belong to the World's largest predators and as such, they have important functions in ecosystems and can constitute indicators of ecosystem health [START_REF] Somaweera | The ecological importance of crocodylians: towards evidence-based justification for their conservation[END_REF]). As apex predators, crocodilians bioaccumulate environmental contaminants that biomagnify across food webs, and thus can be particularly vulnerable to their toxicity [START_REF] Cook | Elevated heavy metal concentrations in captive crocodilians-two cases[END_REF][START_REF] Camus | Elevated lead levels in farmed American alligators Alligator mississippiensis consuming nutria Myocastor coypus meat contaminated by lead bullets[END_REF][START_REF] Rainwater | Metals and organochlorine pesticides in caudal scutes of crocodiles from Belize and Costa Rica[END_REF]. Therefore, they are relevant bioindicators regarding environmental contamination [START_REF] Gross | Developmental abnormalities if the gonad and abnormal sex hormone concentration in juvenile alligator from contaminated and control lakes in Florida[END_REF][START_REF] Manolis | Crocodilians and other reptiles: bioindicators of pollution[END_REF][START_REF] Campbell | Ecotoxicology of crocodilians[END_REF][START_REF] Chumchal | Mercury speciation and biomagnification in the food web of Caddo Lake, Texas and Louisiana, USA, a subtropical freshwater ecosystem[END_REF][START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF]. In addition, such an evaluation is also useful to determine the relatively poorly known detrimental effects of contaminants on this taxon, such as physiological and reproductive impairment and DNA damages [START_REF] Crain | Alligators and endocrine disrupting contaminants: a current perspective[END_REF][START_REF] Siroski | Immunotoxicity of commercial-mixed glyphosate in broad snouted caiman (Caiman latirostris)[END_REF][START_REF] Burella | Oxidative damage and antioxidant defense in Caiman latirostris (Broad-snouted caiman) exposed in ovo to pesticide formulations[END_REF]Lemaire et al. 2021a).

Crocodilians bioaccumulate Hg at various concentrations, depending on the species and on the location [START_REF] Elsey | Mercury levels in Alligator Meat in South Louisiana[END_REF][START_REF] Rumbold | Levels of mercury in alligators (Alligator mississippiensis) collected along a transect through the Florida Everglades[END_REF][START_REF] Campbell | Heavy metal and selenium concentrations in liver tissue from wild American alligator (Alligator mississippiensis) liver near Charleston, South Carolina[END_REF][START_REF] Vieira | Mercury and methyl mercury ratios in caimans (Caiman crocodilus yacare) from the Pantanal areas, Brazil[END_REF]Nilsen et al. 2017a;Lemaire et al. 2021a). However, interspecific variability across the whole group remains still poorly understood to date as most of the available studies on Hg concentrations have disproportionately focused on two species, the American Alligator, Alligator mississippiensis and the Morelet's Crocodile, Crocodylus moreletii [START_REF] Yanochko | Tissue mercury concentrations in alligators (Alligator mississippiensis) from the Florida Everglades and the Savannah River site, Carolina[END_REF][START_REF] Jagoe | Mercury in alligators (Alligator mississippiensis) in the southeastern United States[END_REF][START_REF] Elsey | Mercury levels in Alligator Meat in South Louisiana[END_REF][START_REF] Burger | Metals and metalloids in tissues of American Alligators in three Florida lakes[END_REF][START_REF] Rainwater | Metals and organochlorine pesticides in caudal scutes of crocodiles from Belize and Costa Rica[END_REF][START_REF] Horai | Concentrations of trace elements in American alligators (Alligator mississippiensis) from Florida, USA[END_REF][START_REF] Trillanes | Metals in Caudal Scutes of Morelet's Crocodile (Crocodylus moreletii) from the Southern Gulf of Mexico[END_REF]Nilsen et al. 2017a;[START_REF] Buenfil-Rojas | Mercury and metallothioneins in blood fractions and tissues of captive Morelet's crocodiles in Quintana Roo, Mexico[END_REF][START_REF] Nilsen | Examining toxic trace element exposure in American alligators[END_REF][START_REF] Buenfil-Rojas | Distribution of metals in tissues of captive and wild Morelet's crocodiles and the potential of metallothioneins in blood fractions as a biomarker of metal exposure[END_REF]. Some recent studies, however, have focused on several other species [START_REF] Almli | Hepatic and renal concentrations of 10 trace elements in crocodiles (Crocodylus niloticus) in the Kafue and Luangwa rivbers in Zambia[END_REF][START_REF] Vieira | Mercury and methyl mercury ratios in caimans (Caiman crocodilus yacare) from the Pantanal areas, Brazil[END_REF][START_REF] Lázaro | Non-lethal sampling for mercury evaluation in crocodilians[END_REF][START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF][START_REF] Marrugo-Negrete | Mercury levels and genotoxic effect in caimans from tropical ecosystems impacted by gold mining[END_REF]Lemaire et al. 2021a,b). Furthermore, Hg concentrations in crocodilians have been determined in a variety of tissues (blood, muscles, internal organs or keratinized tissues), depending on the study, which limits robust comparisons between them. It is worth noting that the associated methodologies (e.g., sample preparation and tissues studied) are variable, which also limits straightforward comparisons between studies [START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF]. Further, THg concentrations in these papers are either presented as the concentration relative to the wet-, or dry-weight of sampled tissues. Clearly, such discrepancy prevents direct comparison between studies, especially as the amount of tissue moisture content, and its variation within and across tissues, on Hg concentration has not yet been thoroughly investigated. In addition, one of the most studied tissues in crocodilians is the muscle, most likely because it is used for human consumption [START_REF] Delany | Concentrations of contaminants in muscle of the American Alligator in Florida[END_REF][START_REF] Elsey | Mercury levels in Alligator Meat in South Louisiana[END_REF][START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from Rio Purus, Amazonas, Brazil[END_REF][START_REF] Rivera | Low total mercury in Caiman yacare (Alligatoridae) as compared to carnivorous, and non-carnivorous fish consumed by Amazonian indigenous communities[END_REF].

Although assessing Hg contamination in such tissue can be useful, many species are now classified by the IUCN as in danger of extinction, which supports both the reduction of crocodile hunting per se, and the development of specific, non-lethal sampling methods to assess their Hg contamination levels and its effects (as methods have shown for other reptile species, [START_REF] Day | Monitoring mercury in the Loggerhead Sea Turtle, Caretta caretta[END_REF][START_REF] Lemaire | Determinants of mercury contamination in viperine snakes, Natrix maura, in Western Europe[END_REF][START_REF] Beau | Environmental causes and reproductive correlates of mercury contamination in European pond turtles (Emys orbicularis)[END_REF].

In this context, the goals of the present study were two-fold: first, we assessed the moisture content of different tissues (claws, scutes and total blood) and investigated the influence of sample preparation on total Hg (THg) quantification, in order to provide recommendations to harmonize Hg assessment across multiple matrices. Second, we compared THg concentrations between several tissues (full scutes, scute keratin layers, claws, total blood and red blood cells) to evaluate the use of non-lethal sampling methods to facilitate future comparisons of contamination in crocodilians.

MATERIALS AND METHODS

Sample collection

Our study was conducted in French Guiana between April 2016 and February 2020 (Fig. 1).

We sampled 51 individuals of smooth-fronted caiman, Paleosuchus trigonatus (Schneider, 1801) from 6 different sites, 48 individuals of spectacled caiman, Caiman crocodilus (Linnaeus, 1758) from 2 different sites and 13 individuals of dwarf caiman, Paleosuchus palpebrosus (Cuvier, 1807) from 4 different sites. P. trigonatus were captured in small forest streams, C. crocodilus and P. palpebrosus were captured in marsh or stream habitats.

The snout-vent length (SVL) and total length (TL) of each individual were measured. We took claw (randomly at the posterior legs) and scute samples (in all samplings < 1cm, and never exceeding half of the scute) of all caimans using pliers for clipping the sample. Clipping tail scutes is a common marking method in crocodilians. The scutes which were clipped in order to mark the captured individuals were therefore not the same for all animals as they followed the consecutive identification code. Claw and scute samples were placed in dry plastic containers.

Blood was collected on a subsample of individuals depending on the field possibilities (N = 24 for P. trigonatus, N = 40 for C. crocodilus and N = 7 for P. palpebrosus). Blood samples (0.2 -3 ml) were drawn from the lateral tail vein using 27 gauges -25 mm or 21 gauges -50 mm (depending on the size of the individual animal) heparinized needles (heparin sodium). Each blood sample was separated into two tubes and kept at cold temperatures (4°C) until being processed at the laboratory (always <3h after collection). The first tube, containing total blood, was frozen at -28°C in the lab. The second one was centrifuged at 6500 rpm for 5 minutes to separate red blood cells (RBCs) and plasma, after which both fractions were then frozen at -28°C in the lab until analysis.

After sampling, each individual was released at its capture location.

C. crocodilus, P. trigonatus and P. palpebrosus are protected by the French law (Ministerial decree NOR: TREL1933710A of 08/10/2018) and a permission to capture individuals, draw blood and sample claws and scutes was granted by the French authorities (Direction Régionale des Territoires et de la Mer) after evaluation by the CSRPN, the regional scientific committee (Permit:

R03-2016-06-21-010; R03-2019-01-09-001; R03-2019-10-24-007, www.guyane.developpement-durable.gouv.fr).

Sample preparation

Claws and scutes were cleaned for 5 minutes in an ultrasonic bath of ultrapure water to remove all external dirt and rinsed 3 times, as described in Lemaire et al. (2021a). In order to assess the wet weight of claws and scutes, we weighed cleaned samples after elimination of water surplus with absorbent paper. Because cleaning procedures were relatively short and because caimans are semi-aquatic species which spend most of their lifetime immerged in water, our cleaning procedures were not likely to influence wet weight assessment. In order to assess dry weight, each sample was dried in an oven for 48h at 45°C, which was found to be sufficient to reach stable weight (no difference was found when dried at 45°C for 48h, 72h and 96h, data not shown). Moisture content was calculated as wet weight minus dry weight and expressed as a proportion of wet weight for further analyses. A subsample of dried scutes (N = 26 for C. crocodilus, N = 4 for P. palpebrosus and N = 4 for P. trigonatus) was used to perform comparison of Hg concentrations between these matrices. We separated the external layer, which is composed of keratin (corneoscute), from the underlying connective tissue which is the link between bone/cartilage (osteoscute) and the keratin layer. For the remaining scute samples, only the keratin layer was analysed. In order to assess the moisture of total blood and RBC samples, we weighted the samples before and after the freeze-drying process (48h). Freezedried samples were then ground into a homogeneous powder. The moisture content of total blood and RBC samples was calculated as wet weight minus dry weight and then expressed as a proportion of wet weight.

Instrumental method and quality control

For all samples, total Hg (THg) was determined using an atomic absorption spectrometer AMA- 0.292 ± 0.022 µg.g -1 dw) was performed at the beginning and at the end of the analytical cycle and every 10 samples, for the validation of the method. The TORT-2 measured value was 0.243 ± 0.015 µg.g -1 dw (n=20), giving a recovery of 90.2 ± 5.7 % and the TORT-3 measured value was 0.289 ± 0.007 µg.g -1 dw (n=30) giving a recovery of 99.0 ± 2.3 %. Blanks were included at the beginning of analytical runs and the limit of quantification of the AMA was 0.05 ng. THg concentrations are expressed in µg.g -1 dw.

Statistical analysis

All statistical analyses were performed using the software R, v.3.6.1 (R development Core Team). All data were checked for normality and homogeneity of variances. Depending on the results, parametric or non-parametric tests were used. The differences of moisture content in each tissue between species, as well as the differences of moisture content between different tissues for each species, and the differences of moisture content for each species and each tissue between capture sites were all investigated by Kruskal-Wallis test.

Paired t-tests were used to compare THg concentrations between full scutes (a combination of keratin, bone and connective tissue) and scute keratin layers for each species. To access the relationship between THg concentrations in full scutes and scute keratin layers, we built a predictive equation for C. crocodilus. The predictive equation was built for a significant relationship between both tissues, using slope and intercept derived from the parametric linear regression line. To validate the models, bootstrapping with 1000 iterations were applied [START_REF] Harrell | Regression modeling strategies: with applications to linear models, logistic regression, and survival analysis[END_REF]. We did not build predictive equations for P. palpebrosus and P. trigonatus due to the restricted number of samples.

The difference of THg concentrations between tissues was assessed by Friedman ANOVA and Pairwise Bergmann-Hommel comparisons for each species. We performed Spearman rank tests to assess the relationships between body size (SVL and LT) and THg concentrations in the tissues, independently for each species.

The significance level for statistical analyses was always set at p < 0.05.

RESULTS

Moisture content

The results of the moisture content of blood, claws and scutes for each species are summarized in Table 1. We found significant differences in moisture content between the species for claws (Kruskal-Wallis: X 2 = 22.79, p < 0.001, n = 57) and full scutes (Kruskal-Wallis: X 2 = 10.66, p < 0.005, n = 58), but not for total blood (Kruskal-Wallis: X 2 = 1.11, p = 0.57, n = 31) (Table 1, Fig. 2).

We did not find any geographic variation in moisture content of the different tissues for the three species (all p > 0.18).

THg concentrations in scutes

Our results show significantly higher concentrations of THg in scute keratin layers than full scutes in two species (Paired t-test: C. crocodilus, t = -5.44, p < 0.001; P. trigonatus, t = -4.71, p = 0.02; P. palpebrosus, t = -2.18, p = 0.12, Fig. 3).

Our results do not show significant differences of THg concentration between the two capture sites (site 2, n = 11; site 8, n = 15, see Fig. 1) for full scutes and scute keratin layers of C. crocodilus (t-test: t = 0.24, p = 0.815 and t = 0.15, p = 0.882, respectively). Because no significant differences were found between sites, all C. crocodilus were pooled. A positive significant relationship was found between THg concentrations in full scutes and the scute keratin layers for C. crocodilus (linear regression: F1-24 = 93.67, R 2 = 0.78, p < 0.001, Fig. 4).

The significant level of the predictors and the upper and lower bounds of the 95% confidence interval (CI) are given by the equation (R 2 = 0.788; p < 0.001; 95% CI: 0.518-0.938):

THg scute keratin layers = 0.525 x THg full scute + 0.321

where THg in keratin layers and full scutes are expressed in µg.g -1 dw.

Relation between tissues

Our results show a significant difference of THg concentrations between tissues within each of the three species (Friedman ANOVA: P. trigonatus, X 2 = 20, p < 0.001; C. crocodilus, X 2 = 91.33, p < 0.001; P. palpebrosus, X 2 = 19.04, p < 0.001, respectively, Table 2). The THg concentrations were always higher in claws and scutes than in total blood and RBCs (Fig. 5).

The relationships between body size and THg concentrations in the different tissues, and the relationships between tissues were assessed by Spearman rank test for the three species and summarized in Table 3.

DISCUSSION

Moisture content

Our results show a strong inter-and intraspecific variability of moisture content between tissues (Table 1, Fig. 2). Indeed, claws are mainly composed of alpha-keratin and present the lowest moisture content, while scutes are mainly composed of beta-keratin in association with osteoscutes and connective tissues which increases water content [START_REF] Richardson | Crocodiles: Inside Out: A Guide to the Crocodilians and Their Functional Morphology[END_REF]. In contrast to the study of [START_REF] Yanochko | Tissue mercury concentrations in alligators (Alligator mississippiensis) from the Florida Everglades and the Savannah River site, Carolina[END_REF], in which study sites were separated by a very large distance, we did not find any geographic differences regarding moisture content in the examined tissues, which may be linked to the more restricted area of our study. Our results suggest that the relatively large variation of moisture content in fresh samples can induce variations in terms of trace element quantification, including THg concentration. This seems particularly the case for keratinized tissues, while moisture content in blood samples was less variable. Overall, our assessment of the moisture content in various tissues clearly suggests that the use of dry samples for quantification should be favoured in order to facilitate future largescale comparisons of contamination in crocodilians. Alternatively, the moisture content of the tissues should be specified when trace element concentrations are expressed in wet weight in order to allow straightforward conversion between units.

THg concentration: scute keratin layers vs. full scutes

Clipping tail scutes is a common sampling method in crocodilians which further allows identification of the animal and can be used for DNA, stable isotopes and contaminant analyses [START_REF] Jagoe | Mercury in alligators (Alligator mississippiensis) in the southeastern United States[END_REF][START_REF] De Thoisy | Genetic structure, population dynamics, and conservation of Black caiman (Melanosuchus niger)[END_REF][START_REF] Rainwater | Metals and organochlorine pesticides in caudal scutes of crocodiles from Belize and Costa Rica[END_REF][START_REF] Machkour-M'rabet | Between introgression events and fragmentation, islands are the last refuge for American crocodile in Caribbean Mexico[END_REF][START_REF] Trillanes | Metals in Caudal Scutes of Morelet's Crocodile (Crocodylus moreletii) from the Southern Gulf of Mexico[END_REF][START_REF] Pacheco-Sierra | Genetic and morphological evidence of a geographically widespread hybrid zone between two crocodile species, Crocodylus acutus and Crocodylus moreletii[END_REF][START_REF] Santos | Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus)[END_REF]Lemaire et al. 2021a). The skin of crocodilians is composed of bony scutes covered by connective tissue and keratin layers [START_REF] Richardson | Crocodiles: Inside Out: A Guide to the Crocodilians and Their Functional Morphology[END_REF][START_REF] Alibardi | Immunocytochemistry and keratinization in the epidermis of crocodilians[END_REF]. Although several studies reported concentrations of Hg in crocodilian scutes [START_REF] Jagoe | Mercury in alligators (Alligator mississippiensis) in the southeastern United States[END_REF][START_REF] Lázaro | Non-lethal sampling for mercury evaluation in crocodilians[END_REF][START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF][START_REF] Buenfil-Rojas | Mercury and metallothioneins in blood fractions and tissues of captive Morelet's crocodiles in Quintana Roo, Mexico[END_REF], detailed information on the actual part of the scute which had been used for analyses is generally missing, precluding further comparisons [START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF]. With the exception of 2 animals, our results show that THg concentrations significantly differ between full scutes (a combination of keratin, bone and connective tissues) and scute keratin layer, which can be explained by the poor affinity of Hg for bone tissue [START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF], which results in the highest concentrations in the keratin layer (Fig. 3). In contrast, keratinized tissues of vertebrates such as hair/fur, feathers and claws generally display high Hg concentrations due to the affinity of Hg for the sulfhydryl groups contained in keratins [START_REF] Appelquist | Mercury monitoring: mercury stability in bird feathers[END_REF][START_REF] Schneider | Mercury concentration in the spectacled caiman and black caiman (Alligatoridae) of the Amazon: implications for human health[END_REF][START_REF] Benjamin | Mercury (Hg) concentration in gravid Blanding's Turtles (Emydoidea blandingii) in Northeastern Illinois[END_REF][START_REF] Treu | Correlations between hair and tissue mercury concentrations in Icelandic arctic foxes (Vulpes lagopus)[END_REF][START_REF] Albert | Using blood and feathers to investigate large-scale Hg contamination in Artic seabirds: A review[END_REF].

Standardization of the analysed layers when scutes are used for Hg biomonitoring is therefore required to enable comparison between studies. In this respect, our results suggest that selecting the keratin layer should be favored as it avoids including unknown quantity of other tissue types (e.g., bone, connective tissues), thereby improving the evaluation of the actual environmental contamination status.

By providing a predictive equation of the relationship between THg concentrations found in full scutes and scute keratin layers of C. crocodilus, our study gives an objective tool to compare studies using both tissues for this species (both dry, Fig. 4).

Relationships between tissues

THg concentrations were higher in scutes and claws than in the RBCs and total blood (Table 2; Fig. 5). Blood is involved in the transport of Hg to different organs and represents the recent Hg exposure of the animal via its diet, one source of variation in Hg contamination in crocodilians (Lemaire et al. 2021a). In crocodilians, Hg concentrations in the blood are known to be related to Hg concentrations of internal tissues due to dynamic transfer in tissues involved in elimination (keratinized tissues), excretion (kidneys), detoxification (liver) and storage (muscles) [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from Rio Purus, Amazonas, Brazil[END_REF]Nilsen et al. 2017a). In contrast to blood, keratinized tissues are known to reflect the long-term exposure due to the non-reversible binding of Hg to the sulfhydryl residues of the keratins [START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF][START_REF] Lázaro | Non-lethal sampling for mercury evaluation in crocodilians[END_REF][START_REF] Marrugo-Negrete | Mercury levels and genotoxic effect in caimans from tropical ecosystems impacted by gold mining[END_REF]. A combination of non-lethal sampling of several tissues in caimans provides information on the recent (blood) and the long-term (keratinized tissues) Hg contamination of the individual, as well as the dynamics of Hg contamination over time, which had already been shown in other reptiles [START_REF] Lemaire | Determinants of mercury contamination in viperine snakes, Natrix maura, in Western Europe[END_REF].

Our results show a positive correlation between the body size (SVL and TL) of the three species included in the study and the THg concentrations in claws and scutes (Table 3). In contrast, the THg concentration of RBCs was positively related to SVL in P. trigonatus solely. The THg concentration in total blood showed a positive relationship with the SVL and TL of P.

trigonatus and C. crocodilus, but not with P. palpebrosus (Table 3). The variable relationship between blood Hg and body size is likely related to short-term variations in the diet of the individuals. Accordingly, such relationship was found in some studies on crocodilians [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from Rio Purus, Amazonas, Brazil[END_REF][START_REF] Buenfil-Rojas | Mercury and metallothioneins in blood fractions and tissues of captive Morelet's crocodiles in Quintana Roo, Mexico[END_REF]Lemaire et al. 2021a) but not in others [START_REF] Yanochko | Tissue mercury concentrations in alligators (Alligator mississippiensis) from the Florida Everglades and the Savannah River site, Carolina[END_REF][START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from Rio Purus, Amazonas, Brazil[END_REF][START_REF] Lawson | Nonlinear patterns in mercury bioaccumulation in American alligators are a function of predicted age[END_REF]. This emphasizes the complementarity of assessing Hg concentrations related to both long-term integration (i.e., keratinized tissues such as claws and scutes) and short-term exposure (i.e., blood) in biomonitoring studies. THg values marked with the same letter (a or b) are not statistically different (Pairwise Bergmann-Hommel test, p < 0.05), test performed independently for the three species.

CONCLUSION

  254 (Advanced Mercury Altec®). At least two replicates of 0.3 -1.0 mg dry weight (dw) were analysed for each sample. The reproducibility for duplicate samples was approved when relative standard deviation (RSD) was below 10%. The analyses of certified reference material (CRM) TORT-2 (Lobster hepatopancreas from the National Research Council of Canada; certified Hg concentration: 0.27 ± 0.06 µg.g -1 dw) and TORT-3 (Lobster hepatopancreas from the National Research Council of Canada; certified Hg concentration:

  Our results highlight the need to standardize the evaluation of Hg concentration in crocodilians between studies. The variation in moisture content between tissues and individual animals increases the variation in the reported Hg concentrations, and hence precludes robust comparison between studies. In order to avoid such shortcomings, researchers should perform trace elements quantification to report their results based on dry weight analytical procedures or should provide the information on the tissue moisture contents when studies report concentrations based on wet weight. In addition, our results emphasize the need to analyze scute keratin layer rather than full scutes in order to provide less variable and more reliable values of long-term Hg contamination of caimans. Finally, the simultaneous use of both blood and keratinized tissues in crocodilians offer the possibility to quantify different time scales of Hg exposure by non-lethal sampling.

Figure 1 .Figure 2 .

 12 Figure 1. Location of French Guiana, distribution ranges and capture locations of the spectacled caiman, Caiman crocodilus (sites 2, 8), the smooth-

Figure 3 .

 3 Figure 3. THg concentrations measured in full scutes and scute keratin layers (µg.g -1 dw) of spectacled caiman, Caiman crocodilus (A: n = 26,

Figure 4 .

 4 Figure 4. Linear regression between the mercury (THg) concentration in full scutes and scute keratin layers (in µg.g -1 dw) of the spectacled caiman (Caiman crocodilus; n = 26; F1-24 = 93.67, R 2 = 0.78, p < 0.001). Regression lines (in red) with 95% confidence intervals indicate highly significant

Figure 5 .

 5 Figure 5. THg concentrations measured in claws, scutes, red blood cells (RBCs) and total blood (µg.g -1 dw) of spectacled caiman, Caiman

Table 2 .

 2 Biometric data (cm, Mean ± SD, Min -Max (n)) and THg concentrations (µg.g -1 dw; Mean ± SD, Min -Max (n)) in the tissues of smoothfronted caiman, Paleosuchus trigonatus, spectacled caiman, Caiman crocodilus and dwarf caiman, Paleosuchus palpebrosus, in French Guiana.

	SVL: Snout Vent Length; TL: Total Length; RBCs: Red Blood Cells; n: number of samples.
	Species	SVL	TL	THg claws	THg scutes	THg RBCs	THg Total blood
	Paleosuchus trigonatus	33.32 ± 19.81	62.11 ± 36.42,	2.420 ± 1.905 a ,	3.332 ± 3.066 a ,	0.447 ± 0.270 b ,	0.300 ± 0.178 b ,
		10.90 -81 (51)	22.8 -143 (51)	0.147 -7.509 (50)	0.087 -9.859 (48)	0.049 -0.774 (11)	0.032 -0.738 (24)
	Caiman crocodilus	32.70 ± 13.57	66.60 ± 24.11	2.692 ± 1.608 a	2.638 ± 1.497 a	0.963 ± 0.612 b	0.605 ± 0.380 b
		14.5 -103 (48)	31.0 -176.0 (48)	0.321 -8.807 (48)	0.307 -7.407 (47)	0.145 -2.244 (26)	0.089 -1.532 (40)
	Paleosuchus palpebrosus	38.12 ± 15.29	79.42 ± 33.22	8.351 ± 4.965 a	7.647 ± 4.742 a	2.364 ± 1.884 b	1.376 ± 0.986 b
		16.5 -62 (12)	34.2 -150 (13)	2.028 -20.042 (13)	0.789 -15.628	0.447 -5.775 (6)	0.540 -3.415 (7)
					(13)

Table 3 .

 3 Relationship between body size (cm) and THg concentration (µg.g -1 dw) in tissues, and THg concentration between tissues of smooth-fronted caiman, Paleosuchus trigonatus, spectacled caiman, Caiman crocodilus and dwarf caiman, Paleosuchus palpebrosus. Values refer to  (Spearman rank test) and significant relationships are in bold. Sample sizes are given in parenthesis. TL = Total Length; SVL = Snout Vent Length; RBCs: Red Blood Cells.

		TL	SVL	THg Scutes	THg Claws	THg RBCs
	Paleosuchus trigonatus
	THg Scutes	0.877 (48)	0.885 (48)	-	-	-
	THg Claws	0.842 (50)	0.850 (50)	0.945 (48)	-	-
	THg RBCs	0.451 (11)	0.623 (11)	0.614 (10)	0.614 (10)	-
	THg Total Blood	0.730 (24)	0.769 (24)	0.897 (21)	0.914 (23)	0.771 (6)
	Caiman crocodilus
	THg Scutes	0.459 (47)	0.472 (47)	-	-	-
	THg Claws	0.468 (48)	0.475 (48)	0.909 (47)	-	-
	THg RBCs	0.353 (26)	0.388 (26)	0.894 (25)	0.811 (26)	-
	THg Total Blood	0.386 (40)	0.399 (40)	0.882 (39)	0.870 (40)	0.992 (25)
	Paleosuchus palpebrosus
	THg Scutes	0.831 (13)	0.846 (12)	-	-	-
	THg Claws	0.945 (13)	0.937 (12)	0.857 (13)	-	-
	THg RBCs	0.771 (6)	0.600 (5)	0.829 (6)	0.657 (6)	-
	THg Total Blood	0.750 (7)	0.600 (6)	0.643 (7)	0.607 (7)	0.900 (5)

Acknowledgments

We are grateful to the authorities of Conseil Scientifique Régional du Patrimoine Naturel (CSRPN) and La Direction Générale des Territoires et de la Mer de Guyane (DGTM), which authorized our fieldwork. We would also like to thank the teams of "Réserve Naturelle des Nouragues", "Réserve Naturelle du Mont-Grand Matoury", "Réserve Naturelle Trésor", "Réserve Naturelle de la Trinité", "Réserve Naturelle de Kaw-Roura", "Pripis de Yiyi", the Conservatoire du Littoral, F. Starace, T. LePape, F. Beau and P. Gaucher for their help in the field, and E. Courtois, M. Dewynter and Q. Martinez for providing samples. This work was supported by the Office de l'Eau de Guyane; the Office Française pour la Biodiversité, the Direction Générale des Territoires et de la Mer de Guyane, the Zoo de Paris, the Fondation d'entreprise Hermès and the CNRS. We are grateful to C. Churlaud and M. Brault-Favrou from the "Plateforme Analyses Elémentaires" of LIENSs for their assistance during mercury analyses. The Institut Universitaire de France (IUF) is acknowledged for its support to P.

Bustamante as a Senior Member. We thank the Nouragues Research Field Station (managed by CNRS) which benefits from "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (AnaEE France ANR-11-INBS-0001; Labex CEBA ANR-10-LABX-25-01), and its team for assistance in the field.

Ethics approvaldraw blood and sample claws and scutes was granted by the French authorities (Direction


Ari Saputra

Mireille Del Nero
email: mireille.delnero@iphc.cnrs.fr

Quaranta Gaetana
email: quaranta@unistra.fr

Olivier Courson
email: olivier.courson@iphc.cnrs.fr

M Ari Saputra

Mireille Del Nero

Understanding of Tritium Behavior Toward Sediment in Aquatic System

Keywords: Ecotoxicity, Organically bound tritium, T ritium

come

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INTRODUCTION

Tritium is one of the hydrogen isotopes and can be produced either by a cosmic ray or human activities. When neutron from cosmic radiation interacts with nitrogen, nitrogen-15, which is radioactive, is produced and disintegrates into carbon and tritium. This tritium will reside in the atmosphere in a gaseous form (HT) and as water vapor (HTO). Tritium also can be produced from nuclear facilities such as nuclear power plant (NPP) and nuclear reprocessing facility [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF], [2]. Some chemical industry also can release a little amount of tritium in the environment. This tritium is called "technogenic" tritium because it is mainly released by pharmaceutical industries, in luminous paint residues by the watch industry, or with residues by factories manufacturing luminous safety lighting [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF].

The nuclear power plant is the main producing of tritium. International Atomic Energy Agency (IAEA) reports showing that one nuclear power plant facility can release 11 to 27 TBq/year tritium in liquid form and 0.2 to 0.8 TBq/year tritium in gaseous form depends on types of the reactors [4]. In the environment, it can exist as tritiated water (HTO) and organically bound tritium (OBT). Growing amounts of studies on the environmental behavior of tritium indicate different behaviors dependent on chemical speciation (OBT, NE(non-exchangeable)-OBT, HTO) as shown in the study of Rhone river, France. Latest research has shown that tritium in OBT form can be trapped in the sediment and reside in water [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF], [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF], [START_REF] Ducros | Science of the Total Environment Tritium in river waters from French Mediterranean catchments : Background levels and variability[END_REF]- [START_REF] Péron | Anthropogenic tritium in the Loire River estuary , France[END_REF]. The tritium trapped in sediment has long period biological time, which can lead to an aquatic ecotoxicity potential.

It has thus been found that the influence of a nuclear facility or possibly chemical industry on tritium levels is expressed at a local scale and is visible in the aquatic environment. This study is aiming to get an understanding of tritium behavior in the aquatic system.

TRITIUM FORM IN ENVIRONMENT

In the environment, because tritium is an isotope of hydrogen, tritium is present in all part of the geosphere, including the atmosphere, hydrosphere and lithosphere, the biosphere and the anthroposphere. It is also present in many physical and chemical forms.

A. Tritiated Water (HTO)

Tritiated water (HTO) is the most common form of tritium in the geochemical systems and in living organisms. This form can be found throughout the whole hydrosphere cycle in the environment. It is very mobile since it has similarity with normal water. HTO concentration in hydrosphere depends on many factors such as latitude, season, the age of the water mass, hydrology, drainage basin size, precipitation rate of minerals, and altitude. [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF].

The mean concentration of HTO in French river water [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF]4 Bq/L) is the same with HTO concentration in rainwater [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF][START_REF] Eyrolle | Science of the Total Environment A brief history of origins and contents of Organically Bound Tritium ( OBT ) and 14 C in the sediments of the Rhône watershed[END_REF] Bq/L) due to radioactive decay during the mass transfer from atmosphere to hydrosphere [START_REF] Ducros | Science of the Total Environment Tritium in river waters from French Mediterranean catchments : Background levels and variability[END_REF]. The concentration of HTO is varying because of a rapid transfer of hydrogen and water molecules with different components in the environment. HTO can also be found in the atmosphere in the form of water vapor.

B. Organic Bound Tritium (OBT)

Inorganically bound tritium can be linked either by covalent bound or weak hydrogen bond. Organically Bound Tritium (OBT) initially integrates organic matter cycle mainly during the photosynthesis process. Thus it will move to the biosphere through the food chain. Many research has been done to investigate OBT in the environment. Because tritium is part of the organic matter cycle, OBT is accumulated in organic matter storage media, especially in the sediments of streams and rivers. [START_REF] Eyrolle | Science of the Total Environment A brief history of origins and contents of Organically Bound Tritium ( OBT ) and 14 C in the sediments of the Rhône watershed[END_REF]. Tritium can persist in both terrestrial and aquatic environments according to the recycling rates of organic matter. A study showed that the mean Organically Bound Tritium (OBT) concentration in the sediments of Rhone river in France, is not significantly different from the mean HTO concentration recorded for rainwater (2.4 ± 0.6 Bq/L and 1.6 ± 0.4 Bq/L, respectively) [START_REF] Eyrolle-Boyer | Tritium and 14C background levels in pristine aquatic systems and their potential sources of variability[END_REF]. The authors have the hypothesis that regional differences are expected depending on the biomass inventories affected by the global fallout and the recycling rates of the organic matter in the various affected watersheds. Hence, there are possibilities that seasonal factors also affect OBT concentration.

Several studies have been done on the behavior of tritium in the aquatic systems. Tritium behavior in aquatic systems is influenced by its various forms and biogeochemical exchanges among aquatic components, as can be shown in Figure 2. Tritium is bound to dissolve, colloidal or particulate organic compounds either through exchange reactions or enzymatically catalyzed reactions. In exchange reactions, tritium is bound to oxygen, sulfur, phosphorus or nitrogen atoms through weak hydrogen bonds, resulting in forms that are more or less exchangeable with ambient oxide hydrides known as Exchangeable Organically Bound Tritium (E-OBT). In the case of enzymatically catalyzed reactions, tritium is bound to carbon atoms and is permanently fixed to the resulting molecules until they are metabolized. Such forms are referred to as Non-Exchangeable Organically Bound Tritium (NE-OBT). [START_REF] Cossonnet | Apparent enrichment of organically bound tritium in rivers explained by the heritage of our past d e Fr e[END_REF] Fig. 1. Tritium behavior in aquatic system. (1) NE-OBT will become E-OBT through biodegradation of organic matter after a long time of retention. [START_REF] Cossonnet | Apparent enrichment of organically bound tritium in rivers explained by the heritage of our past d e Fr e[END_REF] E-OBT is tritium bound with sulfur, oxygen, or nitrogen atoms and can be removed by washing with Tissue Free Water Tritium (TFWT). E-OBT equilibrates very quickly with TFWT, which is itself at equilibrium with the water molecules in the surrounding environment [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF], [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF]. Also, E-OBT is in equilibrium with HTO in the plant or animal and behaves the same as HTO. This fraction of OBT depends strongly on the HTO concentration present at the time of sampling and can exchange with water vapor during analysis. [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF]. NE-OBT is tritium covalently bond with carbon atoms, and it makes stronger bound than tritium bound in E-OBT. Unlike E-OBT, tritium in this form has a biological period that depends on the molecules on which it is bound. If the tritium is bound with fast metabolism species of organic matter such as carbohydrates, it can stay in this form from few weeks and few months until it degrades. On the other hand, tritium can be bound with slow metabolism species of organic matter, and in this form, tritium can be bound for several years or decades. [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF], [START_REF] Eyrolle | Science of the Total Environment A brief history of origins and contents of Organically Bound Tritium ( OBT ) and 14 C in the sediments of the Rhône watershed[END_REF].

Recent studies have shown that NE-OBT causes tritium to persist in organic matter and that the fate of NE-OBT depends on the reaction kinetics, particularly the decomposition and recycling dynamics of the organic compounds. Higher OBT concentrations than the HTO concentrations of the surrounding environment have been widely observed in terrestrial plants and river sediments collected away from the influence of releases from nuclear facilities. For example, OBT concentration observed in the Loire river ranges from 10 to 23 Bq/L, and the HTO concentration ranges from 6.9 to 21 Bq/L [START_REF] Péron | Anthropogenic tritium in the Loire River estuary , France[END_REF]. This shows the persistence of tritium initially integrated into the organic matter within these matrices. [START_REF] Kim | Current understanding of organically bound tritium (OBT) in the environment[END_REF], [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF], [START_REF] Ducros | Science of the Total Environment Tritium in river waters from French Mediterranean catchments : Background levels and variability[END_REF], [START_REF] Eyrolle | Science of the Total Environment A brief history of origins and contents of Organically Bound Tritium ( OBT ) and 14 C in the sediments of the Rhône watershed[END_REF]. Another study in Qinshan Nuclear Power Plant, China, showed that for soils collected at ± 2 km from the nuclear power plant, there is a correlation between NE-OBT and HTO ratio in soil samples. They concluded that this could happen because of living organism converting HTO into OBT. [START_REF] Zhang | Studies of particle size distribution of Non-Exchangeable Organically Bound Tritium activities in the soil around Qinshan Nuclear Power Plant[END_REF]. This indicates that NE-OBT exists in an environment not only because of organic matter but also because of living organism. As noted before, tritium in NE-OBT can cause resistance for several years. It o can be bound with many types of organic carbon, and there is a possibility that tritium can be bound in close association with organic molecules sorbed at surfaces of nanoparticles. Also, research showed that around 70% of the tritium bound to the organic matter is in a non -exchangeable form, and the other 30% is tritium is bound with organic matter as E-OBT [START_REF] Eyrolle | An updated review on tritium in the environment[END_REF].

MATERIALS AND METHODS

A. Sample Location

The experiment of tritium adsorption on the sediment was done with extracted organic matter and pre-treated sediment from the Rhine river, located in eastern France. The river is near many industries area including nuclear power plant, chemichal industries, and agriculture area. 300 g sediment was taken in the Rhine river bank (the sample point can be shown in figure 3) with depth ± 5 cm from ground surface. The collected sediment was putted in 500 ml plastic bottle and then stored in refrigerator with temperature 5ºC [START_REF] Eyrolle-Boyer | Tritium and 14C background levels in pristine aquatic systems and their potential sources of variability[END_REF].

B. Sample Processing and Measurement

The sediment collected in the Rhine river was washed three-times using water to remove free tritium. After that, the organic matter was removed using hydrogen peroxide. Then, the model sediment obtained was dried for 6 hours to remove excess water. The sediment without the organic matter was used for two experiments with tritium. The model sediment was used to make two experiments : the first experiment consisted in a 15 mL solution in contact with 20 mg model sediment only, and the second consisted in a 15 mL solution with 20 mg model sediment and 302 µL extracted organic matter from the Rhine river system (the organic matter reconcentrated at a value 623.3 mgC/L). The solution pH was adjusted around 6.5 to 7.5 as measured in the Rhine river system. These two solutions added 10 µL tritium standard with a known concentration (74.1 Bq/L) and then (after shaking during two hours and sediment-solution separation) the supernatants were measured with Liquid Scintillation Counting (LSC). The LSC used for meassurement is Perkin-Elmer QUANTULUS Ultra Low Level Liquid Scintillation Spectrometer. To determine tritium concentration, 8 mL of water samples were mixed with 15 ml high-efficiency LSC cocktail (Perkin-Elmer "Ultima Gold LLT") then the samples were measured with LSC to count the activities of tritium in water samples. The counting time was 7200 s and three-time measurements were done for each sample. The measurement efficiency ε was determined by inner standard source method.

Fig. 4. Scheme of tritium adsorption experiment

The experiment is aiming to see the adsorption of tritium from the Rhine river sediment sample and to reveal whether the organic matter may affect tritium adsorption on the sediment.

RESULTS AND DISCUSSION

The experiment used the "model sediment" sample (sediment from which organic matter was removed) and organic matter extracted from the water of the Rhine River (and reconcentrated at a value of 623.3 mgC/L after extraction). 20 mg of sediment sample was brought in contact with 15 ml solution. One of the solutions was added 302 µL of extracted organic matter solution. The ratio between sediment sediment and organic matter in the solution was 9.06 mgC/g sediment. Both solution then added 0.0196 g of tritium s tandard for solution without organic matter and 0.021 g of tritium standard for solution with organic matter. These tritium standard has activities 0.235 Bq and 0.169 Bq, respectively. After shaking and separation, the supernatant solution was measured by LSC to see is there any difference in tritium concentration. The result of the experiment can be shown in Table 2. The results indicated that the tritium was absorbed by "model sediment" as much 80 %. When the natural organic matter was present in the sample, the adsorption increased to 86 %. To know the sediment characters, Transmission Electron Microscopy (TEM) was done. The TEM analysis can see particles that present on the sediment and predict what elements that are in the particles. The TEM imaging results showed that several types of nanoparticles were present in the sediment sample. The majority of the nanoparticles present are calcite (CaCO 3 ), and clay nanoparticle possibly as smectites (containing iron). The particle sizes vary from 10 nm up to 1 µm, which indicates there are many groups of calcite and smectite in the sample. Figure 5 showed that pictures of nanoparticles that can be found in the sample. The left picture is nanoparticle of clay nanoparticle present in the sample with the size of ±1 µm. The big particle was identified as a clay material consist of aluminum, silica, and iron. The right side picture shows calcite nanoparticle with the size of ±5 µm present on the edge of particle with size of 20 nm. The picture also indicated there are many groups of nanoparticle with different size. We know that surface of smectite bear hydroxyl groups (OH -) so that there may be isotopic exchange between the hydrogen of the surface hydroxyls and tritium [START_REF] Kuligiewicz | The charge of wettable illite-smectite surfaces measured with the O-D method[END_REF]. From TEM analysis, it is known that in the sample we have the presence of smectite. The adsorption of tritium in the sediment might be because of smectite present in the sediment as a nanoparticle or bigger mineral. The smectite can absorb the tritium because of the hydroxyl group in the surface of smectite have isotopic exchange with the tritium in the solution. The result also indicates the organic matter helps to increase sediment tritium adsorption. The organic matter can help the adsorption of tritium possibly because organic matter is adsorbed on the surface of the sediment and organic matter adsorb tritium directly. This result suggests that, besides its association with suspended nanoparticles, tritium is expected to be bound to sediment -and organic sorbed to the sediment sample.

CONCLUSION

The sample collected from the Rhine River system showed that there are present of calcite and smectite nanoparticle. The experiment showed that tritium can be absorbed by sediment, the absorption might be happened due to presence of smectite nanoparticle and possibly with calcite. In addition, the presence of organic matter in the system can help tritium adsorption to sediment. The adsorption of tritium in sediment might be different depends on the characteristics of the sediment. In this case, sediment with the majority consist of smectite can absorb tritium.

Fig. 2 .

 2 Fig. 2. T ritium form in the environment[START_REF] Eyrolle | An updated review on tritium in the environment[END_REF]

Fig. 3 .

 3 Fig. 3. Location of sediment sample, sample was taken in point JR23

Fig. 5 .

 5 Fig. 5. Different size of nanoparticle present on the sample; nanoparticle of clay mineral (left) and calcite (right)

Table 1 .

 1 Performance data of QUANT ULUS Ultra Low Level Liquid Scintillation Spectrometer.

	Isotopes 3 H	Efficiency  Unquenched 3 H ≥	Background  0.4 -1.2 CPM.
		65%.		 Down to 0.5 Bq/l in
		 Water	Sample	500 min counting.
		Efficiency up to
		27 %.
	14 C	 Unquenched 3 H ≥
		95%.
		 Radiocarbon
		dating with PAC
		= 73%.

 <0.3 CPM.

.

Table 2 .

 2 T ritium adsorption result

	Solution Mass of 3 H	NOM added	Mass of sediment	3 H Meassured	3 H Activities	Adsor ption
		added	(µL)	added	(B q/L)	(B q)	(% )
		(g)		(mg)
	Sediment 0.0196	-	21	15.7	0.235	80
	Sediment	0.021	302	20	11.3	0.169	86
	+OM