Vivier Baptiste

David Frank
email: frank.david@live.fr

Marchand Cyril

Thanh-Nho Nguyen

Meziane Tarik

Frank David

Fatty acids, C and N dynamics and stable isotope ratios during experimental degradation of shrimp pond effluents in mangrove water

Keywords: Lipid, stable isotopes, aquaculture, decomposition, denitrification, suspended particulate matter (SPM), mangrove

Intensive shrimp farming generates high loads of wastewaters that are released along tropical coastlines with potential impacts on the ecosystems. In this study, we used an experimental approach to analyze the behavior of shrimp pond effluents released in the Can Gio mangrove waterways (Southern Vietnam). We incubated shrimp pond effluents (EF), river water (RV), and a mixture of both (MI; 90% RV + 10% EF) in a dark room and measured fatty acid (FA) compositions, C and N concentrations and stable isotopes ratios (δ 13 C and δ 15 N) of suspended particulate matter during 16 days.

Fatty acid concentrations rapidly decreased in EF with a 50% loss of FA during the first 24h of the experiment and a 75% loss after 4 days of incubation. Proportions of the FA 18:1ω7 increased in MI during incubation, suggesting that this FA may be used as a tracer of anthropogenic substances release in marine environments.

Introduction

Shrimp farming has strongly grown in the last decades. Global shrimp production was 1.1 billion tons in 2000, 3.4 billion tons in 2008 and reached 4.9 billion tons in 2015 (FAO, 2016). Although shrimp farming generates important social and economic benefits (Primavera, 1997), its huge development is strongly criticized for its negative environmental impacts [START_REF] Anh | Water pollution by intensive brackish shrimp farming in south-east Vietnam: Causes and options for control[END_REF]. Intensive shrimp farming is characterized by high shrimp density, high amount of inputs (food pellets, fertilizers and antibiotics) and high water renewal in ponds (15% every two weeks in Vietnam; [START_REF] Anh | Water pollution by intensive brackish shrimp farming in south-east Vietnam: Causes and options for control[END_REF]. This leads to high effluent releases (including dissolved nutrients, toxic compounds, microorganisms, shrimp excrements, unconsumed food pellets, etc.) associated with highly degraded water quality close to shrimp farming areas [START_REF] Molnar | Impacts of shrimp farm effluent on water quality, benthic metabolism and N-dynamics in a mangrove forest (New Caledonia)[END_REF][START_REF] Páez-Osuna | The environmental impact of shrimp aquaculture: a global perspective[END_REF][START_REF] Páez-Osuna | Environmental Load of Nitrogen and Phosphorus from Extensive, Semiintensive, and Intensive Shrimp Farms in the Gulf of California Ecoregion[END_REF]) that may stimulate algal blooms and create anoxic water conditions [START_REF] Kemp | Temporal responses of coastal hypoxia to nutrient loading and physical controls[END_REF][START_REF] Naylor | Nature's Subsidies to Shrimp and Salmon Farming[END_REF][START_REF] Smith | Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems[END_REF].

Despite potential impacts of shrimp pond effluent discharges in estuarine waters are recognized, their effect is mostly investigated regarding nutrients [START_REF] Páez-Osuna | Environmental Load of Nitrogen and Phosphorus from Extensive, Semiintensive, and Intensive Shrimp Farms in the Gulf of California Ecoregion[END_REF][START_REF] Trott | The impact of shrimp pond effluent on water quality and phytoplankton biomass in a tropical mangrove estuary[END_REF] and other parameters are poorly documented, as for example the nutritional value of impacted water suspended particulate matter (SPM). In New Caledonia, [START_REF] Aschenbroich | Spatio-temporal variations in the composition of organic matter in surface sediments of a mangrove receiving shrimp farm effluents (New Caledonia)[END_REF] have shown that the nutritional quality of mangrove surface sediments was affected by shrimp pond effluents releases (i. e. modified fatty acid composition). Indeed, sediments integrate chronic disturbances on a relatively long-time basis [START_REF] Molnar | Impacts of shrimp farm effluent on water quality, benthic metabolism and N-dynamics in a mangrove forest (New Caledonia)[END_REF], while SPM naturally varies at short spatial scales in mangrove estuaries [START_REF] Trott | The impact of shrimp pond effluent on water quality and phytoplankton biomass in a tropical mangrove estuary[END_REF], making difficult to differentiate changes induced by environmental parameters (e.g. season, salinity, SPM concentration, daylight presence) from those due to shrimp pond effluent releases [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF].

Lipids have been successfully employed to detail the nutritional composition of organic matter (OM) in marine ecosystems [START_REF] Canuel | Relations between river flow, primary production and fatty acid composition of particulate organic matter in San Francisco and Chesapeake Bays: a multivariate approach[END_REF][START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF][START_REF] Meziane | Fatty acids as tracers of organic matter in the sediment and food web of a mangrove/intertidal flat ecosystem, Okinawa, Japan[END_REF]. They are good indicators of microalgae and bacteria relative abundances and allow to evaluate the physiological state of phytoplankton cells [START_REF] Pan | Influence of physiological states of Emiliania huxleyi cells on their lipids and associated molecular isotopic compositions during microbial degradation[END_REF][START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF]. In complement to fatty acids, stable isotopes may inform on the processes occurring during OM decomposition [START_REF] Caraco | Microbial assimilation of DIN in a nitrogen rich estuary: implications for food quality and isotope studies[END_REF][START_REF] Brabandere | δ13C and δ15N dynamics of particulate organic matter in freshwater and brackish waters of the Scheldt estuary[END_REF][START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF]2017). Finally, nitrogen is a major compound of OM that is newly made available for primary producers after OM has been degraded, thus releasing ammonia in the surrounding waters which can fuel nitrification and eutrophication.

A few studies have been examining the degradation of lipids from urban sewages in natural environments [START_REF] Matsumoto | Changes in organic constituents in river water during incubation[END_REF][START_REF] Marty | Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater[END_REF] but none has yet investigated their decomposition in shrimp farm effluents, although the latter are known to cause serious threats to coastal ecosystems in Southeast Asia (e.g. increasing nitrogen influx and change in C/N balance; [START_REF] Lee | From blue to black: Anthropogenic forcing of carbon and nitrogen influx to mangrove-lined estuaries in the South China Sea[END_REF]. The Can Gio mangrove biosphere reserve is located at the downstream end of Ho Chi Minh City (Southern Vietnam) and covers an area of 719.6 km 2 [START_REF] Tuan | Can Gio mangrove biosphere reserve evaluation, 2012: current status, dynamics, and ecosystem services[END_REF]. At the north-east border of the mangrove forest, a transition area between the forested core and the Southern limit of Ho Chi Minh City covers about 300 km 2 where shrimp farming is the main economic activity, releasing wastewaters in the nearby waterways, with potential impacts (e.g. high nutrient loading, spread of diseases, antibiotics accumulation in sediments) on the ecosystem (Le andMunekage 2004, Anh et al., 2010). The release of shrimp ponds effluents is not controlled in this region. Effluents are not being treated and directly released on defined schedules in the nearby rivers throughout the year [START_REF] Anh | Water pollution by intensive brackish shrimp farming in south-east Vietnam: Causes and options for control[END_REF], thus rapidly reaching the 600m wide and 10-20m deep main estuary located a few hundred meters downstream and further flooding the mangrove forest (a map of the Can Gio mangrove biosphere reserve can be found in [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF]).

In the present study, we used an experimental approach based on the one employed by [START_REF] Marty | Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater[END_REF] to examine changes in SPM nutritional quality during decomposition of shrimp pond effluents released in a mangrove river in the Can Gio district. We aimed at characterizing the initial FA composition of shrimp pond effluents and then follow the dynamics of FA once their release in the environment has been simulated. We also measured variations of carbon and nitrogen forms and their isotopic ratios as markers of degradation processes and their kinetics. We expected to measure elevated phytoplanktonic abundances in shrimp ponds, as they generally exhibit high primary production [START_REF] Burford | A synthesis of dominant ecological processes in intensive shrimp ponds and adjacent coastal environments in NE Australia[END_REF], which may induce a strong bacterial development during effluents decomposition in mangrove waters. We sampled water from shrimp ponds and a nearby mangrove river to study the degradation of lipids in SPM in the absence of light (thus imitating natural conditions in the deep and turbid estuary). Pure shrimp pond effluents, pure river water and a combination of both were incubated in controlled conditions during 16 days. We hypothesized that (1) specific bacterial strains (e.g. denitrifying ones) and/or degradation pathways were favored in a mix of shrimp pond effluents with river water compared to both waters taken in isolation (2) specific fatty acids could be used as tracers of shrimp pond effluents release in natural ecosystems.

Materials and methods

Water collection

Water samples were collected in shrimp ponds and a nearby mangrove river of the Can Gio mangrove Biosphere Reserve in February 2017. Surface estuarine water (river water; RV) was collected using a wired bucket from the middle of the Rung Sat bridge (10° 35' 2.94'' N; 106° 49' 32.68'' E) at the beginning of ebb tide, while a composite sample of shrimp pond effluents (EF) was made using water from three different intensive shrimp ponds (40-60 shrimps/m 2 ) located close to the river water sampling point.

Experimental design

Water samples were brought in white plastic jerrycans at ambient temperature to the CARE (Centre Asiatique de Recherche sur l'Eau) facilities in Ho Chi Minh City in February 2017. Jerrycans containing the same water source (EF of RV) were mixed together before being disseminated in 14L plastic buckets. These buckets were meant to test three conditions: (1) pure shrimp pond effluents (EF), (2) pure river water (RV) and, (3) a combination of both (mixed water; MI: 90% RV + 10% EF). Each experimental condition was tested in 4 replicates (12 buckets). The amount of wastewaters released from shrimp ponds to the mangrove river is unknown but probably much below 10% of the discharge of this large estuary (about 300 m 3 s -1 in February, the month we conducted our experiment, [START_REF] Nguyen | Nutrient dynamics and eutrophication assessment in the tropical river system of Saigon -Dongnai (Southern Vietnam)[END_REF]. Proportion of RV and EF in the MI condition (90/10) was chosen in a way to place shrimp pond effluents in the environmental conditions found in river waters, without excessively diluting the FA signal. In addition, previous studies (unpublished data) revealed concentrations of FA roughly 10 times higher in shrimp pond effluents compared to river water. Fatty acids originating from EF and RV are thus expected to be added in similar quantities in the MI condition. All buckets were incubated in a dark room at 30°C and continuously homogenized with 200L h -1 aquarium pumps during 16 days without aeration. Dark conditions are meant to simulate the high turbidity of mangrove waters. The estimated light penetration in the estuary is actually lower than 1m and considering the estuary depth (10-20 m) most of SPM is transported in the dark [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF] although suspended particles may reach the photic zone from time to time due to elevated water turbulence.

Water samples were taken at short intervals at the beginning of the experiment and more spaced thereafter [START_REF] Matsumoto | Changes in organic constituents in river water during incubation[END_REF][START_REF] Marty | Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater[END_REF]. Sampling were performed at T0, and after 8 h, 16 h, 24 h, 2 days, 3 days, 4 days, 6 days, 8 days, 12 days and 16 days of incubation (T8, T16, T24, D2, D4, D6, D8, D12 and D16). Two water subsamples were collected at each sampling time from the 12 buckets and filtered for analysis of fatty acids and stable isotopes of SPM. Filtrations were performed using pre-combusted and pre-weighed (4h at 450°C) 0.7 μm Whatman ® GF/F glass microfiber filters and filtered water was kept for dissolved nutrients analysis. All filters were kept at -25°C and analyzed within two months.

Sample processing

Salinity, dissolved oxygen and pH were measured at the beginning of the experiment (T0) using a multi-parameter probe (WTW 3420®). Dissolved inorganic nitrogen forms (NH4 + , NO2 -and NO3 -) were measured at T0,T8, T24, D2, D4, D8 and D16 only, using a Hach-Lang kit photolab DR 1900 employing standard colorimetric methods (APHA, 1995;[START_REF] Nguyen | Nutrient dynamics and eutrophication assessment in the tropical river system of Saigon -Dongnai (Southern Vietnam)[END_REF]. Reproducibility for replicated measurements was higher than 5%.

Lipids were extracted following a slightly modified method of [START_REF] Bligh | A Rapid Method of Total Lipid Extraction and Purification[END_REF] as described in [START_REF] Meziane | Fate of mangrove organic matter along a subtropical estuary: small-scale exportation and contribution to the food of crab communities[END_REF]. An internal standard (23:0) was added to the samples prior to extraction. Lipids were first extracted using a methanol:water:chloroform mixture (2:1:2 v:v:v). Chloroform, containing lipid extracts, was isolated and evaporated under nitrogen (N2) flux. Dried lipid extracts were then saponified with a solution of methanol and sodium hydroxide (NaOH, 2N) (2:1, v:v) at 90°C for 1h30 min. Subsequently, methylation was performed, transforming all fatty acid esters into fatty acid methyl esters using methanolic boron trifluoride (BF3-CH3OH).

Fatty acids were separated by gas chromatography (Varian 3800), using an Agilent ® VF-WAXms capillary column 30m long, 0.25mm inner diameter and with 0.25 μm film thickness, and quantified using a flame ionization detector. Identification of fatty acids was performed using gas chromatography-mass spectrometry (Varian 450-GC; Varian 220-MS) and comparison of GC retention times with commercial standards (Supelco ® ).

The internal standard (23:0) was used to determine the concentration of each fatty acid (CFA) in µg FA per mg SPM.

Stable isotopes of carbon and nitrogen were measured at T0, T24, D4 and D16. SPM samples were fumigated for 4h using 35% HCl to remove all carbonates. Samples were prepared in tin capsules at the Muséum National d'Histoire Naturelle (MNHN-Paris) and analyzed at the University of California Davis Stable Isotope Facility (Department of Plant Sciences, UC Davis, Davis, California), using a Vario EL Cube elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, U.K.). During analysis, laboratory standards (bovine liver, nylon 6, enriched alanine and glutamic acid) previously calibrated against international standard reference materials were interspersed with samples. Carbon and nitrogen stable isotope results were reported in parts per thousand (‰), using standard delta notation (δ 13 C and δ 15 N) and are relative to V-PDB (Vienna PeeDee Belemnite) and atmospheric air, respectively. The C/N ratio was calculated as the molar ratio of particulate organic carbon (POC) vs. particulate organic nitrogen provided by the isotope mass ratio spectrometer.

4. Data analysis

Degradation rate constants of fatty acids were calculated using an exponential decay model on the form CFAfinal = CFAinitial × e -kt , where CFA is the final or initial concentration of a given FA or group of FA in the treatment, k is the degradation rate constant in days -1 and t is the incubation time in days. Data treatment and graphical representations were performed using R 3.3.2.

Results

Water parameters

Salinity was lower in effluents (10.1 ± 0.0) compared to river water (13.2 ± 0.0) and pH was higher in EF (8.35 ± 0.01) compared to RV (7.45 ± 0.01) (Table 1). As intended, the mixed condition almost kept the conditions displayed by RV (salinity = 12.8 ± 0.0 and pH = 7.53 ± 0.00). Suspended particulate matter concentration was about half as much in EF (91.2 ± 10.3 mg L -1 ) compared to RV (204.6 ± 4.3 mg L -1 ) and C/N ratio was lower in EF (7.0 ± 0.0) compared to RV (9.7 ± 1.4; Table 1). Shrimp pond effluents were enriched in both dissolved inorganic nitrogen (DIN) and total nitrogen compared to RV (NH4 + , NO2 -and NO3 -were summed to get free of the nitrogen cycle steps that are not meant to be discussed in this paper; Fig. 1). Concentrations of DIN slightly increased in EF at the beginning of the experiment (from 1.26 ± 0.04 mmol L -1 at T0 to 1.76 ± 0.19 mmol L -1 at D2) and then dropped from 1.78 ± 0.14 mmol L -1 at D8 to 0.23 ± 0.04 mmol L -1 at D16 (Fig. 1a). Total nitrogen decreased during the experiment, reaching about one third of its initial value at the end of the experiment (2.95 ± 0.05 mmol L -1 at T0 vs. 1.01 ± 0.05 mmol L -1 at D16), while it increased during the same period in MI and RV (from 0.77 ± 0.01 mmol L -1 to 1.03 ± 0.02 mmol L -1 in MI and from 0.60 ± 0.02 mmol L -1 to 1.42 ± 0.11 mmol L -1 in RV; Fig. 1b).

Concentrations of POC were about 3 times higher in EF (10.1 ± 0.8 mg L -1 ) compared to RV (3.3 ± 0.3 mg L -1 ; Fig. 2a). It rapidly dropped to 6.1 ± 0.2 to during the first 24h of the experiment in EF, while it slightly increased in MI and RV, reaching 5.7 ± 0.5 mg L -1 and 6.4 ± 0.8 mg L -1 , respectively in MI and RV at the end of the experiment (Fig. 2a).

Fatty acids

Identified FA in SPM samples included saturated fatty acids (e.g. 16:0 and 18:0), polyunsaturated fatty acids (PUFA; e.g. 18:2ω6 and 20:5ω3), monounsaturated fatty acids (e.g. 16:1ω7 and 18:1ω9), and branched fatty acids (BrFA; e.g. iso-15:0 and anteiso-15:0) (Table 2). Total FA (TFA) concentrations at T0 were about 15 times higher in EF (1.00 ± 0.16 mg L -1 ) compared to RV (0.07 ± 0.02 mg L -1 ; Fig. 2b). High proportions of PUFA were identified in EF (32.9 ± 1.1%), with a dominant contribution of C16 PUFA, 18:3ω3 and 20:5ω3 (Table 2). In contrast, RV was dominated by saturated FA (56.8 ± 4.1%) and exhibited low PUFA proportions (8.8 ± 0.7%; Table 2).

The concentrations of TFA and specific or groups of PUFA rapidly decreased during the first day of incubation, gradually decreased from T24 to D4 and then only slowly degraded over these periods (Fig. 2b and Fig. 3). The degradation of fatty acids thus apparently occurred in three steps, similarly to the observations of [START_REF] Matsumoto | Changes in organic constituents in river water during incubation[END_REF] during the incubation of river water. Consequently, we fitted three degradation rate constants for each treatment. Degradation rate constants were highest in the EF condition, while they were lower or not significantly different from 0 in the MI condition and always not significant in the RV condition (Table 3). A 50% loss of TFA was measured in EF during the first 24h of the experiment, reaching 75% at D4, while TFA remained almost stable or even increased in MI and RV during the entire experiment (Fig. 2b and Table 3). Concentrations of total PUFA, 18:3ω3 and 20:5ω3 followed the same trend as TFA (Fig. 3), while relative contributions remained almost stable between T0 and T24 in EF, and dropped rapidly after T24 (Fig. 3). In contrast, total PUFA and 18:3ω3 relative proportions decreased since the first hours of the experiment in MI (Fig.

3

) and almost did not changed in RV during the entire experiment. Concentrations of BrFA and 18:1ω7 remained almost stable during the entire experiment, while relative proportions increased in all conditions (Fig. 3). Relative proportions of 18:1ω7 showed a sharp increase in MI from 5.3 ± 0.3% at T0 to a maximum of 23.6 ± 0.8% at D4, reaching values higher than in EF (12.8 ± 1.1%) and RV (15.8 ± 2.4%) at the same moment (Fig. 3). δ 13 C ratios of SPM at T0 were lower in EF (-27.89 ± 0.15‰) compared to RV (-25.66 ± 0.04‰; Fig. 4a). It decreased in EF from T0 to T24 (-38.35 ± 0.09‰) and then increased until the end of the experiment, while it slightly increased during the entire experiment in MI and RV (Fig. 4a). δ 15 N ratios of SPM were higher in EF (9.14 ± 0.07‰) compared to RV (5.16 ± 0.73‰; Fig. 4b). It increased until D4 up to 14.2 ± 0.3‰ in EF, and then decreased to 10.0 ± 0.6‰ at D16, while it oscillated between 4.7‰ and 8.4‰ in MI and RV during the entire experiment. High PUFA proportions in EF (32.9 ± 1.1%; Table 2) and C/N ratios close to the Redfield ratio (6.6; Table 1), show that a substantial fraction of SPM is constituted by living phytoplankton [START_REF] Canuel | Relations between river flow, primary production and fatty acid composition of particulate organic matter in San Francisco and Chesapeake Bays: a multivariate approach[END_REF][START_REF] Moynihan | Spatial and temporal dynamics of nano-and pico-size particulate organic matter (POM) in a coastal megatidal marine system: Dynamics of nano-and pico-POM[END_REF][START_REF] Napolitano | Fatty acids as trophic markers of phytoplankton blooms in the Bahía Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)[END_REF].

Shrimp ponds generally exhibit high primary production [START_REF] Burford | A synthesis of dominant ecological processes in intensive shrimp ponds and adjacent coastal environments in NE Australia[END_REF] and the elevated level of DIN we measured in EF (Fig. 1) provides ideal conditions for phytoplankton growth. In our experiment, dominant algal cells were diatoms and green algae, as indicated by the high proportions of PUFA 20:5ω3, C16 PUFA, and PUFA 18:3ω3, indicative, respectively for the two first of Bacillariophyceae, and for the latter of Chlorophyceae [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF]. In contrast, low POC concentrations (Fig. 2a) and low PUFA relative proportions in RV (8.79 ± 0.7%, Table 2) show that SPM was in an advanced state of decomposition [START_REF] Canuel | Relations between river flow, primary production and fatty acid composition of particulate organic matter in San Francisco and Chesapeake Bays: a multivariate approach[END_REF][START_REF] Jaffé | The biogeochemistry of lipids in rivers of the Orinoco Basin[END_REF], as we previously highlighted in this ecosystem [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF]. Lower δ 13 C and higher δ 15 N in EF compared to RV may be attributed to shrimp pellets with high proportion of carbon originating from terrestrial plants with low δ 13 C and high proportion of nitrogen originating from fish trashes with high δ 15 N. However, other measurements in the area revealed high variability of δ 13 C and δ 15 N of SPM in shrimp ponds (-22.9 to -31.8 for δ 13 C and -0.6 to 20.7 for δ 15 N; unpublished data) suggesting that initial values may be poorly informative in this study and that focus is mainly to be given to changes in isotopic values through time. Total concentration of FA in RV at T0 (0.07 ± 0.02 mg L -1 ) was much below the findings of [START_REF] Matsumoto | Changes in organic constituents in river water during incubation[END_REF] who studied the decomposition of organic compounds in a highly polluted river of Japan (about 1 mg of TFA per L).

Actually, although the estuary is subjected to a strong anthropogenic pressure [START_REF] Nguyen | Nutrient dynamics and eutrophication assessment in the tropical river system of Saigon -Dongnai (Southern Vietnam)[END_REF], the sewage effluents released within Ho Chi Minh City are strongly diluted before entering the mangrove area [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF]. In contrast, TFA in EF at T0

(1.00 ± 0.16 mg L -1 ) were about similar to the concentrations measured by [START_REF] Matsumoto | Changes in organic constituents in river water during incubation[END_REF] in river water at the beginning of their experiment, thus possibly allowing relevant comparisons.

Degradation of shrimp pond effluents

The high degradation rate of FA in EF indicates that algal lipids were rapidly degraded once photoautotrophic production was made impossible (in the absence of light). However, the sustaining of PUFA relative proportions during the first 24h of the experiment (from T0 to T24) followed by their gradual decay from T24 to D16 (Fig. 3) suggests that two different processes are successively involved in FA degradation. In complete darkness, living algal cells consume lipids from intracellular storage compartments [START_REF] Brown | Effects of harvest stage and light on the biochemical composition of the diatom thalassiosira pseudonana1[END_REF][START_REF] Eltgroth | PRODUCTION AND CELLULAR LOCALIZATION OF NEUTRAL LONG-CHAIN LIPIDS IN THE HAPTOPHYTE ALGAE ISOCHRYSIS GALBANA AND EMILIANIA HUXLEYI1[END_REF], possibly allowing them to survive during a few days using triacylglycerols and galactolipids as an energy source [START_REF] Manoharan | Acclimation of Prorocentrum Minimum (dinophyceae) to Prolonged Darkness by Use of an Alternative Carbon Source from Triacylglycerides and Galactolipids[END_REF]. In the present study, phytoplankton from shrimp pond effluents most probably survived during the first 24h of the experiment (from T0 to T24), leading to the maintaining of their PUFA relative proportions. In contrast, the decrease of PUFA proportions after T24 suggests the death of algal cells, as usually observed during microbial degradation of phytoplankton [START_REF] Ding | Biochemical degradation of algal fatty acids in oxic and anoxic sediment-seawater interface systems: effects of structural association and relative roles of aerobic and anaerobic bacteria[END_REF][START_REF] Pan | Influence of physiological states of Emiliania huxleyi cells on their lipids and associated molecular isotopic compositions during microbial degradation[END_REF][START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF]. The dynamics of δ 13 C confirms the succession of these two processes, with decreasing values during the first 24h of the experiment and increasing values thereafter (Fig. 4a). [START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF] actually measured decreasing values of phytoplankton δ 13 C during cell respiration, and increasing values during its microbial degradation.

Degradation rate constants k1 of FA during the first 24h of the experiment (Table 3) were much higher than those measured during the respiration phase by [START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF], suggesting that experimental conditions may have conducted algal cells to rapidly depleting their energy stocks. As a consequence, natural populations of microalgae in our experiment died after 24h, while algal cells cultured in laboratory conditions may last weeks in darkness [START_REF] Manoharan | Acclimation of Prorocentrum Minimum (dinophyceae) to Prolonged Darkness by Use of an Alternative Carbon Source from Triacylglycerides and Galactolipids[END_REF][START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF]. The setting up of the experiment may have stressed natural microalgal communities and induced a rapid collapse of their populations. Nevertheless, such situation probably better reflects what happens once effluents are released in the river than the suppression of light alone in sterile laboratory cultures. During the first degradation phase, k1 remained lower than values measured in a highly urbanized river of Japan by Matsumoto (1983; up to 1.5 d -1 ) and the 50% loss of FA was higher than measured during the first 24h in a sewage-seawater mixture by [START_REF] Marty | Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater[END_REF], where initial FA concentration was about half that of our experiment. Such results suggest that decomposition rates may be both sensitive to initial concentration and the conditions in which effluents are placed.

The increase of BrFA and FA 18:1ω7 relative proportions, indicative of bacteria [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Kaneda | Iso-and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance[END_REF][START_REF] Meziane | Fatty acids as tracers of organic matter in the sediment and food web of a mangrove/intertidal flat ecosystem, Okinawa, Japan[END_REF], along with the maintaining of their concentrations (Fig. 2), indicate that the bacterial compartment remained active during the entire experiment. Bacterial OM mineralization led to the release of DIN in EF during the first 2 days of the experiment (Fig. 1a). The preferential consumption of the lighter isotope by heterotrophic bacteria increases δ 15 N in SPM during decomposition [START_REF] Brabandere | δ13C and δ15N dynamics of particulate organic matter in freshwater and brackish waters of the Scheldt estuary[END_REF][START_REF] Caraco | Microbial assimilation of DIN in a nitrogen rich estuary: implications for food quality and isotope studies[END_REF], and most probably induced the δ 15 N increase in shrimp pond effluents SPM during our experiment (Fig. 4b). However, the decrease of DIN concentrations at the end of the experiment (Fig. 1a) was unexpected. This nitrogen was not transferred to the SPM (Fig. 1b) and the nitrogen loss can only be explained by the implementation of a denitrification process, leading to the release of gaseous forms of nitrogen to the atmosphere.

Denitrification generally occurs only in anaerobic condition [START_REF] Chen | Spatial and seasonal variations of atmospheric N2O and CO2 fluxes from a subtropical mangrove swamp and their relationships with soil characteristics[END_REF], which should not have happened in our experiment in which water was continuously mixed with pumps. Nevertheless, mature biofilms may have developed on the bucket walls and in SPM microniches during the experiment and supplied a microbial habitat that contains both aerobic and anaerobic conditions where denitrification could take place [START_REF] Li | Microbial succession in biofilms growing on artificial substratum in subtropical freshwater aquaculture ponds[END_REF].

Mixing of shrimp farm effluents with river water

Conversely to EF, significantly null decomposition rates in RV (Table 3) indicated that organic matter was poorly processed during the experiment and that SPM was in an advanced state of decomposition, as we already evidenced in a previous study [START_REF] David | Short-term changes in the quality of suspended particulate matter in a human impacted and mangrove dominated tropical estuary (Can Gio, Vietnam)[END_REF]). The SPM is thus poorly reactive and the transformation processes measured in MI can be mainly attributed to the input of shrimp pond effluents. In the absence of interaction effect between EF and RV (e. g. RV inhibiting the degradation of EF or EF inducing a priming effect promoting the degradation of RV), we expected measured FA to remain between the values obtained for EF and RV during the entire experiment.

In MI, no maintaining of PUFA relative proportions was measured during the first 24h of the experiment, conversely to EF (Fig. 3), suggesting that phytoplankton cells immediately died and started to decompose just after water mixing. This is confirmed by the δ 13 C of SPM, which immediately increased in MI due to microbial degradation, unlike in EF where cell respiration and microbial degradation occurred successively and showed two different trends in SPM δ 13 C variation (Fig. 4a). This immediate cell death in MI was not due to light depletion, as there was no light available in both MI and EF, but rather to the mixing of the two different water masses. It may have occurred because of a virus present in RV or the sudden change in environmental conditions (pH, salinity, etc.) and/or increased oxidative stress (yet of poorly understood origin; Escher et al.

2013) that may have activated programmed cell death [START_REF] Bidle | Cell death in planktonic, photosynthetic microorganisms[END_REF].

Although the volume of shrimp pond effluents released in the environment is unknown, it is necessarily lower than 10% (the ratio used in our experiment) of this large river discharge. The changes in environmental conditions undergone by the shrimp pond effluents released in mangrove channels are thus at least as intense as in our experiment and immediate cell death would most probably also occur in natural conditions. Decomposition rates k1 were lower than those measured for EF (Table 3) but we may expect the fraction originating from the 90% of river water not to being decomposed. During the second step (T24 to D4), decomposition rates k2 were mostly not significantly different from 0 and total FA even increased (Table 3), along with a twofold increase in POC concentrations (Fig. 2a), suggesting that the input of shrimp pond effluents in river water may have stimulated primary producers and lipid synthesis.

The FA 18:1ω7 showed an interesting dynamic in MI since its relative proportion increased between T0 and D4 up to values that exceed proportions measured in both EF and RV (Fig. 3). Its temporal trend was different from that of the BrFA's one, which never exceeded either that of EF or RV and suggesting that specific bacterial strains may have grown in MI, probably as a result of substrate availability brought by EF and/or the bacterial inoculum brought by RV. The FA 18:1ω7 is commonly used as a bacterial biomarker in natural environments [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Meziane | Fatty acids as tracers of organic matter in the sediment and food web of a mangrove/intertidal flat ecosystem, Okinawa, Japan[END_REF]. [START_REF] Marty | Laboratory study on degradation of fatty acids and sterols from urban wastes in seawater[END_REF] measured the degradation of FA from urban wastes in seawater and also found a significant production of FA 18:1ω7 during the first days of their experiment followed by its decrease after 5 days of incubation. Studying the decomposition of pure phytoplankton strains, [START_REF] Pan | Effects of growth stages, respiration, and microbial degradation of phytoplankton on cellular lipids and their compound-specific stable carbon isotopic compositions[END_REF] found that FA 18:1ω7 became a major component (20-30% of total FAs) during cells microbial degradation.

Such elevated proportions of the FA 18:1ω7 were previously found in phospholipids of alphaproteobacteria, such as Paracoccus denitrificans, which are involved in aerobic denitrification [START_REF] Zelles | Phospholipid fatty acid profiles in selected members of soil microbial communities[END_REF]. Further analysis (e.g. DNA) would be required to investigate the presence of aerobic denitrifying bacteria in decomposing shrimp pond effluents, which may have consequences on the release of greenhouse gases in the atmosphere such as NO and N2O [START_REF] Ji | Aerobic denitrification: A review of important advances of the last 30 years[END_REF][START_REF] Queiroz | Hidden contribution of shrimp farming effluents to greenhouse gas emissions from mangrove soils[END_REF]. In natural conditions, [START_REF] Aschenbroich | Spatio-temporal variations in the composition of organic matter in surface sediments of a mangrove receiving shrimp farm effluents (New Caledonia)[END_REF], found a higher concentration of FA 18:1ω7 in mangrove sediments receiving shrimp pond effluents during the active farm period compared to the non-active period and also attributed this increase to the grow of specific bacterial strains. We thus suggest that the FA 18:1ω7 represents a potential biomarker of anthropogenic substance (i. e. shrimp farm effluents or urban sewage)

releases in natural waters. Finally,shrimp farm effluents are rich in PUFA, that are of high economic value. Developing methods to recover lipids from these effluents may bring additional income to farmers, as well as avoiding excessive inputs of potential polluting substances to the nearby environments.

Conclusion

Our study confirms that shrimp pond effluents are enriched in phytoplankton and nitrogen compared to mangrove waters. Placed in dark conditions, phytoplankton cells first depleted their intracellular storage lipids, leading to a 50% loss of FA during the first 24h of the experiment with little effect on relative proportions. Then, cells death led to changes in FA relative proportions with preferential degradation of PUFA, leading to a total of 75% FA loss after 4 days of incubation. In contrast to shrimp pond effluents alone (EF), the mixture of shrimp pond effluents + river water (MI) induced immediate phytoplankton cells death, most likely due to the sudden change in environmental conditions and representative of the processes that could happen in the natural environment. The degradation of SPM was then accompanied by the growth of specific bacterial communities (yet undetermined and characterized by especially high levels of the FA 18:1ω7), as indicated by higher relative proportions of the FA 18:1ω7 compared to that of EF or RV taken in isolation. This result confirms our hypothesis that the mixing of waters favors specific bacterial strains and/or specific degradation pathways. As a consequence, shrimp ponds effluents release in mangrove waters may impact the nutritional quality of SPM by modifying the balance of fatty acids that filtering organisms are expected to feed on. High proportions of the FA 18:1ω7 in mangrove sediments were also associated to the nearby shrimp farming activity in New Caledonia [START_REF] Aschenbroich | Spatio-temporal variations in the composition of organic matter in surface sediments of a mangrove receiving shrimp farm effluents (New Caledonia)[END_REF]. We suggest that the FA 18:1ω7 represents a potential biomarker of anthropogenic substance (e.g. shrimp farm effluents, urban sewage) releases in natural waters, thus confirming our hypothesis that specific fatty acids may be used as tracers of shrimp farm effluent releases (although not exclusively targeting shrimp farm effluents) in marine environments.

Figure 1 :Figure 2 :

 12 Figure 1: Dynamics of a) dissolved inorganic nitrogen (NH4 + + NO2 -+ NO3 -; mmol L -1 )

Figure 3 :Figure 4 :

 34 Figure 3: Dynamics of relative proportions (%) and concentrations (µg L -1 ) of specific 290

Table 1 :

 1 Water parameters at the beginning of the experiment (T0) in each experimental

	condition
	Parameter	Shrimp pond effluents (EF)	Mixed water (MI)	River water (RV)
		(n = 4)	(n = 4)	(n = 4)
	Salinity	10.1 ± 0.0	12.8 ± 0.0	13.2 ± 0.0
	pH	8.35 ± 0.01	7.53 ± 0.00	7.45 ± 0.01
	SPM (mg/L)	91.2 ± 10.3	175.3 ± 3.7	204.6 ± 4.3
	C/N (mol/mol)	7.0 ± 0.0	9.0 ± 0.4	9.7 ± 1.4

Table 2 :

 2 Relative proportions of main FA in each experimental condition at T0 (mean ± 276 SD). Only FA with proportions > 0.5% in at least one condition are displayed. 277

	Fatty acids (%)	Shrimp pond effluents	Mixed water (MI)	River water (RV)
		(EF) (n = 3)	(n = 4)		(n = 4)
	Saturated
	12:0	0.2 ± 0.1	0.3 ± 0.1	0.6 ± 0.8
	14:0	9.7 ± 0.3	6.8 ± 1.4	4.7 ± 1.4
	15:0	0.9 ± 0.1	1.3 ± 0.2	2.4 ± 0.5
	16:0	22.2 ± 0.6	28.3 ± 1.4	33.9 ± 1.4
	17:0	0.4 ± 0.1	0.9 ± 0.2	1.3 ± 0.4
	18:0	1.9 ± 0.3	4.6 ± 0.8	9.6 ± 1.2
	19:0	0.0 ± 0.0	0.3 ± 0.1	0.8 ± 0.6
	20:0	0.2 ± 0.1	0.4 ± 0.1	0.8 ± 0.1
	22:0	0.2 ± 0.0	0.6 ± 0.1	1.3 ± 0.2
	24:0	0.1 ± 0.0	0.9 ± 0.4	1.0 ± 0.5
	∑SFA	35.9 ± 1.3	44.6 ± 1.6	56.8 ± 4.1
	Monounsaturated
	16:1ω9	2.9 ± 0.1	2.9 ± 0.4	3.6 ± 1.3
	16:1ω7	15.4 ± 0.2	13.1 ± 0.4	9.3 ± 1.3
	16:1ω5	0.4 ± 0.0	0.5 ± 0.0	0.8 ± 0.2
	17:1ω9	0.1 ± 0.0	0.2 ± 0.1	0.6 ± 0.3
	18:1ω11	0.3 ± 0.0	0.2 ± 0.1	1.2 ± 1.3
	18:1ω9	3.6 ± 0.1	5.0 ± 0.7	5.7 ± 0.5
	18:1ω7	3.7 ± 0.2	5.3 ± 0.3	7.3 ± 2.4
	∑MUFA	27.2 ± 0.1	28.2 ± 1.1	29.5 ± 3.7
	Polyunsaturated
	16:2ω6	1.1 ±	0.2	0.5 ±	0.1	0.4 ±	0.2
	16:2ω4	2.3 ± 0.1	1.6 ± 0.1	0.6 ± 0.1
	16:3ω4	2.5 ± 0.1	1.6 ± 0.1	0.5 ± 0.1
	16:3ω3	2.4 ± 0.2	1.4 ± 0.1	0.2 ± 0.0
	16:4ω3	1.3 ± 0.1	0.9 ± 0.2	0.3 ± 0.2
	18:2ω9	tr.		tr.		0.7 ± 0.1
	18:2ω6	3.4 ± 0.1	3.2 ± 0.3	1.1 ± 0.2
	18:3ω3	7.3 ± 0.4	5.2 ± 0.6	0.6 ± 0.1
	18:4ω3	2.6 ± 0.1	1.8 ± 0.1	0.5 ± 0.2
	20:2ω9	tr.		tr.		0.8 ± 0.4
	20:4ω6	1.4 ± 0.1	1.2 ± 0.1	0.5 ± 0.1
	20:5ω3	5.0 ± 0.3	3.4 ± 0.2	1.6 ± 0.3
	22:6ω3	1.5 ± 0.2	0.3 ± 0.4	0.4 ± 0.4
	∑PUFA	32.9 ± 1.1	22.3 ± 0.6	8.8 ± 0.7
	Branched
	15:0iso	1.6 ± 0.0	1.6 ± 0.2	1.5 ± 0.2
	15:0anteiso	0.5 ± 0.0	0.8 ± 0.1	1.2 ± 0.1
	16:0iso	0.4 ± 0.0	0.5 ± 0.0	0.7 ± 0.1
	17:0iso	1.0 ± 0.2	1.5 ± 0.2	0.8 ± 0.1
	∑BrFA	4.0 ± 0.1	4.9 ± 0.3	4.9 ± 0.4
	Others	3.7 ± 0.4	3.0 ± 0.8	3.0 ± 1.6
	tr. = traces
	SFA = saturated fatty acids
	MUFA = monounsaturated FA

Table 3 :

 3 Degradation rate constants (k in d -1 ) of specific fatty acids in the three different treatments. Stable isotopes δ 13 C and δ 15 N

			Total FA	PUFA	C16 PUFA	18:3ω3	20:5ω3
	Shrimp pond effluents k1 (T0 -> T24)	0.73 ± 0.13	0.79 ± 0.14	1.02 ± 0.16	0.77 ± 0.15	0.73 ± 0.15
		k2 (T24 -> D4)	0.26 ± 0.07	0.41 ± 0.06	0.39 ± 0.08	0.46 ± 0.06	0.53 ± 0.06
		k3 (D4 -> D16)	n.s.	0.10 ± 0.01	0.12 ± 0.02	0.20 ± 0.02	0.14 ± 0.02
	Mixed water	k1 (T0 -> T24)	n.s.	0.39 ± 0.12	0.47 ± 0.12	0.76 ± 0.13	0.33 ± 0.14
		k2 (T24 -> D4)	-0.13 ± 0.05	n.s.	n.s.	n.s.	0.15 ± 0.07
		k3 (D4 -> D16)	n.s.	0.03 ± 0.01	0.06 ± 0.02	0.13 ± 0.02	0.09 ± 0.02
	River water	k1 (T0 -> T24)	n.s.	n.s.	n.s.	n.s.	n.s.
		k2 (T24 -> D4)	n.s.	n.s.	n.s.	n.s.	n.s.
		k3 (D4 -> D16)	n.s.	n.s.	n.s.	n.s.	n.s.
	n.s. = not significantly different from 0
	PUFA = polyunsaturated fatty acids
	3.3

Acknowledgements: Authors would like to thank the researchers of the Centre Asiatique de Recherche sur l'Eau, in the University of Technology of Ho Chi Minh City for their warm welcome in their laboratory during the experiment, especially Emilie Strady, Julien Némery, Nicolas Gratiot and Tuyet T.N. Nguyen.

This work was supported by the Muséum National d'Histoire Naturelle (Paris).

Abbreviations

BrFA: branched fatty acid