# Does compost addition affect the mineralisation of 14C-pesticides non-extractable residues in soil ?

Thomas Lerch*†<fnref n="1" /> and Enrique Barriuso<fnref n="2" />

## Abstract

The formation and the fate of pesticide non-extractable residues (NER) in soil are important processes that should be taken into account when assessing pollution risks. However, the underlying mechanisms are still poorly understood. In particular, the compost often applied on agricultural soils to sustain the content in soil organic matter (SOM) is likely to alter the biodegradation of such xenobiotics residues, either by reducing their availability or stimulating the microbial biomass. The main objective of this study was to assess the long and short terms effects of compost on the mineralisation of pesticide NER. To this aim, we set up an incubation with a 5x2x3 factorial design using NER coming from 5 different 14C-labelled pesticides (atrazine, simazine, pendimethalin, metsulfuron-methyl and 2,4-dichlorophenoxy acetic acid), initially formed in a soil amended or not with compost (Barriuso et al., 1996), and incubated with 3 different doses (0, 10 or 20%) of composts during 386 days at 28°C and 80% WHC. During the incubation, total CO2 and 14C-CO2 were trapped in NaOH solution and measured with colorimetric or scintillation counting, respectively. The mineralisation kinetics showed differences among NER, simazine- and pendimethalin-NER being the least mineralised (< 6%) and metsulfuron NER the most (> 12%). The short term effect of compost on NER mineralisation was minor compared to long term effect, especially for 2,4-D and atrazine. These results suggest that the addition of compost strongly affects NER fate when applied at the same time as the pesticides in soil, but has only small effects years after.

**Keywords:** Soil, Pesticides, Bound, residues, Mineralisation, Compost

<footnote n="1">¹Institute of Ecology and Environmental Sciences (IEES-Paris) – CNRS : UMR7618 – Université Paris-Est Créteil Val de Marne (UPEC) Faculté de sciences et technologie Bâtiment P 61, avenue du Général de Gaulle 94010 Créteil Cedex, France</footnote>

<footnote n="2">²Ecologie fonctionnelle et écotoxicologie des agroécosystèmes (ECOSYS) – Institut national de la recherche agronomique (INRA) : UMR1402 – Campus INRA-AgroParisTech 78850 Thiverval-Grignon, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: thomas.lerch@u-pec.fr</footnote>
# Isolation of rhizobacteria from agricultural soils treated with pesticides in the Meknes region, Morocco

Mohamed Maldani*†¹, Laila Nassiri¹, and Jamal Ibijbijen¹

¹Environment Soil Microbiology Unit – Faculty of Sciences, Moulay Ismail University, Meknes, Morocco

## Abstract

Soil quality covers the physical, chemical and biological. the interactions between them result in efficient operation in terms of production. In agricultural soils, contamination by pesticides has become an alarming environmental problem. This problem influences primarily on the biological properties of soils particularly on the microbial community in these soils. several studies have shown that the excessive use of pesticides has a toxic effect on the microbial flora of the soil. The objective of our study was to isolate rhizobacteria from 17 agricultural soils, previously cultivated by legumes and cereals, from the agricultural zone of Moulay Idriss (Meknes, Morocco), and subsequently tested their tolerances and degradation of pesticides. From these soils we isolated 300 strains of bacteria, after the tolerance test we isolated 57 bacterial strains that tolerated high concentration of pesticide. These strains will be identified.

**Keywords:** Rhizobacteria, Soil, Pesticides, Degradation, Morocco

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: maldani.mohamed@hotmail.com</footnote>
# Dispersion of microplastic in various media and its quantification in periphyton by flow cytometry and viSNE data analysis

Stephanie N. Merbt*¹, Linn Sgier¹, Ahmed Tlili†¹, Kristin Schirmer¹, and Renata Behra¹

¹Eawag – Department of Environmental Toxicology, Überlandstrasse 133, 8600 Dübendorf, Switzerland

## Abstract

Microplastic, defined as particles smaller than 1 mm, occurs ubiquitously in aquatic environments. Microplastic can sink in natural waters and potentially accumulates in periphyton, which is an assemblage of embedded algae and bacteria in a matrix of extracellular polysaccharide substances (EPS). With the objective to analyse interactions of microplastic with periphyton, we first aimed to achieve controlled exposure conditions. Therefore, we examined various dispersion media, and developed a method to quantify microplastic within periphyton by flow cytometry. We used various polyethylene microplastics, differing in size and density, and compared the time-dependent stability of the particle suspensions, using a cell counter. For flow cytometry measurements, the particles were dispersed in a periphyton suspension and their abundance was determined using viSNE data analysis. Microplastic was easily dispersed in Tween80, BSA and EPS extracts, unlike in water and fulvic acid where a sonication and strong mixing were needed. As expected, particles with low density buoyed upwards in all media, while particles with high density sedimented. Size of the particles also determined the temporal dynamics of the observed processes. Small particles remained dispersed more than 10 min, while biggest particles accumulated within 1 minute. Based on distinct fluorescence and scattering characteristics of the particles and periphyton organisms, flow cytometry coupled with viSNE data analyses allowed detection and quantification of the microplastic in a highly complex sample. Altogether, this work helps to guide experiments with microplastic under controlled conditions, and adds knowledge to their behaviour in fresh waters.

**Keywords:** Microplastic, periphyton, flow cytometry, fluorescence

<footnote>*Corresponding author:</footnote>

<footnote>†Speaker</footnote>
# Avoidance behaviour of oligochaetes in metal-based fungicide spiked soils: Effect of bacterial consortia

Oluwatosin Oladipo*¹, Pierre De Kock¹, Marco Engelbrecht¹, Carlos Bezuidenhout¹, and Mark Maboeta¹

¹Unit for Environmental Sciences and Management, North-West University, (NWU) – Private Bag X6001, Potchefstroom 2520, South Africa

## Abstract

Metal-based fungicides may influence oligochaetes' behaviour and result in inhibiting habitat function. The presence of bacterial consortia in such polluted soils may be of bioremediative advantage. This study investigated the effect of *Achromobacter spanius* and *Bacillus cereus* isolated from mining sites on the avoidance/preference behaviour of *Eisenia andrei* and *Enchytraeus buchholzi* in copper oxychloride (CO) and mancozeb (MZ) spiked soils. Broth cultures (24-H) of *A. spanius* and *B. cereus* were inoculated into MZ (44 and 1250 mg kg-1) and CO (200 and 1000 mg kg-1) spiked soils. Standardized protocols (ISO, 2007) were used for experimental set-ups. Avoidance behaviour revealed that *Eisenia andrei* and *Enchytraeus buchholzi* demonstrated preference for *A. spanius-B. cereus* inoculated compared to non-inoculated spiked soils. Higher preference was also exhibited by *Enchytraeus buchholzi* than *Eisenia andrei* and MZ compared to CO spiked substrates. In 44 mg kg-1 MZ inoculated substrates, a net response of -76 and -88 % were observed for *Eisenia andrei* and *Enchytraeus buchholzi* while 72 and 68 % were obtained in non-inoculated substrates. For 200 mg kg-1 CO spiked substrates, a net response of 28.57 and -98 % were observed respectively in inoculated substrates while in non-inoculated, 100 and 6.1 % avoidance was obtained. At 1250 and 1000 mg kg-1 MZ and CO concentrations, reduced habitat function was observed, with both oligochaetes revealing higher avoidance especially in non-inoculated and CO substrates. *Achromobacter spanius-Bacillus cereus* consortia thus enhanced *Eisenia andrei* and *Enchytraeus buchholzi* preference behaviour towards fungicide spiked soils.

**Keywords:** Soil pollution, metal, based fungicides, avoidance behaviour, oligochaetes, *Achromobacter* *spanius*, *Bacillus* *cereus* consortia

<footnote>*Speaker</footnote>
# Ecology of bacterial communities during petroleum biodegradation: a molecular analysis on degrading consortia isolated from hydrocarbon polluted soils, sediment and seawater samples

Rahma Omrani*†<fnref n="1" />, Edoardo Puglisi*‡<fnref n="2" />, Giulia Spini<fnref n="2" />, and Dalila Saidane<fnref n="1" />

## Abstract

Environmental microbial communities are key players in the transformation and mineralization of hydrocarbon pollutants. The aim of this study was to assess changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia isolated from different polluted environmental matrix (a petroleum station soil, refinery reservoir soil, harbor sediment and harbor seawater). The microcosms were inoculated for two months with either a defined bacterial consortium and Zarzatine crude oil as only carbon and energy source. The microbial community dynamics was evaluated weekly in microcosms using Illumina 16S rRNA gene sequencing. An average of 7020 sequences per samples were obtained, the measured coverage was higher than 97% and more than 60% of sequence were correctly classified at species level. Degrading consortia enriched from soil and sediments were dominated by species belonging to *Pseudomonas* and *Acinetobacter* genera, while in the seawater-derived consortia *Dietzia*, *Fusobacterium* and *Dicoplana* emerged as dominant genera. We identified specific OTUs whose relative abundances bloomed from small to high percentages and were significantly correlated to total petroleum degradation: an OTU classified as *Dietzia daqingensis* in the seawater microcosms, and three OTUs classified as *Acinetobacter venetianus* in all two soils and sediment derived microcosms. Interestingly, a comparative analysis showed that this was the same OTU in all 3 microcosms which were derived from consortia isolated from different types (soil or sediment) and location. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation.

**Keywords:** petroleum, degrading consortia, Illumina, bacterial ecology

<footnote n="1">¹Laboratory of Analysis, Treatment and Valorization of Environmental Pollutants and Products, Faculty of Pharmacy, Monastir University, Tunisia – Tunisia</footnote>

<footnote n="2">²Istituto di Microbiologia, Università Cattolica del Sacro Cuore (UCSC) – Via Emilia Parmense 84, 29122 Piacenza, Italy, Italy</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: rahmaomrani-114@hotmail.fr</footnote>

<footnote>‡Corresponding author: edoardo.puglisi@unicatt.it</footnote>
# From biodegradation of organic pollutants in soils by saprotrophic fungi ... to bioremediation.

Catherine Rafin*¹ and Etienne Veignie¹

¹Unité de chimie environnementale et interactions sur le vivant (UCEIV) – Université du Littoral Côte d'Opale : EA4492 – Maison de la Recherche en Environnement Industriel 145 avenue Maurice Schumann 59140 Dunkerque, France

## Abstract

Fungi constitute an important group in soil biological diversity and functioning. From the last two decades, our team has explored the natural competence of saprotrophic telluric fungi to degrade persistent organic pollutants (POPs) i.e. polycyclic aromatic hydrocarbons (PAHs) and dioxins/furans for developing soft bioremediation technologies of polluted soils. For bioremediation to be an effective tool for the clean-up of POP-contaminated soils, a greater optimization of factors such as bioavailability and mass transfer of POPs which were once thought to be recalcitrant is required. A collection of filamentous saprotrophic fungi was isolated from various historical contaminated soils (UCEIV mycology collection, Dunkerque, France). We focused our attention on fungi belonging to Ascomycota, Sordariomycetes, Hypocreales, with *Fusarium solani* as a fungal model. The metabolic pathways involved in the degradation of benzo[a]pyrene (BaP) as a model of high molecular weight PAH was explored. Lipid bodies constitute the intracellular storage sites of BaP in *F. solani* in which an energy-dependent uptake of BaP and a cytoskeleton dependent intracellular transport have been demonstrated. The degradative ability of other fungal isolates at laboratory scale (either in mineral medium or in soil microcosms) was also studied. The use of polysaccharides (potato or corn starch) with surfactant properties was considered for enhancing BaP degradation by oxidation. An overview of our current knowledge of saprotrophic telluric fungi mediated degradation/transformation of POPs will be presented. This outlined research performed at different levels of biological organization (from single-species to communities) can be transferred to other polluted ecosystems and other pollutants.

**Keywords:** Benzo[a]pyrene, Biodegradation, Bioremediation, Persistent Organic Pollutants, saprotrophic fungi, soil

<footnote>*Speaker</footnote>
# Environmental impact of natural and synthetic
β-triketone herbicides

Sana Romdhane<fnref n="1" />, Marion Devers-Lamrani, Christophe Calvayrac, Franck Dayan, Cedric Bertrand, Fabrice Martin-Laurent, and Lise Barthelmebs*<fnref n="1" />

## Abstract

In order to limit potential impacts on the use of plant protection products in agriculture, new generation molecules have been developed such as low impact pesticides and pesticides derived from natural substances. Nevertheless, many issues remain to be studied, including the assessment of potential risks and adverse effects related to their use on the environment and human health.

In the frame of the TRICETOX project, we focused on the β-triketone herbicide family, a post-emergence maize selective herbicides belonging to this new generation of molecules, and we worked on sulcotrione, one of the synthetic β-triketone herbicides placed on the market in 1993, and on leptospermone, an allelopathic compound isolated from the bottlebrush plant *Callistemon citrinus*.

A combination of chemical and biological approaches was followed in order to gain new insight for these two molecules, by addressing their soil behaviour, by studying their impact on soil microbial communities, by studying their biotic and abiotic degradation, and by developing new analytical methods.

In this context, our degradation studies revealed the existence of several transformation products. For the first time, we isolated for an arable soil a bacterial strain able to degrade leptospermone, and also, another bacterial strain capable to biodegrade two synthetic β-triketones herbicides. Microcosm studies revealed that leptospermone has a significant ecotoxicological impact on the abundance and diversity of soil bacterial community, similar to this of sulcotrione. We also succeeded in the development of a new bioassay method, based on the use of recombinant *E. coli* strains to quantify β-triketones herbicides in soil.

**Keywords:** β, triketone herbicides, impact, ecotoxicology, biodegradation, analysis, soil.

<footnote n="1">¹Biocapteurs - Analyse- Environnement (BAE) – Université de Perignan Via Domitia – Université de Perignan Via Domitia, 52 Av Paul Alduy, 66860 Perpignan cedex, France</footnote>

<footnote>*Speaker</footnote>
# Enrichment procedures for the isolation of xenobiotic-degrading bacterial and fungal strains: a focus on microbial ecology

Giulia Spini*†<fnref n="1" />, Federica Spina<fnref n="2" />, Anne-Laure Blieux<fnref n="3" />, Tiffanie Regnier<fnref n="3" />, Abdelwahad Echairi<fnref n="3" />, Ilaria Re<fnref n="4" />, Giovanna Varese<fnref n="2" />, and Edoardo Puglisi*‡<fnref n="1" />

## Abstract

LIFE-BIOREST (www.lifebiorest.com) is a EU-project aimed at the remediation of a soil polluted by PHAs, BTEX and alkanes through the application of selected consortia of fungi and bacteria.

In the first phase of the project, we have isolated from the polluted soil about 220 fungal and 140 bacterial strains with degrading abilities. Most of the fungal strains belonged to Ascomycetes (mainly *Aspergillus*, *Cladosporium*, *Fusarium* and *Scedosporium* genera) even though about 20 Basidiomycetes were also isolated. Regarding bacteria, the most abundant genus was *Pseudomonas* followed by *Achromobacter*, *Stenotrophomonas* and *Ochrobactrum*.

The isolates were obtained by means of a liquid enrichment approach on target contaminants (naphthalene, pyrene, phenanthrene, benzene, alkanes and oil extracted from the soil): the enrichment lasted 3 weeks, and sub-culturing was carried out once a week. Since the pollution in the test soil involved several meters of depth, enrichments were carried out separately using as inoculum homogenized soil samples from the 0-1, the 1-2 and 2-3 m depth. A total of 18 enrichment consortia were produced (6 target contaminants X 3 soil depths) for bacteria and other 18 for fungi, and sampled at day 0, 7, 14 and 21, giving a total of 144 samples. For each of these samples, DNA was extracted, amplified respectively for bacterial (16S) and fungal (ITS) biomarkers, and the resulting amplicons sequenced in Illumina. Results will be presented with the aim of assessing the ecological evolution of the microbial communities (bacteria and fungi) during the selective enrichment procedure in presence of different pollutants.

**Keywords:** selective enrichment, microbial diversity, fungal and bacterial consortia

<footnote n="1">¹Istituto di Microbiologia, Università Cattolica del Sacro Cuore (UCSC) – Via Emilia Parmense 84, 29122 Piacenza, Italy, Italy</footnote>

<footnote n="2">²Mycotheca Universitatis Taurinensis, University of Turin, Turin, Italy – Italy</footnote>

<footnote n="3">³SATT Grand-est - Welience Agro-Environnement, Bretenière, France – Societe d'Acceleration du Transfert de Technologie (SATT) Grand Est - Welience – France</footnote>

<footnote n="4">⁴Consorzio Italbiotec – Italy</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: giulia.spini@unicatt.it</footnote>

<footnote>‡Corresponding author: edoardo.puglisi@unicatt.it</footnote>
# The dynamic degradation of pollutants by Nocardia in the infiltration basins

Florian Vautrin*¹

¹Laboratoire d'Ecologie Microbienne (LEM) – Université Claude Bernard - Lyon I (UCBL) – 16 rue Raphael Dubois, 69100 Villeurbanne, France

## Abstract

Infiltration basins concentrate pollutants derived from petroleum of the anthropised catchment basin. Some bacteria degrade these pollutants to grow in these extreme environments, especially actinobacteria, whose *Nocardia* genus belongs to. Some species of this ubiquitous bacteria are pathogen for frail populations and cause nocardiosis, which would affect about a million people each year in the world.

To date, the degradation ability of a given *Nocardia* species for a pollutant in particular remains unknown. The aim of this work is to assess which pollutants that most stimulate the growth of *Nocardia* pathogen species. This will lead to better understand the relationship between pollutants and infectious risk due to pathogenic *Nocardia* species.

We hypothesize that i) non pathogen species better degrade pollutants than pathogenic ones and ii) environmental strains of pathogenic species better degrade pollutants than clinical ones of the same species.

All the environmental and clinical species are able to use pyrene, fluoranthene and nonylphenol as sole carbon source and diuron as sole nitrogen source. These pollutants are degraded in other metabolism byproducts with enhance development of other environmental bacterial species. Bacteria of nonpathogenic species are most abundant in the sediments, which lower the dissemination risk of the pathogenic ones, and impact human health.

**Keywords:** Actinobacteria, Nocardia, urban sediment, infiltration basin, pollutant

<footnote>*Speaker</footnote>
# Bacterial fungal interactions and their role for the degradation of contaminants

Lukas Yvo Wick*¹

¹Helmholtz Centre for Environmental Research - UFZ (GERMANY) (UFZ) – Permoserstrasse 15
D-04318 Leipzig, Germany

## Abstract

Fungal bacterial interactions (FBI) are highly diverse and contribute to many ecosystem processes. Their emergence in structurally heterogeneous soil structures or in environmental stress scenarios still remains mainly elusive. Here we exemplify FBI by the ecosystem function of contaminant biodegradation in soil. The structural heterogeneity of soil results in the formation of a high variety of soil micro-environments and hotspots of microbial diversity and activity. In heterogeneous environments microbial activity can be considered as a 'logistic' problem, as effective biotransformation will evolve only if water, chemicals and nutrients are sufficiently available to actively degrading microbial communities. In order to cope with heterogeneous environments mycelial soil fungi have developed a unique network-based growth form. Unlike bacteria, hyphae spread efficiently in the soil, penetrate air-water interfaces and cross over air-filled pores. We demonstrate the role of mycelial networks for preferential bacterial colonization of subsurface interfaces and discuss its effects on contaminant degradation. In particular, we show that mycelia (i) act as hotspots for horizontal gene transfer, (ii) shape predator-prey interactions and concomitant compound turnover, and (iii) passively and actively enable the functionality of bacterial ecosystems when stressed by low nutrient availability or at low matric potentials. Given the ubiquity and length of up to 500-1000 m g-1 dry soil of hyphae, we conclude that transport and dispersal processes by mycelia not only play a significant role for the ecosystem service of biodegradation of chemicals but also for nutrient and carbon turnover in soil, and, hence, the functional stability of soil ecosystems.

**Keywords:** bacterial fungal interactions, horizontal gene transfer, bioavailability, water, nutrient availability, contaminant

<footnote>*Speaker</footnote>
# Metal pollution effects on ammonia oxidation

Maria Argudo*¹, Helena Guasch¹, Berta Bonet†², Carmen Espinosa†³, and Frederic Gich§¹

¹Universitat de Girona – Spain

²University of Birmingham – United Kingdom

³Universitat de Vic – Spain

## Abstract

Metals from mining are common chemical stressor pollutants from human activities that cause a high impact in natural communities in the Mediterranean rivers. Key biological processes carried out in fluvial biofilms are affected by environmental stressors. In consequence natural communities can be altered both in diversity and activity. The aims of this study are to determine the toxicity of Zn on growth, photosynthetic efficiency and changes on structure and function of the ammonia oxidizing prokaryotes in fluvial epilithic biofilms along three rivers. Accordingly an experiment was set up based on biofilm colonization on artificial substrate during spring along Ter, a river larger and impacted by human activities and two of its tributaries, Osor a contaminated river by an effluent of a blende ((Zn,Fe)S) and galena (PbS) and a control river (Llémena). The effects of Zn on algae and on the abundance of ammonia oxidizing prokaryotes were determined by using amplitude modulated fluorimetry (PAM) and reverse transcription quantitative PCR (qPCR) for 16S rRNA, 16S AOB structural genes and functional AmoA genes. RNA abundances in the biofilm were higher than DNA abundances. Bacteria are more abundant in polluted sites whereas algal growth and photosynthetic activities are greater in the less polluted ones. In relation to structure and function of ammonia oxidizers prokaryotes, the ammonia oxidizing bacteria (AOB) are more abundant than the ammonia oxidizing archaea (AOA). Moreover AOB relative abundance, decrease because of pollution indicating that ammonia oxidation may also be impaired. Effects of pollution on nitrification activities will also be discussed.

**Keywords:** Ter river, pollution, Zn, algal growth, ammonia oxidizing prokaryotes, reverse transcription quantitative PCR.

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: berta.bonet@udg.edu</footnote>

<footnote>‡Corresponding author: u1917287@campus.udg.edu</footnote>

<footnote>§Corresponding author: frederic.gich@udg.edu</footnote>
# Do plant traits matter in the microbial ecotoxicity of metal nanoparticles in soil?

Amélie Cantarel<fnref n="1" />, Jonathan Gervaix<fnref n="1" />, Jean Martins<fnref n="2" />, and Agnès Richaume*†<fnref n="1" />

## Abstract

The use of nanoparticles (NPs) in many common products led to assess their impact on living organisms and microorganisms in various environments. Several studies showed the deleterious effects of metal nanoparticles (NPs) on soil microbial communities and pointed out the importance of organic matter (OM) content in the ecotoxicity of NPs due to its role as dispersing and stabilizing agent. In this study, we assessed the role of plant root exudation in NPs ecotoxicity in soil towards the microbial communities involved in the nitrogen cycle assuming that plant root exudation reduces NPs ecotoxicity in the rhizosphere due to a higher dissolved OM content compared to bulk soil. The experimental design consisted in planted and unplanted soil microcosms contaminated or not with CuO-NPs (1mg and 100 mg/Kg) often entering in the composition of nano-fertilizers or pesticides. Control microcosms spiked with soluble Cu were included. Since the measurement of root exudation is hardly possible in soil, we compared the effect of two types of wheat (Triticum aestivum) exhibiting contrasted traits reflecting contrasted exudation. Ecotoxicological NPs effects were assessed after 30 and 50 days of incubation in a greenhouse by measuring microbial activities (nitrification and denitrification) and microbial abundances by qPCR targeting function genes in rhizosphere and bulk soil. NPs characterizations (surface charges and hydrodynamic diameter) were realized in solutions of rhizosphere and bulk soils. Dissolved OM was measured in both solutions. The microbial ecotoxicological effects of NPs are discussed in the light of plant traits and NPs characteristics.

**Keywords:** metal nanoparticles/ nitrification/ denitrification/ plant traits/ rhizosphere

<footnote n="1">¹Ecologie microbienne (EM) – Institut national de la recherche agronomique (INRA) : UMR1418, CNRS : UMR5557, Université Claude Bernard - Lyon I (UCBL), Ecole Nationale Vétérinaire de Lyon – 43 Bvd du 11 Novembre 1918, 69622 Villeurbanne cedex, France</footnote>

<footnote n="2">²Institute Geosciences Environment (IGE) – CNRS : UMR5001, Université Grenoble Alpes – Université Grenoble Alpes CS 40 700. F-38058 Grenoble cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: agnes.richaume@univ-lyon1.fr</footnote>
# Mixtures effect of antibiotics on soil nitrification and denitrification - an experimental and modelling approach

Viviane David¹,², Celine Roosevelt-Amsaleg², Fabrice Alliot², Marjolaine Bourdat-Deschamps¹, and Olivier Crouzet*†¹

¹UMR 1402 ECOSYS - Institut National de la Recherche Agronomique - INRA, AgroParisTech, Université Paris-Saclay - 78026 Versailles cedex, France

²Milieux Environnementaux, Transferts et Interactions dans les hydrosystèmes et les Sols (METIS) – École Pratique des Hautes Études [EPHE], CNRS : UMR7619, Université Pierre et Marie Curie (UPMC) - Paris VI – UPMC, Case courrier 105, 4 place Jussieu, 75005 Paris, France

## Abstract

Impacts of antibiotic mixtures on microbial ecosystem functions need to be unraveled to improve the environmental risk assessment. This study aims to characterize and predict the toxicity of antibiotic mixtures on soil nitrification and denitrification processes. Different antibiotics (tetracycline, sulfonamide, macrolide, fluoroquinolone) with distinct mechanisms of action, were tested alone and in mixtures by dose-response approaches on soil substrate-induced nitrification (SIN) and minimum inhibitory concentration of soil denitrifier enrichment (MIC-E). Abundance of ammonia-oxidizers (archeal and bacterial) and denitrifiers (clade I and II of N2O-reducers) were investigated by molecular approach. For nitrification, the experimental results for different antibiotic mixture ratio were further analyzed regarding mathematical modeling interpretation based on the concepts of concentration addition (CA) or independent action (IA).

The SIN bioassay highlighted bacteriostatic effects at sulfonamide or macrolides concentrations lower than observed environmental concentrations. The differences in toxicities of antibiotics individually tested were explained by the sorption coefficient of the various antibiotics. Surprisingly, despite different cellular targets of the tested antibiotics, modelling mixture analyses revealed few differences in toxicity prediction between IA or CA concepts. Denitrification showed a higher tolerance than nitrification to the antibiotics and the lowest MIC-E for tetracycline. For nitrification and denitrification, the mixture toxicity was mainly driven by the more toxic compound.

The archeal/bacterial nitrifiers ratio was positively related to the magnitude of nitrification inhibition; archaea showing invariant abundances while the antibiotic doses increased. The abundances of the denitrifier *nosZ* clade II greatly decreased under any antibiotic exposures unlike the *nosZ* clade I guilds.

**Keywords:** Microbial ecotoxicology, antibiotics, mixture effect, N, cycling microbial activities

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: olivier.crouzet@versailles.inra.fr</footnote>
# Assessment of antibiotic resistance of bacteria isolated from Mugil cephalus of the bay of algiers

Souhila Alouache*<fnref n="1" />,<fnref n="2" />, Nawal Touahir<fnref n="1" />, and Rabah Bakour<fnref n="2" />

## Abstract

The study of antibiotic resistance in aquatic organisms is important because it could indicate the extent of change in aquatic ecosystems due to anthropogenic actions. Our study focused on the bacterial flora of *Mugil cephalus*: a fish that frequents coastal waters and mouths; caught in the bay of algiers between May and October 2015.

The evaluation of the resistance of the total flora to antibiotics revealed high rate of resistance to amoxicillin, mean rate to cefotaxime, ceftazidime, ciprofloxacin and tetracycline and low to imipenem and gentamicin.

The identification of 22 gram negative bacteria resistant to amoxicillin or cefotaxime revealed that strains were assigned to: *E.coli* (7), *K.oxytoca* (3), *E. cloacae* (2), *C. freundii* (2), *A. hyrophila* (2), *E. aerogenes* (1), *K. pneumoniae* (1), *R. ornithinolytica* (1), *R. terrigena* (1), *Moell.wisconsensis* (1) and *P.aeruginosa* (1). The antibiogram results were: AMX (100%), TE (31.8%), PIP (27%), CAZ (27%), NA (27%), FOX (22.7%), CTX (18%), ATM: (18%), CFP (13.6%), SXT (13.6%), TMP (9%), CIP (4.5%), GM (4.5%), C (4.5%) et IMP (0%). The antibiotic resistance profiles of these strains range from 1 to 11 antibiotics. The genotyping analysis by PCR showed the presence of the *blaCTX-M* in *K. pneumoniae* (1), the *blaTEM* in *E.coli* (4), *K. pneumoniae* (1), and *C. freundii* (1) and the *blaSHV* in *K. pneumoniae* (2).

The results suggest that anthropogenic input influences the emergence and dessimination of resistant bacteria in coastal waters and in fish that live there, constituting a health and ecological risk.

**Keywords:** antibiotic resistance, bacteria, Mugil cephalus

<footnote n="1">¹High national school of marine sciences and coastal management, LCVRM laboratory (ENSSMAL) – BP19, campus universitaire, Bois des cars, Algiers, Algeria</footnote>

<footnote n="2">²University of sciences and technology Houari boumedienne, laboratory of cellular and molecular biology (USTHB) – BP32 El Alia - Bab ezzouar, Algeria</footnote>

<footnote>*Speaker</footnote>
# New Finding on the Anti-biofilm Activity of Silk Sericin Against Streptococcus mutans Bacteria Residing within Biofilm

Pornanong Aramwit*†¹ and Nipaporn Bang¹

¹Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice - Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

## Abstract

Sericin is considered as a waste from silk industry and causes pollution when discarded into the environment. Sericin is a water-soluble protein component in silk which, recently, shows various biological activities such as anti-oxidation, anti-inflammation, and anti-aging. In this study, sericin was extracted by 4 different methods including heat, acid, alkali, and urea and the anti-biofilm effects and cytotoxicity of each sericin against *Streptococcus mutans* bacteria residing within biofilm were investigated. *S. mutans* bacteria were cultivated in the presence of various concentrations of sericins to evaluate the anti-biofilm formation using cell density assay (inhibition effect before biofilm formed). On the other hand, various concentrations of sericins were added to the biofilm already formed by *S. mutans* bacteria and the viability of bacteria was assessed by MTT assay (disruption effects after biofilm formed). We found that the urea-extracted sericin at all concentrations (12.5-100 mg/mL) showed the highest potential anti-biofilm activity in terms of both inhibition and disruption effects, comparing to the sericins extracted by heat, acid, and alkali. The heat-extracted and acid-extracted sericin were found to reduce the biofilm formation dose-dependently while the alkali-extracted sericin did not show either inhibition or disruption effect on the bacterial biofilm. Due to strong negative charged of urea-extracted sericin, it may instabilize the bacterial cell wall, leading to membrane disintegration and finally to cell death. This new finding on the anti-biofilm activity of sericin would establish further researches on the mechanism and application of sericin as novel anti-biofilm agent.

**Keywords:** Silk protein, sericin, biofilm, S. mutans

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: aramwit@gmail.com</footnote>
# Infection lithiasis inhibition by citrate-aluminum complex

Aissa Belouatek*†1

¹Laboratoire Structure, Elaboration et Application des Matériaux Moléculaires – Faculté des sciences exactes, Université Abdelhamid Ibn Badis, Mostaganem, 27000, Algeria

## Abstract

The Struvite form during urinary infection with urea-splitting bacteria. The precipitation of the phosphates phase from artificial urine at different initial pH values was the object of our investigation. We chose the classical model for the study of phosphate crystallization without inhibitor and with it, in order to assess the inhibiting capacity of any chemical species used. Two solutions of following composition were mixed. In the absence of inhibitor, the crystallization of phosphates at pH = 6.5, led to the formation of brushite and amorphous carbonated calcium phosphates (ACCP), after 6 hours. The struvite and carbapatite are favorites at pH ≥ 7. The capacity of inhibitor was 100% starting from a concentration of mixture (0.05 mM of citrate, and 0.01 mM of the aluminum ions) at pH = 6.5. On the other hand at pH = 7, the addition of mixture concentration (1mM citrate and 0.01 mM of the ions aluminum) caused the total disappearance of struvite and the brushite. The complex citrate-aluminum acts on the growth and the aggregation of the struvite crystals with various concentrations at pH = 8. The complete disappearance of struvite is obtained starting from a concentration of the mixture (6mM ions citrates and 0.07 mm ions aluminium). In this investigation, complex citrate-aluminum proved to be good inhibitors of microorganism. Their effect increases with solution pH but they are more efficient in less acidic or neutral urine than in alkaline one.

**Keywords:** inhibition, pH, citrate, aluminum, brushite, struvite

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: abelouatek@gmail.com</footnote>
# Effect of lanthanides on soil microbial communities

Charlotte Berthelot*†¹, Asfaw Zegeye¹, and Patrick Billard¹

¹LIEC – Université de Lorraine, CNRS : UMR7360, Université de Lorraine – France

## Abstract

Lanthanides (Ln3+) are a group of metals potentially harmful when concentrated in soils such as in mining and industrial areas. However, due to their low bioavailability in soils, Ln3+ have long been considered as biologically inert at low concentration. Recent studies contradict this view, showing that Ln3+ may act as beneficial cofactor in plants and certain bacteria. In the present study, we investigated the promotion role of Ln3+ on soil bacterial communities.

The effect of La3+ (Lanthanum - a model lanthanide) was examined in three soils exhibiting diverse physico-chemical characteristics. Soil spiking was performed to reach concentrations of 50 and 500mg/kg of La3+. Soil parameters (e.g. pH, La3+ availability), enzyme activities related to biogeochemical cycling of C, N and P and bacterial communities (abundance and diversity) were evaluated. Application of La3+ did not alter the soil pH, whereas bioavailability of La3+ was largely dependent on soil properties. Nevertheless, the presence of La3+ at low concentration (50mg/kg) significantly increased microbial respiration (from 129% to 135% of control). Likewise, laccase and cellulase activities were stimulated by the 50 mg/kg treatment whereas β-glucosidase and leucine-amino peptidase were inhibited by 500mg/kg of soil

The impact of La3+ on carbon substrate utilization by microbial communities was studied using NPP method and Biolog® microplates. Consumption of organic acids (e.g. citrate, gluconate) or alcohols (e.g. ethanol) was promoted as a result of La3+ application. Overall, our study provides a first insight into the beneficial effect of lanthanides for bacterial metabolism in soil environments.

**Keywords:** lanthanides, bacterial communities, Lanthanum

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: charlotte.berthelot@univ-lorraine.fr</footnote>
Developing a transcriptomic approach dedicated to
autotrophs in periphytic biofilms

Chloe Bonnineau*<fnref n="1" />, Floriane Larras<fnref n="2" />, Stefan Scholz<fnref n="2" />, Stéphane Pesce<fnref n="1" />, Helena Guasch<fnref n="3" />,
and Mechthild Schmitt-Jansen<fnref n="2" />

Abstract

In the last decade, omics tools dedicated to microbial communities have been developed to characterize the functional groups of those communities and better understand microbial response to contamination. Most of those tools focused on heterotrophic and fungal communities while omics application dedicated to microalgae and diatoms are mostly restrained to diversity assessment (e.g. based on 18S rRNA or rbcL genes). In this study, the possibility of studying gene expression in autotrophic river biofilm communities was explored and we present the proof of concept for a Functional Gene Array (FGA), based on consensus sequences from genes of key physiological processes. Indeed, microarrays offer a comprehensive tool for system-wide analysis of community functioning based on the simultaneous detection of the activity of thousands of genes. The FGA designed in this study include 83 functional genes chosen to reflect several essential biochemical pathways and specific stress response pathways of microalgae. Probes of these genes were designed from consensus sequences from up to 6 microalgal species (diatoms and chlorophytes). Furthermore, species-specific probes were included resulting in 1554 unique oligonucleotide probes for 83 different genes. RNA extracted from *Chlamydomonas reinhardtii*, *Scenedesmus vacuolatus* and multi-species biofilms was then used to test different hybridisation conditions. Signal intensity was affected by sequence divergence but results showed that a hybridisation temperature of 55°C allowed a good compromise between cross-hybridisation and specificity. Due to the reduced number of probes and available sequence information the designed FGAs is at present particularly useful for ecotoxicological laboratory experiments performed with biofilm communities.

**Keywords:** microarray, microalgae, gene expression, community

<footnote n="1">¹Irstea UR Milieux Aquatiques, Ecologie et Pollutions (UR MALY) – Irstea – 5 rue de la Doua BP 32108 F-69616 Villeurbanne Cedex, France</footnote>

<footnote n="2">²Helmholtz Centre for Environmental Research - UFZ (GERMANY) (UFZ) – Leipzig, Germany</footnote>

<footnote n="3">³GRECO, INstitute of Aquatic Ecology, University of Girona – Girona, Spain</footnote>

<footnote>*Speaker</footnote>
# Importance of the sediment microbial diversity for degradation of pharmaceuticals in rivers

Claudia Coll Mora*†<fnref n="1" />, Zhe Li<fnref n="1" />, Raven Bier<fnref n="2" />, Silke Langenheder<fnref n="2" />, and Anna Sobek<fnref n="1" />

## Abstract

Degradation of pharmaceuticals in aquatic systems is impacted by environmental factors. For instance, the attenuation rate of acetaminophen differed by a factor of 40 between rivers Gr'undlach-Germany and Fyris-Sweden [1]. Although data is limited, previous studies show biodiversity may be linked to the degradation potential of pollutants [2]. Therefore, we performed an experiment based on OECD TG308 to assess the influence of bacterial diversity on pharmaceutical degradation.

Water and sediment were collected from rivers Fyris and Gr'undlach, before and after the discharge of waste water treatment plants (WWTP). The sediments were acclimated with synthetic river water, then spiked with 11 pharmaceuticals at two concentration levels (200µg/L and 2mg/L). The compounds represent a wide range of biodegradation rates: acetaminophen, acesulfameK, caffeine, carbamazepine, diclofenac, furosemide, ibuprofen, metformin, oxazepam, tramadol and venlafaxine. The 40-day incubation was performed in the dark at 16°C and enabling an oxygen gradient in the sediment. The water phase was sampled at 10 time points while sediment samples were taken before spiking and at the end of incubation. We are measuring the pharmaceutical concentrations in all samples, and using Illumina-sequencing of bacterial 16SrRNA to analyze the active bacterial community. Subsequently, Rényi's entropy profiles will be calculated.

Our results will show whether microbial communities up- and downstream of a WWTP differ in their potential to degrade pharmaceuticals and whether differences in the composition and diversity can explain the variation in the degradation rates between sampling sites and rivers.
- [1] Li et al. 2016. doi:10.1021/acs.est.5b06327; [2] Johnson et al., 2015. doi:10.1128/AEM.03286-14

**Keywords:** pharmaceutical degradation, microbial diversity, aquatic systems, OECD308

<footnote n="1">¹Department of Environmental Science and Analytical Chemistry, Stockholm University (ACES-SU) – Svante Arrhenius väg 8, SE-11418 Stockholm, Sweden</footnote>

<footnote n="2">²Evolutionary Biology Centre (EBC) – Evolutionary Biology Centre, EBC Norbyvägen 14-18 752 36 Uppsala, Sweden</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: claudia.coll@aces.su.se</footnote>
Effect of nanoceria biotransformation in activated sludge on the microbiota associated to canola roots

Blanche Collin*<fnref n="1" />, Mohamed Barakat<fnref n="2" />, Philippe Ortet<fnref n="2" />, Wafa Achouak<fnref n="2" />, Melanie Auffan<fnref n="1" />, Emmanuel Doelsch<fnref n="3" />, and Catherine Santaella<fnref n="2" />

Abstract

Recent interest in the environmental fate and effects of CeO2 nanomaterials (NMs) has stemmed from its expanded use for a variety of applications (fuel additives, catalytic converters, coating...). The majority of these NMs will end up in wastewater treatment plants (WWTP) where they will partition to sewage sludge during wastewater treatment, and ultimately re-enter the environment through the application of biosolids to agricultural soils. Thus, soil may serve as a primary sink for NMs accumulation in the environment, in which NMs may enter food webs or cause direct toxicity to plants, microbial communities, or other soil organisms. This project aims to study the impact of CeO2 NMs biotransformation on a soil-plant-microbe system using realistic exposure modes. Pristine CeO2-NMs were first aged in a laboratory-scale activated sludge reactor during 5 weeks. The biosolid enriched NMs was then amended to an agricultural soil at environmentally relevant concentration: 1 mg.kg-1 CeO2. Four treatments were performed: control soil, soil amended with 1 mg.kg-1 pristine CeO2-NMs, control biosolid without NMs, biosolid enriched NMs. The selected plant was canola (*Brassica napus*), an oil-producing plant. Bulk Ce L3-edge X-ray absorption spectroscopy was performed in the biosolid before culture in order to evaluate the NMs transformation in the reactor. After the culture, elemental concentrations and distributions were measured in the plant parts by ICP-AES, and laser ablation ICP-MS. Root and rhizosphere bacterial community structure was characterized by sequencing of 16S rRNA gene (Illumina MiSeq) in order to understand the impact of the biotransformation of CeO2-NMs on the microbiota.

**Keywords:** soil, rhizosphere, plant, nanoparticles, bacterial community structure

<footnote n="1">¹Centre européen de recherche et d'enseignement de géosciences de l'environnement (CEREGE) – Institut de Recherche pour le Développement : UMR_D161, *AixMarseilleUniversité* :
*UM34, CollègedeFrance, CentreNationaldelaRechercheScientifique* :
*UMR7330 – AvenueLouisPhilibert – BP80 – 13545Aix – en – Provence, France*</footnote>

<footnote n="2">²Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements extrêmes (LEMIRE) – CNRS : UMR7265, CEA, Aix-Marseille Université - AMU – St-Paul-lez-Durance, France</footnote>

<footnote n="3">³UPR Recyclage et risque (CIRAD) – CIRAD : UPR78 – Aix-en-Provence, Montpellier, Saint-Denis de la Réunion, France</footnote>

<footnote>*Speaker</footnote>
Environmentally relevant copper concentrations
affect microbial composition and community
functions in marine periphyton biofilms

Natàlia Corcoll*<fnref n="1" />, Jianghua Yang<fnref n="2" />, Hadrien Kronenberger<fnref n="1" />, Thomas Backhaus<fnref n="1" />, Xiaowei Zhang<fnref n="2" />, and K. Martin Eriksson<fnref n="3" />

Abstract

Copper pollution is common in coastal areas. In particular, the use of copper-based antifouling paints on ship hulls elevates copper concentrations in these environments. This study assesses the effects of dissolved copper on community structure and function of marine periphyton biofilms. Microbial diversity and community structure was studied by 16S and 18S amplicon sequencing, targeting prokaryotic and eukaryotic organisms, respectively. Community function was studied as impacts on algal biomass, photosynthetic pigment profiles and primary production. Additionally, we studied Pollution-Induced Community Tolerance (PICT) using photosynthesis as the endpoint. Periphyton was exposed for 18 days to five copper concentrations, between 0.01 and 10 µM, in a semi-static test. The amplicon sequencing yielded 7.1 and 5.7 million high quality 16S and 18S reads, respectively. The average numbers of 16S and 18S Operational Taxonomic Units among the samples were 9405 and 1242, respectively. Analysis of Unifrac distances showed that copper significantly changed the eukaryotic community structure at concentrations as low as 0.01 µM. The prokaryotic community structure was changed at slightly higher concentrations (0.06 µM). A total of 23 taxa, including species within *Proteobacteria*, *Bacteroidetes*, *Stramenopiles* and *Hacrobia* classes, were identified as particularly sensitive to copper. Algal biomass, photosynthetic pigments profiles and primary production, were reduced at Cu concentrations of 0.06 µM and higher. PICT measurements confirmed that copper induced community tolerance in exposed communities. Taken together, these findings indicate that negative impacts from copper might be common in coastal ecosystems.

**Keywords:** amplicon sequencing, 16S rRNA, 18S rRNA, biofilms, copper toxicity

<footnote n="1">¹Dept. Biological and Environmental Sciences, University of Gothenburg – Sweden</footnote>

<footnote n="2">²State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University – China</footnote>

<footnote n="3">³Dept. Mechanics and Maritime Sciences, Chalmers University of Technology – Sweden</footnote>

<footnote>*Speaker</footnote>
# Toxicity of nZVI used for nanoremediation towards natural bacterial community from groundwater

Marc Crampon*¹, Catherine Joulian¹, Patrick Ollivier¹, Louis Jolly, and Jennifer Hellal¹

¹BRGM – Bureau de Recherches Géologiques et Minières (BRGM) – 3 avenue Claude Guillemin 45100 Orléans, France

## Abstract

Remediation techniques for toxic/persistent contaminants in groundwater are often technologically difficult. Nanoparticles (NP) such as nZVI (Zero-Valent Iron) applicable as *in-situ* reduction or oxidation agents for groundwater treatment give promising results. However, they may also represent an additional contamination. Our study aims to assess the impact of nZVI on the denitrifying activity, the abundance and the structure of a planktonic nitrate-reducing bacterial community.

Pre-culture of the denitrifying bacterial community was carried out from groundwater samples. It was then allocated in reactors containing substrate (sodium acetate), nitrates and a range of nZVI concentrations (from 0 to 300 mg Fe.L⁻¹). Physical (pH, redox potential), chemical (NO₃⁻ concentrations) and biological (DNA, RNA) parameters were monitored during one week. The size distribution of nZVI and the mortality of bacteria (Live&Dead observations) were also evaluated. Then the bacterial cultures were transplanted in fresh substrates without nZVI to assess the resilience of the bacterial community.

Denitrification activity was stopped in the presence of nZVI at concentrations higher than 30 mg.L⁻¹, demonstrating the toxic effect of nZVI. Live&Dead observations revealed that the viability of bacteria was lower for lower nZVI concentrations. The presence of NP aggregates was higher for higher nZVI concentrations. They could then result in a lower toxicity for bacteria, even if nZVI concentration is high. DNA and RNA analyses allowed us to observe the impact of nZVI on the activity (*narG* gene), abundance and structure of bacterial community. Finally, resilience was worst for concentrations ranging from 50 to 200 mg.L⁻¹.

**Keywords:** Nanoremediation/nZVI/Toxicity/Bacteria/Groundwater

<footnote>*Speaker</footnote>
# The impacts of three herbicides on the fatty acids of the freshwater diatom *Gomphonema gracile*

Floriane Demailly*<fnref n="1" />, Marina Le Guedard<fnref n="2" />, Patrice Gonzalez<fnref n="3" />, Jean-Jacques Bessoule<fnref n="4" />, Nicolas Mazzella<fnref n="1" />, François Delmas<fnref n="1" />, Agnès Feurtet-Mazel<fnref n="3" />, and Soizic Morin<fnref n="1" />

## Abstract

For several years, the intensive use of pesticides caused many problems to the environment, making pesticides major pollutants of aquatic ecosystems (Aydinalp & Porca, 2004). Among biological indicators, biomarkers must be developed in view of their capacity to reveal early toxic effects on organisms of the environment. The aim of this study is to compare the specificities/sensitivities of the fatty acid analysis (composition of saturated, unsaturated and polyunsaturated fatty acids determined by gas chromatography coupled with flame ionization detector) of diatoms, compared to "classical" descriptors analysis (inhibition of photosynthesis, growth, transcriptomic analysis). A model freshwater diatom (*Gomphonema gracile*) was exposed to three herbicides singly, with three different cellular targets, at environmentally relevant and higher concentrations (diuron and S-metolachlor, C1= 1 µg/L and C2= 10 µg/L; glyphosate, C1= 5 µg/L and C2= 50 µg/L). The diatom cultures were incubated during exponential growth at controlled temperature and light intensity of 17oC and 67 µmol.m-2.s-1, respectively. After a 1-week exposure, fatty acids compositions of diatoms were determined in parallel with "classical" descriptors. While no or only slight effects of the pesticides were found with the "classical" descriptors, some fatty acid profiles seemed to be specific of pesticide contamination but in-depth fatty acid analyses are still under progress. This study provides a basis for further use of diatom's fatty acid composition as biomarker of toxic contamination in fresh waters. Aydinalp, C., & Porca, M. (2004). The effects of pesticides in water resource. Journal Central European Agriculture. 5 (1), 5-12.

**Keywords:** Microbial ecotoxicology, fatty acids, pesticides, freshwater diatom, *Gomphonema gracile*

<footnote n="1">¹UR EABX – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA (FRANCE) – 50 avenue de Verdun, 33612 Cestas cedex, France</footnote>

<footnote n="2">²LEB Aquitaine Tansfert – LEB Aquitaine Transfert-ADERA – Bâtiment A3, INRA Bordeaux Aquitaine, 71 avenue Edouard Bourlaux, CS 20032, 33140 Villenave d'Ornon, France, France</footnote>

<footnote n="3">³EPOC, UMR CNRS 5805, Station Marine d'Arcachon – Université de Bordeaux (Bordeaux, France) – Place du Docteur Bertrand Peyneau, 33120 Arcachon Cedex, France</footnote>

<footnote n="4">⁴Laboratoire de Biogenèse Membranaire, UMR CNRS 5200 – Université de Bordeaux (Bordeaux, France) – Bâtiment A3, INRA Bordeaux Aquitaine, 71 avenue Edouard Bourlaux, CS 20032, 33140 Villenave d'Ornon, France</footnote>

<footnote>*Speaker</footnote>
# Soil particles alter the metabolic profile of Pseudomonas putida grown in the presence of phenanthrene: A vibrational spectroscopy approach

Andrea Fanesi*¹, Asfaw Zegeye¹, Christian Mustin¹, and Aurélie Cebron†¹

¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360 – LIEC, Faculté des Sciences et Technologies, BP 70239, 54506 Vandoeuvre-lès-Nancy, France

## Abstract

In soil, the biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) is strongly influenced by the presence of organic matter and mineral particles. Information regarding how the metabolic profile (i.e biochemical composition) of bacteria is affected by carbon source (simple substrates or PAH) in the presence of soil particles (SPs) is scarce. However, it might be of paramount importance for understanding the bioremediation efficiency of polluted sites.

We hypothesized that properties of SPs specifically alter both the micro-niches of bacteria and the availability of the carbon source, affecting in turn PAHs catabolism and cell metabolic profile. To test this hypothesis, the soil dweller *Pseudomonas Putida* was grown in the absence and presence of different SPs (sand, clay and humic acids) using either phenanthrene or glucose as the sole carbon and energy source. The cell metabolic profiles were identified and differentiated using a combination of vibrational spectroscopy techniques (fast and without sample preparation), such as FT-IR and FT-Raman spectroscopy, and chemometric analysis (PLS-DA).

We showed that the metabolic profile of *P. putida*, (i.e. Raman fingerprints) is influenced by the type of SPs present in the medium, with clay and humic acid affecting it the most. The changes of macromolecules regarded mainly proteins, lipids and nucleic acids. Moreover, in the presence of clay and humic acid a more pronounced discrimination between cells grown on glucose or on phenanthrene was observed, suggesting that specific SPs affect the way cells metabolize carbon. Our results provide new information linking PAHs degradation, bacteria physiology and cell surrounding media.

**Keywords:** PAHs bioremediation, Raman spectroscopy, bacteria, metabolic profiles, soil particles

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: aurelie.cebron@univ-lorraine.fr</footnote>
# Availability of mercury to freshwater biofilms: from the gene to the community composition

Severine Le Faucheur¹, Perrine Dranguet¹, Claudia Cosio¹, and Vera Slaveykova*†¹

¹University of Geneva – Department F.-A. Forel for Environmental and Aquatic Sciences, Earth and Environment Sciences, Faculty of Sciences, University of Geneva, Uni Carl Vogt, 66 Bvd. Carl Vogt, CH-1211 Geneva, Switzerland

## Abstract

Bioavailability is a key concept allowing to link the changes in the trace metal concentrations and speciation with the intensity of the induced biological effects. However, the link between mercury speciation, bioaccumulation and effects to biofilms is overlooked. This study focusses on the availability of mercury in freshwater biofilms. The amounts of IHg and MeHg accumulated in biofilms were determined. The changes in bacterial, microalgal and fungal communities were followed simultaneously by amplicon sequencing. Biofilms grown in-situ in the Olt River (Romania) reservoirs impacted by Hg-rich effluents from a chlor-alkali platform as well as biofilms exposed to Hg in microcosm were compared. The results revealed a significant correlations between the IHg accumulation in biofilm and the fraction of dissolved Hg non-complexed by natural organic matter. A shift in algal, bacterial and fungal community composition of the biofilms from Olt River and microcosm was observed. However the principal component analysis revealed that in the in-situ grown biofilm in addition to Hg, other contaminants present in the waste water of chlor-alkali production plant, i.e. Na+, Ca2+ and Cl- as well as some water quality variables, i.e. NO3-, SO42-, DOC and Zn contribute to the community composition alteration. Nevertheless, an increase in the presence of Hg resistance gene (*merA*) in both in-situ and microcosme exposed biofilms with the increase of the accumulated Hg was found. The outcomes of the results contribute to the development of biofilm-based indicators in support to efforts of mercury monitoring programs in aquatic systems.

**Keywords:** mercury, bioavailability, merA, community composition

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: Vera.Slaveykova@unige.ch</footnote>
# Global metabolomic characterizations of *Microcystis* spp. highlights clonal diversity in natural bloom-forming populations and expands metabolite structural diversity

Séverine Le Manach¹, Charlotte Duval¹, Arul Marie¹, Cécile Bernard¹, and Benjamin Marie*†¹

¹Molécules de Communication et Adaptation des Microorganismes (MCAM) – Muséum National d'Histoire Naturelle (MNHN), CNRS : UMR7245, Sorbonne Universités, Sorbonne Universités – France

## Abstract

Cyanobacteria are photosynthetic prokaryotes that are able to synthetize a wild range of secondary metabolites, with noticeable bioactivity (comprising toxicity and anti-bacterial activity) according to the ribosomal and non-ribosomal processes. *Microcystis* represents one of the most common cyanobacteria taxa constituting intensive blooms that arise in freshwater ecosystems worldwide. With the aim of contributing to better understand the variations in bio-actif metabolite production, which are potentially harmful to other aquatic macro- and micro-organisms, between clones of the same cyanobacterial blooms, we investigate the diversity of several *Microcystis* strains isolated from different freshwater bloom-forming populations from various geographical area. Twenty-four strains of *Microcystis* from the Paris' Muséum Collection of cyanobacteria were compared by an integrated approach combining genotyping and metabolites chemotyping. These characterizations comprise analyses on morphology, Internal Transcript Spacer, microcystin synthesis gene, and global secondary metabolite contents. Metabolomics shotgun analyses by mass spectrometry reveal clear discriminant profiles between strains collected from identical or different localities. A global cluster analysis indicates that microcystin synthesis gene presence explains more of the metabolite diversity distribution, than the species or the sample localities parameters do. A global network generated from MS/MS fractionation patterns of the various metabolites detected in all strains performed with GNPS tool indicates that these *Microcystis* strains produce a wide set of chemically diverse metabolites, comprising only few known potent toxins or protease inhibitors, together with a large set of unknown molecules that still remain to be investigated and characterized at their structure as well as at their potential bioactivity/toxicity/antibacterial levels.

**Keywords:** Cyanobacteria, cyanotoxins, ecotoxicology, secondary metabolites, biodiversity

<footnote>*Speaker</footnote>

<footnote>†Corresponding author:</footnote>
Structural and functional response of river sediment
microbial communities to environmental
concentrations of copper and arsenic, alone or in
mixture

Ayanleh Mahamoud Ahmed*†¹,², Emilie Lyautey‡², Aymeric Dabrin¹, Chloé Bonnineau¹, Bernard Motte¹, Rosy Christophe¹, Josiane Gahou, and Stéphane Pesce§¹

¹UR MALY – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture – 5 rue de la Doua, BP32108, 69616 Villeurbanne, France

²Centre Alpin de Recherche sur les Réseaux Trophiques et Ecosystèmes Limniques (CARRTEL) – Institut National de la Recherche Agronomique : UMR0042, Université Savoie Mont Blanc : UMRINRA / USMB – Station d'Hydrobiologie Lacustre, Le Bourget-du-Lac 73376, France

Abstract

While many metals are known to accumulate in sediments, knowledge about the result-
ing ecotoxicological effects on sediment microbial communities is scarce. The present study
aimed at evaluating the structural and functional impact of chronic exposure to environ-
mental concentrations of copper (Cu) and arsenic (As), alone or mixed together, on river
sediment microbial communities. Natural uncontaminated surface sediments collected in a
French River (Ain) were spiked with As and Cu at a nominal concentration of 40 mg/kg and
then were exposed for 21 days in laboratory channels with overlying waters. The response of
heterotrophic microbial communities to metals was evaluated both in terms of genetic struc-
ture (using ARISA analysis) and functional potential (using exo-enzymatic, metabolic and
qPCR analyses). A pollution induced community tolerance (PICT) approach was also per-
formed to assess if the exposure led to an increase in the capacity of microbial communities
to tolerate metals. Our results showed rapid and marked effects of Cu alone on the structure
and the functions of the exposed communities. Chronic Cu exposure also induced an increase
in community tolerance to Cu, as observed by PICT measurement using beta-glucosidase
activity. In contrast, the effects of As were mostly undetectable. Under mixture exposure
(Cu+As), the effects were similar or higher than those provoked by Cu alone, depending
on the measured parameter. Altogether those findings reveal that metals accumulation in
sediments can impact exposed microbial communities thus affecting their functional role in
aquatic ecosystems.

**Keywords:** Benthic communities, metals, microbial ecotoxicology, PICT, mixture

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: ayanleh.mahamoud-ahmed@univ-smb.fr</footnote>

<footnote>‡Corresponding author: emilie.lyautey@univ-smb.fr</footnote>

<footnote>§Corresponding author: stephane.pesce@irstea.fr</footnote>
Natural lake sediment microbial community response
to exposure to environmental PCB concentrations in
microcosms

Ayanleh Mahamoud Ahmed*†1,2, Stéphane Pesce‡2, Emmanuel Naffrechoux3, Nathalie Cottin3, Chloé Bonnineau2, Bernadette Volat2, and Emilie Lyautey§1

Abstract

Sediments act as receptor for contaminants that can accumulate over time thus reaching high concentrations. The main objective of this work was to evaluate the impact of chronic exposure to environmental concentrations of PCB on microbial communities from lake sedi- ments. Sediment cores (n = 52) were collected in Lake Annecy (PCB concentrations < 0.9 ng dw-1 Σ7PCBi) and exposed to 3 treatments: a reference without PCB contamination, a medium contamination (430 ng dw-1 Σ7PCBi) and a high contamination (4300 ng dw-1 Σ7PCBi). Sediments were contaminated using a commercial mixture (Aroclor 1254) and were incubated during 14 days at 6°C with oxygenated overlying water. The resulting effects on communities from depth 0-2 cm, 4-6 cm and 8-10 cm were evaluated independently by assessing microbial enzymatic activities, metabolic potentials, gene abundances and commu- nity structure. While PCB exposure modulated bacterial community structure in microbial communities from 0-2 cm depth, PCB exposure did not affect the other descriptors. Micro- bial communities from 4 to 10 cm depth were not affected by PCB exposure. Our results indicate a limited impact of PCBs on exposed microbial communities suggesting that such exposure would probably not modify their function in lake ecosystems. A low and hetero- geneous contamination of the sediment cores could also explain these results. The present work also pointed out methodological challenges to assess PCB ecotoxicological effects on intact sediment columns. If such approaches are necessary to remain representative of the ecosystem, it still raises questions about the contamination procedure and the bioavailability of the contaminants.

**Keywords:** Benthic communities, persistent organic pollutant, microbial ecotoxicology

<footnote n="1">¹Centre Alpin de Recherche sur les Réseaux Trophiques et Ecosystèmes Limniques (CARRTEL) – Institut National de la Recherche Agronomique : UMR0042, Université Savoie Mont Blanc – Station d’Hydrobiologie Lacustre, Le Bourget-du-Lac, 73376, France</footnote>

<footnote n="2">²Milieux aquatiques, écologie et pollutions (UR MALY) – Irstea – 5 rue de la Doua - BP 32108, F-69616 Villeurbanne, France</footnote>

<footnote n="3">³Laboratoire de Chimie Moléculaire et Environnement (LCME) – Université Savoie Mont Blanc, Université Grenoble Alpes – Campus Scientifique de Savoie-Technolac Le Bourget du Lac Cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: ayanleh.mahamoud-ahmed@univ-smb.fr</footnote>

<footnote>‡Corresponding author: stephane.pesce@irstea.fr</footnote>

<footnote>§Corresponding author: emilie.lyautey@univ-smb.fr</footnote>
# Ecotoxicological impact of leptospermone, a beta-triketone bioherbicide, on fungal community of two arable soils.

Clarisse Mallet*<fnref n="1" />, Sana Romdhane<fnref n="2" />, Jérémie Beguet<fnref n="3" />, Fabrice Martin-Laurent<fnref n="3" />, Christophe Calvayrac<fnref n="2" />, Cédric Bertrand<fnref n="4" />, and Lise Barthelmebs<fnref n="2" />

## Abstract

Ecotoxicological impact of leptospermone, a beta-triketone bioherbicide, was investigated on the abundance and diversity of fungal community which supports important soil ecological functions (i.e. decomposition of organic matter and nutrients recycling) but which is less studied than bacterial community.

A microcosm experiment was carried out on two arable soils (Perpignan and Saint Jean de Fos, France), differing in their physicochemical properties and in history treatment with synthetic beta-triketones. Soil microcosms were treated with 1x or 10x fold recommended dose (FRD) of leptospermone or not (control) and incubated in the dark for 45 days under controlled conditions. Leptospermone dissipation was monitored all along the experiment. Soil DNA was extracted from samples collected at 0, 4 and 45 days and used as template to quantify the abundance of fungi by qPCR targeting 18S rRNA genes and to estimate the diversity of fungal community by pyrosequencing of internal transcribed spacer (ITS) region 2 of the fungal rRNA.

Within the 45 days of incubation, the leptospermone disappeared almost entirely from one of the two soils (i.e., DT50 < 10 days), while 25% remained in the other. In response to leptospermone exposure, the fungal community was significantly modified as compared to control. For both soils, shifts in the fungal diversity was observed in soil microcosms treated with 1x and/or 10x FRD. However, fungal diversity was found to be resilient when leptospermone was dissipated.

**Keywords:** Soil, fungal community, bioherbicide, beta, triketone

<footnote n="1">¹Microorganismes : génome et environnement (LMGE) – Université d'Auvergne - Clermont-Ferrand I, CNRS : UMR6023, Université Blaise Pascal - Clermont-Ferrand II – Université Blaise Pascal, Campus des Cézeaux, 24, avenue des Landais BP 80026 63 170 AUBIERE, France</footnote>

<footnote n="2">²Biocapteurs - Analyse- Environnement (BAE) – Université de Perignan Via Domitia – Université de Perpignan Via Domitia, 52 Av Paul Alduy, 66860 Perpignan cedex, France</footnote>

<footnote n="3">³INRA (UMR Agroécologie) – Institut national de la recherche agronomique (INRA) : UMR1347 – INRA UMR 1347 Agroécologie 17 rue sully BP 86510 21065 Dijon Cedex, France</footnote>

<footnote n="4">⁴CRIOBE USR3278, Université de Perpignan Via Domitia – Univ de Perpignan Via Domitia – 58 Avenue Paul Alduy 66860 Perpignan Cedex, France</footnote>

<footnote>*Speaker</footnote>
# Assessing biofilm pulse exposure to PSII inhibitors using time-response curves

Soizic Morin*¹, Betty Chaumet¹, and Nicolas Mazzella¹

¹Laboratoire Ecosystèmes aquatiques et changements globaux (UR EABX) – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA – 50 avenue de Verdun, 33612 Cestas cedex, France

## Abstract

Diuron-induced photosynthesis inhibition of freshwater biofilms and subsequent EC50s (half maximal effective concentrations) are determined using a 3-hour exposure in average. However, given the known rapid action of diuron, earlier responses can be expected. In this study, we performed different time-response experiments to confirm the short time lag for diuron to reach its cellular target, and to assess whether time-response curves (i.e. photosynthesis inhibition as a function of time, when diuron exposure is kept constant) can improve the assessment of short-time (pulse) pre-exposure to diuron.

Our results demonstrated that:

i) diuron reaches its cellular target in a very short time-span (

ii) the delay required to reach a 50% photosynthesis inhibition is dependent on test exposure concentrations, and

iii) that time-response curves may be useful to assess previous short time exposure.

In particular, we showed that exposure to diuron leads to maximal achievable inhibition within a minute (exposure to diuron-EC100, i.e. > 100µg.L-1) to 7:20 minutes (exposure to EC25).

We were also able to demonstrate previous pulsed (3 hours) exposure to increasing concentrations of diuron (0, 5 and 50µg.L-1) using time-responses curves, providing a new, easy-to-implement, method to assess short-term exposure to PSII inhibitors based on community photosynthesis.

**Keywords:** Biofilm, Freshwater, Diuron, Dose, response curves, Time, response curves

<footnote>*Speaker</footnote>
# Towards easy-handling tools to assess functional effects of contaminants on natural microbial and invertebrate sediment communities

Stéphane Pesce*†<fnref n="1" />, Sophie Campiche<fnref n="2" />, Ayanleh Mahamoud Ahmed<fnref n="1" />,<fnref n="3" />, Carmen Casado-Martinez<fnref n="2" />, Chloé Bonnineau<fnref n="1" />, Aymeric Dabrin<fnref n="1" />, Emilie Lyautey<fnref n="3" />, and Benoit J.d. Ferrari<fnref n="2" />

## Abstract

Sediments play a critical role in biogeochemical cycling and in the maintenance of the biodiversity. They are natural sinks for many contaminants which can accumulate over time and impact exposed communities, thereby disturbing the functioning of aquatic ecosystems. However, knowledge about the functional effects of contaminants on benthic communities is scarce and there is a need of ecosystem functioning indicators of ecotoxicological impacts. In the present microcosm study, we assessed the individual and combined effects of Cu and As (40 mg kg dw-1 each, 21 days of exposure) on the capacity of natural micro- and macro-organism communities from an uncontaminated river sediment to consume and decompose particulate organic matter using the bait lamina method (ISO 18311) as well as artificial tablets consisting of cellulose, bran flakes and active coal embedded in an agar matrix. The sediment toxicity was also evaluated using the standardized ostracod toxicity test (ISO 14371:2012). The two tested substrates (i.e. bait lamina and artificial tablets) showed similar results with low effects of As on feeding activity and organic matter breakdown whereas Cu demonstrated a strongest functional effect. When the two metals were combined, a total functional inhibition was observed, whatever the kind of substrate. The ostracod toxicity test also showed high toxicity of Cu-spiked and mixture-spiked sediments and low toxicity of As-spiked sediments. Our results highlight the relevance of artificial organic matter substrates to assess the functional effects of contaminants on sediment microbial and invertebrate communities, opening new perspectives to assess the functional integrity of contaminated sediments.

**Keywords:** Bait Lamina, Functional ecotoxicology, Benthic communities, Mixture, Metals

<footnote n="1">¹UR Milieux Aquatiques, Ecologie et Pollutions (UR MALY) – Irstea – 5 rue de la Doua BP 32108 F-69616 Villeurbanne Cedex, France</footnote>

<footnote n="2">²Swiss Centre for Applied Ecotoxicology (Eawag-EPFL) – EPFL-ENAC-IIE-GE, Station 2, CH-1015 Lausanne, Switzerland</footnote>

<footnote n="3">³Centre Alpin de Recherche sur les Réseaux Trophiques et Ecosystèmes Limniques (CARRTEL) – Institut National de la Recherche Agronomique : UMR0042, Université Savoie Mont Blanc : UMRINRA / USMB – Station d'Hydrobiologie Lacustre, Le Bourget-du-Lac 73376, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: stephane.pesce@irstea.fr</footnote>
Amplicon pyrosequencing of bacterial and fungal
community structure in Norwegian agricultural
sandy loam soil - assessment of chemical mixture
effects and implications for soil and plant health

Marianne Stenrød*¹ and Erik Lysøe¹

¹Norwegian Institute of Bioeconomy Research (NIBIO) – Høgskoleveien 7, NO-1433 Aas, Norway

Abstract

The main objective of this study was to assess the effects of the single chemicals and mixtures of the fungicide picoxystrobin and the chemical 4-n-nonylphenol frequently occurring in sewage sludge, on soil microbial diversity in an agricultural sandy loam soil. The choice of compounds was based on their individual occurrence and effects in the environment, expected potential for co-occurrence in the field, and expected independent effect mechanisms due to differences in mode of action. Effects of the chemicals were assessed by amplicon pyrosequencing of the bacterial and fungal microbiota on soil samples at 0, 7, 28 and 70 days after chemicals treatment (DAT).

The soil bacterial community was affected by chemicals treatment in a way causing a tran-
sient increase in abundance of *Proteobacteria* and decrease in *Acidobacteria* that was reduced
through the 70 d incubation period back to a level comparable to the original soil sample. For
the fungal community the most apparent effect was found for treatment with the fungicide
picoxystrobin at 7 DAT, when *Sordariomycetes* were increased in abundance. The results
for the picoxystrobin treatment indicated a slower or lack of recovery, with comparable a-
diversity levels at 7 and 70 DAT.

Identification of the main differentially abundant OTUs further indicated effects on abundance of plant pathogenic fungi, and bacteria and fungi of importance for decomposition and nutrient cycling, underlining the need for in-depth studies of the effects of single chemicals and mixtures to assess the potential effects of agrochemicals on soil quality and function.

**Keywords:** pesticides, amplicon pyrosequencing, soil quality

<footnote>*Speaker</footnote>
# Examining the impact of plastic leachates on key marine photosynthetic bacteria

Indrani Sarker¹, Verena Schrameyer¹, Liam Elbourne¹, Ian Paulsen¹, and Sasha Tetu*†¹

¹Chemistry and Biomolecular Sciences – Macquarie University, NSW, 2109, Australia

## Abstract

Plastic pollution in the marine realm is an issue of growing concern; one understudied risk related to such pollution is that plastic debris can leach a variety of potentially toxic chemicals. Currently little is known regarding how plastic leachates impact marine organisms particularly primary producers such as photosynthetic marine cyanobacteria. *Prochlorococcus* species are significant players in oceanic primary production and global carbon cycling. Exposure to plastic leachate from common plastic debris items: plastic bags (HDPE) and plastic matting (PVC), impacted *in vitro* growth and primary productivity of *Prochlorococcus marinus* strains. The strains tested, MIT 9312 and NATL2A (model high light and low light-adapted ecotypes), showed distinct differences in their sensitivity to each leachate. Genome-wide transcriptomic analyses revealed a suite of genes were differentially expressed following leachate exposure, including many linked to primary production and stress responses. Understanding how key marine primary producers are affected by plastic leachate is critical to determining potential future impacts of plastic pollution on marine ecosystems as a whole.

**Keywords:** Prochlorococcus, cyanobacteria, plastic pollution, leachate, transcriptomics

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: sasha.tetu@mq.edu.au</footnote>
# Isolation of Heavy Metal Resistant Bacteria from Contaminated industrial Effluent in North Algeria

Ouelhadj Akli*<fnref n="1" />, Kuschk Peter<fnref n="2" />, and Djamel Djenane<fnref n="3" />

## Abstract

The resistance of bacteria strains isolated from contaminated industrial effluent at the site of ENIEM, Tizi-Ouzou Algeria to four heavy metals was investigated and the antagonistic effect against six pathogenic bacteria and three fungal strains were analyzed. Eight bacterial strains have been isolated and identified on the basis of the morphological and biochemical characters. On this whole, the metal resistance and the antagonistic activity of five strains have been studied. The MICs of heavy metals were similar for each strain: 10 mg/ml of zinc; 5 mg/ml of aluminium; 3.125 mg/ml of copper and 2.5 mg/ml of chromium, except for *Pseudomonas* sp.1 inhibited to 1.25 mg/ml of chromium. The general order of resistance to the metals was found to be as Cr < Cu < Al < Zn and the toxic effects of these metals increased with the increase of the concentrations. It can be concluded that all isolates were resistant to Cr but were highly resistant to Zn and Al. The antimicrobial activity was determined in six bacterial strains and three fungal strains, according to the agar duel method. All isolates tested showed an inhibitory effect at least on one of the tested microbial strains, confirming that the isolated bacterial strains are endowed with antimicrobial properties.

**Keywords:** Bacterial resistance, Heavy metals, Chromium, Zinc, Copper, Aluminium, Antimicrobial activity.

<figure><img src="image_59.png" /></figure>

<footnote n="1">¹Laboratory of food quality and safety – University of Mouloud Mammeri, Tizi Ouzou, Algeria</footnote>

<footnote n="2">²Department of Environmental Biotechnology – UFZ, Leipzig, Germany</footnote>

<footnote n="3">³Laboratory of food quality and safety – University of Mouloud Mammeri, Tizi Ouzou, Algeria</footnote>

<footnote>*Speaker</footnote>
# Herbicide mixture of mesotrione, nicosulfuron and s-metolachlor: biotransformation pathway and toxicity.

Louis Carles<sup>2,1</sup>, Muriel Joly<sup>1,2</sup>, Frédérique Bonnemoy<sup>1</sup>, Martin Leremboure<sup>2</sup>, Florence Donnadieu<sup>1</sup>, Isabelle Batisson*†<sup>1</sup>, and Pascale Besse-Hoggan<sup>2</sup>

<sup>2</sup>Institut de Chimie de Clermont-Ferrand (ICCF) – CNRS : UMR6296, Université Blaise Pascal - Clermont-Ferrand II, Ecole Nationale Supérieure de Chimie de Clermont-Ferrand – 24 Avenue des Landais, 63177 Aubière Cedex, France

<sup>1</sup>Laboratoire Microorganismes : Génome et Environnement, UMR CNRS 6023 (LMGE) – Université Clermont Auvergne – France

## Abstract

To reduce pesticide consumption, the new-generation molecules, currently used in agriculture, are sprayed at lower dosages but often in mixtures to increase their efficiencies. Despite the great environmental concern of unintentional mixture of pesticides coming from agricultural lands, little data is available concerning the effects of these cocktails on the fate and impact of each molecule. We focused our work on a mixture of three herbicides composed of mesotrione (β-triketone), nicosulfuron (sulfonylurea) and S-metolachlor (chloroacetanilide), commonly applied on maize crops in a short period of time. A *Bacillus megaterium* Mes11 strain [1], isolated from an agricultural soil, was found to be able to biotransform mesotrione and nicosulfuron, alone or in cocktail, but not S-metolachlor. Although the biodegradation pathways were not affected in the case of binary or ternary herbicide mixtures, kinetics of nicosulfuron disappearance as well as those of mesotrione and nicosulfuron metabolite formation were strongly modulated.

The toxicity of the parent compounds and metabolites was evaluated for individual compounds and mixtures with the standardized Microtox® test. Synergistic interactions were evidenced for all the parent compound mixtures, while synergistic, antagonistic or additive toxicity was obtained for metabolite mixtures, depending on the mixture. The results obtained highlight the need to take into account the active ingredient and metabolites all together for the determination of environmental fate and toxicity of pesticide mixtures and thus to improve the Environmental Risk Assessment of pesticide cocktail.

**Keywords:** Pesticide, Mixture, Simultaneous biotransformation, Metabolites, Microbial toxicity

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: isabelle.batisson@uca.fr</footnote>
# Toxicity of POCIS passive sampler extracts from a vineyard area in Bordeaux towards two marine microalgae.

Valentin Dupraz*†<fnref n="1" />, Dominique Ménard<fnref n="1" />, Farida Akcha<fnref n="1" />, Hélène Budzinski<fnref n="2" />, and Sabine Stachowski-Haberkorn<fnref n="1" />

## Abstract

Pesticides used in viticulture end up in aquatic systems downstream of vineyard plots. Concentrations of single substances are often below the maximum residue level imposed by the WFD but few is known about their potential toxicity when present in mixtures. According to their chemical properties, pesticides accumulate over time in POCIS passive samplers deployed in contaminated environments and toxicity of the whole POCIS extracts can be tested after extraction. As phytoplankton plays a key role in the aquatic food web, this study aimed to assess the toxicity of POCIS extracts towards the growth of two marine microalgae, *Tisochrysis lutea* (haptophyte) and *Skeletonema marinoi* (diatom). POCIS were placed in three stations along a contamination gradient in small watercourses close to the vineyards plots over four one-month periods. The toxicity tests consisted in 96-h exposures of microalgae to i) POCIS extracts and ii) single substances expected to be present in the extracts, in sterile 48-well microplates incubated under controlled conditions. Each species was exposed to five dilutions of POCIS extracts and six concentrations of single substances. The toxicity was assessed by calculating the growth rate inhibition. The toxicity tests and analyses to chemically characterize the extracts are currently ongoing. The first results showed a dose-response trend for the two microalgae species, with subtle differences in the toxicity depending on the extracts. The testing of complex mixtures from the environment provides additional information regarding the contamination of aquatic ecosystems and enlightens the necessity of focusing on mixtures effects, along with single compound toxicity.

**Keywords:** microalgae, passive samplers, toxicity, mixtures, vineyards

<footnote n="1">¹Laboratoire d'Écotoxicologie (LEX) – Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Université de Nantes, Université de Nantes – rue de l'île d'Yeu, BP 21105, 44311 Nantes, France</footnote>

<footnote n="2">²Environnements et Paléoenvironnements OCéaniques (EPOC) – Centre National de la Recherche Scientifique - CNRS, École Pratique des Hautes Études [EPHE], Université Sciences et Technologies - Bordeaux I, Observatoire Aquitain des Sciences de l'Univers – Av. des Facultés, 33405 Talence, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author:</footnote>
# Ecotoxicology of Herbicide Exposure on Harmful Estuarine Phytoplankton Under Varying Nutrient Conditions

Stacie Flood*†<fnref n="1" />,<fnref n="2" />, Joann Burkholder<fnref n="1" />, and Gregory Cope<fnref n="3" />

## Abstract

Anthropogenic inputs of macronutrients (nitrogen and phosphorus) are frequently associated with developing harmful algal blooms (HABs), but little is known about how estuarine phytoplankton assemblages respond to multiple, co-occurring chemical contaminants in chronically disturbed habitats. The available evidence suggests that off-target herbicide exposures can significantly affect phytoplankton assemblage composition, but questions remain about how estuarine phytoplankton respond to environmental stressors and why some strains respond by producing phycotoxins that can kill fish and other aquatic life. The goals of this research were to establish a robust protocol for testing the concentration-related effects of atrazine, a ubiquitous herbicide, on growth and toxin production in potentially harmful estuarine phytoplankton, and then to use that protocol to compare the effects of atrazine exposure both with and without nutrient enrichment. The protocols were developed using the benign estuarine/marine alga, *Dunaliella tertiolecta* (Chlorophyta) and the HAB species tested included the ichthyotoxic haptophyte *Prymnesium parvum* (Haptophyta) and the toxigenic raphidophyte *Chattonella subsalsa* (Raphidophyceae). Species sensitivity in nutrient-replete media followed the general ranking of *D. tertiolecta* > *P. parvum* > *C. subsalsa*, but responses were modulated by salinity, strain origin and exposure duration. Imbalanced nutrient regimes alleviated the growth-inhibiting effects of atrazine for all tested taxa to varying extents. Production of hemolytic substances by the HAB species increased under co-occurring nutrient stress and herbicide exposure, and the greatest toxicity was observed under combined imbalanced nitrogen/phosphorus levels and high atrazine exposures. These findings advance knowledge about how nutrient regimes and herbicides interact to control harmful estuarine phytoplankton population dynamics.

**Keywords:** atrazine, herbicide, phytoplankton, harmful algal blooms, algal toxins

<footnote n="1">¹Center for Applied Aquatic Ecology – Department of Applied Ecology, North Carolina State University, Raleigh, NC, 27606, United States</footnote>

<footnote n="2">²National Center for Computational Toxicology – United States Environmental Protection Agency, Research Triangle Park, NC, 27709, United States</footnote>

<footnote n="3">³Environmental Toxicology Division – Department of Applied Ecology, North Carolina State University, Raleigh, NC, 27695, United States</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: Flood.Stacie@epa.gov</footnote>
Simultaneous removal of azo-dyes and hexavalent chromium by a metal and salt tolerant Pseudomonas sp. ZM183 harboring plant growth promoting traits

Sabir Hussain*†<fnref n="1" />, Zahid Maqbool<fnref n="1" />, Faisal Mahmood<fnref n="1" />, Tanvir Shahzad<fnref n="1" />, and Muhammad Shahid<fnref n="2" />

**Abstract**

In this study, a metal and salt tolerant dyes decolorizing bacterial strain, *Pseudomonas* sp. ZM183, was isolated from an agricultural soil polluted with textile wastewater containing the dyes, metal ions and salts. This strain had the potential to decolorize different azo dyes in the presence of a mixture of heavy metal ions (Cr2+, Cd2+, Zn2+, Pb2+). The individual and interactive impact of four different variables (viz. NaCl content, pH, concentration of yeast extract and level of multi-metal mixture) on decolorization of reactive red-120 by *Pseudomonas* sp. ZM183 was estimated following response surface methodology (RSM). Based on RSM model, optimal decolorization of reactive red-120 by the strain ZM183 was predicted in the medium containing 12.5 g L-1 of NaCl, pH 8.0, 7.5 g L-1 of yeast extract and a multi-metal mixture (Cr: 20 mg L-1; Pb: 40 mg L-1; Cd: 20 mg L-1; Zn: 40 mg L-1). This strain was also found highly efficient in simultaneous removal of hexavalent chromium (25 mg L-1) and different reactive dyes (100 mg L-1). Moreover, the strain ZM183 was also found to harbor the azoreductase gene amplified through PCR using specific primers. In addition to dye decolorization and hexavalent chromium reduction, this strain was also found to have plant growth promoting traits including inorganic phosphate solubilization and indole acetic acid production. Hence, the isolation and characterization of multi-functional *Pseudomonas* sp. ZM183 might serve as a potential bio-resource to develop the strategies for integrated bioremediation and plant growth promotion in agricultural soils contaminated with textile wastewaters.

**Keywords:** Azo, dyes decolorization, multi, metal stress, Pseudomonas sp. ZM183, Plant growth promotion, Azoreductase gene, RSM Modelling

<figure><img src="image_60.png" /></figure>

<footnote n="1">¹Department of Environmental Sciences Engineering, Government College University, Faisalabad – Pakistan</footnote>

<footnote n="2">²Department of Bioinformatics and Biotechnology, Government College University, Faisalabad – Pakistan</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: sabirghani@gmail.com</footnote>
# Cyanotoxins role for cyanobateria in stress conditions

Sandra Kim Tiam*†<fnref n="1" />, Katia Comte<fnref n="1" />, Caroline Dalle<fnref n="2" />, Chakib Djediat<fnref n="1" />, Charlotte Duval<fnref n="1" />, Kathleen Feilke<fnref n="3" />, Muriel Gugger<fnref n="2" />, Diana Kirilovsky<fnref n="3" />, Sahima Hamlaoui<fnref n="1" />, Séverine Le Manach<fnref n="1" />, Benjamin Marie<fnref n="1" />, Pierre Setif<fnref n="3" />, Claude Yéprémian<fnref n="1" />, and Cécile Bernard<fnref n="1" />

## Abstract

Cyanobacteria are photosynthetic prokaryotes having a key role in ecosystem functioning but are also a source of concern for managers since they can form toxic blooms. Environmental risk assessment related to cyanotoxin exposure is particularly complex since the toxin concentrations cannot be predicted based on the cyanobacteria species forming the bloom or on the bloom intensity. Cyanotoxin biologic role characterization could be a key factor for concentration prediction and a better management of environmental risks. Ecological advantages offered or not by toxins in stress condition (high light and high temperature), through their role in photosynthesis and photoprotection, were studied. Five Planktothrix strains have been selected: green (Planktothrix agardhii) or red (Planktothrix rubescens) ecotypes, non-microcystins or microcystins producers and a wild-type green strain and its mutant for toxin production. A 24 hours experiment was realized in laboratory controlled conditions and was composed of two different phases: stress exposure phase (4H) followed by a recovery phase. The cultures were exposed in triplicate to high light intensity (150 µE/m2/s) or high temperature (33°C) and controls (20°C, 6 µE/m2/s) were run in parallel. Along the experiment, responses of cyanobacteria were evaluated with: i) biophysical measurements (photosynthesis and photoprotective mechanisms), ii) transcriptomic analyses (genes involved in toxin synthesis, photosynthesis, photoprotection, temperature tolerance and oxidative stress regulation), iii) metabolomics analyses (secondary metabolites synthesis), iv) protein analysis (orange carotenoid protein) and v) immunolocalization analysis (microcystins).

**Keywords:** Cyanobacteria, toxins, microcystins, photoprotection, Planktothrix.

<footnote n="1">¹Molécules de Communication et Adaptation des Micro-organismes, équipe "Cyanobactéries, Cyanotoxines et environnement" (MCAM) – Muséum National d’Histoire Naturelle (MNHN), CNRS : UMR7245 – 12 rue Buffon - RCP 39, 75231 Paris Cedex 05, France</footnote>

<footnote n="2">²Collection des Cyanobactéries – Institut Pasteur de Paris – 28 rue du Dr Roux, 75724 Paris, France</footnote>

<footnote n="3">³Institut de Biologie Intégrative de la Cellule (I2BC) – Université Paris-Sud - Paris 11, Commissariat à l'énergie atomique et aux énergies alternatives : DSV/I2BC, Université Paris-Saclay, Centre National de la Recherche Scientifique : UMR9198 – 1 avenue de la Terrasse, 91198 Gif/Yvette cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: sandra.kim-tiam-fook-chong@mnhn.fr</footnote>
Light modulated effects of toxicants toward river
biofilm photosynthesis: an ecophysio-toxicological
approach

Martin Laviale*†1, Anne Créach2, Evane Thorel3, Johann Lavaud4, and Soizic Morin3

¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360,
Université de Lorraine – France

²Unité de Glycobiologie structurale et fonctionnelle (UGSF) – CNRS : UMR8576, Université Lille I -
Sciences et technologies – Bâtiment C9 59655 VILLENEUVE D ASCQ CEDEX, France

³Réseaux épuration et qualité des eaux (UR REBX) – Irstea – 50 avenue de Verdun - Gazinet, F-33612
Cestas, France

⁴CNRS/Université Laval UMI 3376 (TAKUVIK) – Canada

Abstract

Traditional ecotoxicological bioassays using diatoms are often based on growth or pho-
tosynthetic activity inhibition under stable and standardized conditions. However, their re-
sponses to toxic stress may be modulated by variations in environmental parameters. Light,
which is the most important factor for photosynthetic organisms, may modify algal sensitivity
to contaminants. Daily light fluctuations, indeed, have consequences on algal photophysiol-
ogy. The capacity to cope with both toxicants and swift changes in light conditions should
thus be taken into consideration. In order to increase the complexity and the realism of
ecotoxicological tests, it is necessary to take into account multistress situations.

In this context, short-term experiments (< 24h) were carried out to investigate the com-
bined effects of light and toxicants presenting different modes of action (isoproturon, diuron,
copper, norflurazon) on the photophysiology of intact natural biofilms by measurements of
chlorophyll fluorescence and pigment composition. Firstly, biofilms were exposed to toxicants
under constant light at different irradiance levels (25–300 µmol m–2 s–1). Secondly, biofilms
were exposed using more environmentally realistic conditions (i.e. daily light cycle). The
obtained results lead to the design of an ecotoxicological protocol based on the so-called ”
light stress recovery experiment ” which is typically used in ecophysiology.

This protocol offers interesting perspectives in environmental risk assessment, providing a
more generic and ecologically realistic framework than traditional approaches currently used
in ecotoxicology.

**Keywords:** multi, stress, light, toxicants, biofilms, diatoms, photosynthesis

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: martin.laviale.bio@gmail.com</footnote>
# Biocidal coatings triggers low diversified Alteromonadaceae dominated biofilm community

Thomas Pollet¹,², Lyria Berdjeb³, Cédric Garnier³, Benjamin Misson³, Christine Bressy¹, Jean-François Ghiglione⁴, and Jean-François Briand*¹

¹MAPIEM-EA 4323 – Université de Toulon – France

²INRA-UMR BIPAR, Maisons-Alfort, France – Institut national de la recherche agronomique (INRA) – France

³PROTEE – Université de Toulon – France

⁴LOMIC – Université Pierre et Marie Curie - Paris 6 – France

## Abstract

Identifying microbial successions are crucial to understand the antifoulants impact on the dynamics of marine biofilm. We investigated the density (flow cytometry) together with the community succession (Metabarcoding) in coastal marine biofilms in Toulon Bay (France), during 75 days of immersion of 2 antifouling coatings (a biocidal named SPC and the other based on physical properties named FRC) and a reference surface (PVC).

Based on seven sampling performed (triplicates), density showed 3.5 times lower value on SPC and FRC compared to PVC. In addition, bacterial succession showed that although pioneer taxa were dissimilar on all the three surfaces, the community on PVC and FRC tend to converge with time whereas biocidal coating tends to diverge.

If Flavobacteriaceae represented critical members of diversified biofilms on PVC, Alteromonadaceae accounted for more than 90% of the community on SPC after 4 days and until 12 days of immersion. Then, bacterial diversity increased progressively, keeping Alteromonadaceae as one of the co-dominant taxa until 75 days of immersion. If pioneer stage on the FRC were also dominated by Alteromonadaceae (around 60%), the rapid increase in diversity reached a higher level than for SPC and promoted the co-dominance of other bacterial families (Rhodobacteraceae, Flavobacteriaceae, ...).

A second Mediterranean site, in the bay of Banyuls exhibited dissimilar metal contamination and hydrodynamics especially), allowed demonstrating a higher efficacy of SPC after 75 days of immersion. Furthermore, SPC exhibited dissimilar diversified communities at the 2 immersion sites. In conclusion, community shaping was mainly driven by biocide but also site.

**Keywords:** Biofilm, biocide, marine microbial ecotoxicology, antifouling

<footnote>*Speaker</footnote>
# Shifting in the structural diversity of archaeal communities with contrasted hydrodynamic regimes in Mediterranean coastal rivers

Brice Reoyo-Prats*¹, Mégane Noyer¹, and Carmen Palacios¹

¹Centre de Formation et de Recherche sur les Environnements Méditérranéens (CEFREM) – Université de Perpignan Via Domitia, Centre National de la Recherche Scientifique - CNRS, Université de Perpignan Via Domitia – Perpignan, France

## Abstract

Assessing the impact of anthropogenic contaminants in aquatic environments represents nowadays a fundamental issue. Pollutants concentrations in watercourses mainly depend on land use and hydrometeorological phenomena. These phenomena are particularly contrasted in Mediterranean regions, where regimes of droughts alternate with heavy rainfalls. We have demonstrated that typical storm events in these regions are at the origin of multipollution episodes in Mediterranean coastal rivers. In this study, we examine the impact of these sudden changes in contaminants on microorganisms attached to suspended matter of the Têt River, a major coastal watercourse in the South-East of France. Microbial biofilms are the first to interact with contaminants, degrading or transforming them. This has been mainly studied in bacterial and algal biofilms. Here, we focus in the archaeal component of biofilms attached to particles. Water quality measurements, including the analyses of more than 350 micropollutant molecules, were performed from upstream to foreshore waters in two complete drought-flood-drought hydrological cycles. A high frequency sampling during heavy rain periods at two contrasted stations was also carried out. The diversity of Archaea was studied using Illumina targeted metagenomics analysis on the 16S rRNA gene. Their communities were mainly composed of two phyla, Thaumarchaeota and Euryarchaeota, and sometimes a third phyla, Crenarchaeota. However, a great quantity of sequences did not match anything in the databases. Variations in phylogenetic composition could be directly linked to changes in pollutants levels, particularly during heavy rainfalls. These results indicate the importance of the archaeal compartment in aquatic ecosystems reactivity to pollutants.

**Keywords:** Microbial ecotoxicology, multipollution, aquatic ecosystems, Mediterranean, archaeal communities, structural diversity.

<figure><img src="image_61.png" /></figure>

<footnote>*Speaker</footnote>
# Combined effect of a realistic pesticide mixture and climate change-related stressors on river biofilms.

Ferran Romero*¹, Vicenç Acuña¹, Anna Freixa†¹, Julio C. López-Doval¹, Maria Casellas¹, Natalia Mingorance¹, and Sergi Sabater¹,²

¹Catalan Institute for Water Research (ICRA) – Spain

²Institute of Aquatic Ecology - University of Girona (IEA - UdG) – Spain

## Abstract

Chemical stressors such as agricultural pesticides are known to significantly impact community composition and functioning in stream ecosystems. Moreover, agricultural streams are often subjected to climate change-related stressors, such as warming and flow reduction. Since these stressors are often investigated separately, uncertainty remains over the combined impacts that they may pose to freshwater communities. An additional knowledge gap results from the comparison between the responses on a temporal scale, and to which extent the interactions observed at short term remain when the stressors combination is applied at long-term (e.g. one month). Using river biofilms and microfauna (micrometazoans and ciliophora) as target communities, we evaluated the individual and combined effects of a realistic pesticide mixture and two climate change-related stressors (i.e. flow reduction and warming). The experimental procedure followed a full-factorial design using 24 artificial indoor channels. The biofilm response was evaluated both at short and long-term. Biofilm and microfauna analyses covered a wide range of physiological and structural variables, from molecular approaches to ecosystem metabolism measurements. Overall, our results show that climate change-related stressors drove the overall effects and shaped the response of the river biofilm and the microfaunal community to agricultural pesticides. We conclude that the response of a freshwater community to the combined action of chemical and climate change-related stressors cannot be predicted straightforwardly based on the individual effects.

**Keywords:** multiple stressors, river biofilm, microfauna, pesticides, climate change.

<footnote>*Corresponding author:</footnote>

<footnote>†Speaker</footnote>
Responses of two strains of the marine microalga
Tetraselmis suecica to binary mixtures of two PSII
inhibitors

Rossana Sussarellu*<fnref n="1" />, Valentin Dupraz<fnref n="1" />, Dominique Ménard<fnref n="1" />, Hélène Budzinski<fnref n="2" />, and
Sabine Stachowski-Haberkorn<fnref n="1" />

Abstract

Irgarol (triazine) and diuron (phenylurea) are two biocides commonly used in copper-
based antifouling paints. They both act as photosystem II (PSII) inhibitors due to their
binding action on the D1 protein in PSII. They are thus particularly toxic to photosynthetic
organisms such as microalgae. This study aimed to describe the interactions between the
two compounds in binary mixtures towards two strains of *Tetraselmis suecica*: a wild strain
(TW) and a mutant strain (TM) resistant to diuron.

The toxicity tests consisted in 96-h exposures to contaminants in 48-well microplates. Each strain was exposed to each substance at 6 concentrations. The binary mixtures were achieved following a fixed-ratio ray design with 5 mixture ratios. The toxicity was assessed by calculating EC50 values based on the 96-h growth rate inhibition. The results were analysed using Concentration Addition (CA) reference model, and Helwett and Volund models for synergy and antagony.

The EC50 obtained were: 4.2 ± 0.13 µg.L-1 diuron and 0.65 ± 0.027 µg.L-1 irgarol for TW, when EC50 for TM were 20-fold higher for diuron and almost twice for irgarol. For binary mixtures, a slight but significant concentration-dependent synergy was demonstrated in both strains. To further investigate the mechanisms responsible for such effects, transcriptome analyses (RNAseq HiSeq 3000, paired-end 2x150 bp) were performed from cDNA libraries of both strains in control condition and in condition with 50 % growth rate inhibition after exposure to a mixture. The analyses are currently ongoing.

**Keywords:** microalgae, PSII inhibitors, resistance, toxicity, binary mixtures, transcriptomic

<footnote n="1">¹Laboratoire d'Ecotoxicologie, Institut Français de Recherche pour l'Exploitation de la Mer (LEX, IFREMER) – Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER) – Rue de l'Ile d'Yeu - BP 21105 - 44311 Nantes Cedex 03, France</footnote>

<footnote n="2">²CNRS, UMR 5805, EPOC, Laboratoire de Physico Toxico Chimie de l'environnement (UMR EPOC, LPTC) – Université de Bordeaux (Bordeaux, France) – 351 Cours de la Libération, 33405 Talence, France</footnote>

<footnote>*Speaker</footnote>
Quick screening of isolates relevant in microbial
ecotoxicology by tandem mass spectrometry

Béatrice Alpha-Bazin*†<fnref n="1" />, Karim Hayoun<fnref n="1" />, Gerard Steinmetz<fnref n="1" />, Pauline Petit<fnref n="2" />, Corinne Rivasseau<fnref n="2" />, Olivier Pible<fnref n="1" />, and Jean Armengaud<fnref n="1" />

Abstract

We developed a new strategy to quickly identify microorganisms based on tandem mass spectrometry and characterize their main metabolic potential. Following plate isolation, a single colony may be used for extracting bacterial proteins. These are then proteolyzed with trypsin, and the resulting peptides are sequenced by high-resolution tandem mass spectrometry. A home-developed bioinformatics pipeline allows from a simple 60 min measurement identifying the microorganisms present in the sample either at the genus, species or sub-species level, certifying if the isolate is pure or present in mixture with some contaminants, and obtaining its functional characterization based on the identified proteins. Dereplication of isolates, taxonomical identification or functional focus on the most interesting microorganisms are quickly achieved. We applied this methodology for exploring bacteria present in the cooling pool for spent fuel in a nuclear reactor. We used different culture media to isolate bacteria. The quick screening approach will be illustrated with an overview of the numerous identified bacteria. This methodology is applicable to a large variety of samples and thus has a great potential in microbial ecotoxicology.

**Keywords:** tandem mass spectrometry, taxonomical identification, screening

<footnote n="1">¹Laboratory Innovative technologies for Detection and Diagnostics (LI2D) – CEA - Direction de la Recherche Fondamentale - Institute Frederic Joliot – CEA Marcoule, 30207 Bagnols sur Ceze, France</footnote>

<footnote n="2">²Laboratoire de Physiologie Cellulaire Végétale (LPCV) – CEA - Direction de la Recherche Fondamentale - Biosciences Biotechnology Institute of Grenoble, Centre National de la Recherche Scientifique - CNRS, Institut National de la Recherche Agronomique - INRA, Université Grenoble Alpes – CENG 17 rue des Martyrs, 38054 Grenoble, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: beatrice.alpha-bazin@cea.fr</footnote>
# Development of a method to test soil protists in ecotoxicology and its application to an herbicide. Euglypha rotunda (Rhizaria) proposed as model organism

Nathalie Amacker*†<fnref n="1" />,<fnref n="2" />, Edward Mitchell<fnref n="1" />,<fnref n="3" />, and Nathalie Chèvre<fnref n="2" />

## Abstract

Many ecotoxicological tests have been elaborated the last decades to address the effect of toxic compounds in environment with a special focus on metazoans. The protists (micro-eucaryotes) that constitute the major part of eukaryotic diversity and drive ecosystems functions (such as biogeochemical cycles) are clearly under-represented. To our knowledge not a single model organism for soil ecotoxicology exists among the Rhizaria, a highly diverse group or terrestrial and aquatic protists.

We aim to fill this gap by developing a robust ecotoxicity test for the common soil testate amoeba *Euglypha rotunda*. In particular, we aim in finding out i) the variables that influence the growth of the amoeba in the laboratory, and ii) the environmental conditions allowing reproducible growth of the organisms, so that the effects of chemical on that growth can be highlighted. In a second time, we investigated the effects of an herbicide, *S*-metolachlor on the growth of the amoeba and of the bacteria *Escherichia coli* (co-inoculated as carbon source). The *S*-metolachlor is a chloroacetamide that affects the production of very-long-chain-fatty acids (VLCFAs). The protozoan growth is expected to be influenced as VLCFAs are important players of cell division that is the most common reproduction strategy of testate amoebae.

Conditions supporting reproducibility could be defined and the effect of *S*-metolachlor evaluated. The protozoan growth was reduced when exposed at around 15 µg/L of *S*-metolachlor but not at higher concentration. The bacteria were not affected at all.

**Keywords:** testate amoebae, E. coli, small trophic chain, S, metolachlor

<footnote n="1">¹Laboratory of Soil Biodiversity, Université de Neuchâtel (UniNE) – Emile-Argand 11 CH-2000 Neuchâtel, Switzerland</footnote>

<footnote n="2">²Institut des dynamiques de la surface terrestre, Université de Lausanne (IDYST, UNIL) – UNIL Mouline CH-1015 Lausanne, Switzerland</footnote>

<footnote n="3">³Jardin botanique de Neuchâtel – CH-2000 Neuchâtel, Switzerland</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: nathalie.amacker@gmail.com</footnote>
# Environmental surveillance of marine oil releases using an autonomous ecogenomic sensor – development of a surface plasmon resonance based biosensor for Oleispira antarctica

Andrea Bagi*†<fnref n="1" />, Knapik Kamila<fnref n="1" />, Mari Mæland Nilsen<fnref n="1" />, Adriana Krolicka<fnref n="1" />, Catherine Boccadoro<fnref n="1" />, and Thierry Baussant<fnref n="1" />

## Abstract

Environmental changes and pollution monitoring from expansion of oil and gas exploration activities pose a practical challenge in remote marine environments, e.g. pristine Arctic region. Timely delivery of critical information for mitigating environmental impacts in such areas are necessary but manpower and access to infrastructure are limited. There is a need for automatized low-manpower monitoring and rapid communication of critical data. This poster presentation will introduce the GENOMAPE project lead by the International Research Institute of Stavanger (IRIS), Norway. The central technology is the ESP (Environmental Sample Processor, MBARI), a cutting-edge autonomous genosensor that uses molecular detection of microorganisms from water samples. Here, nucleic acid (primarily rRNA) signals from hydrocarbon degrading microorganisms will be used to infer occurrence of small to medium oil releases. Our aim is to develop molecular assays targeting obligate hydrocarbon-degrading bacteria using phylogenetic genes in a manner compatible with the analytical modules of the ESP. Surface plasmon resonance (SPR), a highly sensitive, label-free and rapid optical sensing technology is one analytical module that will be integrated in ESP in the future. We are working on adapting a portable SPR unit for the detection of bacterial rRNA, using morpholino probes immobilized gold surface. Currently, a single probe targeting 16S rRNA of *Oleispira antarctica* and synthetic oligonucleotides are used for method development. The poster will present the vision and workplan of GENOMAPE and describe in detail the ongoing SPR assay development.

**Keywords:** surface plasmon resonance, hydrocarbon degrading bacteria, marine environmental monitoring

<footnote n="1">¹International Research Institute of Stavanger – Environment Department, Mekjarvik 12, Randaberg, N-4070, Norway</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: Andrea.Bagi@iris.no</footnote>
Impact of pharmaceutical industry effluents on
microorganism biodiversity inside river biofilms

Cédric Bernarde¹, Anne Goubet¹, Pierric Jeannin*†², and Jean-Jacques Pernelle¹

¹UR HBAN, Equipe EPURE – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA – 1, rue Pierre-Gilles de Gennes CS 10030 92761 Antony Cedex, France

²SANOFI – SANOFI Recherche – route d'Avignon 30390 ARAMON, France

Abstract

The continental water quality evaluation is currently achieved with normalized indexes
based on observations, like the French "indice biologique global normalisé (IBGN)" or the
"indice biologique diatomique (IBD)". These complex processes need the intervention of
experimented specialists for taxonomic assignment. The development of next-generation
sequencing (NGS) would open a new way for a tool based on molecular inventory of the
microbiota of rivers' biofilms. Such a tool could efficiently support the evaluation of water
quality by a regular monitoring biofilm population and the differences that could be identi-
fied between the rivers upstream and downstream a discharge point.

Sanofi initiate collaboration with IRSTEA to propose a tool allowing estimating quickly the impact of effluents of wastewater treatment plants (WWTP) on the receiving environment. This project will allow defining supports for biofilm development, choosing primer sets in order to describe microbiological diversity with the most exhaustive manner. We will also define the number of sample and the immersion duration. The project covers also the instal- lation of the "pipeline" for the analysis and data filtration.

We present here some details of the project and very first results.

**Keywords:** biofilm, impact, next, generation sequencing

<figure><img src="image_62.png" /></figure>

<footnote>*Speaker</footnote>

<footnote>† Corresponding author:</footnote>
# Development of whole-cell bacterial sensors for lanthanides bioavailability

Charlotte Berthelot*¹, Asfaw Zegeye¹, and Patrick Billard¹

¹LIEC – Université de Lorraine, CNRS : UMR7360, Université de Lorraine – France

## Abstract

Lanthanides (Ln) are economically important metals with wide applications in technology and industry. With their increasing release into the environment, there is a need for the development of new tools for the monitoring of Ln bioavailability. Here we describe the design of bacterial bioreporters and their use in liquid- or solid-phase bioassays to measure the presence of Ln in a variety of samples. Bioreporters were constructed by coupling the Ln-responsive methanol dehydrogenase genes *xoxF* and *mxaF* from *Methylobacterium extorquens* (Vu et al., 2016) and two alcohol dehydrogenase counterparts from *Pseudomonas putida* (Wehrmann et al., submitted) to the *luxCDABE* reporter genes. In both strains, the expression of the two dehydrogenase genes were inversely regulated in response to available Ln concentrations. This transcriptional response was specifically triggered by light Ln (La, Ce, Pr, Nd) in *M. extorquens*, while *P. putida* bioreporters additionally responded to medium Ln (Sm, Gd). In optimized assay conditions, as low as 1 nM Lncould be detected within 4 hours of incubation. As a proof-of-principle application, the bioassays were tested on contaminated soil samples as well as with Ln-containing solids such as Nd-magnets. Our results indicated that the bioreporter are sensitive enough to detect Ln in these samples, thus making them potential a ecotoxicological tool for Ln monitoring. Vu HN, Subuyuj GA, Vijayakumar S, Good NM, Martinez-Gomez NC, Skovran E. 2016. Lanthanide-Dependent Regulation of Methanol Oxidation Systems in *Methylobacterium extorquens* AM1 and Their Contribution to Methanol Growth. J Bacteriol 198: 1250-1259

**Keywords:** lanthanides, bioreporter, Ln monitoring

<footnote>*Speaker</footnote>
# Exposures of soil microbial communities to realistic antibiotics concentrations: key parameters for environmental risk assessment

Olivier Crouzet*<fnref n="1" />, Anaïs Goulas<fnref n="1" />, Claire-Sophie Haudin<fnref n="1" />, Pierre Benoit<fnref n="1" />, Alain Hartmann<fnref n="2" />, Géraldine Depret<fnref n="2" />, Agnès Richaume-Jolion<fnref n="3" />, Thomas Pommier<fnref n="3" />, and Sylvie Nazaret<fnref n="3" />

## Abstract

While the occurrence and persistence of antibiotics in environment are recognized as major environmental and human health issues, their ecotoxicological impacts on soil microbial communities are still overlooked for environmental risk assessment. Most available studies highlight effects on soil microbial communities at antibiotic concentrations or under exposure pathways that are far from realistic conditions. To investigate the relationships between SMX (sulfamethoxazole) availability and impact on microbial nitrogen-cycling communities and antibiotic resistance genes (ARGs), a dose-effect approach was performed, with sludge compost or farmyard manure amended soil microcosms. The organic wastes were contaminated before mixing in soils, to obtain SMX concentration surrounding those usually measured in amended agricultural soils. Microbial descriptors were monitored during 84 days following amendments. The total and available fractions of SMX were quantified by UPLC-MS/MS. At these low but environmentally realistic concentrations, only nitrification was impaired by SMX, in compost-amended soils and little consistent effect was observed on denitrification. However, SMX availability was similar in both compost- or manure-amended soils, thus not explain the short-term effects. A temporal decrease of SMX availability contributed to the nitrification recovery, in compost-amended soils. The dynamics of ARGs were partly explained by SMX gradient, but the duration of the experiment and the type of organic waste influenced the ARGs responses to SMX. Although it is necessary to define normal operating ranges of these descriptors, for further interpretations of their suitability as indicators in risk assessment, nitrification and abundances of ARGs are sensitive descriptors of AB effects on soil microbial functioning.

**Keywords:** soil microbial communities, antibiotics, functional endpoint, resistance genes

<footnote n="1">¹UMR 1402 ECOSYS - Institut National de la Recherche Agronomique - INRA, AgroParisTech, Université Paris-Saclay - 78026 Versailles cedex, France</footnote>

<footnote n="2">²Agroécologie - Agrosup, Institut national de la recherche agronomique (INRA) : UMR1347, Université de Bourgogne - BP 86510, F-21000 Dijon, France</footnote>

<footnote n="3">³Ecologie microbienne (EM) - CNRS : UMR5557, Ecole Nationale Vétérinaire de Lyon, INRA : UR1193, Université Claude Bernard - Lyon I - La Doua, 69622 Villeurbanne, France</footnote>

<footnote>*Speaker</footnote>
# Can biodegradation of cis-1,2-dichloroethene and vinyl chloride by bacterial consortiums of a contaminated aquifer be directly linked to functional gene abundance?

Catherine Joulian¹, Camille Bourdier¹, Jérémie Denonfoux², Gwenaël Imfeld³, Stéphanie Ferreira², Stéphane Vuilleumier⁴, Louis Hermon¹,⁴, and Jennifer Hellal*¹

¹Environmental Biogeochemistry and Water Quality Unit (D3E/BGE) – Bureau de Recherches Géologiques et Minières (BRGM) – France

²Genoscreen – Institut Pasteur de Lille – 1 rue du Professeur Calmette, Lille 59000, France

³Laboratoire d'Hydrologie et de GEochimie de Strasbourg (LHYGES) – Ecole Nationale du Génie de l'Eau et de l'Environnement – UMR7517 CNRS EOST/UdS, 1, rue Blessig 67084 Strasbourg, France

⁴Génétique moléculaire, génomique, microbiologie (GMGM) – université de Strasbourg, CNRS :
UMR7156 – 28 Rue Goethe 67083 Strasbourg, France

## Abstract

The extensive use of aliphatic chlorinated hydrocarbons (e.g. tetrachloroethene and its degradation products) has resulted in their accumulation and persistence in aquifers worldwide, representing a serious risk for human health and the environment. Polluted sites are generally diagnosed and monitored using classical physico-chemical assessment of the plume. In addition, the evaluation of molecular biomarkers reflecting a biodegradation potential may enhance the evaluation of *in situ* attenuation and assist site diagnosis. The aim of this study was to evaluate whether the biodegradation of *cis*-1,2-dichloroethene (cDCE) and vinyl chloride (VC) by the bacterial consortiums of a contaminated aquifer could be directly linked to the abundance of functional genes involved in their degradation (*tceA*, *vcrA* & *bvcA*). This approach may help interpret the abundance patterns of functional genes on-site. Groundwater samples contaminated with cDCE and VC were incubated under i) oxic conditions to evaluate the potential of oxidative pathways, ii) anoxic conditions with addition of H2 to evaluate reductive dechlorination, and iii) anoxic conditions with addition of N2 and 10 mM Fe(III), since Fe(II) was present in the aquifer. Degradation was most effective in the Fe(III) condition, with significant reduction of DCE and formation of VC after approximately 3 months, following Fe(III) and sulfate reduction. The *vcrA* gene was present during the entire experiment and its abundance did not vary significantly. In contrast, *tceA* gene abundance was only detected during the active phase of DCE and VC degradation, suggesting that it may be a good biomarker of DCE and VC degradation.

**Keywords:** vinyl chloride, dichloroethene, biomarkers, reductive dechlorination, functional genes

<footnote>*Speaker</footnote>
# Hospital effluents, not an exclusive source of contaminant spread in sub-saharian urban rivers

Amandine Laffite*¹, Vera Slaveykova¹, and John Poté†¹

¹Forel Departement for Environmental Sciences – Bd Carl Vogt 66, CH-1211 Geneva, Switzerland

## Abstract

The contamination of freshwater resources with anthropogenic pollutant is particularly alarming in developing countries where most rivers and lakes are receiving urban and hospital wastewater without any prior treatment. Cumulative with the knowledge gaps in antibiotic prescription and the free access to last resort antibiotics, under-developing countries may represent a broth culture to antibiotic resistance emergence, maintenance and dissemination. In this study, we investigate the contamination of sediment and the resistance profile of extending-spectra β-lactamase *E. coli* (ESBLECs) isolated from 4 urban river subjected to hospital outlet pipes (HOP) discharge in order to determine the effect of HOP discharge on urban river quality and the pattern of antibiotic resistance dissemination. ARGs (*blaTEM*, *blaCTX-M*, *blaSHV* and *aadA*) and selected bacterial species (i.e. *E.coli*, *Enterococcus*, Pseudomonas *spp.*) where quantified in sediments using quantitative PCR (qPCR), toxic metals content were quantified by ICP-MS, and ESBLECs isolates where subjected to pulse-field electrophoresis to assess their clonality and characterized for their β-lactams resistance. The results highlight the great concentration of toxic metals (i.e. Cr, Cu, Zn, Cd, Pb and Hg) in sediments and the high content of FIB and ARGs copy number in all sampling sites. The analysis of ESBLECs profile showed the high diversity of clone dissemination and a high resistance profile against β-lactams. These findings indicates the human and environmental potential risk link to exposure to these contaminants and the need of developing strategies to limit the spread of these emerging contaminant.

**Keywords:** hospital effluents, emerging contaminant, antibiotic resistance, metal resistance

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: John.Pote@unige.ch</footnote>
Integrating functional responses of autotrophic
microorganisms to chemicals in ecological risk
assessment

Floriane Larras*†<fnref n="1" />, Elise Billoir<fnref n="2" />, Stefan Lips<fnref n="1" />, and Mechthild Schmitt-Jansen<fnref n="1" />

Abstract

Microorganisms (e.g. bacteria, fungi and algae) are implied in various ecosystem func-
tions such as biogeochemical cycles or pollutants degradation. That means that they are
crucial for ecosystem functioning, here considered as the overall performance of a system.
In the environment, organisms are exposed to anthropogenic pressures which are known to
potentially induce structural and functional changes. Many studies report causal links be-
tween chemicals exposure and functional responses, but little is known about the involved
biochemical pathways supporting specific functions. Moreover, most of *a priori* ecological
risk assessment (ERA) tools are based on structural endpoints and do not necessarily ensure
the protection of functional endpoints. The recent raise of OMICs approaches (e.g. tran-
scriptomics and metabolomics) opens the perspective in ecotoxicology to explore pathways
involved in ecological functions. Based on OMICs approaches, we identified functional re-
sponses of microorganisms across different biological organization levels to improve linkage
of pathways, responsive to chemical exposure, and ecosystem functions. Such results present
a great potential for the development of an integrative ERA tool based on microorganisms
functions sensitivity at different biological levels. The challenge remains how to include
these data in ERA strategies. As a first step, we developed an approach based on multi-level
sensitivity distributions. We exemplify a first version of this tool, using the transcriptomic
and metabolomic responses of *Scenedesmus vacuolatus* to triclosan. As a perspective, the
tool will be transposed and adapted to benthic microbial communities in order to release an
integrative ERA tool considering ecological systems.

**Keywords:** Microorganisms, Functions, OMICs, Ecological Risk Assessment

<footnote n="1">¹Helmholtz Centre for Environmental Research, Department Bioanalytical Ecotoxicology (UFZ) –
Leipzig, Germany</footnote>

<footnote n="2">²Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360,
Université de Lorraine – Metz, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: floriane.larras@ufz.de</footnote>
Lignin degradation pathway in Scedosporium species.

Wilfried Poirier*†¹, Amandine Gastebois¹, Patrick Vandeputte¹, Jean-Philippe Bouchara¹,², and Sandrine Giraud¹

¹Groupe d'Étude des Interactions Hôte-Parasite (GEIHP) – Université d'Angers : EA3142 – UFR Sciences pharmaceutiques et ingénierie de la santé 16, Bd Daviers 49045 Angers cedex 01, France

²Laboratory of Parasitology-Mycology – Centre Hospitalier Universitaire – Angers, France

Abstract

Scedosporium are usually soil saprophytes. Among the ten species recognized within the genus, three have been reported regularly causing infections in human, i.e. Scedosporium apiospermum, Scedosporium aurantiacum and Scedosporium boydii. Environmental studies were conducted to get a better knowledge on the ecology of these opportunistic molds. Scedosporium species are abundant in human-made environments and have been associated with nutrient-rich substrates, in relation with specific traits of the fungi: ability to survive at low oxygen pressure, to tolerate high salt concentration and to degrade hydrocarbons. Nevertheless, the natural habitat of these fungi is still unknown. Recent experiments performed in our laboratory highlighted the Scedosporium capacity to grow on media containing the different components of lignocellulose as sole carbon source, suggesting lignocellulolytic properties.

Here we attempted to define the metabolic pathways involved in the degradation of lignocellulose in Scedosporium species, focusing on S. apiospermum and S. aurantiacum since genomic data were available only for these two species. A literature search was performed in order to identify enzymes involved in lignin degradation in other fungi and conserved sequences which were then used to screen Scedosporium genomic data by tBLASTn (Basic Local Alignment Search Tool) searches. 15 candidates were identified. qPCR experiments demonstrated that 8 of them exhibited an important increase in their expression level in media containing lignocellulose components. Moreover, similar results were obtained in presence of 4-hydroxybenzoate, suggesting the involvement of this pathway in adaptation of these fungi to polluted environments.

**Keywords:** Scedosporium, human, impacted areas, polluted environments, lignin degradation, 4-hydroxybenzoate.

<figure><img src="image_63.png" /></figure>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: wilfried.poirier94@gmail.com</footnote>
Impact of repeated treatments of the pesticides
chlorpyrifos or tebuconazole on soil microorganisms
using alternative methods

Veronika Storck<fnref n="1" />, Nadine Rouard<fnref n="1" />, David Bru<fnref n="1" />, Jeremie Beguet<fnref n="1" />, Lucio Botteri<fnref n="2" />, Ayme Spor<fnref n="1" />, Marion Devers<fnref n="1" />, and Fabrice Martin-Laurent*<fnref n="1" />

Abstract

Pesticides can harm non-target organisms such as soil microorganisms involved in im-
portant ecosystem functions. In the European Union, during pesticide authorization process
conducted by European Food Safety Authority (EFSA), potential risks on soil microorgan-
isms are solely evaluated by estimating its impact on mineralization of nitrogen which is
a too general test to detect pesticide ecotoxicity towards microorganisms. In consequence,
pesticide risk assessment conclusions on soil microorganisms edited by EFSA can differ from
academic researches carried out with more advanced techniques allowing to monitor the
abundance, diversity and activity of microbial community. This is the case for the insecti-
cide chlorpyrifos (CHL) and the fungicide tebuconazole (TCZ) studied for their microbial
ecotoxicological impact using new experimental design and advanced techniques.

A new experimental design consisting in repeated exposure of soil microcosms to CHL or TCZ was used to evaluate (i) pesticide biodegradation, (ii) the impact on purification functions, and (iii) on the diversity of soil bacteria.

In response to repeated CHL treatments, the capability of soil microorganisms to degrade it increased. On the contrary, soil microorganisms kept a low ability to degrade TCZ. Soil purification functions were slightly (CHL) or not (TCZ) affected by pesticide treatments. However, the soil bacterial diversity changed in response to repeated exposure to both pesticides. *Nitrospira* (reducing effects) and *Lysobacter* (enhancing effects) were the two genera the most importantly modified in their abundance.

This study provides new insight in the fate and ecotoxicological impact of CHL and TCZ on
soil microorganisms.

**Keywords:** pesticide, microorganisms, microbial ecotoxicology

<footnote n="1">¹Agroécologie – Institut National de la Recherche Agronomique : UMR1347 – INRA - 17 rue Sully - BP 86510 - 21000 Dijon, France</footnote>

<footnote n="2">²AEIFORIA srl (AEIFORIA) – Fidenza, Italy</footnote>

<footnote>*Speaker</footnote>
Predictive assessment of the impact of biocidal
substances on soil microbial respiration and nitrogen
transformation

Anne Straczek\*†1 and Béatrice Chion¹

¹French Agency for Food, Environment and Occupational Health Safety. Regulated products assessment Department, – ANSES – 14 Rue Pierre et Marie Curie, 94 701 Maisons-Alfort Cedex, France

Abstract

The purpose of the Biocidal Products Regulation (BPR) is to improve the free movement of biocidal products within the Union while ensuring a high level of protection of human, animal health and the environment. The approach is based on, first the approval of the active substance (inclusion in the Union list) and afterwards the authorisation of products that contain such active substances.

For the environment, the risk is mainly assessed for the aquatic and the terrestrial com-
partments. For each compartment, the predicted environmental concentration (PEC) is
compared to the predicted no effect concentration (PNEC). The PNEC is derived from the
lowest endpoint for the compartment taken into account and the appropriate assessment
factor. Soil ecotoxicity endpoints can be provided for earthworms, plants and soil microor-
ganisms. Ecotoxicity data for soil microorganisms are often coming from two OECD Tests
Guidelines: Carbon (OECD 217) and Nitrogen (OECD 216) Transformation Tests.

These tests guidelines are also used for the risk assessment of the Plant Protection Pro-
ucts. Nevertheless, different requirements and interpretation of the results are applied for
each regulation. These differences deal with the threshold of toxicity, the number of tested
concentrations to derive the endpoints, the duration of the tests... These adaptations are
either directly proposed in the tests guidelines, or have been included in the Guidance on
the BPR (volume IV Environment, Part B).

This poster presents the OECD 216 and 217 guidelines and their adaptations for the BPR.
Moreover, the case of the assessment of microbial biocidal substance is discussed.

**Keywords:** biocide product, soil, respiration, nitrogen transformation, OECD Guidelines for the
Testing of Chemicals

<figure><img src="image_64.png" /></figure>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: Anne.STRACZEK@anses.fr</footnote>
# A pilot experimental installation to evaluate the efficiency of a pharmaceutical industry treatment plant and the resulting decrease in effluents toxicity to aquatic microbial biofilms

Vincent Tardy*†<fnref n="1" />, Chloe Bonnineau<fnref n="1" />, Agnès Bouchez<fnref n="2" />, Christophe Rosy<fnref n="1" />, Bernard Motte<fnref n="1" />, Pierric Jeannin<fnref n="3" />, and Stéphane Pesce<fnref n="1" />

## Abstract

Pharmaceutical wastewaters are usually characterized by a complex mixture of active substances and metabolites with variable characteristics and composition throughout the year. Accordingly, assessing the efficiency of pharmaceutical industry treatment plants and the resulting decrease in effluent ecotoxicity and ecological risk throughout the treatment process remains very challenging.

Microbial biofilms have been proven to be early warning natural assemblages to detect acute and long-term effects produced by toxic substances, including pharmaceuticals. Being composed of both autotrophic and heterotrophic microorganisms exhibiting a large range of sensitivity to many toxicants, biofilms are thus relevant models to assess ecotoxicological risks of effluents by studying effects on microbial structure, diversity and functions.

In this context, in collaboration with the pharmaceutical firm SANOFI, we installed a pilot system, connected to the wastewater treatment plant of a SANOFI production factory, to evaluate, throughout the treatment steps, the decrease in chronic and acute toxicity of pharmaceutical production effluents for natural biofilms. The system consists of five artificial outdoor channels, each sub-divided into three compartments to ensure replication, continuously filled with wastewater collected after secondary, tertiary and quaternary treatments, respectively, as well as with stream water collected at the immediate upstream and downstream from the discharge. The chronic and acute toxicity of effluents on natural biofilms will be evaluated by combining structural and functional analysis as well as a pollution community tolerance (PICT) approach using pharmaceutical mixtures directly extracted from passive samplers (Polar Organic Chemical Integrative Samplers, POCIS) immersed for several weeks in the different channels.

**Keywords:** Ecotoxicological risk, Microbial diversity, Microbial functions, PICT

<footnote n="1">¹Irstea UR Milieux Aquatiques, Ecologie et Pollutions (UR MALY) – Irstea – 5 rue de la Doua BP 32108 F-69616 Villeurbanne Cedex, France</footnote>

<footnote n="2">²UMR CARRTEL – Institut National de la Recherche Agronomique - INRA (FRANCE) – 75 av. de Corzent, 74203 Thonon-les-Bains, France</footnote>

<footnote n="3">³SANOFI – SANOFI Recherche – route d'Avignon 30390 ARAMON, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author:</footnote>
# Development of a new functional marker for β-triketone herbicides exposure in agricultural soils

Clémence Thiour Mauprizez<fnref n="1" />,<fnref n="2" />, Fabrice Martin-Laurent<fnref n="2" />, Christophe Calvayrac<fnref n="1" />, and Lise Barthelmebs†<fnref n="1" />

## Abstract

The β-triketone herbicides are maize selective herbicides that have been largely applied in replacement of atrazine, banned in Europe in 2004. Their mode of action lays on the inhibition of the p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the carotenoid biosynthesis.

In recent studies, we showed that within the soil bacterial community, many microorganisms possess a functional HPPD enzyme involved in tyrosine metabolism. These "non-target organisms" harbor the target of the β-triketone herbicides and consequently may be affected in response to its exposure. From this point of view, the bacterial community harboring the *hppd* gene might be a relevant marker to assess the ecotoxicological impact of β-triketone herbicides on soil bacterial functional diversity.

Within this context, the objective of our work is to check for the interest of *hppd* bacterial community as a marker of exposition and/or of impact sensitive to β-triketone herbicides. This will require the development of a molecular tool box to assess the abundance, activity and diversity of the *hppd* bacterial community in various arable soils exposed to β-triketone herbicides. The abundance and the activity of the *hppd* bacterial community will be monitored from the nucleic acids extracted directly from soils, and the diversity of the *hppd* community will be evaluated by a metagenomic study based on the high-throughput sequencing of *hppd* amplicons. Our results will lead to the selection of a set of characteristic *hppd* sequences, allowing the development of *hppd* DNA chips to assess the ecotoxicological impact of β-triketone herbicides on soil microorganisms.

**Keywords:** β-triketone herbicides, impact, ecotoxicology, marker, soil

<footnote n="1">¹Biocapteurs-Analyse-Environnement (BAE-LBBM) – Université de Perpignan Via Domitia : USR3579 – F-66860, Perpignan, France</footnote>

<footnote n="2">²Agroécologie – Institut National de la Recherche Agronomique : UMRINRA 1347, Université Bourgogne Franche-Comté, Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement – 17 rue Sully - BP 86510 - 21000 Dijon, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: barthelm@univ-perp.fr</footnote>
# Biodiversity of an opportunistic pathogen in the water cycle

Florian Vautrin*¹

¹Laboratoire d'Ecologie Microbienne (LEM) – Université Claude Bernard - Lyon I (UCBL) – 16 rue Raphael Dubois, 69100 Villeurbanne, France

## Abstract

Rising urbanization waterproofs cities. To avoid flooding, many infrastructures have been set up to manage pluvial water flows, whose infiltration basins. Rainwater loads on dust and pollutants during its passage in the atmosphere and on the ground. These particles rich in pollutants such as PAH, PCB, pesticides... end up on the surface of the infiltration basin to form the so-called "urban sediments". This represents a new ecological niche for microorganisms, such as *Nocardia*, which are able to degrade these compounds and use it as carbon or nitrogen sources for its growth.

*Nocardia* is an actinomycete opportunistic pathogen which causes nocardiosis, a granulomatous and suppurative infection and would affect about 1 million persons each year around the world. The target patients are mainly immunocompromised.

The aim of this study was to assess the impact of human activity on the enrichment and biodiversity of the pathogenic actinobacteria in anthropised environment such as infiltration basins.

The high rate of pollution in anthropised environment lead to a modification of the bacterial communities in enhancing the proliferation of some bacterial species, because of their ability to integrate pollutants in their metabolism. Urban sediments constitute a new ecological niche for some pathogenic species, and so concentrate these bacteria and increase the risk of their dissemination in the environment.

**Keywords:** Infiltration basin, biodiversity, bacterial community, opportunistic pathogen, urban pollution

<footnote>*Speaker</footnote>
Identification, isolation and purification of two toxic
Cyanobacteria of Al-Mactaa lake (Mostaganem, west
Algeria)

Aicha Zellal*†1 and Sidi Mohammed El-Amine Abi-Yad¹

¹Laboratory of Aquaculture Bioremediation (AquaBior) – Department of Biotechnology (I.G.M.O campus). Faculty of Natural Sciences and Life, University of Oran, BP 1524, EL M'Naouer - 31000 Oran., Algeria

Abstract

The frequent dominance of eutrophic freshwaters by cyanobacteria (blue-green algae) is of increasing concern because these organisms can produce several types of potent toxins (cyanotoxins). This paper presents the first data on the identification, isolation and purification of some cyanobacteria, collected from a eutrophic lac (Al-Mactaa, Mostaganem, west Algeria) from July to August 2014 and isolated strain cultivated under laboratory conditions. Images of the cyanobacteria specimens were subsequently analyzed and matched with cyanobacteria in authoritative references and literatures on cyanobacteria taxonomy. The result of the taxonomical analysis showed many different species of cyanobacteria like: Anabaena sp, phormidium-chalybeum, Spirulina sp, Oscillatoria sp, Calothrix sp, Microcystis sp, Chroococcus turgidus, Merismopedia sphagnicola, Aphanocapsa sp. Culture samples of cyanobacteria on the solid medium BG11 and the successive subcultures helped purify two toxic strains of cyanobacteria (Aphanocapsa sp and Anabaena sp), cells were harvested by centrifugation, frozen, freeze-dried and kept at -20°C until further use.

**Keywords:** Cyanobacteria, Eutrophic lake, Aphanocapsa sp., Anabaena sp., West Algeria

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: zellalaicha@yahoo.fr</footnote>
Interaction between dark-septate-endophytes and
ectomycorrhizal fungi in the context of trace-element
contaminated sites

Charlotte Berthelot¹, Julie Foulon², Thierry Béguiristain¹, Michel Chalot², Corinne Leyval¹, and Damien Blaudez*†¹

¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360,
Université de Lorraine – Faculté des Sciences et Technologies, Université de Lorraine F-54506
VANDOEUVRE-LES-NANCY, France

²Laboratoire Chrono-environnement (LCE) – Centre National de la Recherche Scientifique : UMR6249,
Université de Franche-Comté – UFR Sciences et Techniques - 16, route de Gray - 25030 Besançon
Cedex, France

Abstract

Dark septate endophytes (DSE) are ascomycetous fungi that colonize living plant without causing apparent negative effects. They are characterized by melanized hyphae and microsclerotia within plant roots. High tolerance of DSE to metal pollution and their relatively high abundance in contaminated habitats suggest that DSE might have an important function for host survival in these conditions. Therefore, in the context of phytoremediation assisted by symbiotic fungi, we studied this group of fungi into more details. We first isolated seven DSE strains from poplar roots from metal-polluted sites. Fungal isolates were identified as members of the *Phialophora*, *Phialocephala*, *Cadophora* and *Leptodontidium* genera. A screening of the strains was performed to select the best promising symbiont. Their were characterized for their plant-growth-promoting abilities through different tests such as production of indol-3-acetic acid, release of volatile organic compounds or production of antifungal compounds. For the most promising strains, an inoculation experiment was performed to monitor the effect of the fungi on the growth of poplar and birch on metal-contaminated soils. Moreover, we also investigated the interaction between DSE and endomycorrhizal fungi through a dual-inoculation of host plant. Finally the use of DSE in the phytomanagement of metal-contaminated soils will be discussed.

**Keywords:** Phytomanagement, endophyte, trace, elements, plant growth promoting abilities

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: damien.blaudez@univ-lorraine.fr</footnote>
# Impacts of elevated ozone and temperature on wheat rhizosphere microbial communities

Frédérique Changey¹, Matthieu Bagard¹, and Thomas Lerch¹*

¹Institut d'Ecologie et des Sciences Environnementales de Paris (IEES-Paris) – CNRS : UMR7618 – Université Paris-Est Créteil Val de Marne (UPEC) Faculté de sciences et technologie Bâtiment P 61, avenue du Général de Gaulle 94010 Créteil Cedex, France

## Abstract

Ozone (O3) has increased significantly in recent decades as the result of anthropogenic activities. This gas is responsible for reductions in crop yields and plant biomass production of forest and grassland ecosystems. Rhizosphere microorganisms are in close relation with plant roots. They feed organic compounds released by roots and, as a feed-back, they are involved in improvement of plant fitness. Little is known on the impact of elevated O3 on rhizosphere microbial communities. Our objectives were to access O3 and temperature impacts on wheat physiology, soil parameters and rhizosphere microbial communities. The experiment consisted on a 3 week fumigation with or without O3 (70ppb) of two different varieties of wheat (Premio and Soissons) at two temperatures (20°C and 30°C). Samples without plant were used as controls. The effect of O3 was measured on plant physiological parameters (biomass, CO2 assimilation...), on soil parameters (pH and DOC). Microbial communities' structure, abundance and functions were evaluated by T-RFLP, qPCR and MicroRespTM techniques. The results show a significant effect of ozone on plants (in particular by reducing their root biomass) in interaction with temperature (Premio was less resistant to O3 at 30°C). Shifts in the microbial communities' structure were also observed under elevated ozone conditions, with a reduction of bacteria and increase of fungi. These changes were largely shaped by plant physiology and soil properties including pH and DOC. This study provides new insight into our understanding of the influence of O3 and temperature on the interaction between plants and soil microbial communities.

**Keywords:** Ozone, Temperature, Rhizosphere, microbial communities

<footnote>*Speaker</footnote>
Interactive impacts of silver and phosphorus on
autotrophic biofilm elemental and biochemical
quality for a macroinvertebrate consumer

Michaël Danger*<fnref n="1" />, Alexandre Bec<fnref n="2" />, Clément Crenier<fnref n="1" />, Vincent Felten<fnref n="1" />, Jessica Ferriol<fnref n="3" />, Aridane Gonzalez<fnref n="4" />, Fanny Perrière<fnref n="2" />, Loïc Ten-Hage<fnref n="3" />, and Joséphine Leflaive<fnref n="3" />

Abstract

Autotrophic biofilms are fundamental biological compartments of many aquatic ecosystems, representing notably a major resource for many invertebrate consumers. To date, most studies have tried to understand the impacts of stressors on microbial communities or on functional processes taking place into the biofilm mat. Far less studies investigated the indirect effects of stressors on upper trophic levels through alterations of the quality of biofilms. We investigated, through a laboratory study, the single and combined effects of phosphorus (P) availability and silver contamination on the elemental (C:N:P ratios) and biochemical (fatty acid profiles) compositions of a diatom-dominated biofilm. We hypothesized that 1) P would enhance the elemental quality while 2) P and silver, through the replacement of diatoms by more tolerant primary producer species, would reduce the biochemical quality of biofilms for their consumers. The quality of biofilms for consumers was assessed for a common crustacean species, *Gammarus fossarum*, by measuring organisms survival and growth rates. Results mainly showed that species replacement induced by both stressors affected biofilm fatty acid compositions, and that P immobilization permitted to achieve low C:P biofilms, whatever the level of silver contamination. Gammarids growth and survival were not significantly impacted by the ingestion of silver-contaminated resource. On the contrary, we found a significant positive relationship between the biofilm P-content and gammarids growth, while biofilm fatty acid contents were unrelated to this parameter. This study underlines the large indirect consequences stressors could play on basal resources quality for consumers, and, in turn, on the whole food web.

**Keywords:** Diatoms, fatty acids, ecological stoichiometry, Gammarus fossarum

<footnote n="1">¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – Université de Lorraine,
Centre National de la Recherche Scientifique : UMR7360 – 15 Avenue du Charmois, 54500
Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="2">²Laboratoire Microorganismes: Génome et Environnements, UMR CNRS 6023 (LMGE) – Université Clermont Auvergne – France</footnote>

<footnote n="3">³Laboratoire Ecologie Fonctionnelle et Environnement (EcoLab) – CNRS : UMR5245, Observatoire Midi-Pyrénées, PRES Université de Toulouse, Université Paul Sabatier (UPS) - Toulouse III, Institut National Polytechnique de Toulouse - INPT – 118 Route de Narbonne 31062 Toulouse, France</footnote>

<footnote n="4">⁴Institut Universitaire Européen de la Mer (IUEM) – CNRS : FR2195, Université de Bretagne Occidentale (UBO) – Technopôle Brest-Iroise, Place Copernic, 29280 Plouzané, France</footnote>

<footnote>*Speaker</footnote>
# Carbon nanotubes shape the bacterial communities associated with the diatom Nitzschia linearis

Yehia El-Temsah, Eric Pinelli¹, Laurent Verneuil¹, Jérôme Silvestre¹, Florence Mouchet¹, Laury Gauthier¹, and Maïalen Barret*†¹

¹Laboratoire Ecologie Fonctionnelle et Environnement (EcoLab) – CNRS : UMR5245, Observatoire Midi-Pyrénées, PRES Université de Toulouse, Université Paul Sabatier (UPS) - Toulouse III, Institut National Polytechnique de Toulouse - INPT – 118 Route de Narbonne 31062 Toulouse, France

## Abstract

Carbon nanotubes (CNTs) are nanomaterials with a wide range of current and developing applications. Their increasing use raises the question of their ecotoxicological impact. In aquatic ecosystems, diatoms account for more than 25% of primary productivity. Diatoms are intimately associated with bacterial communities in benthic biofilms. Diatoms-Bacteria interactions condition the carbon and nutrient cycling. Here, the impact of CNTs on a model ecosystem was assessed. This ecosystem was sampled in the Garonne river, simplified by cultural methods, and was finally constituted by the diatom *Nitzschia linearis* and a bacterial consortium. This model ecosystem was exposed to CNTs at 0, 0.1, 1 and 10 mg/L. After 6 days, cells were harvested either by filtration at 0.45 µm (to collect all bacteria and diatoms), or by filtration at 5µm (diatoms/"attached" bacteria) followed by filtration at 0.45 µm (planktonic bacteria). DNA was extracted from these filters to measure the abundance of bacteria (qPCR) and to determine the structure of bacterial communities (high-throughput sequencing). A negative effect of CNTs on the abundance of bacteria was evidenced at 50mg/L. Only the planktonic fraction was significantly affected. Besides, CNTs exposure modified the structure of bacterial communities in this planktonic fraction from 1 mg/L. While species affiliated to Bacteroidetes phylum were dominant in planktonic community of control incubations, they were strongly lower at 1 and 10 mg/L. On the contrary, the Proteobacteria phylum increased. These results demonstrate that CNTs impact the bacterial communities at relatively low concentration, with potential disruption of their functions in aquatic ecosystems.

**Keywords:** Diatoms, bacteria, nanoparticle, 16S rRNA, metabarcoding

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: maialen.barret@ensat.fr</footnote>
# Potential use of a biopolymer extracted from marine waste as antifungal agent

Hassiba Laribi-Habchi*†¹ and Ibtissem Galizia¹

¹Functional analysis laboratory of chemical processes – University of Blida 1, Algiers, Algeria

## Abstract

For a long time, agriculture across the world was dependent on pesticides. Today, it is influenced by a movement that favours practices that are more sustainable and environmentally safe. To meet these new demands, producers wishing to exploit natural resources and make them profitable must turn to new agronomic practices that combine culture performance and protection at a low environmental cost. In this context, the development of biological polymer able to protect our natural resources is a strategy that is attracting more attention. Natural and healthy for the environment since it comes from the shells of shell-fish, polymer of chitosan is recognized in several fields and areas of activity, especially for its antifungal properties. Currently used extensively in agriculture, it is possible to think that chitosan can also be used in forestry to meet several needs. This study helps to highlight the antifungal properties of chitosan at different concentrations against *Aspergillus Niger*. The results show a highly significant inhibition at dose of 1 g / l.

**Keywords:** Chitosan, Organic polymer, Environment

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: larhassiba@yahoo.fr</footnote>
# Gastrointestinal microbial community changes in Atlantic cod (Gadus morhua) exposed to crude oil

Daniela Pampanin*<fnref n="1" />,<fnref n="2" />, Andrea Bagi†<fnref n="1" />,<fnref n="2" />, Even Riiser<fnref n="3" />, Hilde Molland<fnref n="2" />, Bastiaan Star<fnref n="3" />, Thomas Haverkamp<fnref n="3" />, and Magne Sydnes<fnref n="2" />

## Abstract

Expansion of offshore oil exploration increases the risk of marine species being exposed to oil pollution in currently pristine areas. The adverse effects of oil exposure through toxic properties of polycyclic aromatic hydrocarbons have been well studied in Atlantic cod (Gadus morhua). Nevertheless, the fate of conjugated metabolites in the intestinal tract and their effect on the diversity of intestinal microbial community in fish is less understood. Here, we investigated the intestinal microbial community composition of Atlantic cod after 28 days of exposure to crude oil (concentration range 0.0-0.1 mg/L). Various evidence for altered microbial communities was found in fish exposed to high (0.1 mg/L) and medium (0.05 mg/L) levels of oil concentration. First, altered banding patterns were observed on denaturing gradient gel electrophoresis for samples pooled from each treatment group. Secondly, based on 16S rRNA sequences, higher levels of oil exposure were associated with a loss of overall diversity of the gut microbial communities. Furthermore, 8 operational taxonomic units (OTUs) were found to have significantly different relative abundances in samples from fishes exposed to high and medium oil concentrations when compared to samples from fishes exposed to low oil concentration or no oil. Among these, only one OTU, a Deferribacterales, had increased relative abundance in samples from fish exposed to high oil concentration. The results presented here contribute to a better understanding of the effects of oil contamination on the gut microbial community changes in fish and highlight the importance of further studies into the area of host-microbiome interactions.

**Keywords:** gut microbiota, Atlantic cod, oil contamination

<footnote n="1">¹International Research Institute of Stavanger (IRIS-Environment) – N-4070, Stavanger, Norway</footnote>

<footnote n="2">²Department of Mathematics and Natural Sciences – University of Stavanger, N-4036, Stavanger, Norway</footnote>

<footnote n="3">³Centre for Ecological and Evolutionary Synthesis – Department of Biosciences, University of Oslo, PO Box 1066, Blindern, N-0316 Oslo, Norway</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: Andrea.Bagi@iris.no</footnote>
<figure><img src="image_65.png" /></figure>
Participant list

<table><thead><tr><td>Name</td><td>Affiliation</td><td>Country</td><td>e-mails</td></tr></thead><tbody><tr><td>Souhila Alouache</td><td>ENSSMAL</td><td>Algeria</td><td>alouache.enssmal@gmail.com</td></tr><tr><td>Beatrice Alpha-Bazin</td><td>Li2D</td><td>France</td><td>beatrice.alpha-bazin@cea.fr</td></tr><tr><td>Nathalie Amacker</td><td>Unine</td><td>Switzerland</td><td>nathalie.amacker@gmail.com</td></tr><tr><td>Pornanong Aramwit</td><td>Chulalongkorn Univ.</td><td>Thailand</td><td>aramwit@gmail.com</td></tr><tr><td>Maria Argudo</td><td>Girona Univ.</td><td>Spain</td><td>maria.argudo@udg.edu</td></tr><tr><td>Jean Armengaud</td><td>Li2D</td><td>France</td><td>jean.armengaud@cea.fr</td></tr><tr><td>Charlotte Arnal</td><td>Veolia</td><td>France</td><td>charlotte.arnal@veolia.com</td></tr><tr><td>Muhammad Arslan</td><td>UFZ</td><td>Germany</td><td>arsilan324@gmail.com</td></tr><tr><td>Joan Artigas</td><td>LMGE</td><td>France</td><td>joan.artigas_alejo@uca.fr</td></tr><tr><td>Andrea Bagi</td><td>IRIS</td><td>Norway</td><td>andrea.bagi@iris.no</td></tr><tr><td>Louise Barberis</td><td>LEHNA</td><td>France</td><td>louise.barberis@univ-lyon1.fr</td></tr><tr><td>Maïalen Barret</td><td>Ecolab</td><td>France</td><td>maialen.barret@ensat.fr</td></tr><tr><td>Diana Barros</td><td>CBMA</td><td>Portugal</td><td>dianamcbarros@gmail.com</td></tr><tr><td>Lise Barthelmebs</td><td>BAE</td><td>France</td><td>barthelm@univ-perp.fr</td></tr><tr><td>Isabelle Batisson</td><td>LMGE</td><td>France</td><td>isabelle.batisson@uca.fr</td></tr><tr><td>Patrick Baudy</td><td>Koblenz-Landau Univ</td><td>Germany</td><td>baudy@uni-landau.de</td></tr><tr><td>Christine Bazin</td><td>INSAVALOR</td><td>France</td><td>christine.bazin@provademse.com</td></tr><tr><td>Fabrice Bedin</td><td>APAVE</td><td>France</td><td>fabrice.bedin@apave.com</td></tr><tr><td>Aissa Belouatek</td><td>Abdelhamid Ibn Badis Univ.</td><td>Algeria</td><td>abelouatek@gmail.com</td></tr><tr><td>Annette Berard</td><td>EMMAH</td><td>France</td><td>annette.berard@inra.fr</td></tr><tr><td>Emmanuelle Bergeron</td><td>Ecologie Microbienne</td><td>France</td><td>emanuelle.bergeron@univ-lyon1.fr</td></tr><tr><td>Cecile Bernard</td><td>MCAM</td><td>France</td><td>cbernard@mnhn.fr</td></tr><tr><td>Charlotte Berthelot</td><td>LIEC</td><td>France</td><td>charlotte.berthelot@univ-lorraine.fr</td></tr><tr><td>Patrick Billard</td><td>LIEC</td><td>France</td><td>patrick.billard@univ-lorraine.fr</td></tr><tr><td>Elise Billoir</td><td>LIEC</td><td>France</td><td>elise.billoir@univ-lorraine.fr</td></tr><tr><td>Julien Blaise</td><td>Eurofins</td><td>France</td><td>JulienBlaise@eurofins.com</td></tr><tr><td>Damien Blaudez</td><td>LIEC</td><td>France</td><td>damien.blaudez@univ-lorraine.fr</td></tr><tr><td>Anne-Laure Blieux</td><td>Welience</td><td>France</td><td>anne-laure.blieux@welience.com</td></tr><tr><td>Berta Bonet</td><td>GEES - Uob</td><td>UK</td><td>b.bonet@bham.ac.uk</td></tr><tr><td>Chloe Bonnineau</td><td>IRSTEA</td><td>France</td><td>chloe.bonnineau@irstea.fr</td></tr><tr><td>Philippe Bouchard</td><td>LMGE</td><td>France</td><td>philippe.bouchard@uca.fr</td></tr><tr><td>Agnes Bouchez</td><td>CARRTEL</td><td>France</td><td>agnes.bouchez@inra.fr</td></tr><tr><td>Emilie Bourgeois</td><td>Ecologie Microbienne</td><td>France</td><td>emilie.bourgeois@sfr.fr</td></tr><tr><td>Kristian Koefoed Brandt</td><td>University Of Copenhagen</td><td>Denmark</td><td>kkb@plen.ku.dk</td></tr><tr><td>Jean-François Briand</td><td>MAPIEM</td><td>France</td><td>briand@univ-tln.fr</td></tr><tr><td>Cecile Capdeville</td><td>Ecolab</td><td>France</td><td>cecile.capdeville1@univ-tlse3.fr</td></tr><tr><td>Louis Carles</td><td>Clermont Auvergne Univ.</td><td>France</td><td>louis.carles64@gmail.com</td></tr><tr><td>Fernanda Cassio</td><td>CBMA</td><td>Portugal</td><td>fcassio@bio.uminho.pt</td></tr><tr><td>Aurelie Cebron</td><td>LIEC</td><td>France</td><td>aurelie.cebron@univ-lorraine.fr</td></tr><tr><td>Virginie Chapon</td><td>CEA</td><td>France</td><td>virginie.chapon@cea.fr</td></tr><tr><td>Betty Chaumet</td><td>IRSTEA</td><td>France</td><td>betty.chaumet@irstea.fr</td></tr><tr><td>Giulia Cheloni</td><td>UNIGE</td><td>Switzerland</td><td>giulia.cheloni@unige.ch</td></tr><tr><td>Clement Coclet</td><td>PROTEE</td><td>France</td><td>clement.coclet@univ-tln.fr</td></tr><tr><td>Blanche Collin</td><td>CEREGE</td><td>France</td><td>collin@cerege.fr</td></tr><tr><td>Nathalie Coquille</td><td>IFREMER</td><td>France</td><td>nathalie.coquille@gmail.com</td></tr><tr><td>Natalia Corcoll</td><td>Gothenburg Univ.</td><td>Sweden</td><td>natalia.corcoll@gu.se</td></tr><tr><td>Benoit Cournoyer</td><td>Ecologie Microbienne</td><td>France</td><td>benoit.cournoyer@univ-lyon1.fr</td></tr><tr><td>Sophie Courtois</td><td>Suez</td><td>France</td><td>sophie.courtois@suez.com</td></tr><tr><td>Marc Crampon</td><td>BRGM</td><td>France</td><td>m.crampon@brgm.fr</td></tr><tr><td>Cristiana Cravo-Laureau</td><td>EEM</td><td>France</td><td>cristiana.cravo-laureau@univ-pau.fr</td></tr><tr><td>Aurélien Croze</td><td>Lyon Univ.</td><td>France</td><td>aurelien.croze@hotmail.fr</td></tr><tr><td>Olivier Crouzet</td><td>ECOSYS</td><td>France</td><td>olivier.crouzet@inra.fr</td></tr><tr><td>Armelle Crouzet</td><td>Agroécologie</td><td>France</td><td>armellecrouzet@gmail.com</td></tr><tr><td>Michael Danger</td><td>LIEC</td><td>France</td><td>michael.danger@univ-lorraine.fr</td></tr><tr><td>Marie-Laure Delignette-Muller</td><td>LBBE</td><td>France</td><td>ml.delignette@gmail.com</td></tr></tbody></table>
<table><thead><tr><td>Name</td><td>Affiliation</td><td>Country</td><td>e-mails</td></tr></thead><tbody><tr><td>Floriane Demailly</td><td>IRSTEA</td><td>France</td><td>floriane.demailly@irstea.fr</td></tr><tr><td>Clement Descarpentries</td><td>UCA</td><td>France</td><td>clement descarpentries@gmail.com</td></tr><tr><td>Werner Desiante</td><td>Eawag</td><td>Switzerland</td><td>werner.desiante@eawag.ch</td></tr><tr><td>Anna Dupont</td><td></td><td></td><td>annaadupont@gmail.com</td></tr><tr><td>Valentin Dupraz</td><td>IFREMER</td><td>France</td><td>valentin.dupraz@ifremer.fr</td></tr><tr><td>Robert Duran</td><td>Pau Univ.</td><td>France</td><td>robert.duran@univ-pau.fr</td></tr><tr><td>Marie-Jose Durand</td><td>GEPEA</td><td>France</td><td>marie-jose.durand-thouand@univ-nantes.fr</td></tr><tr><td>Elizabeth Edwards</td><td>Toronto Univ.</td><td>Canada</td><td>elizabeth.edwards@utoronto.ca</td></tr><tr><td>Emilie Egea</td><td>FCS Rovaltain</td><td>France</td><td>e.egea@fcsrovaltain.org</td></tr><tr><td>Talaat El Sebai</td><td>NRC</td><td>Egypt</td><td>talaatelsebai@gmail.com</td></tr><tr><td>Martin Eriksson</td><td>Chalmers Univ.</td><td>Sweden</td><td>martin.eriksson@chalmers.se</td></tr><tr><td>Sonja Fagervold</td><td>Obs Banyuls</td><td>France</td><td>fagervold@obs-banyuls.fr</td></tr><tr><td>Andrea Fanesi</td><td>LIEC</td><td>France</td><td>andrea.fanesi@univ-lorraine.fr</td></tr><tr><td>Sabine Favre-Bonte</td><td>Ecologie Microbienne</td><td>France</td><td>sabine.favre-bonte@univ-lyon1.fr</td></tr><tr><td>Stacie Flood</td><td>Raleigh Univ.</td><td>USA</td><td>flood.stacie@epa.gov</td></tr><tr><td>Laurence Fraissinet-Tachet</td><td>Ecologie Microbienne</td><td>France</td><td>fraissin@univ-lyon1.fr</td></tr><tr><td>Anna Freixa</td><td>ICRA</td><td>Spain</td><td>afreixa@icra.cat</td></tr><tr><td>Nicolas Gallois</td><td>CEA</td><td>France</td><td>nicolas.gallois@cea.fr</td></tr><tr><td>Marion Garacci</td><td>ECOLAB</td><td>France</td><td>marion.garacci@etu.ensat.fr</td></tr><tr><td>Jeanne Garric</td><td>IRSTEA</td><td>France</td><td>jeanne.garric@irstea.fr</td></tr><tr><td>Philippe Garrigues</td><td>CNRS</td><td>France</td><td>philippe.garrigues@u-bordeaux.fr</td></tr><tr><td>Jean-François Ghiglione</td><td>MAPIEM</td><td>France</td><td>ghiglione@obs-banyuls.fr</td></tr><tr><td>Nicolas Grosjean</td><td>LIEC</td><td>France</td><td>nicolas.grosjean4@gmail.com</td></tr><tr><td>Abdulsamie Hanano</td><td>AECS</td><td>Syria</td><td>ashanano@aec.org.sy</td></tr><tr><td>Alain Hartmann</td><td>Agroécologie</td><td>France</td><td>alain.hartmann@inra.fr</td></tr><tr><td>Jennifer Hellal</td><td>BRGM</td><td>France</td><td>j.hellal@brgm.fr</td></tr><tr><td>Pascaline Herbelin</td><td>EDF R&amp;D</td><td>France</td><td>pascaline.herbelin@edf.fr</td></tr><tr><td>Louis Hermon</td><td>BRGM</td><td>France</td><td>louis.hermon@etu.unistra.fr</td></tr><tr><td>Marina Hery</td><td>HSM</td><td>France</td><td>marina.hery@umontpellier.fr</td></tr><tr><td>Kevin Hidalgo</td><td>INRA</td><td>France</td><td>kevin.hidalgo@gmx.com</td></tr><tr><td>Anna Hua</td><td>GEPEA</td><td>France</td><td>anna.hua@univ-nantes.fr</td></tr><tr><td>Sabir Hussain</td><td>Government College Univ.</td><td>Pakistan</td><td>sabirghani@gmail.com</td></tr><tr><td>Pauline Jacob</td><td>Veolia</td><td>France</td><td>pauline.jacob@veolia.com</td></tr><tr><td>Pierric Jeannin</td><td>SANOFI</td><td>France</td><td>pierric.jeannin@sanofi.com</td></tr><tr><td>Dimitrios Karpouzas</td><td>Thessaly Univ.</td><td>Greece</td><td>dkarpouzas@bio.uth.gr</td></tr><tr><td>Ahmad Khodr</td><td>L'ORÉAL</td><td>France</td><td>akhodr@rd.loreal.com</td></tr><tr><td>Sandra Kim Tiam</td><td>MCAM</td><td>France</td><td>sandra.kim-tiam-fook-chong@mnhn.fr</td></tr><tr><td>Apostolos Koutsaftis</td><td>ADAMA Deutschland Gmbh</td><td>Germany</td><td>apostolos.koutsaftis@adama.com</td></tr><tr><td>Amandine Laffite</td><td>UNIGE</td><td>Switzerland</td><td>amandine.laffite@unige.ch</td></tr><tr><td>Hassiba Laribi-Habchi</td><td>Blida Univ.</td><td>Algeria</td><td>larhassiba@yahoo.fr</td></tr><tr><td>Elia Laroche</td><td>Montpellier Univ.</td><td>France</td><td>elia.laroche@umontpellier.fr</td></tr><tr><td>Floriane Larras</td><td>UFZ</td><td>Germany</td><td>floriane.larras@ufz.de</td></tr><tr><td>Martin Laviale</td><td>LIEC</td><td>France</td><td>martin.laviale.bio@gmail.com</td></tr><tr><td>Marie Le Jean</td><td>LIEC</td><td>France</td><td>marie.lejean@univ-lorraine.fr</td></tr><tr><td>Florian Lemmel</td><td>LIEC</td><td>France</td><td>florian.lemmel@univ-lorraine.fr</td></tr><tr><td>Thomas Lerch</td><td>IEES-Paris</td><td>France</td><td>thomas.lerch@u-pec.fr</td></tr><tr><td>Corinne Leyval</td><td>LIEC</td><td>France</td><td>corinne.leyval@univ-lorraine.fr</td></tr><tr><td>Emilie Lyautey</td><td>CARRTEL</td><td>France</td><td>emilie.lyautey@univ-smb.fr</td></tr><tr><td>Ayanleh Mahamoud-Ahmed</td><td>IRSTEA</td><td>France</td><td>ayanleh.mahamoud-ahmed@univ-smb.fr</td></tr><tr><td>Mohamed Maldani</td><td>Moulay Ismail Univ.</td><td>Morocco</td><td>maldani.mohamed@hotmail.com</td></tr><tr><td>Clarisse Mallet</td><td>LMGE</td><td>France</td><td>clarisse.mallet@uca.fr</td></tr><tr><td>Benjamin Marie</td><td>MCAM</td><td>France</td><td>benjamin.marie@mnhn.fr</td></tr><tr><td>Samantha Marshall</td><td>Syngenta</td><td>UK</td><td>samantha.marshall@syngenta.com</td></tr><tr><td>Fabrice Martin-Laurent</td><td>Agroécologie</td><td>France</td><td>fabrice.martin@inra.fr</td></tr><tr><td>Jean Martins</td><td>IGE</td><td>France</td><td>jean.martins@univ-grenoble-alpes.fr</td></tr><tr><td>Shiny Martis B</td><td>INSA</td><td>France</td><td>shiny mb@gmail.com</td></tr><tr><td>Michael-Thomas Marx</td><td>BAYER</td><td>Germany</td><td>michaelthomas.marx@bayer.com</td></tr></tbody></table>
<table><thead><tr><td>Name</td><td>Affiliation</td><td>Country</td><td>e-mails</td></tr></thead><tbody><tr><td>Leïla Meistertzheim</td><td>LOMIC</td><td>France</td><td>dussud@obs-banyuls.fr</td></tr><tr><td>Caroline Michel</td><td>BRGM</td><td>France</td><td>c.michel@brgm.fr</td></tr><tr><td>Jean-Michel Monier</td><td>ENOVEO</td><td>France</td><td>jm.monier@enoveo.com</td></tr><tr><td>Soizic Morin</td><td>IRSTEA</td><td>France</td><td>soizic.morin@irstea.fr</td></tr><tr><td>Christian Mougin</td><td>INRA</td><td>France</td><td>christian.mougin@inra.fr</td></tr><tr><td>Sylvie Nazaret</td><td>Ecologie Microbienne</td><td>France</td><td>sylvie.nazaret@univ-lyon1.fr</td></tr><tr><td>Julie Neury-Ormanni</td><td>IRSTEA</td><td>France</td><td>julie.neury-ormanni@irstea.fr</td></tr><tr><td>Oluwatosin Oladipo</td><td>North-West Univ.</td><td>South Africa</td><td>tosin1oladipo@gmail.com</td></tr><tr><td>Rahma Omrani</td><td>Monastir Univ.</td><td>Tunisia</td><td>rahmaomrani-114@hotmail.fr</td></tr><tr><td>Akli Ouelhadj</td><td>Mouloud Mammeri Univ.</td><td>Algeria</td><td>ouelhadj_akli@hotmail.com</td></tr><tr><td>Rosa Padilla</td><td>Ecologie Microbienne</td><td>France</td><td>ropagui@hotmail.com</td></tr><tr><td>Frederic Palais</td><td>Solvay</td><td>Belgium</td><td>frederic.palais@solvay.com</td></tr><tr><td>Daniela Pampanin</td><td>IRIS-Environment</td><td>Norway</td><td>daniela.pampanin@iris.no</td></tr><tr><td>Claudia Pascoal</td><td>CBMA</td><td>Portugal</td><td>cpascoal@bio.uminho.pt</td></tr><tr><td>Olivier Perceval</td><td>AFB</td><td>France</td><td>olivier.perceval@afbiodiverseite.fr</td></tr><tr><td>Stephane Pesce</td><td>Irstea</td><td>France</td><td>stephane.pesce@irstea.fr</td></tr><tr><td>Ines Petric</td><td>RBI</td><td>Croatia</td><td>ipetric@irb.hr</td></tr><tr><td>Wilfried Poirier</td><td>GEIHP</td><td>France</td><td>wilfried.poirier94@gmail.com</td></tr><tr><td>Thomas Pollet</td><td>INRA</td><td>France</td><td>thomas.pollet@vet-alfort.fr</td></tr><tr><td>Lorenzo Proia</td><td>ESA - ULB</td><td>Belgium</td><td>proialorenzo@hotmail.it</td></tr><tr><td>Edoardo Puglisi</td><td>UCSC</td><td>Italy</td><td>edoardo.puglisi@unicatt.it</td></tr><tr><td>Catherine Rafin</td><td>UCEIV</td><td>France</td><td>rafin@univ-littoral.fr</td></tr><tr><td>Rick Relyea</td><td>RPI</td><td>USA</td><td>relyer@rpi.edu</td></tr><tr><td>Brice Reoyo-Prats</td><td>CEFREM</td><td>France</td><td>brice.reoyo@univ-perp.fr</td></tr><tr><td>Cecile Revellin</td><td>Agroécologie</td><td>France</td><td>cecile.revellin@inra.fr</td></tr><tr><td>Agnes Richaume</td><td>Ecologie Microbienne</td><td>France</td><td>agnes.richaume@univ-lyon1.fr</td></tr><tr><td>Jennifer Rocca</td><td>Duke Univ</td><td>USA</td><td>jennifer.rocca@duke.edu</td></tr><tr><td>Francesco Romaniello</td><td>UCSC</td><td>Italy</td><td>francesco.romaniello@unicatt.it</td></tr><tr><td>Florent Rossi</td><td>LMGE</td><td>France</td><td>florent.rossi@univ-bpclermont.fr</td></tr><tr><td>Sergi Sabater</td><td>IRCA</td><td>Spain</td><td>pilar.eijo@udg.edu</td></tr><tr><td>Wilfried Sanchez</td><td>FCS Rovaltain</td><td>France</td><td>w.sanchez@fcsrovaltain.org</td></tr><tr><td>Catherine Santaella</td><td>LEMIRE</td><td>France</td><td>catherine.santaella@cea.fr</td></tr><tr><td>Mechthild Schmitt-Jansen</td><td>UFZ</td><td>Germany</td><td>mechthild.schmitt@ufz.de</td></tr><tr><td>Dorina Seitaj</td><td>Frontiers</td><td>Switzerland</td><td>dorina.seitaj@frontiersin.org</td></tr><tr><td>Claire Sergeant</td><td>CENBG</td><td>France</td><td>sergeant@cenbg.in2p3.fr</td></tr><tr><td>Marie Simonin</td><td>Duke University</td><td>USA</td><td>marie.simonin@duke.edu</td></tr><tr><td>Vera Slaveykova</td><td>Geneva Univ.</td><td>Switzerland</td><td>vera.slaveykova@unige.ch</td></tr><tr><td>Federica Spina</td><td>Turin Univ.</td><td>Italy</td><td>federica.spina@unito.it</td></tr><tr><td>Giulia Spini</td><td>UCSC</td><td>Italy</td><td>giulia.spini@unicatt.it,</td></tr><tr><td>Sabine Stachowski-Haberkorn</td><td>IFREMER</td><td>France</td><td>sabine.stachowski.haberkorn@ifremer.fr</td></tr><tr><td>Marianne Stenrod</td><td>NIBIO</td><td>Norway</td><td>marianne.stenrod@nibio.no</td></tr><tr><td>Anne Straczek</td><td>ANSES</td><td>France</td><td>anne.straczek@anses.fr</td></tr><tr><td>Jürg Oliver Straub</td><td>Hoffmann-La Roche</td><td>Switzerland</td><td>juerg.straub@roche.com</td></tr><tr><td>Rossana Sussarellu</td><td>IFREMER</td><td>France</td><td>rossana.sussarellu@ifremer.fr</td></tr><tr><td>Cyril Sweetlove</td><td>L'ORÉAL</td><td>France</td><td>csweetlove@rd.loreal.com</td></tr><tr><td>Vincent Tardy</td><td>Irstea</td><td>France</td><td>vincent.tardy@irstea.fr</td></tr><tr><td>Sasha Tetu</td><td>Macquarie Univ.</td><td>Australia</td><td>sasha.tetu@mq.edu.au</td></tr><tr><td>Clemence Thiour-Mauprizez</td><td>BAE-LBBM</td><td>France</td><td>thiourmauprizezclemence@gmail.com</td></tr><tr><td>Ahmed Tlili</td><td>Eawag</td><td>Suisse</td><td>ahmed.tlili@eawag.ch</td></tr><tr><td>Edward Topp</td><td>Agriculture And Agri-Food</td><td>Canada</td><td>ed.topp@agr.gc.ca</td></tr><tr><td>Florian Vautrin</td><td>Ecologie Microbienne</td><td>France</td><td>florianvautrin@gmail.com</td></tr><tr><td>Quentin Vincent</td><td>LSE-LIEC</td><td>France</td><td>quentin.vincent@univ-lorraine.fr</td></tr><tr><td>Stephane Vuilleumier</td><td>Strasbourg Univ.</td><td>France</td><td>eh_torabi@ut.ac.ir</td></tr><tr><td>Lukas Yvo Wick</td><td>UFZ</td><td>Germany</td><td>lukas.wick@ufz.de</td></tr><tr><td>Regina-Michaela Wittich</td><td>Frontiers</td><td>Switzerland</td><td>r.wittich@csic.es</td></tr><tr><td>Aicha Zellal</td><td>Aquabior</td><td>Algeria</td><td>zellalaicha@yahoo.fr</td></tr><tr><td>Jochen Zubrod</td><td>Koblenz-Landau Univ.</td><td>Germany</td><td>zubrod@uni-landau.de</td></tr></tbody></table>