Keywords: Performance, niche overlap, coexistence, sex dimorphism, short-tailed mouse (Mus spretus), wood mouse (Apodemus sylvaticus

Comparative bite force in two syntopic murids (Rodentia) suggests lack of competition for food resources

Samuel Ginot, Camille Le Noëne, Jacques

samuel.ginot@univ-montp2.fr camille.le-noene@univ-montp2.fr jacques.cassaing@univ-montp2.fr *Corresponding author: samuel.ginot@umontpellier.fr. D r a f t 3 spretus Lataste 1883) sont deux espèces de rongeurs muridés trouvés en syntopie dans le sud de la France. Nous avons mesuré la force de morsure chez des specimens sauvages des deux espèces pour tester de potentielles différences de performance. Bien que le mulot sylvestre soit plus gros, sa force de morsure n'est que légèrement plus haute que celle de la souris à queue courte. Nous n'avons pas détecté de dimorphisme sexuel marqué au sein des deux espèces, cependant parmi les souris à queue courte mâles, il apparaît deux groupes en termes de force de morsure. Ceux à la morsure plus forte pourraient représenter des mâles dominants, tandis que ceux à la morsure plus faible seraient des subordonnés. Généralement, les deux espèces ont des forces de morsures similaires, et par conséquent montrent un grand chevauchement dans les ressources qu'elles peuvent utiliser. D'autres facteurs pourraient exister qui créeraient une différenciation de niche entre le mulot sylvestre et la souris à queue courte. Une explication alternative pourrait être la présence de nourriture en abondance, qui supprimerait la compétition entre les deux espèces à ce niveau.

Keywords

Performance ; short-tailed mouse (Mus spretus) ; wood mouse (Apodemus sylvaticus) ; niche overlap ; coexistence ; sex dimorphism D r a f t Introduction Syntopic species (sensu [START_REF] Rivas | A reinterpretation or the concepts "Sympatric" and "Allopatric" with proposal or the additional terms "Syntopic" and "Allotopic[END_REF], i.e. species sharing the micro-habitats, have long been known to differentiate in their ability to cope with food types and hardnesses to reduce competition (e.g. [START_REF] Grant | Bill size, body size, and the ecological adaptations of bird species to competitive situations on islands[END_REF][START_REF] Grant | Interspecific competition in rodents[END_REF][START_REF] Verwaijen | Relationships between head size, bite force, prey handling efficiency and diet in two sympatric lacertid lizards[END_REF]Yamashita et al. 2009). [START_REF] Grant | Interspecific competition in rodents[END_REF] particularly highlighted that there was often significant disparities in size among coexistent species with similar needs and that this was probably due to ecological and evolutionary factors. His synthesis ended by the hypothesis that the advantage to the larger species may be the free access to food, due to dominance, and the ability to deal with larger and/or harder seeds; while the smaller species might have a greater efficiency in energy extraction from the foods it exploits, and being better at avoiding predators. Such a dichotomy can be achieved either by evolution toward a greater body size, or by changes in the masticatory apparatus (e.g. [START_REF] Van Daele | Biting Performance in Teeth-Digging African Mole-Rats (Fukomys, Bathyergidae, Rodentia)[END_REF]. Therefore, comparing pairs of syntopic species at their natural localities can be very fruitful in highlighting ecological performance differences and niche separation. Thus, [START_REF] Verwaijen | Relationships between head size, bite force, prey handling efficiency and diet in two sympatric lacertid lizards[END_REF] correlated bite force and prey hardness in two species of lacertid lizards, and proposed that differences in bite force are an important factor in prey handling efficiency and also influence prey selection in nature. In a large set of turtle species, Herrel et al. (2002) demonstrated that in vivo bite force was correlated with trophic ecology, as well as head height. Similar results were found in large Neotropical cats [START_REF] Kiltie | Size ratios among sympatric Neotropical cats[END_REF]) which differenciate from each other in terms of skull morphology, gape and bite force under the influence of ecological character displacement. In Neotropical bats, the amazing diversity of skull shapes among the phyllostomid radiation was also explained in terms of bite force and dietary niche [START_REF] Aguirre | Ecomorphological analysis of trophic niche partitioning in a tropical savannah bat community[END_REF]. Yamashita and colleagues (2009) found that sympatric (living in the same national park, but not necessarily syntopic) lemurs from Madagascar, all feeding on bamboo, specialized on different part of the plant D r a f t 5 and accordingly segregated in their feeding behaviours. Finally, comparing two Cricetidae rodent species with different sizes and diets Williams et al. (2009) highlighted the importance of gape in the biting performance. They showed that the larger species, which is also carnivorous, could maintain a large proportion of its maximal bite force at wide gape angles due to a more derived and advantageous condition of the jaw muscles (notably a lower stretch factor).

According to the various studies cited above, the measure of bite force appears to be a great tool to address the evolution of food niche dimension and separation. The wood mouse Apodemus sylvaticus (L., 1758) and the short-tailed mouse Mus spretus (Lataste, 1883)-two murid species often found in syntopy (i.e. caught in the same trap lines) in southern Franceare good candidates to run such a study. The two species share several habitats and most food items [START_REF] Bauduin | Interactions between the shorttailed mouse Mus spretus and the wood mouse Apodemus sylvaticus: diet overlap revealed by stable isotopes[END_REF][START_REF] Cassaing | Interactions between two Mediterranean rodent species: habitat overlap and use of heterospecific cues[END_REF]. Therefore, they may be considered to be in a situation of competition for food, yet they seem to coexist at least since the Holocene period. The wood mouse is about 1.5 times larger than the short-tailed mouse. In an experimental setting in the wild, it has been shown that these rodents can carry and eat larger seeds than equivalent individuals of the short-tailed mouse [START_REF] Muñoz | Are you strong enough to carry that seed? Seed size/body size ratios influence seed choices by rodents[END_REF]. The question remains as whether it also displays differences in terms of ingestion and comminution of food items. For its part, the short-tailed mouse demonstrates great skills to retrieve food sources. Notably, this species appears to use inadvertent information released by others [START_REF] Valone | Group foraging, public information and patch estimation[END_REF][START_REF] Doligez | When to use public information for breeding habitat selection? The role of environmental predictability and density dependence[END_REF][START_REF] Danchin | Public Information: From Nosy Neighbors to Cultural Evolution[END_REF][START_REF] Parejo | The heterospecific habitat copying hypothesis: can competitors indicate habitat quality?[END_REF]), even heterospecifics [START_REF] Cassaing | Interactions between two Mediterranean rodent species: habitat overlap and use of heterospecific cues[END_REF]). This may be highly expensive for the wood mouse if it gets its food caches used by the short-tailed mouse. To our knowledge, there has been no evidence so far that the short-tailed mouse does not achieve the second part of [START_REF] Grant | Interspecific competition in rodents[END_REF] prediction mentioned above (i.e. that it is better at energy extraction and predator avoidance).

D r a f t 6

We will focus here on the first step of ingestion by measuring the bite force of both species in the wild. This measure, which notably depends on the biomechanics of the skull and associated muscles, is a good proxy of the diet-and competition-related ecological performance, at the inter-and intraspecific levels [START_REF] Davis | Predicting bite force in mammals: two-dimensional versus three-dimensional lever models[END_REF][START_REF] Santana | Mechanics of bite force production and its relationship to diet in bats[END_REF]. Myomorphous rodents, such as the species studied here, have been hypothesized to be "highperformance generalists", according to [START_REF] Cox | Functional Evolution of the Feeding System in Rodents[END_REF]. If this holds true, we may expect strong bite forces in the two murids studied here, compared to values reported in the literature for non-murid rodents. Among them, we expect bite force to correlate body size, both at the interspecific [START_REF] Freeman | Measuring bite force in small mammals with a piezoresistive sensor[END_REF][START_REF] Ginot | Skull size and biomechanics are good estimators of in vivo bite force in murid rodents[END_REF], and intraspecific level [START_REF] Becerra | Bite force and jaw biomechanics in the subterranean rodent Talas tuco-tuco (Ctenomys talarum) (Caviomorpha: Octodontoidea)[END_REF]. Within species, we may find sex dimorphism in bite force, associated with intra-sex competition, as reported by Becerra and colleagues (2011) in another species of rodent (Ctenomys talarum). Interspecifically, the wood mouse is much larger than the short-tailed mouse and it should display much stronger bites. Because both species occur in syntopy -and feed mostly on the same items [START_REF] Bauduin | Interactions between the shorttailed mouse Mus spretus and the wood mouse Apodemus sylvaticus: diet overlap revealed by stable isotopes[END_REF][START_REF] Cassaing | Interactions between two Mediterranean rodent species: habitat overlap and use of heterospecific cues[END_REF])-the difference in bite force may be expected to be large, reducing competition for food (i.e. by giving access to harder and larger items to the largest species). On the other hand, the difference may be reduced if resources are widely available, therefore producing no competition between both species, and if other factors (e.g. behaviour differences, intra-specific competition) do not influence bite force.

Materials & methods

Individuals of both species were first sampled in a garrigue near Montpellier (43° 34' 38" N, 03° 43' 06" W) , with a mix of custom-built multi-catch traps (described in [START_REF] Cassaing | Les captures multiples chez les Rongeurs : fait du hasard ou phénomène social ?[END_REF]) and Victor® Tin Cat® traps set up in a 7 x 7 grid. The traps were 25 m apart, so the D r a f t 7 grid covered 2.25 ha. An exhaustive description of the site can be found in [START_REF] Cassaing | Interactions between two Mediterranean rodent species: habitat overlap and use of heterospecific cues[END_REF]. At this site, we captured 45 specimens of wood mice and 27 short-tailed mice. Additional samplings (wood mouse n = 49 ; short-tailed mouse n = 8) were carried out at the Lunaret zoo in Montpellier in a large mixed wood (Holm oak, Aleppo pines) with dense underneath vegetation, setting the same traps every 10 meters along a 100 meter-long line. Although caught within the zoo area, these specimens were wild, living in unkept spaces between the enclosures. Six specimens of wood mice were also captured in the Caroux moutain range (Hérault, France) near the village of Douch (altitude 700m), using the same trap density as for the zoo samples. Wood mice and short-tailed mice were caught in the same trap lines (sometimes even in the same traps), except in the Douch locality, where only wood mice were caught, probably because it is at the limits of the short-tailed mouse's range.

We determined the rodents' age on the basis of their weight, which is known to have a good correlation with genuine age (Pearson's correlation: male R²=0.88, female R²=0.79, with p<0.05 for both sexes according to Frynta and Zižková 1992). Broad age categories were defined as follows: for the short-tailed mouse, juveniles <10g ; subadults 10 to 13g ; adults >13g; for the wood mouse, juveniles <15g ; subadults 15 to 20g ; adults >20g. We recorded their sex and their apparent reproductive status by morphological features (e.g. testis position, opening of the vagina, nipple condition, suspected gestation).

Shortly after capture we measured the animals' voluntary bite force at the incisors using a piezoelectric force transducer (Kistler, type 9203, range 0-500 N, accuracy 0.01-0.1 N; Amherst, NY, USA ; calibrated by the constructor at 25 °C and 36% humidity) attached to a handheld charge amplifier (Kistler, type 5995, Amherst, NY, USA ; [START_REF] Herrel | Sexual dimorphism of head size in Gallotia galloti: testing the niche divergence hypothesis by functional analyses[END_REF]). The force transducer was mounted between two steel bite plates as described in [START_REF] Herrel | Sexual dimorphism of head size in Gallotia galloti: testing the niche divergence hypothesis by functional analyses[END_REF]. We adjusted the distance between the bite plates by measuring it with a caliper, and D r a f t 8 by increasing or decreasing it via the micrometer head, so that each individual had a gape angle of approximately 30° [START_REF] Dumont | The effects of gape angle and bite point on bite force in bats[END_REF], at which we found the rodents bit most consistently. All animals bit directly onto steel at the same spot on the plates (i.e. at the tip), to ensure a consistent out-lever length. We recorded three trials in a row for each individual, and the maximal score was used in the analyses. Body mass (g) was recorded using a Pesola® LightLine tubular weighing scale. Bite force over body weight ratios were also computed (Bite Force Quotient, or BFQ, Table 1), after converting mass (g) to weight (N), by dividing it by 1000 g/kg and multiplying by 9.8 m/s². All field procedures were under the Approval No. A34-172-042 (Hérault Prefecture).

The animals were gently handled, and when necessary, marked by toe-tattooing (e.g. [START_REF] Leclercq | A permanent marking method to identify individual small rodents from birth to sexual maturity[END_REF] to avoid duplicated measurements. All individuals were released at the location of their capture after manipulations.

Difference in mean bite forces, mass and BFQ between species, as well as differences in bite force between sexes were tested using two-tailed Student's t-tests. Correlations between bite force and weight were assessed by fitting least-squares linear regressions, and differences between the slopes and intercepts were tested using an ANCOVA. Allometric trajectories of log bite force against log body mass were tested against the expected slope of 2/3 for isometric scaling by linear regressions. Distribution of bite forces within sexes were visually inspected and tested for multimodality using Hartigan's dip test. All analyses were run in R (R Core Team 2017).

Results

Both species have a similar bite force, barely higher for the wood mouse than for the short-tailed mouse (mean=9.08, max=12.66, min=3.50 for the wood mouse; mean=8.31, D r a f t 9 max=11.20, min=5.13 for the short-tailed mouse, Table 1). Although apparently negligible, the difference between mean absolute bite force values was significant (Student's t-test: t=2.13, df=64, p<0.05). When comparing only adult specimens with sexes pooled between both species (mean=9.73 ± 2.01 for adult wood mouse ; mean=8.93 ± 1.75 for adult shorttailed mouse), the difference was not significant df=30,p=0.123).

On the other hand, interspecific differences of mean body mass (g) values were significant either when looking at the whole dataset (Student's t-test: t=10.60, df=81, p<0.01) or only at adults (Student's t-test: t=11.30, df=33, p<0.01). In accordance with the literature, the wood mouse was almost 1.5 times bigger than the short-tailed mouse (20.42 g and 13.72 g respectively on average). Therefore, BFQ mean value was significantly higher for the short-tailed mouse than the wood mouse (Student's t-test: t=5.70, df=43, p<0.01).

When body mass was plotted against bite force (Fig. 1), both species showed similar ranges in bite force and body mass. Both linear regressions showed a significant relationship, however with fairly low coefficients of determination (short-tailed mouse: R²=0.14, df=32, p<0.05 ; wood mouse: R²=0.24, df=99, p<0.01). The ANCOVA run on both species showed that as a whole, body mass had a significant effect on bite force (F=39.22, df=1, p<0. 001) and that the slopes were almost exactly identical to each other (F=0. 00, df=1, p=0.988, i.e. the relationship between bite force and body mass is the same for both species). Furthermore, the intercepts were not significantly different between both species (F=2.90, df=1, p=0.091), although the regression line for the short-tailed mouse was slightly higher than that of the wood mouse. Tests of the slopes of log bite force against log body mass showed no significant deviation from isometry (p=0.096 for the wood mouse, p=0.202 for the shorttailed mouse). D r a f t 10 Looking at adults within species, we found that the female short-tailed mice did not bite significantly harder than males df=14,p=0.069). No sex dimorphism was found in terms of body mass df=7.37,p=0.526). Likewise, in the wood mouse there was no difference in either bite force df=36.67,p=0.725), or body mass df=30.10,p=0.176) between sexes.

Visual inspection (Fig. 2) of the distribution of bite forces in short-tailed subadult and adult males reveals two groups, the first one with 14 individuals (11 subadults and 3 adults), had a mean of 6.59 N, and a range from 5.13 N to 7.82 N. The other group, with 7 individuals (2 subadults and 5 adults), had a mean of 10.38 N, with a range from 9.33 N to 11.20 N.

Despite the large gap between both groups, Hartigan's dip test for multimodality showed that the distribution was not significantly different from unimodality (D=0.10, p=0.33). Comparing short-tailed mouse adult and subadult males with the higher bite forces to conspecific adult and subadult females, we found a significant difference in mean bite force (Student's ttest: t=3.61,df=18,p<0.01).

Discussion

The two murids we studied showed a consistent bite force compared with published data on murid rodents. [START_REF] Cox | Functional Evolution of the Feeding System in Rodents[END_REF] for example, reported an average in vivo bite force of 31.1 ± 10.75 N in laboratory R. norvegicus, which is much bigger than the species studied here. Between our species, the difference in bite force appeared to be small compared to the difference in body mass, and was not signicant in adults. As shown by its greater average BFQ (Table 1), the short-tailed mouse M. spretus had a greater bite force relative to its size, compared to the wood mouse A. sylvaticus. Within species, a significant positive relationship D r a f t 11 was found between bite force and size (represented here by body mass), as was found other vertebrates [START_REF] Herrel | Ontogeny of performance in vertebrates[END_REF]. Indeed, the lightest individuals, likely the youngest, showed an absolute bite force lower than that of the heavier ones (Fig. 1). Furthermore, we found that bite force scaled isometrically with body mass in both our species, showing that, relative to their mass, the lighter individuals do not have lower bite force than heavier individuals.

Short-tailed mouse adult females bit on average harder than males, although not significantly. Furtermore, adult males in this species appeared to be split in two groups in terms of bite force (Fig. 2). However, the distribution was not significantly different from unimodality, perhaps due to small sample size. Still, this result is in line with those of staged dyadic encounters reported by [START_REF] Cassaing | Interactions intra-et inter-spécifiques chez les souris sauvages du Midi de la France, Mus musculus domesticus et M. spretus: conséquences sur la compétition entre les espèces[END_REF]. That study suggested some behavioural differences in males due to social hierarchy, and this may influence bite force as well. The adult males with the weaker bite forces could be the subordinate ones, while the other group would comprise the more dominant males. The mean bite force of the latter group is significantly higher than the one of subadult and adult females. So the potentially more dominant males appear to bite harder than the females, in accord with to previous results on rodents (e.g. [START_REF] Becerra | Bite force and jaw biomechanics in the subterranean rodent Talas tuco-tuco (Ctenomys talarum) (Caviomorpha: Octodontoidea)[END_REF]. Altough our data may fit with [START_REF] Cassaing | Interactions intra-et inter-spécifiques chez les souris sauvages du Midi de la France, Mus musculus domesticus et M. spretus: conséquences sur la compétition entre les espèces[END_REF] and Hurst's (1994) hypotheses that male short-tailed mice display hierarchical relationships (with dominant or subordinate status), it is not sufficent to explain or quantify them, and it was not the goal of this study. One way to assess how bite force and social status may be linked to each other in the short-tailed mouse would be by measuring bite force in dominant and defeated individuals of male-male encounters (e.g. [START_REF] Husak | Bite-Force Performance Predicts Dominance in Male Venerable Collared Lizards (Crotaphytus antiquus)[END_REF].

Our results show a suprisingly close bite force for the two syntopic murines studied here. As far as bite force is concerned, their abilities to break down hard seeds seem to be D r a f t 12 similar. Even if the wood mouse could handle larger acorns thanks to its greater body sizeas showed by [START_REF] Muñoz | Are you strong enough to carry that seed? Seed size/body size ratios influence seed choices by rodents[END_REF] in Central Spain-it should be noted that the higher range of acorns occurring in Spain is missing in garrigues in southern France where animals were captured. It seems that in our case both species eat the same seeds [START_REF] Bauduin | Interactions between the shorttailed mouse Mus spretus and the wood mouse Apodemus sylvaticus: diet overlap revealed by stable isotopes[END_REF] and share the same habitat [START_REF] Cassaing | Interactions between two Mediterranean rodent species: habitat overlap and use of heterospecific cues[END_REF], therefore displaying a great niche overlap, despite their important size differences. This would suggest that, in the areas we studied, the competition for food is not a limiting factor. This may partially explain why the wood mouse seems to show no attempt of pushing the short-tailed mouse aside, despite being used by the latter as a cue to resource abundance.

It seems that niche partitioning between our species, if any, does not occur through a qualitative difference in the access to harder foods. Resource partitioning may be produced by other factors such as the level of the habitat being exploited (strictly on the ground, or at least partially above and partially below it), the proportion of the animal parts in the diet, and the metabolic assimilation rate of both species. However, our data, as well as those of Bauduin and colleagues (2013) seem to point toward a lack of competition for resources, perhaps due to a great abundance of food in the localities studied rather than toward .

D

  Ginot* (1), Camille Le Noëne (1), Jacques Cassaing (1) (1) Institut des Sciences de l'Evolution de Montpellier -UMR 5554. Université Montpellier 2, CNRS, IRD, EPHE -cc 064. F 34095 Montpellier cedex 05.

Figure 1 :

 1 FIGURE LEGENDS

Figure 2 :Figure 1 :Figure 2 :

 212 Figure 2 : Histogram of bite force in adult and subadult males of Mus spretus, representing

Table 1 :

 1 Peiffer, E., and Ford, S. 2009. Gape and Bite Force in the Rodents Onychomys leucogaster and Peromyscus maniculatus: Does Jaw-Muscle Anatomy Predict Performance? J. Morphol. 270: 1338-47. Doi: 10.1002/jmor.1076 Yamashita, N., Vinyard, C. J., and Tan, C. L. 2009. Food mechanical properties in three sympatric species of Hapalemur in Ranomafana National Park, Madagascar. Am. J. Bite force, body mass, and bite force quotient (bite force/weight ratio) measured in two syntopic species of murine rodents, the short-tailed mouse Mus spretus and the woodmouse Apodemus sylvaticus. Values shown in the table represent the mean ± standard deviation for each subset. Sex was recorded based on the presence of testicles or nipples, age class is based on body mass (for M. spretus : juveniles < 10g ;

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https://mc06.manuscriptcentral.com/cjz-pubs

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology

Acknowledgements

The authors are grateful to workers of the Zoo de Lunaret in Montpellier, as well as François Catzeflis, who allowed us to collect data during the trapping sessions at the zoo. We also thank Pierre-Henri Fabre who organized fieldwork in the Caroux moutains and Thomas Tixier, who helped during fieldwork at the zoo as well as in Murviel-les-Montpellier. We are very grateful to two anonymous reviewers for their exhaustive and in depth comments and corrections, which greatly improved the quality of the manuscript. The authors received no funding for this study. This publication is a contribution of the Institut des Sciences de l'Evolution de Montpellier (UMR5554 -UM2 + CNRS + IRD) No. ISEM 2017-255.


Stéphane Pesce
email: stephane.pesce@inrae.fr

Sophie Campiche

Carmen Casado-Martinez

Ayanleh Mahamoud Ahmed

Chloé Bonnineau

Aymeric Dabrin

Emilie Lyautey

J D Benoit

Ferrari

Emilie Lyautey D , Benoît

J D Ferrari

Towards simple tools to assess functional effects of contaminants on natural microbial and invertebrate sediment communities

Keywords: Bait-lamina test, Functional ecotoxicology, Invertebrates, Metals, Microbial communities, Organic matter

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In river ecosystems, sediment is a habitat that supports large biological diversity (from viruses to fish) as well as a functional compartment that plays a major role in biogeochemical cycles [START_REF] Zoppini | Importance of extracellular enzymes for biogeochemical processes in temporary river sediments during fluctuating dry-wet conditions[END_REF]. However, with increasing nutrients, metals, and persistent organic pollutants released into human-dominated watersheds, a large amount of sediments accumulate various contaminants along the river continuum, particularly those exhibiting a strong affinity for particulate matter [START_REF] Burton | Assessing sediment toxicity: past, present, and future[END_REF]. Significant deposition of such contaminants can represent a risk for benthic communities and disrupt the functioning of aquatic ecosystems. It is therefore crucial to evaluate sediment ecotoxicological quality. This can best be achieved by means of integrated laboratory-and field-based approaches (i.e., weight-of-evidence studies) combining not only exposure-based tools but also effectbased metrics at different levels of biological organization (e.g., [START_REF] Chapman | The sediment quality triad approach to determining pollution-induced degradation[END_REF][START_REF] Vignati | Laboratory-to-field extrapolation in aquatic sciences: conceptual frameworks are needed to narrow the gap between laboratory-and field-based research[END_REF]. However, despite a number of ecotoxicological approaches available for evaluating sediment quality, there is a limited number of tools developed at community level [START_REF] Höss | Nematode species at risk -a metric to assess pollution in soft sediments of freshwaters[END_REF][START_REF] Castro-Català | Ecotoxicity of sediments in rivers: invertebrate community, toxicity bioassays and the toxic unit approach as complementary assessment tools[END_REF][START_REF] Pesce | Looking at biological community level to improve ecotoxicological assessment of freshwater sediments: report on a first French-Swiss workshop[END_REF]. Accordingly, sediment ecotoxicological studies performed at community level are still scarce in comparison with those concerning the soil and water compartments. Indeed, while the ecological role of sediment communities is widely recognized, there are still big gaps in knowledge on the direct and indirect effects of contaminants on their ecological traits and functions and on the resulting consequences for aquatic ecosystem functioning [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF][START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF].

Besides the need for further knowledge on the potential toxicity of contaminants on ecological functions and processes performed by benthic communities, a major challenge in environmental risk assessment of sediment pollution still lies in establishing a causal relationship between field exposure and functional effects at benthic community level [START_REF] Faupel | The functional response of a freshwater benthic community to cadmium pollution[END_REF][START_REF] Pesce | Looking at biological community level to improve ecotoxicological assessment of freshwater sediments: report on a first French-Swiss workshop[END_REF]. The need for indicators of ecotoxicological impacts on ecosystem functioning has driven a surge of interest into the use of natural or artificial organic substrate breakdown and decomposition to assess the functional integrity of contaminated aquatic [START_REF] Young | Organic matter breakdown and ecosystem metabolism: functional indicators for assessing river ecosystem health[END_REF][START_REF] Piscart | Leaf litter recycling in benthic and hyporheic layers in agricultural streams with different types of land use[END_REF][START_REF] Brosed | Litter breakdown for ecosystem integrity assessment also applies to streams affected by pesticides[END_REF] and terrestrial ecosystems [START_REF] Filzek | Metal effects on soil invertebrate feeding: measurements using the bait lamina method[END_REF][START_REF] Niemeyer | Functional and structural parameters to assess the ecological status of a metal contaminated area in the tropics[END_REF]. In sediment, the decomposition and consumption of organic matter by microbial and invertebrate benthic communities can be measured via several methods, such as using buried leaf litter bags [START_REF] Piscart | Leaf litter recycling in benthic and hyporheic layers in agricultural streams with different types of land use[END_REF] or buried artificial substrates like the DECOTAB tablet consisting of cellulose powder embedded in an agar matrix [START_REF] Kampfraath | DECOTAB: a multipurpose standard substrate to assess effects of litter quality on microbial decomposition and invertebrate consumption[END_REF]. In soil ecosystems, the use of the standardized bait-lamina test (ISO 18311, 2016) as a functional indicator of soil ecological integrity and/or as a tool for ecological risk assessment of soil contamination (ISO 19204, 2017) is now a well-established method [START_REF] Griffiths | Selecting cost effective and policy-relevant biological indicators for European monitoring of soil biodiversity and ecosystem function[END_REF][START_REF] Welsch | How many samples? Soil variability affects confidence in the use of common agroecosystem soil indicators[END_REF]. Beyond their valuable ecological relevance, these methods measuring global organic matter loss have the advantages of being relatively inexpensive and easy to use, and thus offer useful perspectives for routine ecotoxicological monitoring. The potential effects of contaminants on the capacity of benthic communities to degrade particulate organic matter in sediments has been poorly studied. Using mesocosm studies and testing realistic (i.e., several dozen mg/kg) to very high (i.e., several hundred mg/kg) exposure scenarios, [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF] and [START_REF] Sutcliffe | Microbial communities are sensitive indicators for freshwater sediment copper contamination[END_REF] observed no chronic effect of copper sediment contamination on the decomposition of leaf litters, cotton strips, and/or cellulose papers deployed on the surface of sediments and/or buried 1-2 cm below this surface. [START_REF] Piscart | Leaf litter recycling in benthic and hyporheic layers in agricultural streams with different types of land use[END_REF] also showed a lack of effect of agricultural contamination (pesticides and metals, including copper) on the decomposition of leaf litters buried 20 cm below the sediment surface in two French rivers. However, the potential toxicity of environmental concentrations of copper on the diversity of natural benthic microbial communities and on their capacity to perform several functions has been recently demonstrated [START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF][START_REF] Sutcliffe | Diverse fungal lineages in subtropical ponds are altered by sediment-bound copper[END_REF][START_REF] Sutcliffe | Microbial communities are sensitive indicators for freshwater sediment copper contamination[END_REF]. There is therefore a need for studies to evaluate the sensitivity of different methods assessing particulate organic matter breakdown and decomposition to detect the effects of environmental contaminant exposure on this important ecological function in the sediment compartment.

To this aim, we performed a 21-day laboratory microcosm experiment using two different kinds of artificial organic matter substrate containing cellulose powder, bran flakes, and active coal: one enclosed in a PVC strip (i.e., adapted from the bait-lamina method used for soil) and one encapsulated in agar (i.e., artificial tablet). These two different tools are easy to deploy in the field and are easy to interpret using respectively a visual analysis procedure (bait-laminas) and a weight loss measurement (tablets).

Given their frequent occurrence in many river sediments (e.g., [START_REF] Farag | Concentrations of metals in water, sediment, biofilm, benthic macroinvertebrates, and fish in the Boulder River watershed, Montana, and the role of colloids in metal uptake[END_REF][START_REF] Kohušová | A long-term survey of heavy metals and specific organic compounds in biofilms, sediments and surface water in a heavily affected river in the Czech Republic[END_REF], copper (Cu) and arsenic (As) were chosen as model contaminants. It has been shown that Cu and As can reach concentrations up to several hundred mg kg-1 dw in sediments collected in contaminated rivers [START_REF] Farag | Concentrations of metals associated with mining waste in sediments, biofilm, benthic macroinvertebrates, and fish from the Coeur d'Alene River Basin, Idaho[END_REF][START_REF] Farag | Concentrations of metals in water, sediment, biofilm, benthic macroinvertebrates, and fish in the Boulder River watershed, Montana, and the role of colloids in metal uptake[END_REF][START_REF] Ancion | Metal concentrations in stream biofilm and sediments and their potential to explain biofilm microbial community structure[END_REF], thus posing an ecotoxicological risk for benthic organisms [START_REF] Macdonald | Development and evaluation of consensus-based sediment quality guidelines for freshwater ecosystems[END_REF]. Here, we assessed the individual and combined effects of Cu and As on the capacity of previously unexposed natural sediment communities to consume and decompose particulate organic matter. We considered a worst-case but realistic exposure scenario by choosing a theoretical nominal concentration of 40 mg kg-1 dw for each metal. These concentraRons are respectively representative of high (i.e., 3rd quartile) and very high (i.e., 9th decile) levels of sediment contamination with Cu and As in French aquatic ecosystems (INERIS, 2010;[START_REF] Dreal-Remipp | Micropolluants dans les sédiments de la région Rhône-Alpes[END_REF]. The toxicity of the sediment following Cu and/or As treatment was also assessed using the standardized ostracod toxicity test (ISO 14371, 2012). This study allowed us to evaluate the usefulness of assessing particulate organic matter loss (i.e., consumption of organic matter through breakdown and decomposition activities) as a functional ecotoxicological endpoint, and to discuss the relevance of the two kinds of artificial organic matter substrates tested for developing functional ecotoxicological studies at community level in the sediment compartment.

Materials and methods

Sediment sampling and spiking with metals

About 100 L of surface sediments (0-10 cm) were collected in June 2016 at the "Pont de Chazey" sampling station (latitude 45° 54′ 38″ N-longitude 5° 14′ 11″ E) on the Ain river, which is one of the main tributaries of the Upper Rhône River (upstream of the city of Lyon) in France. This station was chosen as reference station as it presents a low level of metal contamination [START_REF] Dabrin | Coupling geochemical and biological approaches to assess the availability of cadmium in freshwater sediment[END_REF]. Immediately after sampling, the sediment was sieved at 2 mm to eliminate debris and coarse particles and to remove large macrobenthic organisms. The sieved sediment, containing < 2 mm natural communities (i.e., microorganisms, meiofauna, and small macrofauna) was homogenized and stored at 4 °C overnight until spiking. Aliquots of 13 kg (wet weight) of sediment were spiked or not with copper (Cu) and/or arsenic (As), at a targeted nominal concentration of 40 mg kg-1 dw for each metal, using a protocol slightly modified from [START_REF] Dabrin | Coupling geochemical and biological approaches to assess the availability of cadmium in freshwater sediment[END_REF] after preliminary spiking tests and according to the sediment characteristics (i.e., 70% of 250-300 μm particles, 18% of 70-80 μm particles, 2% of 10-20 μm particles, < 1% clays, and 8% silts). Further details on the spiking procedure can be found in Mahamoud [START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF].

Experimental design and microcosm setup

The experiment was performed in 13 glass indoor channels (length × width × height = 83 cm × 11 cm × 10 cm; Fig. 1 a andb) containing 3.5 kg wet weight of previously spiked sediment (equivalent to about a 3-cm-thick sediment) and a 6 L mixture of demineralized water and uncontaminated drilled groundwater (75:25). Each channel was connected to a 20-L glass tank (i.e., one independent tank per channel) through an aquarium pump (NEWA MJ 750) delivering water recirculation at a flow velocity of 1.5 L min-1. The microcosm experiment was conducted for 21 days (with weekly surface water renewals) under ambient laboratory temperature conditions (about 21 °C) and following a 13:11-h light/dark cycle. This exposure duration is in line with the recommendations formulated in the baitlamina ISO protocol, which stipulates that the bait lamina exposure time in soil lasts on average 10 to 20 days in temperate zones (ISO 18311, 2016).

Four experimental conditions were tested, i.e., (i) "Ref," (ii) "Cu," (ii) "As," (iv) "Mix," using three independent replicate channels filled at day 0 with the sediment previously spiked with (i) uncontaminated water, (ii) Cu (nominal concentration of 40 mg kg-1 dw), (iii) As (nominal concentration of 40 mg kg-1 dw), and (iv) a mixture of Cu and As (nominal concentraRon of 40 mg kg-1 dw for each metal), respecRvely. Furthermore, the remaining channel, which served as a sterile control microcosm (i.e., "Sterile"), was filled with the sediment previously spiked with uncontaminated water and further autoclaved at 120 °C for 1 h.

Metal analysis in sediments and water

Cu and As concentrations in sediment were checked just after the spiking procedure (day 0, d0) and at the end of the experiment (d21). Accordingly, composite samples of sediment were collected after spiking (d0, one replicate per spiking condition) and in each channel (d21, three replicates per experimental condition) and stored in polypropylene tubes. After drying in an oven at 40 °C, the sediments were grinded and homogenized with an agate ball mill (PM100, Retsch) and then kept dry in a desiccator. Approximately 300 mg of crushed and homogenized sediment was then mineralized with aqua regia (2 mL HNO3 and 6 mL HCl SUPRAPUR quality) in a microwave digester (MARS-6 from CEM) using teflon reactors (T°: 180 °C for 15 min). After mineralization, the samples were diluted in ultrapure water (Veolia) to obtain a volume of 50 mL. Cu and As concentrations in the sediment samples were measured by inductively coupled optical emission spectrometry (ICP-OES 720 ES, series 700, Agilent) according to AFNOR NF EN ISO 11885. The water content was measured in order to correct the results if necessary. The quantification limits were 0.66 mg kg-1 dw for Cu and 1.6 mg kg-1 dw for As.

Metal concentrations in the overlying water samples were measured at d0 (within the first 2 h after the installation of the artificial channels), d7, d14 (before water renewals), and d21. Samples (30 mL) were filtered using a polypropylene syringe and a cellulose acetate filter (0.45 μm porosity and 30 mm diameter). The filtered water was collected into polypropylene tubes, acidified with 0.5% (vol/vol) HNO3 (65%, Suprapur, Merck), and placed in the dark at 4 °C until analysis. Cu and As concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS, X7, Thermo Electron Series II) according to AFNOR NF EN ISO 17294.2. The quantification limits were 0.05 μg L-1 for Cu and 0.01 μg L-1 for As.

Ostracod test

The sediment toxicity test using the ostracod Heterocypris incongruens was performed using the commercial kit available from MicroBioTests and in accordance with the corresponding ISO standard 14,371 (ISO 2012). Briefly, 56 h before the start of the tests, ostracod cysts were incubated in standard freshwater at 25 °C under continuous illumination. After 48 h of incubation, the neonates were prefed with algal food (Spirulina) and incubated for 4 more hours. Each well of the microplate was filled with 2 mL of standard freshwater and 1 mL of sediment. Then, 2 mL of algal food, Scenedesmus spp., was added as extra food during testing to prevent starvation and its possible influences on ostracod growth or mortality. Then, 10 freshly hatched ostracods were transferred from the hatching Petri dish to each well. Each test sediment and each control was tested in six replicates. The microplates were incubated at 25 °C for 6 days in darkness in a humidity chamber. On d0, the length of 10 neonates was measured using CellD microscope software (Olympus). At the end of the exposure period, the ostracods were recovered from the microplates and transferred to a glass slide for counting and length measurement. Length at the end of the 6-day exposure was converted to growth (difference between length at the end and at the start of exposure) and to relative values with respect to the control sediment (% control response).

Sediments were tested for toxicity on d0 and d21. Only one sediment was tested on d0, corresponding to a sample collected after spiking, whereas one composite sample per replicate was collected and tested on d21. The clean sand provided with the commercial kit was used as control sediment in all trials.

Organic matter loss

The functional effects of metal contamination were assessed on the natural benthic communities present in the sieved sediments (i.e., microorganisms and macro-invertebrates < 2 mm) by studying the consumption of particulate organic matter (i.e., organic matter breakdown and decomposition) using (i) the bait-lamina method (ISO 18311, 2016) adapted to the sediment compartment and (ii) artificial tablets modified from the DECOTABs model [START_REF] Kampfraath | DECOTAB: a multipurpose standard substrate to assess effects of litter quality on microbial decomposition and invertebrate consumption[END_REF].

The bait-lamina material (Von [START_REF] Törne | Assessing feeding activities of soil living animals. I. Bait lamina tests[END_REF][START_REF] Kratz | The bait-lamina test[END_REF] consisted of PVC plastic strips of 160 × 6 × 1 mm perforated with 16 holes at 5-mm intervals (Fig. 1c). The strips were purchased from Terra Protecta® GmbH. Stick holes were filled with a standard mix made of cellulose powder (Fluka 11363), bran flakes (ground down and sieved to < 500 μm), and active coal (Merck 1.02186) (70:27:3 w/w) following the Terra Protecta protocol (www.terra-protecta.de). The principle of the test consists in exposing the bait-lamina in the sediment and assessing the loss of bait material consumed by the organisms (percentage of emptied apertures in comparison to the total number of holes after retrieval). In each replicate, 5 bait-lamina strips were distributed over the entire length of the channel. They were buried horizontally about 1 cm below the sediment surface (instead of vertically for soil; see ISO 18311) and exposed for 21 days. At the end of the experiment, the strips were removed, then examined by eye under bright light conditions, and the number of pierced holes was recorded. Bait holes were categorized into three classes, i.e., not pierced (scored 0), partially pierced (scored 1; light passing through a portion of the bait) or totally pierced (scored 2). Organic matter loss was expressed as a percentage of perforated holes for the 5 bait-lamina per channel.

The artificial tablets (Fig. 1d) were made using a protocol inspired from [START_REF] Kampfraath | DECOTAB: a multipurpose standard substrate to assess effects of litter quality on microbial decomposition and invertebrate consumption[END_REF]. Purified agar (3 g, agar-agar Merck 1.01614) was dissolved in 150 mL of demineralized water at 100 °C for a few minutes. After cooling to about 50 °C, 9 g of cellulose powder (Fluka 11363), 3 g of pre-ground bran, and 0.4 g of active coal (Merck 1.02186) was added to the suspension, which was poured into a multiwell polycarbonate mold (20 mm diameter, 5 mm height) to cast tablets with a final volume of about 1885 mm3. After solidification of the agar, the tablets were dried for 48 h at 70 °C before being weighed with an analytical balance (Sartorius A200S). Mean dry mass of the 65 tablets used in the microcosm experiment was 213.99 ± 9.32 mg. Each tablet was covered with an individual 5 mm meshsize plastic net, and 5 tablets were buried about 1 cm below the sediment surface in each channel. At the end of each experiment, the tablets were removed, dried at 70 °C for at least 3 days, and weighed. Changes in dry mass (DM) were expressed as % mass remaining relative to initial DM (measured individually for each tablet at the start of the experiment). The loss of DM in the sterile microcosm was used as a control to estimate the abiotic degradation of the tablets under the tested experimental conditions. This control was not needed for bait-lamina as we had previously checked the absence of abiotic degradation after several weeks in similar microcosms (unpublished data).

Organic matter loss measured both by the bait-lamina test and the use of artificial tablets and expressed in percentages were transformed as asin (squareroot(x/100) prior to statistical analyses to meet the assumptions of normality and homoscedasticity. Significant differences between treatment were detected by one-way analysis of variance (ANOVA) followed by a Tukey test [START_REF] Yandell | Practical data analysis for designed experiments[END_REF], where a p value < 0.05 was considered to indicate a significant difference between treatments.

Results and discussion

Sediment metal concentrations and ecotoxicity

Sediment samples containing natural communities were collected in a section of river known to be relatively uncontaminated by metals. This was confirmed by the chemical analysis (Table 1) which showed initial concentrations of As comprised between 2.8 and 3.3 mg kg-1 dw and concentraRons of Cu close to 1.8 mg kg-1 dw. Measured As and Cu concentrations in the natural sediment used were thus respectively bracketed in the low (i.e., 1st quartile) and very low (i.e., 1st decile) sediment contamination levels in French aquatic ecosystems (INERIS, 2010). Moreover, the ostracod toxicity test showed no initial toxicity of the natural sediment, with mortality (Table 1) and growth (results not shown) in the range of that observed for control sediments (less than 20% mortality and negative growth inhibition percentages with respect to controls, ISO 14371). These findings attest that the studied communities were not previously exposed to high As and Cu concentrations, ensuring that the component invertebrates [START_REF] Posthuma | Heavy-metal adaptation in terrestrial invertebrates: a review of occurrence, genetics, physiology and ecological consequences[END_REF][START_REF] Clements | Metal tolerance and predator-prey interactions in benthic macroinvertebrate stream communities[END_REF] and microorganisms [START_REF] Tlili | Use of the MicroResp™ method to assess pollution-induced community tolerance to metals for lotic biofilms[END_REF] were not preadapted to As and Cu metals, thus justifying the use of natural sediment from the Ain River as reference in the present experiment. After spiking, As (31.2-31.3 mg kg-1 dw) and Cu (55.1-56.6 mg kg-1 dw) concentraRons were respectively lower and higher than the target concentrations (40 mg kg-1 dw), without notable difference between individual and combined spiking for each metal. At the end of the experiment, there was still no significant difference in measured concentrations between individual and combined treatments for both As and Cu concentrations. However, the in-sediment concentrations of both metals decreased over the 21 days, with mean values ranging between 26.2 mg kg-1 dw and 24.6 mg kg-1 dw for As and 43.6 mg kg-1 dw and 47.8 mg kg-1 dw for Cu at the end of the experiment. Both the sediment spiked with Cu and the sediment spiked with the mixture of Cu and As caused 100% mortality of ostracods on d0 and d21. The mortality rate observed in Cu-contaminated sediments (alone or combined with As) was very high compared to the study of [START_REF] Torokne | Evaluation of the toxicity of river and creek sediments in Hungary with two different methods[END_REF], who observed less than 60% mortality in natural sediments containing up to 273 mg Cu kg-1 in mixture with other contaminants. Our results thus point to a very high bioavailability of Cu, probably linked to a high level of Cu release from the spiked sediment under our experimental conditions. However, this hypothesis is only partially supported by the chemical analyses performed weekly in the overlying water samples, which showed that dissolved Cu concentrations fluctuated between 38.9 and 60.1 μg L-1 over the course of the experiment (Table 2). In comparison, [START_REF] Sevilla | Comparison of aquatic and dietary exposure of heavy metals Cd, Cu, and Zn to benthic ostracod Heterocypris incongruens[END_REF] The As-spiked sediment initially showed intermediate levels of toxicity with 16.6% mortality and 28.3% growth inhibition on day 0. Toxicity decreased over time, and by d21 there was no growth inhibition and the mortality percentages were very low (2 ± 5%, no significant difference from the reference sediment). It suggests a lack of toxicity of As under our experimental conditions despite very strong As release from the sediments based on the dissolved As concentrations measured throughout the study (i.e., 760 to 2829 μg L-1, Table 2). The results are consistent with [START_REF] Kudlak | Determination of EC50 toxicity data of selected heavy metals toward Heterocypris incongruens and their comparison to "direct-contact" and microbiotests[END_REF] who showed that dissolved arsenic (spiked as As2O3) was the least toxic among many metals tested (including Cu, Hg, Cd) using this same ostracod testing protocol and species.

Table 1. Concentrations of As and Cu (mg kg-1 dw) in sediments aber spiking (day 0, n = 1) and aber 21 days (mean ± SD, n = 3) in the four treatments, and resulting toxicity to ostracods (control-normalized mortality percentage). Toxicity results on day 0 are averages of 6 replicates per sample, with one single sediment sample per treatment. Results on day 21 are means ± SD of 3 sediment samples per treatment (6 replicates per sample).

Table 2. Concentrations (means ± SD, n = 3) of Cu and As in the overlying water (μg L-1) at d0 (within the first 2 h after the installation of the artificial channels), d7, d14 (before water renewals), and d21 in the four treatments.

Relevance of artificial organic matter substrates for assessing the functional effects of As and Cu in the tested conditions

The main aim of this study was to test the relevance of two methods for assessing ecotoxicological effects on the consumption of particulate organic matter by benthic communities in contaminated sediments. To this aim, two kinds of artificial substrates containing cellulose powder, bran flakes and active coal were used-one enclosed in a PVC strip (i.e., bait-lamina method), and the other encapsulated in agar (i.e., artificial tablet). The bait-lamina method was first introduced by Von [START_REF] Törne | Assessing feeding activities of soil living animals. I. Bait lamina tests[END_REF] to assess the consumption of organic matter in soils. Its use in soil ecology is now wellestablished and standardized, and the method was recently proposed as a relevant and cost-effective tool that should be included in a set of indicators for European monitoring of soil ecosystem functions [START_REF] Griffiths | Selecting cost effective and policy-relevant biological indicators for European monitoring of soil biodiversity and ecosystem function[END_REF]. Several studies have also evidenced that bait-lamina can serve as a tool to assess the effects of contaminants, including metals and pesticides, in the organic matter consumption activity of soil communities [START_REF] Filzek | Metal effects on soil invertebrate feeding: measurements using the bait lamina method[END_REF][START_REF] Andre | Bait-lamina assay as a tool to assess the effects of metal contamination in the feeding activity of soil invertebrates within a uranium mine area[END_REF][START_REF] Niemeyer | Do recommended doses of glyphosate-based herbicides affect soil invertebrates? Field and laboratory screening tests to risk assessment[END_REF]. Along these lines, [START_REF] Bart | How to assess the feeding activity in ecotoxicological laboratory tests using enchytraeids?[END_REF] proposed the use of bait-lamina to perform ecotoxicological laboratory tests on the feeding activity of soil enchytraeidae. Because of its simplicity, its applicability in both microcosm or field studies, and the fact that organic matter breakdown is one of the most integrative processes in soil ecosystems, the bait-lamina method was identified as an appropriate tool for refined screening (tier II) for soil environmental risk assessment (ERA) (ISO 19204, 2017;[START_REF] Jensen | Ecological risk assessment of contaminated land-decision support for site specific investigations[END_REF][START_REF] Casabe | Ecotoxicological assessment of the effects of glyphosate and chlorpyrifos in an Argentine soya field[END_REF]. Given the importance of microorganisms and invertebrates in organic matter breakdown in both soil compartment and sediment compartment, the bait-lamina may be equally suitable for sediment ERA. However, to the best of our knowledge, the applicability of the baitlamina in sediments has not yet been demonstrated.

In the present study, bait-lamina organic matter loss in the reference sediment without metal spiking reached 33.5 ± 8.1% after 21 days (Fig. 2). The very low percentage of bait holes totally or partially pierced in the Mix treatment (1.0 ± 1.8% of organic matter loss) showed no significant influence of abiotic processes (e.g., physical or chemical constraints that may lead to the dissolution or the detachment of the substrates) in the degradation of bait-lamina in the sediment compartment of the laboratory channels, confirming our previous observation on sterile sediments from a preliminary (unpublished) experiment. Taken together, these results showed that the bait-lamina method can be used to measure the breakdown and decomposition activities of particulate organic matter by sediment communities (i.e., microorganisms and macro-invertebrates < 2 mm here). We also assessed the feasibility and relevance of using bait-lamina as an integrative ecotoxicological indicator of the functional impacts of Cu and As, alone or in mixture, on sediment communities. In our experimental conditions, bait-lamina organic matter loss was not significantly reduced in the As-spiked sediment (29.4 ± 12.9%) but was significantly decreased in the Cu-spiked sediment (11.6 ± 3.7%, p = 0.05) compared to the reference channels (but without significant difference between As and Cu treatments, p = 0.12). When both metals were co-present in mixture, the bait-lamina organic matter loss was almost totally inhibited (1.0 ± 1.8%), with a significantly higher combined effect than the effect of Cu alone (p = 0.04).

Whereas bait-lamina probably did not undergo abiotic degradation, the artificial tablets buried in the autoclaved sediment showed a weight loss of 15.8 ± 1.7% (data not shown). This result points to the possible occurrence of abiotic degradation processes and/or of decomposition activities performed by persistent microorganisms despite autoclaving. Based on use of the tablets, the consumption of organic matter was thus assessed by considering normalized values, obtained by subtracting the mean tablet degradation percentage observed in the autoclaved sediment (Fig. 3).

In the reference uncontaminated sediments, tablet degradation was 14.7 ± 1.6% higher than the mean abiotic degradation observed in sterile sediments, thus confirming the relevance of this kind of artificial substrate for estimating the consumption of organic matter by sediment communities. Compared to the reference channels, both As (p < 0.01) and Cu (p < 0.001) treatments significantly inhibited the tablet organic matter loss, without significant difference between the two metals (p = 0.23). As observed with bait lamina, the inhibition in tablet organic matter loss was almost total when Cu and As were combined, and the Mix treatment was significantly different from the As (p < 0.01) and Cu (p = 0.03) treatments alone.

The effects of spiked metals on natural sediment communities were evidenced with both bait lamina and artificial tablets, both of which have the advantages of being relatively inexpensive, simple to prepare, and easy to use. However, and contrary to the bait-lamina method, the tested tablets were significantly degraded by abiotic factors, as shown by the roughly 15% weight loss observed in the sterile sediment. This emphasizes the need to use controls in experimental studies and suggests possible limitations when comparing organic matter breakdown and decomposition activities of communities from sediments characterized by different abiotic properties, especially during in situ surveys. Whatever the substrate used (i.e., bait lamina or tablets), Cu had a significant effect on organic matter loss. This result contradicts findings from previous mesocosm studies [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF][START_REF] Sutcliffe | Microbial communities are sensitive indicators for freshwater sediment copper contamination[END_REF], which showed no effect of sediment Cu contamination (from 46 to 650 mg kg-1) on the consumption of organic matter, whatever the kind of substrate tested (i.e., leaf litter, cotton strip, or cellulose paper). Differences between the present results and those of [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF] and [START_REF] Sutcliffe | Microbial communities are sensitive indicators for freshwater sediment copper contamination[END_REF] could be due to methodological differences including the particulate organic matter substrates used, exposure time and substrates deployment. Indeed, most of the substrates used in the previous studies were left on the surface, except for some leaf litterbags that were buried in the sediment in [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF]. Moreover, it cannot be excluded that the Cu toxicity we reported on organic matter consumption in the contaminated sediments was mainly due to the high quantity of Cu released from the sediment particulate fraction to the pore water. As mentioned above to explain the high Cu toxicity we observed with the ostracod test, the strong Cu effect on organic matter consumption also points to a very high bioavailability of Cu in our exposure conditions. While [START_REF] Gardham | Direct and indirect effects of copper contaminated sediments on the functions of model freshwater ecosystems[END_REF] observed Cu concentrations close to 5 and 3 μg/L in pore water and overlying water respectively, in the present study Cu concentrations in the surface water were much higher and varied from 60.1 ± 5.4 μg L-1 at day 0 to 38.9 ± 0.7 μg L-1 at day 21 (Table 2).

In contrast, the effect of As applied alone was moderate in comparison to Cu despite concentrations of As in the overlying water ranging between 760 and 2829 μg L-1 depending on the sampling Rme (Table 2). This effect was only significant on tablet organic matter loss, and not on the bait-lamina. The apparent difference in the sensitivity of the two methods to the As toxicity was probably due in part to the higher between-replicate variability observed with the bait-lamina method. This may be also partially attributable to the lack of sensitivity of the method used to interpret the results, which is only based on three classes defined according to visual analysis of the holes (i.e., not pierced, partially pierced, or totally pierced) and/or to the surface area-to-volume ratio differences in the two tools (i.e., the bait-lamina method has two exposed sides while the tablet is fully exposed). Given this methodological limitation, the tablet method, which is based on weight loss measurement, is arguably more sensitive for assessing low or moderate effects on the consumption of particulate organic matter.

Whatever the method used, the toxicity induced by the mixture of Cu and As was higher than expected by a simple additive effect, leading to an almost total inhibition of the organic matter consumption in mixture-exposed sediments. Co-occurrence of different metals in aquatic ecosystems is frequent [START_REF] Altenburger | Understanding combined effects for metal co-exposure in ecotoxicology[END_REF] and previous studies have reported sediment and particulate contamination by metal mixtures including Cu and As [START_REF] Rabiet | Transfer of metal(loid)s in a small vineyard catchment: contribution of dissolved and particulate fractions in river for contrasted hydrological conditions[END_REF][START_REF] Castro-Català | Ecotoxicity of sediments in rivers: invertebrate community, toxicity bioassays and the toxic unit approach as complementary assessment tools[END_REF]. However, knowledge of the ecotoxicological effects of such joint exposure in sediment is still scarce [START_REF] Castro-Català | Ecotoxicity of sediments in rivers: invertebrate community, toxicity bioassays and the toxic unit approach as complementary assessment tools[END_REF][START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF]. Assessing the toxicity of river sediments contaminated by chemical mixtures (including metals and pesticides), De Castro-Català et al. (2016) showed that Cu was one of the main contributors of the effects they observed from ecotoxicity bioassays and from the description of the natural invertebrate communities. In a previous study (Mahamoud [START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF], we demonstrated that the effects of the mixture described in the present study on selected microbial activities were similar or slightly stronger than the effects of Cu alone. Moreover, the possible combined effects were not reflected in the bacterial community structure, which was similar in sediments exposed to Cu alone or to the mixture of Cu and As (Mahamoud [START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF]. These results suggest that the interaction between Cu and As had limited effects on the microbial functional potential while not modifying the community structure. The two methods tested in the present study, which allow the assessment of ecotoxicological effects on the effective activity of the sediment microbial and invertebrate communities in terms of organic matter breakdown and decomposition, were thus more sensitive to detect the combined effects of Cu and As than the assessment of the microbial functional potential. This can be due, at least partially, to the fact that As alone had no effect on the sediment bacterial structure and on the microbial functional potential [START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF] while it inhibited the consumption of particulate organic matter as shown from the use of the tablets in the present study (Fig. 3). It could suggest that As toxicity in the tested exposure conditions mainly affect invertebrates, thus confirming the necessity for developing ecotoxicological studies with organisms from different organizational levels to consider different sensitivities to contaminants, according among others, to their mode of action [START_REF] Castro-Català | Ecotoxicity of sediments in rivers: invertebrate community, toxicity bioassays and the toxic unit approach as complementary assessment tools[END_REF].

How can artificial organic matter substrates improve the ecological relevance of sediment environmental risk assessment?

The use of artificial organic matter substrates offers a cheap and easy approach [START_REF] Bart | How to assess the feeding activity in ecotoxicological laboratory tests using enchytraeids?[END_REF] that can help improve the ecological relevance of sediment environmental risk assessment for several reasons. First, it enables functional ecotoxicological assessment to be performed at the benthic community level, which is the intermediate biological level between populations and ecosystems. Moreover, it enables investigations of ecotoxicological effects on an important ecological function (i.e., particulate organic matter breakdown and decomposition) involving different microorganisms (mainly hyphomycete fungi and heterotrophic bacteria) and macroorganisms. In microbial ecotoxicology [START_REF] Ghiglione | Microbial ecotoxicology: an emerging discipline facing contemporary environmental threats[END_REF], the functional endpoints investigated to assess sediment heterotrophic community response to contaminants include, among others, various enzymatic activities, basal or substrate-induced respiration, and metabolic activities related to the nitrogen cycle, such as denitrification (e.g., [START_REF] Widenfalk | Effects of the pesticides captan, deltamethrin, isoproturon, and pirimicarb on the microbial community of a freshwater sediment[END_REF]Zoppini et al., 2016, Jaiswal and[START_REF] Jaiswal | Impact of heavy metal on activity of some microbial enzymes in the riverbed sediments: ecotoxicological implications in the Ganga River (India)[END_REF][START_REF] Ahmed | Environmental concentration of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF]. However, these approaches are based on measurements of functional potential, which are performed under optimized laboratory conditions and so do not necessarily reflect the effective activity of these communities in the real-world environment. In comparison, direct study of organic matter breakdown and decomposition processes in sediments via the use of bait lamina or artificial tablets can give relevant information on the ecotoxicological risks and effects on this crucial ecosystem process. This kind of approach could thus be viewed as a powerful tool for developing predictive functional ecotoxicology in the sediment compartment, by considering the ecological role of both microorganisms and benthic invertebrates as consumers of organic matter. Reaching further forward, it should be possible to dissociate microbial-driven decomposition from invertebrate feeding, for example by enclosing the tablets in small-mesh-size bags (barring access to invertebrates) and large-mesh-size bags (giving access to invertebrates), as frequently used in studies assessing leaf-litter breakdown [START_REF] Brosed | Litter breakdown for ecosystem integrity assessment also applies to streams affected by pesticides[END_REF][START_REF] Rossi | Structural and functional responses of leaf-associated fungal communities to chemical pollution in streams[END_REF][START_REF] Rossi | Stimulation or inhibition: leaf microbial decomposition in streams subjected to complex chemical contamination[END_REF]. Finally, the methods tested here could be proposed for standardization to further improve sediment ERA protocol, as already done in the soil compartment through the TRIAD approach (ISO 19204, 2017).

Conclusions

The two methods tested allowed the assessment of functional impacts of metal exposure in the sediment through the study of particulate organic matter consumption by benthic microorganisms and invertebrates. Both methods are inexpensive and easy to handle, and they carry both advantages and limits. Indeed, the bait-lamina method offers the main advantages of not being subjected to abiotic degradation in sediments and of being a standardized method that is already used for the assessment of the biological status of soils (ISO 18311, 2016) and for environmental risk assessment of soil contamination (ISO 19204, 2017). However, the current interpretation method, which is based only on a simple definition of three classes according to visual analysis of the holes, is arguably not sensitive enough to detect low or moderate ecotoxicological effects or to specifically assess effects on microbial decomposition. In contrast, the observed abiotic degradation of the artificial tablets used in the present study highlights the need for reference controls, which points to possible limitations when comparing sediments characterized by different abiotic properties. That said, the artificial tablet substrate method, which is based on weight loss measurement, may be more relevant for assessing low or moderate effects on feeding activities and for specifically focusing on microbial decomposition if the tablets can be isolated in small-mesh-size bags barring access to invertebrates.

In summary, the proposed methods offer potentially powerful tools for developing functional ecotoxicology in the sediment compartment by considering the ecological role of both microorganisms and macroinvertebrates as consumers of organic matter. Note too that such a simple tool could be applicable in both soil and sediment ecotoxicology, opening interesting perspectives for ecotoxicological studies to better integrate the soil-sediment-water continuum.

Fig. 1 .

 1 Fig. 1. Pictures of the indoor microcosms (a and b) and selected illustrations (from other experiments) of the bait lamina (c, freshly retrieved in situ from river surface sediments and covered by oligochetes) and tablet (d) tools..

  observed 100% mortality of H. incongruens ostracods exposed for 6 days to dissolved Cu concentrations from 260 up to 2600 μg L-1, and Kudlak et al. (2011) found a median lethal concentration (LC50, 6 days) of 953 μg Cu L-1.

Fig. 2 .

 2 Fig. 2. Mean mass loss (percentage of the initial mass) measured at day 21 with the bait-lamina test (n = 3 ± SD) in the four treatments (5 replicates per sample). Different letters indicate significant betweentreatment differences in baitlamina mass loss (Tukey test, p < 0.05)..

Fig. 3 .

 3 Fig. 3. Mean normalized mass loss (normalized percentage of the initial mass) measured at day 21 with the tablets (n = 3 ± SD) in the four treatments (5 replicates per sample).Values were normalized by subtracting mean mass loss observed in sterile sediment (horizontal lines give 95% confidence intervals for mean tablet degradation percentage).

Acknowledgments

The authors thank Bernadette Volat, Josiane Gahou, Bernard Motte and Christophe Rosy for their valuable technical support.

Funding

This work was funded by a grant from the Auvergne-Rhône-Alpes regional council (a Coopera grant for research and an Explora Pro grant for mobility awarded to Stéphane Pesce at the Ecotox Centre in Lausanne).