ADEME Bioindicateur Phase 2 Program

Protocol and sampling procedures under the BIOindicateurs II program
The sampling protocol applied under the program has been collectively discussed in order to reach the goals of all teams. It has been applied on each site by a single team of samplers (ensuring the quality of samples), the whole sampling process being coordinated by the University of Rennes 1 (UMR EcoBio, Daniel Cluzeau) which had previously conducted the same work under the RMQS BioDiv program (Cluzeau et al., 2012). The plan was based on the recommendations of European program ENVASSO’s working group (Bispo et al., 2007; Kibblewhite et al., 2008) as well as on the measures applied in the RMQS BioDiv national program (Cluzeau et al., 2009; Cluzeau et al., 2012) and complied with the ISO 2311-6:2012 standard for sampling procedures. Each sampling zone has therefore been subject to the same sampling strategy in order to compare data collected from the different sites. Samples were collected on a 100 square-meter-wide area (10m x 10m) for each modality, subdivided into 4 replications of 25 square meters (see figure below). For experimental sites organised in blocks (cases of QualiAgro-Feucherolles, BioREco-Gotheron and Thil), replications have been distributed across every block. Considering the limitations of indicators under analysis, soil sampling (for microorganisms and microfauna) and fauna sampling (meso- and macrofauna) have been conducted simultaneously (in the spring) while flora has been sampled afterward, at the most optimal stage of development (May-June). For the same reasons, the analyses of “snail” and “micromammals” bioindicators have been realized in May-June.

In order to optimise the measurement of certain biological parameters, soil samples have been systematically collected on sites at the beginning of the week so as to ship them as soon as possible to laboratories (Chronopost delivery including cold accumulators to maintain optimal temperatures). As a result, under this program, only one site could be sampled every week. The different stages of sampling are detailed hereafter.
Step 1 : Positioning of the sampling zone, and localization by GPS
		Based on the advice of site managers, the sampling area is positioned and singled out by construction site tape; similarly, replications are marked out and their external corner is recorded using GPS. Before any samples are collected, in each replication, the location of future samples is identified by a marker to avoid any future disruption (trampling).
Step 2 : Composite soil samplings
		Soil core samples (7 cm diameter) taken from 0-15 cm deep (in forest systems, the OL+OF horizons are removed, their respective width is written down; soil samples are therefore collected by integrating the OH and A horizons). Twelve cores are drilled randomly by replication, before they are assembled and distributed evenly to obtain a composite sample that will then be subdivided into equal parts. In order to meet the needs of different biological parameters: part of the subsamples is sieved to 6 mm, which corresponds to the following parameters: nematods, microbial biomass and DNA extraction, other soil samples, not sieved, preserve their initial structure for the measurement of other parameters, namely: fungal molecular biomass, microbial abundance and biomass (PLFA, Pseudomonas), enzyme activities, potential metabolic diversity, physical chemistry and archiving. Samples are weighed and shipped to different laboratories for analysis. DNA extraction was carried out by a single laboratory (GenoSol) which then redistributed DNA to relevant laboratories, allowing for the analysis of the following parameters: molecular fingerprint of microbial communities, microbial taxonomic diversity, microbial or fungal molecular biomass.

Step 3: Soil sampling of mesofauna
		It is conducted according to standardised ISO 23611-2:2006 procedures. Samplings (one per replication) are conducted using Plexiglas (6 cm diameter) at a depth of 5 cm. Samples are directly sent to the laboratory in charge of mesofauna extraction and its determination at the functional group level.
Step 4: Sampling of earthworms

		They are collected in accordance with the ISO 23611-1:2006 standard which combines chemical extraction (formalin) and handsorting. One sampling is carried out for each replication. Collected earthworms are brought to the laboratory where they are finely determined at the specific level and individually weighed.
For the analysis of earthworm biomarkers, a formalin solution (Bouché, 1972) is applied on the soil surface; the collected earthworms are thoroughly rinsed. 12 live individuals (preferably adults) from the same species are sent in plastic boxes containing their original soil, then analysed at the laboratory.
Step 5: Sampling of total macrofauna (samples collected by the IRD Bondy or the INRA-PESSAC teams). The samples are collected using the TSBF (Tropical Soil Biology and Fertility) method which has been modified to be adapted to temperate habitats (Lavelle, 1988; Anderson et Ingram, 1993) (ISO 23611-5:2011 standard). Five replications are conducted per modality. This method combines formalin extraction (1.5 litres of 0.2% solution poured twice over a ten minute interval) and manual sorting of a soil block (25 x 25 cm at a 15 cm depth). Collected organisms are preserved in a 4% solution and brought to the laboratory where they are determined at the level of order, and, if possible, at the level of family or genus. The following steps (6 to 8) were undertaken by the teams in charge of the biological parameter, and according to a calendar and protocols adapted to this parameter.
Step 6: Study of the Snail bioindicator Bioaccumulation was studied on snails from the Cantareus aspersus (syn. Helix aspersa) species, placed in microcosms (25 x 25 cm stainless-steel cylinder) on the ground. The snails remained on the site for a limited time (28 days) before they were taken back to the laboratory for analysis (absorption spectrometry, SAA or ICPMS).
Step 7: Study of the Micromammal bioindicator Bioaccumulation on micromammals was specifically studied in the Auzon site on 3 sectors with a gradient of contamination on areas adapted to the life of these vertebrate animals. Micromammals are sampled by lethal trapping. Caught micromammals are frozen and brought to the laboratory for analysis.
Step 8: Study of plant parameters The list of sites on which plant parameters have been studied is reported in the table below.

First, the vegetation of each modality was prospected in order to identify the main dominant species representative of the studied areas. Plant sampling was then conducted depending on the distribution of plant species present and the relevant indicator: Bioaccumulation of ETM : 2 sampling strategies were used: i) the first one (on Métaleurop, Auzon) consisted in studying metal concentrations in foliage on samples that were collected individually. ii) the second strategy (on all sites) consisted in studying metal concentrations in foliage on composite samples (“pool”): the aerial parts of 5 different species present in the same area were collected and this basic sampling was replicated 5 times on each modality, by using, when possible, mixes of different species between each replica. Oméga 3 : 4 to 6 species, to the extent possible, common to each plot, were identified. A piece of leaf from each plant species is cut using scissors and collected foliar tissues are placed in glass tubes containing a mix of methanol and sulphuric acid, before they are brought back to the laboratory for analysis. Photosynthesis : 5 different plant species common to each modality were identified. Parameters characterising the operation of photosynthetic systems were directly measured on the ground: Net photosynthesis (Pn) was measured on each sample; the other parameters (qP, PSII, Fv/Fm…) were measured on unwilted leaves. Dendrochemistry : On the Metaleurop site, 5 trees were sampled (Quercus robur and Acer pseudoplatanus) on several modalities and 10 trees (Quercus robur) were sampled on the whole Auzon site. For each tree, two wooden cores (superimposed) to a 1.30 m depth were collected from the trunk, completed by one bark sample (with a blanking tool). Microelementary measures were performed at the laboratory by energy dispersion spectrometry (EDS) and wavelength-dispersive spectroscopy (WDS). These techniques allow for a semi-quantitative measurement of a large range of elements, coupled with an imaging interface (scanning electron miscroscope).
General organisation In view of the significant number of sites, and considering the sampling constraints related to biological parameters which make the latter only measurable during specific activity periods (spring or autumn), it has been decided to conduct a first sampling campaign in the spring of 2009 (sampling of sites located in the northern half of France) and to sample in the spring of 2010 the sites located in the southern half; the ANDRA site has been sampled in the spring of 2011.
Bibliography Bispo A, Peres G, Cluzeau D, Graefe U, Römbke J, Rutgers M, Fuchs M, Sousa JP, Schulte R, Dombos M, Simon B, Gál A, Cortet J, Chaussod R, Ritz K, Creamer R, Winding A, English M, Boixadera J. Rubio JL. 2007. ENVASSO (Environmental assessment of soil for monitoring) WP 5 – Decline in soil biodiversity. EU Contract No. 022713, 22 pp. Cluzeau D., M. Guernion, R. Chaussod, F. Martin-Laurent, C. Villenave, J. Cortet, N. Ruiz-Camacho, C. Pernin, T. Mateille, L. Philippot, A. Bellido, L. Rougé, D. Arrouays, A. Bispo, G. Pérès. 2012. Integration of biodiversity in soil quality monitoring: Baselines for microbial and soil fauna parameters for different land-use types. European Journal of Soil Biology. Volume 49, 63-72 Cluzeau D., Pérès G., Guernion M., Chaussod R., Cortet J., Fargette M., Martin-Laurent F., Mateille T., Pernin C., Ponge J-F., Ruiz-Camacho N., Villenave C., Rougé L., Mercier V., Bellido A., Cannavacciuolo M., Arrouays D., Boulonne L., Jolivet C., Lavelle P., Velasquez E., Plantard O., Walter C., Foucaud-Lemercier B., Tico S., Giteau J-L., Bispo A. 2009. Intégration de la biodiversité des sols dans les réseaux de surveillance de la qualité des sols : exemple du programme-pilote à l’échelle régionale, le RMQS BioDiv. Etude et Gestion des Sols, Volume 16, 3/4, 187-201. ISO 23611-1. 2006. Qualité du sol -- Prélèvement des invertébrés du sol -- Partie 1: Tri manuel et extraction au formol des vers de terre. ISO 23611-2. 2006. Qualité du sol -- Prélèvement des invertébrés du sol -- Partie 2: Prélèvement et extraction des micro-arthropodes (Collembola et Acarina). ISO 23611-3. 2007. Qualité du sol -- Prélèvement des invertébrés du sol -- Partie 3: Prélèvement et extraction des enchytréides. ISO 23611-4. 2007. Qualité du sol -- Prélèvement des invertébrés du sol -- Partie 4: Prélèvement, extraction et identification des nématodes du sol. ISO 23611-5. 2011. Qualité du sol -- Prélèvement des invertébrés du sol -- Partie 5: Prélèvement et extraction des macro-invertébrés du sol. ISO 23611-6.2012. Qualité du sol Prélèvement des invertébrés du sol Partie 6 : Lignes directrices pour la conception de programmes d’échantillonnage des invertébrés du sol. Kibblewhite, M.G., Jones, R.J.A., Baritz, R., Huber, S., Arrouays, D., Micheli, E., Stephens, M. (2008) ENVASSO final report part 1: scientific and technical activities. ENVASSO project (contract 022713) coordinated by Cranfield university, UK for scientific support to policy, European Commission 6thFramework Research Programme.

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