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Multi-substance indicators based on caged Gammarus bioaccumulation reveal the influence of chemical contamination on stream macroinvertebrate abundances across France Benjamin Alric, Olivier Geffard, Andre Chandesris, Martial Ferreol, Adeline François, Olivier Perceval, Jeremy Piffady, Bertrand Villeneuve, and Arnaud Chaumot Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01271 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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Environmental Science & Technology

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Multi-substance indicators based on caged Gammarus

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bioaccumulation reveal the influence of chemical contamination on

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stream macroinvertebrate abundances across France

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Benjamin Alric†, O. Geffard†, A. Chandesris‡, M. Ferréol‡, A. François†, O. Perceval§, J.

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Piffady‡, B. Villeneuve‡, A. Chaumot*†

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†Irstea,

UR RiverLy, Laboratoire d’écotoxicologie, centre Lyon-Villeurbanne, 5 rue de la

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Doua CS 20244, F-69625, Villeurbanne, France

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‡Irstea,

UR RiverLy, Laboratoire d’hydrobiologie quantitative, centre Lyon-Villeurbanne, 5

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rue de la Doua CS 20244, F-69625, Villeurbanne, France

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§Agence

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Vincennes, France

française pour la biodiversité, site de Vincennes, 5 Square Felix Nadar, 94300

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*Corresponding author: Arnaud Chaumot, Irstea, UR RiverLy, Laboratoire

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d’écotoxicologie, centre de Lyon-Villeurbanne, 5 rue de la Doua CS 20244, F-69625,

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Villeurbanne, France. [email protected]

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Running tittle: Biota for assessing pollutants in the ecosystem

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ACS Paragon Plus Environment

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Abstract

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Most of anthropogenic stressors affecting freshwater systems are qualitatively known.

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But the quantitative assessment of contaminant exposure and effects to aquatic

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communities is still difficult, limiting the understanding of consequences on aquatic

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ecosystem functioning and the implementation of effective management plans. Here,

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multi-substance indicators based on caged gammarid bioaccumulated contamination

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data are proposed (for metals and persistent organic pollutants, POPs) to map the

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bioavailable contamination level of freshwater ecosystems at a large spatial scale. We

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assessed the ability of these indicators to highlight the relationships between chemical

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exposure gradients and alteration in the abundance of macroinvertebrate populations,

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on a data set of 218 watercourses distributed throughout France. We identified spatial

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regional heterogeneities in the levels of bioavailable contamination of metals (18

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compounds) and POPs (43 compounds). Besides, a degradation of Gammaridae,

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Ephemeridae, and Hydrobiidae densities with increasing levels of metal contamination

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are identified relative to Baetidae, Chironomidae, and Hydropsychidae. We show here

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that active biomonitoring allows the establishment of multi-substance indicators of

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bioavailable contamination, which reliably quantify chemical exposure gradients in

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freshwater ecosystems. Our ability to identify species-specific responses to chemical

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exposure gradients demonstrates the promising possibility to further decipher the

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effects of chemical contamination on macroinvertebrate assemblages through this type

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of indicators.

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Introduction

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Human-made chemical contaminants released into aquatic environments have attracted

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widespread attention during recent years owing to the increasing anthropogenic

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loading1 as well as their persistence, biological accumulation and potential toxic

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effects,2,3 which can compromise the future provisioning of vital ecosystem services.4,5

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Given that water is regarded as the most essential of natural resources,6,7 a forward-

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looking system of chemical status assessment for surface water bodies has been

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implemented in Europe and embodied in the Water Framework Directive (WFD8). The

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measurement of contaminant concentrations in biota is increasingly recommended for

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assessing the environmental exposure of biological communities to chemicals.9,10

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Bioaccumulated concentration data collected in biomonitoring surveys provide time-

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integrated records of the fraction of contaminants potentially toxic for organisms, i.e.,

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bioavailable and bioaccumulative fraction.11,12 More recently, active biomonitoring

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(caged organism transplantation) has been proposed as an alternative approach to the

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field collection of wild organisms, which may improve spatial and time comparisons of

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contamination levels. Active biomonitoring obviously allows one to avoid the potential

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lack of native species in studied sites, but also to minimize some confounding factors

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known to influence the accumulation of contaminants in organisms (e.g., variability in

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the exposure time, age and size of sampled organisms).13,14 Among freshwater sentinel

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species, recent interest has been focused on the genus Gammarus because of its

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widespread distribution throughout the Northern Hemisphere,15,16 and its important

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ecological role as a trophic link between the base of food webs, as shredder, and higher-

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order consumers, as food for fish, amphibians and birds.17,18 Also, an efficient

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accumulation of metal and organic contaminants has been reported in gammarids at


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environmental exposure levels,19,20 making it a suitable candidate for identifying the

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sources and origins of contamination in freshwater ecosystems.10,21–23

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Evaluating the effects of contamination in aquatic ecosystems requires tools that

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quantitatively link the exposure to chemical contaminants to the impairment of

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biological communities. Relationships between the contamination level of individual

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metal measured in native organisms and the abundance of macroinvertebrates known

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to be sensitive to metals have been described in the literature. This was reported at the

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scale of single watersheds affected by occasional and local chemical contaminations

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(e.g., mining discharge),24–26 but also at larger spatial scales (Flanders, Belgium) with a

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lower ecological quality when levels of bioaccumulated individual pollutants in native

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eels were elevated.9 Recently, active biomonitoring with caged gammarids was

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successfully carried out at the French national scale to identify the possible link between

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exposure to three metal contaminants and native gammarid densities.27 All these

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findings highlight the effect of bioavailable chemical contaminations on aquatic

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populations and communities. Nevertheless, in terms of management, the establishment

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of such relationships obtained separately for each contaminant can be inefficient to

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quantify the link between contamination gradient and ecological degradation because

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the exposure to multiple contaminants seems to be the rule rather than the

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exception.28,29

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Thus, this study aimed to propose multi-substance indicators, based on the

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aggregation of concentrations accumulated by caged gammarid biomonitors, to

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demonstrate their relevance for mapping the contamination level of aquatic systems and

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to describe responses of macroinvertebrates to chemical pressure. This work benefited

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from caged gammarid contamination data at the French national scale acquired in the

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WFD regulatory context by regional water agencies, considering 18 metals and 43


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persistent organic pollutants (POPs), and records of benthic macroinvertebrate

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abundance, in 218 French river sites. Thanks to multi-substance indicators developed in

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this study, we sought in a first step to establish a global snapshot of metal and POP

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bioavailable contamination of watercourses at the national scale, and in a second step, to

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evaluate the response of resident macroinvertebrates to bioavailable chemical

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contamination. According to our previous study,27 we thus assumed that a multi-

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substance indicator could allow us to describe quantitative relationships between metal

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or POP environmental contamination gradients and native gammarid abundances.

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Finally, taking advantage of such an integrated indicator of metal contamination, we

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inspected the covariation between the estimated metal bioavailable contamination

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gradient and the distribution of abundances of five additional widely distributed

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invertebrate families (Ephemeridae, Hydrobiidae, Baetidae, Chironomidae, and

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Hydropsychidae), known to present contrasting toxicological sensitivities toward metals

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in ecotoxicological surveys.

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Materials and methods

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Chemical contamination and ecological bioindication datasets

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This study was based on two datasets gathering information on (a) the bioavailable

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chemical contamination in 218 wadeable river sites distributed throughout France, and

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(b) the abundances of benthic macroinvertebrates in these sites. Chemical and biological

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sampling dates (one sampling/year) cover the period 2009–2016 and the sampled sites

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belong to two major surveying networks established by French regional water agencies

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implementing the WFD requirements: the surveillance monitoring network (SMN) and

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the operational monitoring network. The former aims to assess long-term changes in the

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overall surface water status within each catchment and sub-catchment of the river basin


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district, while the latter aims to follow the status of French waterbodies identified as

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being at risk of failing to meet good quality objectives. Hence, available ecological data

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were obtained by a normalized protocol, applied once a year to sample benthic

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macroinvertebrates for this purpose.30 This protocol is based on twelve sample units

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distributed along different predefined habitats in reaches by regarding both their

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relative coverage but also their hosting capacity (i.e., the capacity of a given substrate to

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host a rich and diverse invertebrate assemblage). These sample units, performed with a

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normalized Surber net (sampling area 0.05 m2, mesh size 500 µm), are distributed

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evenly into three groups according to their habitat representativeness. The first, group

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A, focused on ‘marginal habitats’ (i.e., with an individual share of less than 5% coverage)

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with a high hosting capacity, while the other eight sample units, split in two groups (B

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and C), are taken from ‘major habitats’ (i.e., with an individual share of at least 5%

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coverage). The last two groups are distinguished by their hosting capacity and the

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relative coverage of major mesohabitats. From these samples, benthic

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macroinvertebrate assemblages are characterized by identifying and counting

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individuals at the genus level.

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Bioavailable chemical contamination data (contamination levels in caged

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Gammarus fossarum) were acquired either through French regional water agencies in

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the WFD context, or through additional research programs carried out by our own

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laboratory (ANR CESA program GAMMA021 02; ANR Blanc program Multistress0004-

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03). The methodology used for caged organism transplantation is presented in previous

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studies.27,31 Briefly, it consists in transplanting during 7 days 20 individuals G. fossarum,

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which have previously been acclimated 3 weeks in the laboratory under controlled

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housing parameters. To limit the influence of body size and sex on contaminants

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accumulation, size-calibrated male individuals were exposed. To avoid the influence of


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starvation on survival rate and contamination uptake, alder leaves were supplied into

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cages. Exposed gammarids came from a source population, commonly employed by our

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laboratory, and characterized by very low concentration levels for both metals and

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POPs. Hence, a dataset of 313 sampling events at 218 sites was obtained for metals and a

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dataset of 196 sampling events at 165 sites for POPs. There were 136 common

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samplings between metal and POP data sets.

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Physical state and land use data

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The surface water bodies studied in the present work are categorized and characterized

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according to System B of Annex II of the WFD by considering a climatic, hydrogeological

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and landform hydro-ecological classification.32 By comparing the distribution of our

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study sites in the different classes of physical state and land use types with the one

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determined on the SMN (𝑛 = 1501 sites) we can evaluate how far our set of sites

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encompass the diversity of rivers over the whole metropolitan French territory. The

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physical state of rivers was based on the Strahler number and the geological nature of

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the watershed soils. Digital land cover information, obtained from the CORINE Land

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Cover 2006 database (CLC2006),33 was used in order to identify land use types in the

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watersheds. On the basis of river catchment delimitations estimated beforehand with a

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digital field model, land use cover proportions were calculated at the scale of entire

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watersheds using ArcInfo analytical tools (ArcGis 10.5 software). We focused on two

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groups of land use categories likely to induce chemical contamination: urbanization of

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watersheds (i.e., urban areas, industrial and commercial areas, roads and highways,

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mines, dumps and construction sites, non-agricultural artificial green spaces), and

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intensive agriculture (i.e., arable lands, permanent crops, orchards, vineyards, annual

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crops associated with permanent crops, cropping and field systems).


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Multi-substance indicators of bioavailable contamination

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In order to represent the overall contamination pressure on aquatic ecosystems,

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indicators of integrated bioavailable contamination (IBC) are proposed, one for metals

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(IBCmetals) and one for POPs (IBCPOPs). The IBC calculated for each site studied takes into

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account contamination levels observed in caged organisms in regard to a threshold

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value determined for each compound (BBAC: bioavailable background assessment

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concentrations), as defined by Besse et al. (2013)31 and Ciliberti et al. (2017)27. First, the

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BBACs for all elements considered in the present study (Table 1–2) were determined by

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implementing the same statistical approach described by Besse et al. (2013)31 with the

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present national database. From this, the indicators IBCmetals and IBCPOPs computed a

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given site only consider the compounds whose concentrations recorded in G. fossarum

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significantly exceed their respective BBAC. Second, the concentrations were normalized

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according to an average contamination level to avoid scale effects among compounds.

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Hence, the IBC indicator is given as: 𝑛

∑𝑘 = 1 186

𝐼𝐵𝐶 =

𝑐𝑘 > 𝐵𝐵𝐴𝐶𝑘 𝜑𝑘 𝑛

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where 𝑐𝑘 is the concentration of compound k measured in gammarids (only when the

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measured concentrations exceed their respective BBAC), 𝜑𝑘 is the average

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contamination level defined as the mean between BBAC value of compound k and the

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90th percentile of concentrations of the compound measured over the whole dataset,

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and 𝑛 is the number of analyzed compounds. A square-root transformation of compound

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contributions was applied to reduce the asymmetry of the distribution of concentration

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values between sites.


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Link between chemical bioavailable contamination and macroinvertebrate

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abundances

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To inspect whether IBC indicators could predict effects on population densities at a

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national scale, macroinvertebrate abundances recorded at corresponding sites should

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be previously weighted by an ecoregional reference abundance level (i.e., carrying

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capacity in undisturbed watercourses) in order to make the comparison possible

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between geographically distant watercourses. Following the procedure previously

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applied by Ciliberti et al. (2017)27, macroinvertebrate abundances recorded at the sites

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defined in the French reference monitoring network (𝑛 = 392 with 3,127 sampling

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events performed between 2005 and 2013) were grouped according to the physico-

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chemical typology used in France. This typology classes streams based on alkalinity,

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altitude, and nutrient conditions.34 Six classes make up this typology (hard water

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streams: H1, H2, H3; and soft water streams: S1, S2 and S3). Reference levels of

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abundance for a given taxon in H1, H2, H3, S1, S2, S3 streams were determined like the

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95th percentile of the abundances recorded in the reference sites belonging to the six

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classes respectively. The macroinvertebrate abundances recorded in our study sites

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were then weighted by these reference levels which stand for a proxy of the optimal

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carrying capacity of the respective physico-chemical class of each site. Data for six

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macroinvertebrate families were collected and analyzed; Baetidae, Chironomidae,

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Ephemeridae, Gammaridae, Hydrobiidae, and Hydropsychidae. Abundance values were

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expressed at the family scale as a total of the genus level abundances in sampling units

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of groups B and C so as to ensure fine representative sampling of the site. Following our

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previous study,27 Gammaridae abundances were confronted either to IBCmetals or to

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IBCPOPs. In a last step, taking advantage of IBCmetals as an integrated indicator of metal


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contamination, the relationships between Baetidae, Chironomidae, Ephemeridae,

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Hydrobiidae, and Hydropsychidae abundances and IBCmetals were assessed. These widely

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distributed macroinvertebrate families were selected because they are regarded to

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present contrasting toxicological sensitivities to metals in the ecotoxicological

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literature.35–37

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Quantile regression analyses were used to estimate the relationships between

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IBC and changes in the density of macroinvertebrates. This type of regression analysis

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developed to estimate rates of changes in all parts of the distribution of a response

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variable is well suited to examine relationships between an environmental driver and a

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descriptor of biological communities in cases where other influencing factors are

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unmeasured and unaccounted for.38 Since other factors than chemical contamination

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may also affect the macroinvertebrate abundances (e.g., habitat impairment, life history

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disruption, loss or excess of food availability, etc.), a quantile regression model was

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constructed to estimate the abundance of macroinvertebrate as a function of IBCmetals or

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IBCPOPs by using the quantreg package39 of the R software. Quantile regression

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coefficients were estimated for percentiles (𝜏) from 80% to 95% by increments of 5%.

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Linear and curvilinear (exponential) models were built to each percentile and selection

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procedure based on Akaike Information Criteria (AIC) was used to determine which

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model (linear or curvilinear) best fits the data.

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Results

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Study site dataset

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Out of 218 sites sampled and listed in the database, 211 sites were sufficiently

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contextualized to be classified in the 6-class national general physicochemical classes

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(H1: 𝑛 = 24, H2: 𝑛 = 70, H3: 𝑛 = 47, S1: 𝑛 = 8, S2: 𝑛 = 23, S3: 𝑛 = 39). These 211 sites


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then were grouped in relation to watercourse size, geological nature of the watershed

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soils, and land use types (Figure S1 of the Supporting Information, SI). The comparison

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with sites of the entire SMN (𝑛 = 1,501 sites) shows that our set of sites was diverse and

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represented with reliability the hydrographic network at the national scale. The

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proportions of sites allocated to the different classes in the variables of agricultural land

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cover and watercourse size conform with those calculated for the sites of the French

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SMN (agricultural land cover: 𝜒2 = 1.11, 𝑝 = 0.57; watercourse size: 𝜒2 = 0.87, 𝑝 = 0.93).

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In our database, the proportions of sites in the urbanization intensity classes are slightly

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different than in the national SMN (𝜒2 = 7.72, 𝑝 = 0.02). Sites with a medium and high

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urban land cover are, respectively, underrepresented and overrepresented (44% and

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42% vs. 63% and 26% in the national SMN, respectively). The highest number of shifts

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in the distribution of the sites among the classes is observed for the variable geology (𝜒2

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= 18.52, 𝑝 = 9.50 × 10 ―5), with an underrepresentation of mixed profiles in our set of

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sites (2% vs. 18% in the national SMN, respectively) while the proportion of sites with a

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sedimentary profile is higher (80% vs. 56% in the national SMN, respectively).

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National scale snapshot of bioavailable contamination in French watercourses

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supplied by IBCmetals and IBCPOPs

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The list of metal and POP compounds considered, respectively, in IBCmetals and IBCPOPs, is

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presented in Tables 1 and 2. Considering metals, 17 compounds are quantified in more

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than 90% of the sites, while one compound [mercury (Hg)] is quantified in only 45% of

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sites (Table 1). For POPs, there is a more variable quantification among compounds,

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with 15, 14, and 14 compounds quantified, respectively, on average in 90%, 62%, and

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33% of sites (Table 2). The accumulation capacity of G. fossarum is assessed by the ratio

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of maximal to minimal recorded concentrations. All compounds exhibited a ratio higher


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than 2, with values higher than 100 for some metal contaminants [chromium (Cr) and

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nickel (Ni)]], some DDT isomers (4,4’-DDE), some PAHs (benzo(a)pyrene,

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benzo(e)pyrene, chrysene, perylene), and some PCB congeners (n° 101, 138, 153 and

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180).

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Overall, the geographic distribution of our study sites covers the whole territory,

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but with an underrepresentation in the central area of France (Figure 1). Mapping

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shows that weak and high metal contamination levels exhibited a homogeneous spatial

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repartition at the territory scale (Figure 1a). Nonetheless, a striking case is the western

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part of the Loire-Bretagne (LB) basin, where all sites exhibited contamination values

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higher than the median of the national IBCmetals values, and 75% of them are higher than

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the 75th percentile of the national IBCmetals values. Similar observations were made for

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the hydro-ecoregion of Cévennes (situated on the south-western border of the Rhône-

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Méditerranée-Corse (RMC) basin; 100% of IBCmetals values > median, 57% of IBCmetals

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values > 75th percentile) and Vosges (situated on the eastern border of the Rhin-Meuse

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(RM) basin; 100% of IBCmetals values > median, 83% of IBCmetals values > 75th percentile).

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To a lesser extent, high values of IBCmetals are measured in the eastern area of the hydro-

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ecoregion of Massif Central (situated on the eastern border of the LB basin; 62.5% of

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IBCmetals values > median and 40% of them > 75th percentile). For POPs, 85% of sites

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with a contamination value higher than the 75th percentile of the national IBCPOPs values

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are located in the eastern part of France, RMC and RM basins, and north of France, in the

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Artois-Picardie (AP) basin (Figure 1b). A spatial heterogeneity between metal and POP

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contamination levels is shown by the lack of a correlation between the spatial

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repartition of IBCmetals and IBCPOPs (𝑐𝑜𝑟 = ―0.036, 𝑝 = 0.655). For instance, sites

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presenting higher values (> 90th percentile) of IBCmetals (respectively IBCPOPs) had null or

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very low POP (or metals) contamination scores (Figure 2).


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Co-variation between IBCmetals, IBCPOPs and gammarid population abundances

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The relationships between the bioavailable chemical contaminations with the river-

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typology-corrected abundance of gammarids are depicted in Figure 2. Curvilinear model

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was the best fitted model of the relationships between the abundance of free-ranging

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gammarids and the IBCmetals and ICBPOPs values. One significant reduction in the

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abundance of free-ranging gammarids is apparent with the increase in values of metal

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contaminations for all tested percentiles from 80% to 95% (Tables S1). The quantile

302

regression for 𝜏 = 90 is plotted on Figure 2a. A scatter plot revealed a similar trend

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toward a decrease in the relative abundance of free-ranging gammarids as a function of

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the increase in values of POP contaminations (Figure 2b), but the relationship in this

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latter case with fewer study sites (n=196 sampling events) was not significant whatever

306

the considered percentiles (Tables S2).

307 308

Population abundances of six macroinvertebrate families toward the national

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IBCmetals gradient

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Figure 3 shows the distribution of river-typology-corrected abundances of six

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macroinvertebrate families with a widespread distribution in French watercourses

312

(including gammarids) along the increasing gradient of IBCmetals values. For the two

313

families Ephemeridae and Hydrobiidae a significant negative relationship in quantile

314

regression analysis is detected between IBCmetals and corrected abundance as seen for

315

Gammaridae (Figure 3a, 3c, 3e). The best fitted model was curvilinear for Gammaridae

316

and Hydrobiidae, and linear for Ephemeridae (Table S1). For Hydrobiidae, regression

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slopes were significant only for the highest percentiles (𝜏 = 90–95, Table S1). By

318

contrast, no significant relationship is detectable for Baetidae, Chironomidae, and


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Hydropsychidae whatever the considered percentile (Figure 3b, 3d, 3f and Table S1). In

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addition, the proportions of sites with zero abundance values are significantly different

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among the six macroinvertebrate families in sites with the highest contamination scores,

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for instance, the sites with IBCmetals > 0.39, i.e., the 90th percentile of the national

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distribution (light gray/white areas in Figure 3, 𝜒2 = 35.08, 𝑝 = 1.452 × 10 ―6).

324 325

Discussion

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Gammarid caging for monitoring the bioavailable contamination in freshwater

327

ecosystems

328

This study confirms and expands previous results that showed the relevance of caged G.

329

fossarum for monitoring chemical contamination in watercourses.27,31,40 Data from the

330

present study show that, during environmental exposure, a great diversity of

331

contaminants were accumulated in tissues of G. fossarum at concentrations exceeding

332

their respective BBAC (Tables 1 and 2). On the whole of contaminants quantified, 35

333

were identified like WFD priority substances. A total of 18 metals, whose eight had not

334

been detected in our previous study,31 were quantified. Also, 43 POPs were measured,

335

whose 24 compounds were different to Besse et al. (2013)31. For metal contaminants, in

336

common with our previous studies of the caged G. fossarum biomonitor, we report here

337

similar or higher frequencies of quantification (except for Hg27,31). For these same

338

metals, the ratios between maximal and minimal concentrations are higher in our study

339

than reported earlier,31 indicating that the enrichment of our database allows us to

340

cover a wider range of metal concentrations at the national scale. Regarding POPs, 19

341

contaminants are common to the regional study of Besse et al. (2013)31, and a difference

342

in the frequency of quantification is observed for only two contaminants (2,4’-DDT and

343

4,4’-DDD) for which we found lower frequencies, 14% and 46% instead of 100%,


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respectively. This result suggests that the weaker quantification of these substances in

345

our national study, compared with the regional study, is explained by limited

346

contamination of the study sites by these contaminants rather than by low accumulation

347

in G. fossarum. These findings show the relevance of using the biota as a storage matrix

348

for metals and POPs to study the contamination levels, but also as an integration matrix

349

over time, giving a best representation of the contamination of aquatic ecosystems.

350 351

Spatial heterogeneity of bioavailable contamination revealed by IBCs at a national

352

scale

353

Numerous studies confirmed that contaminants co-occur in mixtures with many

354

chemical compounds in aquatic environments28,29,41 warranting the development of

355

multi-substance indicators. But the integration of many compounds into a single

356

indicator raises questions about how to manage the specific variability of each

357

compound, taking into account the background concentration levels of each and the

358

scale effect induced by ranges of contamination levels variable between compounds. By

359

considering in the IBC only the concentrations above the defined BBACs and by

360

weighting these concentrations according to the range of concentration values observed

361

at the national scale, we provided an approach allowing to graduate and compare

362

reliably the spatial and temporal levels of bioavailable contamination in streams.

363

The regional hotspots of high metal contamination observed in the LB basin, RM

364

basin, and RMC basin can be compared to the physicochemical characteristics of

365

watercourses. The water geochemistry influences the bioavailability of metal

366

contaminants (e.g., decrease in bioavailable of cadmium (Cd), nickel (Ni) and lead (Pb)

367

under raised water calcium concentration), which can reduce their bioaccumulation in

368

organisms.42–44 Also, biomonitoring integrates the influence of the water physico-


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369

chemistry on the bioavailability of contaminants.45,46 Given that sites of these regions

370

belong to soft water classes of the physico-chemical typology, the low water hardness,

371

which makes metals relatively more bioavailable, explains the high metal bioavailable

372

contamination recorded thereinto. These findings reinforce the hypothesis that IBC,

373

because it is based on bioaccumulation data, exposes the vulnerability of communities

374

faced with exposure to metals in relation to national physicochemical typology.

375

Moreover, in the same hydro-ecoregion with soft water, profiles of contaminants vary

376

between study sites. For instance, in the western part of the LB basin, apart from the

377

cobalt (Co) and nickel (Ni) that were present (albeit at various concentrations) in all

378

study sites, cadmium (Cd) prevailed in five sites, while lead (Pb) and/or chromium (Cr)

379

prevailed in three other sites, and selenium (Se) in another site, suggesting different

380

sources of metal contamination. Similar reasoning can be followed to account for the

381

spatial heterogeneity in organic contamination levels. It can be argued that the

382

differentiated urbanization rates between regions explain the contrast in the

383

distribution of POP contamination among eastern and northern/western zones of the

384

territory, with contamination levels and urban land use higher in the latter (Figure S2),

385

while a more extensive urbanization releases a higher quantity of persistent organic

386

substances in aquatic ecosystems.47 Consequently, by converting a multitude of chemical

387

measurements into a single value, IBC indicators allow us to qualify sites relative to each

388

other and to identify those most subject to bioavailable contamination and/or the most

389

vulnerable, which can make them relevant prioritization tools for managers.

390 391

Relevance of multi-substance IBC indicators to decipher the effect of chemical

392

contaminations on macroinvertebrate populations


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393

Previous studies showed a sensitivity of macroinvertebrate populations to bioavailable

394

chemical contaminants, but they considered one contaminant at a time.48–50 Dealing with

395

water concentrations of multiple substances, some studies have used alternative metrics

396

to evaluate the ecological effect of multiple contaminants, for instance, by using the axis

397

scores from an ordination method to express a contamination gradient51 or by

398

developing models such as the cumulative criterion unit (CCU, which corrects for the

399

hardness of the water and therefore does correct the metal availability because of water

400

quality52). However, the use of data of contaminant concentrations measured in the

401

water column may limit the ability to establish a link to ecological effects. Ignoring

402

bioavailability could partly explain the weak relationships between CCU and the

403

macroinvertebrate richness.53 As such, we evaluated the relevance of the indicators,

404

which integrate over one week the bioavailable chemical contamination measured in

405

transplanted organisms (G. fossarum), as proxies of freshwater ecosystem chemical

406

contamination to which biological communities are exposed.

407

Analyzing the on-site abundances of Gammaridae against the IBCmetals or IBCPOPs

408

values shows a negative effect of bioavailable contamination on populations (Figure 2).

409

This reinforces the findings of our previous work carried out separately on three

410

individual metal contaminants27 and confirms that the ecosystem chemical

411

contamination such as that proposed through IBC indicators is reflected by a response of

412

the population abundance of this sensitive species. IBC indicators express the influence

413

of bioavailable contamination as a gradient, in contrast to Ciliberti et al. (2017)27, who

414

depicted a binary response of Gammaridae abundance in relation to a threshold of

415

contamination by each single metal element. It offers the opportunity to establish a

416

quantitative link between the chemical contamination exposure and the impairment of

417

populations. The exposure to chemical contamination can take various forms; most of


17


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418

the contaminated study sites displayed a complex pattern of bioavailable contamination

419

with concentrations above BBACs for many substances, simultaneously for metals and

420

organics. There was no correlation among the spatial repartition of metal and organic

421

contamination with the sites presenting the higher values (>90th percentile) of IBCmetals

422

(IBCPOPs) and having no or very low organic (metal) contamination (Figure 2); this

423

shows that the patterns of Gammaridae abundance degradation with increasing

424

bioavailable contaminations of metals or POPs are independent at this national scale.

425

Regarding the five other macroinvertebrate families, contrasting patterns were

426

reported in their response to the metal contamination of ecosystems (expressed from

427

IBCmetals, Figure 3). Two families (Ephemeridae, Hydrobiidae), in addition to

428

Gammaridae, were found to be sensitive to metal contamination, as expressed by a

429

significant negative relationship in quantile regression analysis, while the other three

430

families seem tolerant to metal contamination (i.e., Baetidae, Chironomidae,

431

Hydropsychidae). Interestingly, the family-specific sensitivity patterns observed for the

432

six families are consistent with results of earlier experimental studies in which

433

Ephemeridae and Hydrobiidae were described with Gammaridae as being more

434

sensitive to metal toxicity relatively to Baetidae, Chironomidae, and Hydropsychidae.35–

435

37

436

metals is yet recognized.54–56 Therefore the relative tolerance observed here in several

437

populations should certainly be explained by ecological factors such as demographic

438

recovery or adaptation abilities, also already documented in experimental studies.57–59 A

439

similar pattern was hypothesized to explain the resilience of gammarid populations

440

exposed to insecticides in agricultural landscapes.60,61

441 442

This statement should be nuanced for Baetidae for which physiological sensitivity to

These results validate the relevance of the caging gammarid approach to decipher the effect of ambient chemical contaminations on macroinvertebrate


18

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443

populations, by considering the complex link between the source of contamination and

444

population exposure at a large spatial scale. Further work is currently in progress to

445

validate the hypothesis of the effect of chemical contaminations, expressed through IBC

446

indicators, on macroinvertebrate community assemblages, expressed through

447

community metrics. Another prominent aspect is the potential of these indicators as

448

explicative variables in pressure-impact models62 to better understand the link between

449

pressure and ecological status, which is an essential step for developing effective river

450

management plans and shaping future environmental policy.63

451

In conclusion, providing tools allowing for a reliable assessment of the chemical

452

risk of aquatic ecosystems remains a challenging issue. The successful application of the

453

proposed multi-substance indicators over an entire country reinforces that resorting to

454

biota for large-scale monitoring of bioavailable contaminants in continental waters

455

constitutes a highly valuable approach. Because the bioavailable contamination is

456

expressed relative to reference thresholds defined at a large spatial scale, such

457

indicators allow us to reliably quantify levels of bioavailable contamination in aquatic

458

ecosystems. Our findings demonstrate that they are relevant in tracing the chemical

459

pressure to which some components of biological communities are subjected, for

460

instance, by highlighting species-specific responses to chemical exposure gradients. It

461

paves the way for further studies to examine the link between aquatic ecosystem

462

exposure to chemicals and potential changes in the structure of macroinvertebrate

463

communities and the functions they sustain.

464 465

Acknowledgments

466

We are grateful for financial support from AFB (the French National Agency Grant

467

agreement for action program 2016-2018 for Water and Aquatic Ecosystems). This


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468

work benefited also from the support of two programs: ANR CESA program GAMMA021

469

02 and Blanc program Multistress0004-03.

470 471

Authors’ contributions

472

ArC, OG and OP conceived the idea and designed methodology; ArC, AF and OG collected

473

the chemical data; AnC collected the land use data; MF, JP and BV gathered data on

474

macroinvertebrates; BA analyzed the data; BA led the writing of the manuscript. BA, ArC

475

and OG contributed critically to the drafts and all authors gave final approval for

476

publication.

477 478

Supporting Information

479

Figures showing the distribution of the whole 211 study sites according to their physical

480

state and land use data, and into eastern and northern/western regions of the French

481

territory. Tables showing the fit statistics for linear and curvilinear quantile regression

482

models describing the river-typology-corrected abundance organisms as a function of

483

the chemical bioavailable contamination (expressed from IBCmetals and IBCPOPs) for six

484

families of macroinvertebrates.

485

If paper is accepted for publication, supplementary data will be found online on the

486

website of the journal.


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Figures Figure 1. Distribution throughout France of sites where active biomonitoring (caged G. fossarum) is applied to evaluate the level of (a) bioavailable metal contamination (𝑛 = 218 sites) and (b) bioavailable POP contamination (𝑛 = 165 sites). Dot color corresponds to the chemical contamination expressed either from IBCmetals values or IBCPOPs values. The color gradient (metals: from green to blue, POPs: from green to red) indicates growing level of chemical contamination. When IBCmetals equals zero (green points), all measured values for each contaminant are above their respective BBACs. Geographic delimitations on the map represent the boundaries of six French regional environmental agencies; Adour-Garonne (AG), Artois-Picardie (AP), LoireBretagne (LB), Rhin-Meuse (RM), Rhône-Méditerranée-Corse (RMC).


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Figure 2. River-typology-corrected abundance of free-ranging Gammaridae (expressed as a percentage of the 95th percentile of the abundance observed on reference sites with the same physico-chemical typology) as a function of (a) the metal bioavailable contamination (IBCmetals) and (b) the POP bioavailable contamination (IBCPOPs). When IBCmetals or IBCPOPs equal zero (green points in both panels), all measured values for each contaminant are above their respective BBACs. In the top panel, dot colors correspond to the chemical contamination expressed from IBCPOPS. Black dots correspond to sampling sites for which data of POPs contamination are not available. In the bottom panel, dot colors correspond to the chemical contamination expressed from IBCmetals. Gray lines indicate regressions fitted at 90th percentile distributions. Significant relationships are shown with solid lines, and non-significant relationships are shown with dashed lines. According to the AIC value, curvilinear model was the best to fit the relationships between relative abundance of Gammaridae and IBCmetals or IBCPOPs values (Tables S1, S2). 𝑝 values were computed by bootstrapping with 200 replications.


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Figure 3. River-typology-corrected abundance of free-ranging organism estimates as a function of the metal bioavailable contamination expressed from the computation of IBCmetals for six families of macroinvertebrates: (a) Gammaridae, (b) Chironomidae, (c) Hydrobiidae, (d) Hydropsychidae, (e) Ephemeridae, and (f) Baetidae. Points in the white part of the scatter plot exhibit a value of metal contamination higher than the 90th percentile of the distribution of IBCmetals values. Gray lines indicate regressions fitted at 90th percentile distributions. Significant relationships are shown with solid lines, and non-significant relationships are shown with dashed lines. According to the AIC value, curvilinear model was the best to fit the relationships between relative abundance of Gammaridae (a), Hydrobiidae (c), and Baetidae (f) and IBCmetals vlaues, while linear model was the best for Ephemeridae (e), Chironomidae (b), and Hydropsychidae (d) (Table S1). 𝑝 values were computed by bootstrapping with 200 replications.


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Tables Table 1. Concentrations of metals, limits of quantification (LQ) and frequency of quantification (% data ≥ LQ) in caged G. fossarum after 7 days of exposure for the 218 sites. Concentrations and LQ are expressed in µg g-1 dw (dry weight). Compounds Concentration (µg g-1 dw) Frequency (%) Mean Med Min 90th Max LQ Agb 0.18 0.14 0.04 0.301 1.02 0.02 90 Al 600.24 463.40 36.10 1300 2311.00 2.00 100 B 3.99 3.54

Multi-substance indicators based on caged ... - ACS Publications

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