Ecodynamics and bioavailability of metal contaminants in a constructed wetland within an agricultural drained catchment

Ecological Engineering 136 (2019) 108–117

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Ecodynamics and bioavailability of metal contaminants in a constructed wetland within an agricultural drained catchment

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Jérémie D. Lebruna,b, , Sophie Ayraultb,c, Aymeric Droueta,c, Louise Bordierc, Lise C. Fechnerd, Emmanuelle Uhera, Cédric Chaumonta, Julien Tournebizea,b ⁎

a

Irstea, UR HYCAR – Artemhys, CS 10030, 92761 Antony Cedex, France Federation of Research FIRE, FR-3020, 75005 Paris, France c Laboratoire des Sciences et de l'Environnement (LSCE), UMR 8212 (CEA/CNRS/UVSQ), Université Paris-Saclay, Gif-sur-Yvette, France d Ministery for the Ecological and Inclusive Transition, General Directorate of Risk Prevention, Paris, France b

ARTICLE INFO

ABSTRACT

Keywords: Nature-based solution Buffer zone DGT Metal speciation Bioaccumulation Biomonitoring

Constructed wetlands are designed to mitigate nutrient and pesticide fluxes from agricultural catchments. Nevertheless, information on their efficiency in removing non-degradable contaminants such as metals is still scarce. This study aimed to explore the metallic signature and fate of metals within the Rampillon wetland (France) receiving water from a drained 355-ha catchment under intensive agriculture. Original monitoring coupling classic, time-integrated and bioaccumulation-based tools was achieved to characterise spatiotemporal dynamics of various metals (As, Cd, Cr, Co, Cu, Mn, Ni, Pb, Sb, Se and Zn). To assess metal inflows and mitigation, samples of dissolved and particulate metals were collected bimonthly at the inlet and outlet of the wetland over 3 months. Simultaneously, time-integrated (sediment traps and passive samplers) and bioaccumulation-based (caged gammarids and biofilms) tools were deployed to monitor temporal changes in metal speciation and bioavailability. To gain insight into the spatial distribution of metals between abiotic and biotic matrices, sediments and indigenous invertebrates with contrasted ecologies were sampled in different cells of the wetland. The results showed time-integrated tools were more suitable than bimonthly samples to quantify metal mitigations because of temporal fluctuations and low contamination levels. Significant mitigations were thus observed in trapped sediments for all metals (ranged 11–23%, except Mn) as well as in the DGT-labile fraction for Cd, Cr, Co, Mn and Ni (ranged 13–51%). Bioaccumulation levels in biofilms also revealed a decrease in metal bioavailability at the outlet. Furthermore, the spatial survey supported the central role of sediments in metal trapping and the beneficial effect of this wetland for local biodiversity in terms of exposure. To conclude, this study provides valuable information on the ecodynamics and bioavailability of metals required for sustainable management of such artificial ecosystems and furthermore, of agricultural areas.

1. Introduction As artificial landscape interfaces, constructed wetlands (CWs) designed to intercept and trap contaminants from drainage and/or runoff waters offer promising nature-based solutions to mitigate agricultural impacts. Implementation of CWs in agricultural drained catchments reduces nutrients (e.g. nitrates, phosphates) and pesticide fluxes circulating in the hydrosphere, hence contributing to improving freshwater and groundwater quality (Tournebize et al., 2017). Removal efficiencies of CWs result from natural combined processes (e.g. photolysis, biodegradation, sequestration) and depend on the local spatiotemporal conditions (hydrology, season, retention time, etc.) as

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well as the physicochemical properties of contaminants such as nitrates and pesticides (Tournebize et al., 2017; Vymazal and Březinová, 2015). However, the efficiency of such artificial ecosystems in the removal of non-degradable contaminants such as metals remains poorly documented, especially in the agricultural context. Metals are often studied in wetlands used in contexts related to the runoff/remediation of wastewater heavily contaminated by mining and industrial and/or urban activities (Arroyo et al., 2013; Leung et al., 2017; Marchand et al., 2010; Mulkeen et al., 2017). Usually considered as tracers of urban or industrial contaminations, metals are also significantly introduced into cultivated soils by the use of pesticides, fertilisers and various amendments such as the spreading

Corresponding author at: Irstea, UR HYCAR – Artemhys, 1 rue Pierre-Gilles de Gennes, CS 10030, F-92761 Antony Cedex, France. E-mail address: [email protected] (J.D. Lebrun).

https://doi.org/10.1016/j.ecoleng.2019.06.012 Received 11 April 2019; Received in revised form 13 June 2019; Accepted 20 June 2019 0925-8574/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Map showing the localization of the constructed wetland of Rampillon (Seine-et-Marne, France) within the Seine River basin. Crosses are sampling sites at the inlet and outlet of the wetland for temporal monitoring of metallic contamination levels where water fractions and trapped sediment were collected bimonthly, and caged gammarids, biofilms and thin film technique devices were exposed. Circled numbers indicate the different cells separated by bunds and considered for the spatial sampling (i.e. bed sediment and indigenous macroinvertebrates).

use of soils on the whole of the catchment (Tournebize et al., 2017; Tournebize et al., 2012). This CW was implemented upstream of direct discharges of drainage/runoff waters into one of the main underground drinking water sources for the Paris region, i.e. about one million inhabitants. The area is representative of intensive agricultural areas in Europe. An original monitoring campaign was designed to evaluate the spatiotemporal dynamics of metals by combining classic, time-integrated and bioaccumulation-based approaches. To assess metal inflows and their abatement in outflows, grab samples of dissolved and particulate metals were collected bimonthly at the inlet and outlet of the CW over 3 months (March–May 2015). At the same time, chemical (sediment traps and passive samplers) and biological (caged gammarids and biofilms) time-integrated tools were deployed to monitor temporal changes in metal speciation and bioavailability (Faburé et al., 2015; Lebrun et al., 2015; Priadi et al., 2011b). To provide an overview of the spatial distribution of metals between abiotic and biotic matrices, sediments and indigenous invertebrates with contrasted ecologies (habitats, diet, etc.) were sampled in different cells of this artificial ecosystem.

of sewage sludge or even by atmospheric fallouts (Joimel et al., 2016; Lebrun et al., 2012; Thévenot et al., 2007). For example, the use of phosphorus fertilisers is a major source of contamination of soils cultivated in Europe by arsenic (As), cadmium (Cd) and chromium (Cr) (Nziguheba and Smolders, 2008). Thus, metals are likely to contaminate receiving media, including CWs, notably in the presence of subsurface drainage systems facilitating hydraulic transfers from the soil towards aquatic media (Tournebize et al., 2012). Nevertheless, very little information is currently available on the influx of metals from agricultural sources, the contamination levels and their fate within CWs. Closely related to water column physicochemistry, speciation governs the circulation and distribution of metals between and within abiotic and biotic compartments, their sequestration in the receiving media as well as their bioavailability, i.e. the metallic fraction available and potentially toxic to aquatic organisms (Tercier-Waeber et al., 2012). Because of the persistence of metals, their accumulation in sediments and along trophic chains may then disrupt ecological functions. Indeed, diffuse multi-metallic contaminations are reported to generate adverse effects on organisms, disturbances in faunal assemblage and environmental processes in freshwaters such as leaf decomposition (Fechner et al., 2012; Kellar et al., 2014; Schultheis et al., 1997). Characterising the ecodynamics of metals in CWs is therefore crucial to assess their ecological vulnerability to metallic pressure as well as to promote their sustainable management for preserving ecosystem services rendered by these landscape interfaces, e.g. pollution mitigation or biodiversity (Thorslund et al., 2017). In this regard, metal determination in indigenous biota provides an integrated measurement of ambient metallic exposures over a recent period in the local habitat (Lebrun et al., 2014; Rainbow, 2007). Nevertheless, the contamination levels of organisms vary from one species to another according to their physiology, diet and trophic level. As dissolved species, metals can be directly internalised in exposed organisms via gills. In addition, contaminants bound to abiotic compartments (e.g. suspended particulates, litter or sediments) are also exposure routes for filter-feeders, benthic organisms or burrowers (Bourgeault et al., 2011; Hadji et al., 2016). This study aimed to investigate the efficiency a CW draining a 355ha area under intensive agriculture (Rampillon, France) in mitigating fluxes of a wide panel of metals (i.e. As, Cd, Cr, Co: cobalt, Cu: copper, Mn: manganese, Ni: nickel, Pb: lead, Sb: antimony, Se: selenium and Zn: zinc) by exploring the metallic signature and the biological-chemicalphysical fate of metals trapped in the CW. The study site exhibits both environmental and health issues because of the intensive agricultural

2. Materials and methods 2.1. Study site The Rampillon CW (48°32′19.5″N; 3°03′46.7″E; 70 km southeast of Paris, France) is located on the Brie plateau subjected to intensive agriculture. The CW was implemented in 2010 to meet local environmental and health issues. This artificial site was designed to collect drainage and run-off waters from an agricultural catchment covering 355 ha before their falling into sinkholes directly connected to the Champigny aquifer, which constitutes the one of main drinking water resources for the Paris region. Because of waterlogged soils, agricultural land required subsurface drainage for the whole catchment area (buried perforated pipes spaced 10 m apart about 90 cm deep). The 355-ha agricultural catchment receives an annual mean rainfall of 689 mm and the annual mean drained flow is 228 mm. Farmers mainly grow winter wheat, sugar beet, corn, beans and rape. The 0.53-ha constructed wetland consists of ecologically engineered sub-basins separated by bunds to enhance water retention time and mitigation processes (Tournebize et al., 2012). A first sedimentation basin is 100 cm deep and 300 m3 (Fig. 1; i.e. cell 1). The 4000-m2 intermediate zone is a shallow sub-basin a maximum 50 cm deep, including cells 2, 3 and 4 (i.e. 1680, 1450 and 870 m2 respectively). About 20, 60 and 50% of 109

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cells 2, 3 and 4 was covered by vegetation in 2015: reed (Phragmites australis), bulrush (Juncus spp.) and sedge (Carex spp.). Note that this last plant was not present on cell 2. A final 1000-m3 basin (i.e. cell 5) was implemented that is 80 cm deep before the outlet. Since 2012, the Rampillon wetland is instrumented to continuously monitor nutrient and pesticide fluxes as well as major water physicochemical parameters (flow, temperature, major ions) at the inlet and outlet of the site (Fig. 1). The typical monitoring stations comprise a flowmeter based at the water level and a Doppler (Sigma 950, Hach), a multi-parameter spectrophotometer (Spectrolyser UV–vis, S::can), for hourly measurements of turbidity and nitrates, and an automatic sampler managed for bi-monthly flow-weight sampling strategies. In the present study, the sampling period for metallic monitoring was March–May 2015 when the spring rain events can lead to sporadic high inflows of both drainage and runoff waters into the CW. The retention time was calculated from hourly data as the ratio of the total volume of the CW to the outlet flow and averaged over the study period.

achieved and stored at −80 °C for metal determination. Biofilms were grown by in situ colonisation on PES membranes (30 cm × 10 cm) vertically attached to plastic crates immersed at both Rampillon CW sampling sites (Faburé et al., 2015). On each sampling date, five colonised membranes were collected at each site and carried back to the laboratory in 250-mL glass bottles filled with local water. They were then carefully hand-scraped in 250 mL of 0.45-µm filtered local water to avoid cellular stress of the sampled microbial community related to water chemistry changes. Triplicates of 5- to 10-mL of homogenised biofilm suspensions were then transferred on pre-weighed cellulose nitrate filters and stored at −80 °C for metal determination. The same procedures were performed from biofilm suspensions in the presence of 0.28 M EDTA as a metal chelator in order to distinguish the metallic fraction internalised in biofilms from the total fraction, where metals can be internalised, bound to the biofilm surface and trapped to extracellular polysaccharides. Control filters were performed by filtration of local water without biofilms.

2.2. Grab samples and deployment of time-integrated tools

2.3. Spatial distribution of metals within the Rampillon CW

To characterise metallic fluxes, bimonthly samples of water were collected at the inlet and outlet of the Rampillon CW over 3 months in 2015, i.e. six sampling dates as follows: t0 = March 4th , t1 = March 18th, t2 = April 1st, t3 = April 18th, t4 = April 28th and t5 = May 11th. For the dissolved metal fraction, 30 mL of bulk water was directly filtered on-field through a 0.45-µm PES syringe filter (Millipore) in a 50-mL polypropylene (PP) tube (Uher et al., 2018). The water filtrates were acidified with HNO3 at 1% v/v immediately after the return to the lab (3–4 h after collection) and stored at 4 °C before analysis. For the particulate fraction, bulk water was collected in 2-L PP bottles, brought back in a cool box for filtration through pre-weighed 0.45-µm quartz filters (Millipore) in the laboratory. Filters were then dried at 110 °C and weighed to determine the dried mass of total suspended particulate matter (SPM). At the same time as these bimonthly samples were collected, sediment traps and the diffusive gradient in thin film technique devices (DGTs) were deployed for time-integrated monitoring of metal speciation (Priadi et al., 2011b). DGTs and sediment traps were thus immersed using wooden stakes so that these tools stayed in water column and did not derive. These sediment traps comprised perforated 2-L bottles, which were hung from the pegs at the inlet and outlet of the Rampillon CW to collect suspended sediments, as described elsewhere (Priadi et al., 2011b). Trapped sediments were retrieved every 2 weeks at the same times as grab samples (i.e. five deployment periods) and brought back to the laboratory for metal determination. As passive samplers of free inorganic metals and weak organic complexes, three DGTs were also deployed and replaced by new ones to monitor DGTlabile metals from t0 to t1, from t1 to t2 and from t2 to t4. DGTs comprised 0.8-mm-thick restrictive diffusive gels and a 0.4-mm PC filter (DGT Research, Lancaster, UK) (Uher et al., 2011). Complementary to this classic monitoring strategy on water fractions, we also deployed bioaccumulation-based tools to quantify the metal fraction that was mobile and available for biota. Metal bioavailability was assessed by measuring metals bioaccumulated in tissues of well-known biomonitors, i.e. gammarids and biofilms (Faburé et al., 2015; Lebrun et al., 2015). Gammarids (Gammarus fossarum sp.) were collected on a reference site and size-calibrated in the field using sieves (Gismondi et al., 2017). Then gammarids were caged in transparent PVC tubes (195 mm long, 80 mm in diameter) closed with 0.5-mm nylon mesh. Caged gammarids were fed ad libitum with local mixtures of poplar, hornbeam, alder and hazel leaves, immersed beforehand on site for 2 weeks so they would be impregnated with local contaminations. Three cages of 40 individuals fixed in perforated plastic crates were deployed on both the Rampillon CW sampling sites (Fig. 1), retrieved and renewed at the same times as grab samplings. At the end of the in situ deployments, triplicate pools of five caged gammarids were

Following a survey of the invertebrate species present on the Rampillon wetland performed 1 month before, isopod and amphipod crustaceans (Asellus and Gammarus spp.), insect larvae (Chironomus spp.) and snails (Lymnaea spp.) were collected on 31 March 2015 at the five-cell level, as illustrated in Fig. 1. The individuals sampled were directly placed in triplicates of 50-mL PP tubes. For snails, only the soft bodies were kept. Despite their occurrence during the survey, sites 3 and 4 were devoid of chironomids and site 5 of snails, suggesting temporal population dynamics. At each cell, bottom sediments were also sampled (8 cm deep) during the survey and placed in plastic bags. Indigenous biota and sediments were brought back to laboratory in the cool box and stored at −80 °C until metal analysis. Note that metals contents in emergent vegetation would have provided valuable information on the spatial fate of metals and their absorption by plants (Leung et al., 2017; Mulkeen et al., 2017). Nevertheless, no plant species homogeneously distributed to the sites could be sampled for relevant comparisons in metal bioavailability. 2.4. Metal determination in environmental matrices For metal determination in SPM, quartz filters were digested by treatment with 6 mL of HF (48.9%, for trace metal, Fisher) and 3 mL of HClO4 (65–71%, for trace metal, Fisher) for 2 h at 110 °C, in closed PTFE vessels on a hotblock (Digiprep, SCP Science). A second treatment with aqua regia followed, i.e 1.25 mL of HNO3 (69.5%, for trace analysis, Fluka) and HCl (> 37%, for trace analysis, Fluka) for 100 min at 100 °C. Then 1 mL of HNO3 was added and the liquid was brought to near dryness (repeated 3 times). The liquid was then brought to 20 mL with HNO3 0.5 N. The same procedure was applied to 2-mm sieved bottom and trapped sediments from 100 mg of lyophilised matter, except that the final volume was 50 mL. The digestion included a 100-mg aliquote of the certified reference material (CRM) (Lake Sediment SL1 AIEA) and a digestion blank to control mineralization quality. Digested as well as dissolved samples were stored at 4 °C until they were analysed by inductively coupled plasma mass spectrometry (ICPMS; X series 2, Thermo Fisher Scientific) to determine As, Cd, Cr, Co, Cu, Mn, Ni, Pb, Sb, Se and Zn concentrations using the Collision Cell Technology mode using gas input (H2 (7%) and He (93%)). The correction of instrumental drift was based on the deviation observed on internal standards (Ge, 10 µg L-1). For DGT, resins were eluted in 1 mL of HNO3 (1.2 M) and directly analysed by ICP-MS. The DGT-labile metal concentrations were calculated using the method adopted by Uher et al. (2011) taking into account the deployment time. For all the matrices analysed, values were above the limits of quantification (LQ) except for Cr in the dissolved fraction (< 0.5 µg/L) and 4 out of 11 dissolved Cd samples (< 0.01 µg/L). A river water CRM (NIST 1640a) was analysed 110

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repeatedly and the results were within 94–108% with relative standard deviations < 5% between the different analysis dates. Good agreement was observed between the data obtained and the certified values for SL1 measurements (87–108%). For frozen biological matrices, biofilm filters, caged gammarid pools as well as indigenous species were lyophilised, weighed and digested by adding nitric acid and hydrogen peroxide (Faburé et al., 2015; Lebrun et al., 2015). Digested samples were analysed by ICP-MS to determine metal concentrations in organisms and expressed in µg of metal per g of dry weight (µg/gdw). The digestion method was successfully validated by a reference material (Mussel Tissue ERM-CE278). Cr was not quantified in either caged gammarids or indigenous macroinvertebrates because of values below LQ.

during and at the end of flooding periods, where the water flow within the wetland exceeded 30 L/s in the two cases. 3.2. Temporal monitoring of metal contamination by grab samples Dissolved and particulate metal concentrations monitored bimonthly at the Rampillon CW inlet and outlet over 3 months (March–May 2015) are presented in Fig. 3. For both sampling sites, all the metals tested exhibited significant variability between sampling times both in dissolved (RSD range, 25–135%) and particulate (RSD range, 25–90%) fractions, except Se (RSD < 15% for both fractions). These temporal fluctuations suggest significant dynamics of metals within the drained catchment, probably related to climatic and hydrological conditions (Meite et al., 2018). Nevertheless, no obvious link was established between temporal fluctuations in metallic levels and variations in water flow within the Rampillon CW (Fig. 2), except for the particulate fraction at the t0 sampling time (4th March 2015). For all metals (As, Mn and Sb excluded), the highest metallic concentrations in suspended particulate matter (SPM) were observed at this date included in a period of flooding, notably at the inlet of the CW. Even if the concentrations were not the highest, the particulate contents in As, Mn and Sb were elevated at t0 and higher at the inlet than at the outlet. These results highlighted the role of SPM in metallic inflows as the carrier phase and suggested a possible mitigation of particulate metals by sedimentation of SPM. Metal levels in dissolved and SPM fractions were overall comparable to those measured at the Marnay station (data not available for As, Sb and Se) as a reference site of the Seine River that drains intensive agricultural and slightly urbanised areas upstream of the Paris Megacity (Barjhoux et al., 2018; Lebrun et al., 2015; Priadi et al., 2011b). The major difference was observed for dissolved Zn that had levels onequarter those of the reference levels. From average concentrations for all sampling times combined, the preference of metals for the particulate fraction ranged as follows: Cr, Pb > Zn > Cd > Co > Cu > Sb > Mn > Ni > As, Se (see metal partitioning in SI; Fig. S1). This order was in agreement with other Seine reference data, excluding As, Sb and Se for which information is not available (Lebrun et al., 2015; Priadi et al., 2011b). As expected, the metallic signature of the Rampillon CW is typical of aquatic media located in catchments subjected to intensive agriculture (Baize and Sterckeman, 2001). For all the metals tested, particulate concentrations are higher than natural contents defined at the scale of the Seine basin (data not available for Se) without exceeding three times these references (ratios

2.5. Statistical analysis Statistical analysis was performed using XLStat (Addinsoft). The significant differences in metal levels in grab samples as well as chemical and biological integrative tools during the monitoring between the inlet and outlet of the Rampillon CW were tested with a t-test paired by sampling date (P < 0.05). 3. Results and discussion 3.1. Hydrology during the study period The sampling period for metallic monitoring was performed in March–May 2015 during which the nutrients were applied to soils, as potential sources of metallic contaminations. During this period, 82.4% of drainage/run-off water from the agricultural catchment, namely about 70 365 m3, was intercepted by the Rampillon wetland (i.e. 10.3% of the annual volume from Sept. 2014 to August 2015). The continuous monitoring of water flow and associated time retention of water within the wetland are presented in Fig. 2. The mean retention time was 4.9 days and the hydraulic loading rate was 0.19 m/d over the study period. However, this retention varied from a few hours to twenty days as a result of climatic conditions in that rain events led to sporadic high inflows into the CW. Therefore, the bimonthly sampling integrated different hydrological regimes of the wetland, alternating with periods of flooding and low water. For instance, a substantial low-water period associated with long retention times (> 5 days) was observed in April, comprising the t4 sampling time (i.e. 28th April). By contrast, the t0 and t2 sampling times (i.e. 4th March and 1st April) were performed

Fig. 2. Water flow (blue) and retention time (red) within the constructed wetland of Rampillon in March–May 2015. Black arrows are sampling dates performed bimonthly during this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 111

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Fig. 3. Metal concentrations in dissolved (A) and particulate (B) fractions sampled bimonthly (from 4 March to 11 May 2015; n = 6 sampling dates) at the inlet (black symbol) and outlet (white symbol) of the Rampillon constructed wetland. Letters indicate significant differences between both sampling sites, i.e. “a” for higher metal concentrations at the inlet than outlet, and “b” for the inverse case (P < 0.05, paired t-test). Dotted lines are concentrations measured on the reference site of Marnay upstream of the Seine River basin (Priadi et al., 2011). Grey lines are European (Cd, Cu, Ni, Pb and Zn) or French (As, Cr, Co, Mn, Sb and Se) quality standards for the dissolved fraction. Black lines are natural references of particulate metals for the Seine basin (Thévenot et al., 2007). For more clarity, scaling factors are applied to some metals.

ranged from 1.5 [Cu] to 2.9 [Mn]), except Zn (ratio > 3.8) (Le Cloarec et al., 2011; Thévenot et al., 2007). This supports an inflow of particulate metals from agriculture that is likely due to the leaching of cultivated soils, as can be noted in the case of Zn during rainfall events (Horowitz et al., 1999; Meite et al., 2018). Nevertheless, metal contents in SPM were 3–4 times lower than those measured at sites downstream of Paris for Cd, Cu, Pb and Zn, which are metals known as urban tracers (Priadi et al., 2011a; Priadi et al., 2011b). To assess the potential toxicity of metals, dissolved metal concentrations were compared to environmental quality standards (EQS) defined by European (for Cd, Cu, Ni, Pb and Zn) or French (as predicted no-effect concentrations or PNEC for As, Cr, Co, Mn, Sb and Se) regulations for the protection of surface waters (Directive 2013/39/UE, 2013; INERIS, Website). Only average dissolved Mn and Se exceeded their respective PNEC, by about 1.5-fold, i.e. 15 and 0.9 µg/L, respectively. Although these metals are essential elements in aquatic organisms, they can be toxic when in excess (Peters et al., 2011). Overall, the compliance of dissolved levels with EQSs suggests low impacts of metals circulating into the Rampillon CW on the local aquatic life. However, metals in mixture can induce sublethal effects, even if their levels

are individually in compliance with EQSs, as demonstrated in the freshwater amphipod G. fossarum (Lebrun et al., 2017). For all metals, no significant mitigation in the dissolved or particulate fraction was observed between the inlet and outlet of the Rampillon CW, except dissolved Se, which had the lowest temporal fluctuations. Anecdotally, dissolved levels of Co were significantly higher downstream than upstream. Nevertheless, the interpretation of comparisons in metallic levels between both sampling sites can be hampered by the fact that metals are sampled bimonthly and subject to high temporal fluctuations, as previously discussed. Moreover, the retention time varies with hydrological conditions (Fig. 2), hence leading to possible shifts in contamination levels between the inlet and outlet of the CW. Consequently, using chemical time-integrated tools should be more suitable than grab samples to assess whether or not metals are effectively mitigated in outflows (Priadi et al., 2011b; Uher et al., 2018). 3.3. Time-integrated monitoring of metal contamination and its mobility Metal concentrations determined from in situ deployment of chemical time-integrated tools, i.e. DGTs and sediment traps, are presented 112

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Fig. 4. Time-integrated metal concentrations in DGTs (A) and trapped sediments (B) at the inlet (black symbol) and outlet (white symbol) of the Rampillon constructed wetland. DGT values are means of triplicates. In some cases, metal concentrations are multiplied by scaling factors for more clarity. The letter “a” indicates that metal concentrations are significantly higher upstream than downstream (P < 0.05, paired T-test, with n = 9 for DGTs and n = 5 for traps).

in Fig. 4. Except Mn, all metals in trapped sediments were significantly mitigated at the outlet of the constructed wetland with mean efficiency ranging from 11 to 23% (Table 1). For each metal, the highest efficiency of mitigation was observed in the period from t4 to t5 when the hydraulic retention time was maximal, i.e. 11.4 days (Figs. 2 and 4B; Se and Sb excluded). As regards DGTs, significant mitigations were observed for five out of eight metals analysable by DGT devices, i.e. Cd, Cr, Co, Mn and Ni (range, 13–51%, Table 1). Note that the increase in dissolved Co previously observed downstream of the Rampillon CW by grab sampling was not supported by the passive samplers. Vegetated wetlands have been shown to efficiently remove metals such as As, Cd, Cu, Pb and Zn in contexts related to the remediation of wastewaters heavily contaminated by industrial, mining or urban discharges (Arroyo et al., 2013; Leung et al., 2017; Mulkeen et al., 2017). In addition, the efficiency of CWs in removing metals has been reported to depend strongly on inlet metal concentrations and hydraulic loading (Marchand et al., 2010). Here, we provide evidence of efficient removal for various cationic and anionic metals in a drained agricultural context, where contamination levels are low and subjected to temporal fluctuations. Unlike grab samples, using time-integrated tools thus offers better sensitivity for quantifying mitigation efficiency of metals in both dissolved and particulate fractions. Furthermore, these results also

Table 1 Metal mitigation by the wetland of Rampillon in trapped sediments and DGTlabile fractions during the study period. Bold values are significant mitigations from upstream to downstream of the wetland (P < 0.05; paired t-test with n = 5 for traps and n = 9 for DGTs). Metals not analysed by DGTs are indicated by n.a. Metal mitigation (%)

As Cd Cr Co Cu Mn Ni Pb Sb Se Zn

Trapped fraction

Labile fraction

23.4 ± 13.7 13.3 ± 8.2 18.3 ± 7.7 20.1 ± 6.4 11.3 ± 7.5 −6.5 ± 23.8 19.7 ± 6.7 16.8 ± 9.0 15.0 ± 12.7 16.4 ± 14.7 12.5 ± 8.0

n.a. 50.5 ± 3.3 13.2 ± 8.7 43.8 ± 7.8 −4.5 ± 18.7 19.0 ± 15.1 40.7 ± 13.5 4.3 ± 9.4 n.a. n.a. 9.9 ± 8.0

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Metal bioaccumulation in both gammarids and biofilms are reported to be efficient indicators of multi-metallic contaminations even at low exposure levels and sensitive to seasonal fluctuations (Faburé et al., 2015; Lebrun et al., 2015). In our study case, biofilms were revealed to be more relevant than gammarids to record changes in metal bioavailability. The absence of net bioaccumulation in gammarids can be explained by active mechanisms of regulation and elimination of internalised metals not exceeded in a context of low exposure, i.e. physiologically acceptable levels for gammarids (Lebrun et al., 2014; Rainbow, 2007). Furthermore, it is possible that gammarids are less responsive than biofilms to changes in metal loads in the particulate fraction. Indeed, biofilms produce polymeric substances forming an extracellular matrix able to sorb suspended matter (Faburé et al., 2015; Morin et al., 2008). Thus, Cd and Zn contents in biofilms have been shown to be more correlated with the particulate metals in water than with the dissolved fraction (Morin et al., 2008). In the present study, significant linear correlations were obtained between total metal contents in biofilms and in trapped sediments for Cr, Mn, Ni, Pb and Zn (R2 = 0.56, 0.62, 0.65, 0.55 and 0.72, respectively, with n = 10 for both sites combined; P < 0.05), and to a lesser extent for Cd, As and Se (R2 = 0.38, 0.39 and 0.33, respectively, with P < 0.10). Similar correlations were obtained when the metallic fraction internalised in biofilms was considered. By contrast, no significant correlations were obtained when metal levels in biofilms were plotted against DGT-labile concentrations. This confirms that the particulate fraction was more representative of the fraction available to biofilms than the dissolved fraction. Thus, the decrease in bioaccumulation levels in biofilms, related to reduced loads of particulate metals at the outlet, argues in favour of a beneficial effect of the Rampillon CW for local microbial communities in terms of exposure.

reveal changes in metal dynamics and speciation occurring within the CV. For instance, decreases in DGT-labile metals observed upstream of the wetland highlight decreases in free inorganic metals and weak organic complexes, which has been shown to be representative of the bioavailable fraction for aquatic organisms exposed by aqueous route (Ferreira et al., 2013; Uher et al., 2011). Insofar as metals are not (bio)-degradable, metal mitigations observed in the dissolved and particulate fractions suggest metal transfers from water column to bed sediment and/or to vegetation in the Rampillon wetland. These transfers can result from various processes: adsorption to bed sediments, precipitation as insoluble salts, absorption by vegetation and/or deposition of suspended solids in a context of low water flow (Arroyo et al., 2013; Marchand et al., 2010; Mulkeen et al., 2017). Whatever the combination of processes involved in our context, this potentially generates metal accumulation in sediments and biological components, with possible environmental issues over the long term (source/sink of metals releasable in the dissolved phase, dredging and reallocation of sediments on soils, biomagnification in trophic chains, etc.). They must be considered for sustainable and proper management of CWs. 3.4. Biological monitoring and metal bioavailability During the study period, metal bioavailability was monitored at the individual and community scale through the caging of gammarids and sampling of biofilms. Except Mn, contamination levels in biofilms were higher at the inlet than the outlet of the CW, demonstrating a decrease in bioavailability of metals crossing in this artificial zone (Fig. 5; not significantly for Co and Sb). Similar patterns were obtained when only the fraction of internalised metals was considered in biofilms (data shown in SI, Fig. S2). These results are in agreement with the decrease in metal lability and loads of trapped sediments previously observed during the monitoring campaign (Fig. 4). In respect with gammarids, the only significant change in bioaccumulation levels was observed for Mn, where levels were higher downstream than upstream (Fig. S2), as also noted in biofilms. This was controversial with decreasing DGT-labile Mn observed at the outlet (Fig. 4). The particular behaviour of Mn can be attributed to its high responsiveness to reducing conditions, which favour its release from colloidal oxides or fine organic complexes of the dissolved phase and subsequently its bioavailability (Marchand et al., 2010; Tercier-Waeber et al., 2012). Furthermore, Mn was the only metal for which no mitigation was observed in trapped sediments (Fig. 4).

3.5. Ecodynamics of metals in different environmental matrices 3.5.1. Sediments Metallic contamination levels of bed sediments sampled in the various cells of the Rampillon CW are presented in Fig. 6A. For all the metals tested, excluding Mn, the highest levels were found in downstream cells (4 or 5), whereas the lowest were in upstream cells (1 or 2), with relatively low amplitudes (< 1.7-fold). The metal levels measured at Rampillon are close to natural values in sediments for the Seine Basin (Le Cloarec et al., 2011; Thévenot et al., 2007), as discussed above. This illustrates low rates of metal accumulation within the wetland since its inception in 2010, to be associated with low metallic inflows from

Fig. 5. Metal concentrations in biofilms sampled at the inlet (grey bars) and outlet (white bars) of the Rampillon constructed wetland during the study period. The letter “a” indicates that metal concentrations are significantly higher upstream than downstream, and “b” for the inverse case (P < 0.05, t-test paired by sampling date). 114

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Fig. 6. Metal levels in bed sediments (A) and indigenous macroinvertebrates (B) sampled in different functional cells of the constructed wetland of Rampillon. Values are means expressed in µgmetal/gdw of sediments (n = 2) or biological tissues (n = 3, except chironomids with n = 1 pool of about thirty individuals). For more clarity, values are classed by a colour code increasing with metallic concentrations (green < light green < yellow < orange < red). Grey boxes mean no metal analyses because of the absence of a species at the studied cell. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

agricultural sources. Unlike the dissolved fraction, there are no sediment quality standards in the European legislation. By way of comparison with Canadian guidelines for the top 5-cm layer sediments, levels did not exceed the quality levels for Cd, Cu, Pb and Zn (i.e. 0.6, 35.7, 35.0 and 123 µg/gdw, respectively) (Väänänen et al., 2018). To a lesser extent, levels were exceeded for As only for cell 5 (> 5.9 µg/gdw) and for Cr for the downstream sites 3, 4 and 5 (> 37.3 µg/gdw). The current status of contamination levels of sediments seems to be neither harmful to aquatic life, nor inconsistent with their reuse on watershed soils following a dredging operation for the maintenance of the wetland.

The spatial dynamics of metals observed within the Rampillon wetland suggests a gradual sequestration of metals in bed sediments along waterways, likely associated with sedimentation of fine suspended particles downstream of the wetland. Metals are indeed often highly concentrated in fine particles such as clays with large specific surfaces compared to coarse fractions (Le Cloarec et al., 2011). This is in accordance with the depletion of metals in suspended sediments at the outlet, as observed above (Fig. 4). Moreover, sedimentation can be promoted at the downstream cells because of the presence of plants and low non-turbulent flows, unlike the upstream cells (1 and 2) exhibiting low plant densities. This assumption was supported by additional 115

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granulometry analyses performed subsequently to this study. The analyses in spatial granulometry confirmed a gradual drop in the proportion of coarse elements (sand type > 50 µm; ranging from 20 to 5%) and an increase in silt elements (2–50 µm; range, 49–66%) from the upstream cells to the downstream cells. Nevertheless, a low spatial variability in clay content was observed (about 28 ± 3% for all sediments; data not shown). The fact remains that the retention of metals in sediments contributes in part to their removal in our context. For instance, this process of sequestration has been reported to be much more efficient than metal absorption by plants in a heavily metal-contaminated CW (Leung et al., 2017).

for various cationic and anionic metals in a drained agricultural context, where contamination levels are low and subjected to temporal fluctuations. Consequently, using chemical time-integrated tools is more suitable than grab sampling for quantifying the mitigation efficiency of metals in both dissolved and particulate fractions. Bioaccumulation-based tools confirmed temporal changes in metal speciation and bioavailability occurring within the CW. Furthermore, the spatial survey supports the important role of sediments in metal trapping as well as the beneficial effect of the wetland for local biodiversity in terms of exposure. Here the results provide valuable information on ecodynamics and bioavailability of metals required for further sustainable management of such artificial ecosystems. Finally, the study confirms the potential ability of CWs as effective tools to mitigate intensive agriculture impacts including the particulate fluxes, and the inorganic and organic contaminant fluxes released to aquatic systems. Further seasonal investigations should provide complementary information on long-term performance of the CW (e.g. metal absorption related to vegetation turnover, release of sediment-bound metals and, temperature and flow effects on metal mitigation…).

3.5.2. Indigenous biota To assess spatial trends of metal bioavailability in the Rampillon CW, bioaccumulation levels were determined in macroinvertebrates exhibiting different ecologies, i.e. detritivore crustaceans (Asellus and Gammarus spp.), insect larvae living in surface sediments (Chironomus spp.) and herbivore snails (Lymnaea spp.). In general, the results showed that the indigenous populations of gammarids, asellids and larval chironomids were less contaminated downstream than those exposed upstream to drainage discharges, in a less obvious way for snails because of their absence at cell 5 (Fig. 6B). This spatial trend supports a decrease in metal bioavailability, as previously observed in biofilms (Fig. 5), whatever the exposure routes (aqueous/dietary) of targeted macroinvertebrates. This trend reflecting spatial changes in metal speciation and distribution within the wetland is also in agreement with decreasing metal lability and loads in suspended sediments at the outlet (Fig. 4). Nevertheless, this is rather surprising for chironomids as organisms closely in contact with bed sediments, for which metal levels were inversely higher at the outlet (Fig. 6A). This can be explained by a negligible contamination of larvae by the epidermal route compared to the dietary route insofar as larval chironomids feed on SPM and micro-detritus. It is known that bioaccumulation levels vary from one species to another according to their behavioural characteristics (e.g. feeding habits), the exposure route (aqueous/dietary) and species-specific physiological mechanisms involved in regulation of internalized metals (Hadji et al., 2016; Rainbow, 2007; Veltman et al., 2008). The highest bioaccumulation levels were observed in larval chironomids for 6 out of 11 metals quantified in their bodies (i.e. Cd, Co, Ni, Pb, Sb and Zn), in snails for As and Mn and in isopods for Cu and Se. Despite lower metal contents, gammarids, however, exhibited significant spatial variations in bioaccumulation levels ranging 2- to 30-fold (max/min ratio, except for Ni and Zn with a ratio of 1.4) when compared to other species (ratios < 2.5, except Ni in chironomids with a 7.5 ratio and Cd in snails and asellids with ratios of 3.1 and 3.2, respectively). However, bioaccumulation levels in caged gammarids demonstrated no spatial differences between the inlet and outlet of the wetland. This can be explained by caging exposure that is not representative of natural conditions (e.g. access limited to local food, caging time too short to integrate low contamination levels, etc.) and/or size differences between indigenous and caged gammarids (Besse et al., 2012). Caged gammarids were 1-cm adults, whereas indigenous gammarids measured 0.5 cm because of the collection period, i.e. juvenile individuals. Nevertheless, metal bioaccumulation provides relevant insights of the exposure of ambient metals over a recent period in the local habitat (Rainbow 2007, Lebrun et al. 2015). Consequently, decreases in bioaccumulation levels at the outlet argue in favour of the constructed wetland being beneficial for local macro-biodiversity in terms of exposure.

Declaration of Competing Interest The authors declare no interest conflict. Acknowledgments The authors are sincerely grateful to Nicolas Hette (Irstea, HEF Team, Antony) for his expertise in collection and identification of indigenous invertebrates. The authors also thank the Laboratoire d'Analyse des Sols (INRA, Arras) for the granulometry analyses of sediments. The work was funded by the PIREN-Seine Research Program (Phase VII) and the FIRE Federation of Research (FR-3020). We also would like to thank Linda Northrup for her linguistic review of this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoleng.2019.06.012. References Arroyo, P., Ansola, G., Sáenz de Miera, L.E., 2013. Effects of substrate, vegetation and flow on arsenic and zinc removal efficiency and microbial diversity in constructed wetlands. Ecol. Eng. 51, 95–103. Baize, D., Sterckeman, T., 2001. Of the necessity of knowledge of the natural pedo-geochemical background content in the evaluation of the contamination of soils by trace elements. Sci. Total Environ. 264, 127–139. Barjhoux, I., Fechner, L.C., Lebrun, J.D., Anzil, A., Ayrault, S., Budzinski, H., Cachot, J., Charron, L., Chaumot, A., Clérandeau, C., Dedourge-Geffard, O., Faburé, J., François, A., Geffard, O., George, I., Labadie, P., Lévi, Y., Munoz, G., Noury, P., Oziol, L., Quéau, H., Servais, P., Uher, E., Urien, N., Geffard, A., 2018. Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River. Environ. Sci. Pollut. Res. 25, 23404–23429. Besse, J.P., Geffard, O., Coquery, M., 2012. Relevance and applicability of active biomonitoring in continental waters under the Water Framework Directive. Trac-Trends Anal. Chem. 36, 113–127. Bourgeault, A., Gourlay-France, C., Priadi, C., Ayrault, S., Tusseau-Vuillemin, M.H., 2011. Bioavailability of particulate metal to zebra mussels: biodynamic modelling shows that assimilation efficiencies are site-specific. Environ. Pollut. 159, 3381–3389. Directive 2013/39/UE, 2013. Directive of the European Parliament and the Council of the European Union for environmental quality standards in the field of water policy (WFD). Off. J. Eur. Commun. URL: http://eur-lex.europa.eu/. Faburé, J., Dufour, M., Autret, A., Uher, E., Fechner, L.C., 2015. Impact of an urban multimetal contamination gradient: metal bioaccumulation and tolerance of river biofilms collected in different seasons. Aquat. Toxicol. 159, 276–289. Fechner, L.C., Gourlay-Francé, C., Bourgeault, A., Tusseau-Vuillemin, M.-H., 2012. Diffuse urban pollution increases metal tolerance of natural heterotrophic biofilms. Environ. Pollut. 162, 311–318. Ferreira, D., Ciffroy, P., Tusseau-Vuillemin, M.H., Bourgeault, A., Gamier, J.M., 2013. DGT as surrogate of biomonitors for predicting the bioavailability of copper in freshwaters: an ex situ validation study. Chemosphere 91, 241–247. Gismondi, E., Thomé, J.P., Urien, N., Uher, E., Baiwir, D., Mazzucchelli, G., De Pauw, E.,

4. Conclusions The metallic contamination signature of the Rampillon CW is typical of aquatic media located in catchments subjected to intensive agriculture pressure. Moreover, this study demonstrates efficient removal 116

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