# Exposures of soil microbial communities to realistic antibiotics concentrations: key parameters for environmental risk assessment

Olivier Crouzet, Anais Goulas, Claire-Sophie Haudin, Pierre Benoit, Alain Hartmann, Géraldine Depret, Agnès Richaume-Jolion, Thomas Pommier, Sylvie Nazaret

<figure><img src="image_1.png" /></figure>

To cite this version:

Olivier Crouzet, Anais Goulas, Claire-Sophie Haudin, Pierre Benoit, Alain Hartmann, et al.. Exposures of soil microbial communities to realistic antibiotics concentrations: key parameters for environmental risk assessment. 1ère conférence internationale d'Ecotoxicologie Microbienne, EcotoxicoMic, Nov 2017, Lyon, France. hal-01604497

HAL Id: hal-01604497

https://hal.science/hal-01604497

Submitted on 2 Jun 2020

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EcotoxicoMic 2017

1st International Conference on
Microbial Ecotoxicology

21st-24th November, 2017
Lyon, FRANCE

<figure><img src="image_4.png" /></figure>

ecotoxicoMic

The emerging Network of
Microbial Ecotoxicology

Book of Abstracts

https://ecotoxicomic.sciencesconf.org

ecotoxicomic@sciencesconf.org

<figure><img src="image_5.png" /></figure>

FONDATION ROVALTAIN

RECHERCHE EN ECO-TOXICOLOGIE

Pour un monde sans pollution
<figure><img src="image_6.png" /></figure>
# Welcome to EcotoxicoMic 2017!

We are very proud to welcome you to Lyon for the first International Conference on Microbial Ecotoxicology.

Organized by the emerging Network of Microbial Ecotoxicology (EcotoxicoMic) & the Rovaltain Foundation, the conference will focus on the main research advances on Microbial Ecotoxicology, which is an emerging branch of Science at the crossroad between microbial ecology, microbial toxicology, ecotoxicology, physics and chemistry.

This event will gather for the first time more than 160 microbial ecotoxicologists representing 23 countries from around the world and belonging for both the private and academics sectors. It will address important issues related to increasing demands from worldwide politics and society rising with the threats on environmental and human health caused by intense anthropogenic activities.

Thanks to the support of our many partners, the quality of the presentations and the presence of world-renowned speakers we hope that this event will be a success and will start a long series of passionate conferences dedicated to Microbial Ecotoxicology!

We wish you a very fruitful conference with numerous exciting communications, discussions and prospects of collaboration and a very pleasant stay in Lyon.

Stéphane Pesce, Irstea
Co-Funder of the EcotoxicoMic Network

Philippe Garrigues
President of the Rovaltain Foundation

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<figure><img src="image_8.png" /></figure>

## Who we are.

### The Emerging Interdisciplinary Network of Microbial Ecotoxicology

The EcotoxicoMic Network aims at enhancing visibility and structuring microbial ecotoxicology community by developing transverse actions between studied ecosystems and researchers belonging to various research organizations.

<figure><img src="image_9.png" /></figure>

ecotoxicMic

### Rovaltain Foundation

Created in July 2013 by the Ministry of Higher Education and Research, the Rovaltain Foundation's main purpose is to support high-level scientific research by promoting interdisciplinary research, particularly in the fields of environmental toxicology and ecotoxicology.

<figure><img src="image_10.png" /></figure>
<table><caption>Programme</caption><thead><tr><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Tuesday November 21</td><td></td><td></td><td></td></tr><tr><td>08:40 - 09:00</td><td></td><td></td><td></td></tr><tr><td>09:00 - 09:20</td><td></td><td></td><td></td></tr><tr><td>09:20 - 09:40</td><td></td><td></td><td></td></tr><tr><td>09:40 - 10:00</td><td></td><td></td><td></td></tr><tr><td>10:00 - 10:20</td><td></td><td></td><td></td></tr><tr><td>10:20 - 10:40</td><td></td><td></td><td></td></tr><tr><td>10:40 - 11:00</td><td></td><td></td><td></td></tr><tr><td>11:00 - 11:20</td><td></td><td></td><td></td></tr><tr><td>11:20 - 11:40</td><td></td><td></td><td></td></tr><tr><td>11:40 - 12:00</td><td></td><td></td><td></td></tr><tr><td>12:00 - 12:20</td><td></td><td></td><td></td></tr><tr><td>12:20 - 12:40</td><td></td><td></td><td></td></tr><tr><td>12:40 - 13:00</td><td></td><td></td><td></td></tr><tr><td>13:00 - 13:15</td><td></td><td></td><td></td></tr><tr><td>13:15 - 13:40</td><td></td><td></td><td></td></tr><tr><td>13:40 - 14:00</td><td></td><td></td><td></td></tr><tr><td>14:00 - 14:20</td><td></td><td></td><td></td></tr><tr><td>14:20 - 14:40</td><td></td><td></td><td></td></tr><tr><td>Wednesday November 22</td><td></td><td></td><td></td></tr><tr><td>Keynote Conference<br>How do aquatic microbes respond to emergent contaminants?<br>F.CASSIO</td><td></td><td></td><td></td></tr><tr><td>Gallois (Gallois et al. Insights into uranium tolerance of Microbacterium oleivorans A9 by proteogenomic analyses)</td><td></td><td></td><td></td></tr><tr><td>Meistertzheim (Dussud et al. Influence of plastic litters on marine microbial life)</td><td></td><td></td><td></td></tr><tr><td>Coffee Break</td><td></td><td></td><td></td></tr><tr><td>Chaumet (Chaumet et al. Transfer and distribution of diuron in biofilms and joint toxic effects)</td><td></td><td></td><td></td></tr><tr><td>Cheloni (Cheloni et al. Morphological plasticity in Chlamydomonas reinhardtii: an adaptive response to micropollutant stress)</td><td></td><td></td><td></td></tr><tr><td>Grosjean (Grosjean et al. Genome-wide analyses of rare earth elements responsive genes in microbial model organisms)</td><td></td><td></td><td></td></tr><tr><td>Poster Corner session (N cycle, bioremediation, genomic)</td><td></td><td></td><td></td></tr><tr><td>Poster session</td><td></td><td></td><td></td></tr><tr><td>Lunch</td><td></td><td></td><td></td></tr><tr><td>Simonin (Simonin et al. Novel nanopesticides introduce new challenges to microbes in agro-ecosystems and downstream wetlands)</td><td></td><td></td><td></td></tr><tr><td>Lemmel (Lemmel et al. Diversity and functions of microbial communities in historically multi-contaminated soils)</td><td></td><td></td><td></td></tr><tr><td>OPENING SESSION</td><td></td><td></td><td></td></tr><tr><td>Opening Conference<br>Microbial ecotoxicology : Past, present, and future,<br>E. TOPP</td><td></td><td></td><td></td></tr><tr><td>Brandt (Brandt- Improving ecotoxicological assessment of chemicals using soil microbial communities)</td><td></td><td></td><td></td></tr></tbody></table>
# Detailed poster corners programme

Wednesday, November 22, 2017 @ 11:40-12:20

## Bioremediation

* Bioremediation of an aged dioxins and furans contaminated soil by saprotrophic telluric fungi and a mild chemical oxidation, Isabelle Delsarte

* On-farm bioremediation of leftover pesticide solutions using biobed, Talaat Elsebai

* Dichloromethane degradation in multi-contaminated groundwater: coupling biomolecular and compound-specific isotope analyses, Louis Hermon

* Microbiology of Acid Mine Drainage: from natural attenuation to bioremediation, Marina Héry

* Microbial resources management for bioremediation, Jean-Michel Monier

## Ecotoxicological effects on N cycle

* Metal pollution effects on ammonia oxidation, Maria Argudo

* Do plant traits matter in the microbial ecotoxicity of metal nanoparticles in soil?, Agnès Richaume

* Mixtures effect of antibiotics on soil nitrification and denitrification - an experimental and modelling approach, Olivier Crouzet

* Impact of pesticides on denitrification in groundwaters, Caroline Michel

* Long Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Bacteria Occupying Nodules of Soybeans (Glycine max), Alain Hartmann

## Genomic approaches in microbial ecotoxicology

* SYNAQUA: A French-Swiss program to modernize the environmental biomonitoring of Lake Geneva and transboundary rivers, Agnès Bouchez

* Environmental persistence of pharmaceutical residues from hospital and urban effluents and their impacts on environmental aquatic microbial communities, Agnès Bouchez

* Shifts in planktonic and biofilm prokaryotic community composition along a chemical multicontamination gradient in a north-western coastal mediterranean environment (Toulon bay, France), Clément Coclet

* Combination of high-throughput sequencing and geochemistry methods for evaluating the impact of single and multi-metal pollutions in soil at the aggregate scale, Jean Martins
Thursday, November 23, 2017 @ 11:40-12:20

Omics approaches in microbial ecotoxicology

* Quick metaproteomics for environmental risk assessment, Jean Armengaud

* From the screening of soil eukaryotic metatranscriptomes to the functional characterization of new metallothioneins, Laurence Fraissinet-Tachet

* Volatolomics in microbial ecotoxicology, a pathway for detecting metabolic signatures of exposure to xenobiotics, Kevin HIDALGO

Innovative microbial biomarkers and bioassays for environmental risk assessment

* Development of a standardized aquatic hyphomycete laboratory assay for fungicide risk assessment, Patrick Baudy

* Integrated biomarker and ecological response (IBER) index, Helena Guasch

* Microbial molecular and isotopic biomarkers for evaluating natural attenuation of chlorinated hydrocarbons in multi-contaminated sites, Jennifer Hellal

* High throughput microbial array for complex environmental sample assessment, Anna Hua
## Conference Dinner

The conference dinner will be held on Thursday evening, 23rd November. The organizing committee invites you to admire the most beautiful urban landscapes aboard the Hermès restaurant boat of the Lyon City Boat company!

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## PROGRAM

### DEPARTURE & RETURN

Boarding time at 8:00 pm
In front of the 16 dock Claude Bernard, Lyon

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DON'T BE LATE!!!

Left bank-side of the Rhône at the foot of the University bridge

Departure at 8:30 pm – Dinner served on board

Return around 11:00 pm

During the ILE BARBE TOUR, we will sail along the Rhône docks to the confluence neighborhood where the Saône & the Rhône meet. The travel will stretch through the historic city & up to the Barbe island.

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## Partners

### Platinum Partners

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**Irstea**

The National Research Institute of Science and Technology for Environment and Agriculture is a research organization which, since more than 30 years, has been working on major issues of a responsible agriculture and territory sustainable planning, water management and related risks, drought, floods, inundations, the biodiversity and complex ecosystem study in their interrelation with human activities.

**Auvergne Rhône Alpes Region**

With major national research centres based in the region, 14 competitivity hubs and 18 clusters, Auvergne-Rhône-Alpes files the second largest number of patents in France, and has huge potential for innovation. Very much open to forging international ties, the Auvergne-Rhône-Alpes region has over the years made connections with other areas, both in Europe and across all continents – thus supporting international projects, ensuring its citizens – In particular young people – are free to move around, and encouraging regional businesses to spread their knowledge and experience.

<figure><img src="image_15.png" /></figure>

### Gold Partners

AGENCE FRANÇAISE
POUR LA BIODIVERSITÉ

ÉTABLISSEMENT PUBLIC DE L'ÉTAT

**AFB**

The French Agency for Biodiversity is a public institution of the Ministry of the Environment, Energy and Sea. It supports the implementation of public policies in the fields of knowledge, preservation, management and restoration of biodiversity of terrestrial, aquatic and marine environments. It supports public actors but also works in close partnership with socio-economic actors. It also aims to reach out to the public to mobilize citizens in favor of biodiversity.

**IRD**

A crucial player in providing scientific advice on the major challenges of development. The Institut de recherche pour le développement, an internationally recognised multidisciplinary organisation, is a French public research establishment operating under the joint authority of the French Ministry for Higher Education, Research and Innovation and the French Ministry for Europe and Foreign Affairs. It takes an original approach to research, expertise, training and knowledge-sharing for the benefit of countries and regions, making science and innovation key drivers in their development.

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INRA
SCIENCE & IMPACT

## INRA

The French National Institute for Agricultural Research is Europe's top agricultural research institute and the world's number two centre for the agricultural sciences. Its scientists are working towards solutions for society's major challenges for food, nutrition, agriculture and the Environment.

The Department Environment and Agronomy of INRA is supporting the 1st international congress in Microbial Ecotoxicology as one of his actions to encourage new and innovative researches to monitor the impact of agricultural practices on natural resources.

## ECOTOX, the INRA's network of ecotoxicologists

Set up in 2009, ECOTOX, INRA's network of ecotoxicologists, has since broadened to include the French community active in this field and currently involves around 150 members from some twenty INRA research units and from external partners (INERIS, IRSTEA, IRSN, CNRS, CEA, MNHN, universities and specialized schools, etc.).

<figure><img src="image_18.png" /></figure>

## BRGM, the French geological survey

The Bureau de recherches géologiques et minières is France's reference public institution for Earth Science applications in the management of surface and subsurface resources and risks.

<figure><img src="image_19.png" /></figure>

Géosciences pour une Terre durable

brgm

## Ifremer

The Institut français de recherche pour l'exploitation de la mer is an oceanographic institution in France. Ifremer contributes, through studies and expert assessments, to the knowledge of the oceans and their resources, the monitoring of marine environment and the coastline and sustainable development of maritime activities.

<figure><img src="image_20.png" /></figure>

Ifremer

## Frontiers

Frontiers is a community-rooted, open-access academic publisher. Our grand vision is to build an Open Science platform where everybody has equal opportunity to seek, share and generate knowledge, and that empowers researchers in their daily work. **Frontiers in Microbiology** is the #2 most cited open-access journal in the category of Microbiology, and overall the #8 most cited journal in Microbiology.

<figure><img src="image_21.png" /></figure>

frontiers

## Bronze Partners

<figure><img src="image_22.png" /></figure>

## CNRS

The National Center for Scientific Research, or CNRS, is a public organization under the responsibility of the French Ministry of Education and Research. As the largest fundamental research organization in Europe, the CNRS carries out research in all fields of knowledge, through its ten institutes.
## Genoscreen

GenoScreen is a French biotech company. Thanks to its 30 highly experienced staff GenoScreen offers innovative services to public and private sector research groups and develops dedicated solutions based on DNA/RNA information. Our innovation strategy has generated a stream of successful projects, including top-level expertise in the characterization of microorganisms. GenoScreen supports companies of the environmental sector in various fields of application: water and air treatment, diagnostic and decontamination of polluted soils, monitoring of microbial biodiversity and ecosystem and biosafety.

## FST Lyon 1

La Faculté des Sciences et Technologies est une composante de l'Université Claude Bernard Lyon 1. Elle regroupe l'ensemble des acteurs (enseignants-chercheurs, chercheurs, personnels administratifs et techniques, étudiants) autour d'un projet de formation et de recherche en sciences et technologies à l'Université ; son activité se concentre principalement sur le campus Lyon Tech La Doua ouvert sur la ville de Villeurbanne et la métropole de Lyon.

## BioEnviS Research Federation

The FR BioEnviS is a research federation regrouping several research units from the Lyon scientific community, operating in the field of environmental biology and its implications in the field of global health (human as well as animal or plant). It includes the agronomic, veterinary or health concerns of the non-anthropized environments.

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## Microbial Ecology Laboratory

The Research Unit UMR 5557 has title and theme for Microbial Ecology, discipline at the crossroads of two major research fields: ecology and microbiology. Microbial ecology aims to know the physiology of microorganisms and the influence of their activities on other organisms and the environment in general, and, symmetrically, to clarify the impact of biotic and abiotic factors of the environment of these organisms, from the individual to the community in an ecosystem.
<figure><img src="image_24.png" /></figure>
Committees

**Scientific committee**

<figure><img src="image_25.png" /><figcaption>Jean-François Ghiglione
(CNRS)</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>Cécile Bernard
(MNHN)</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>Christophe Leboulanger
(IRD)</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>Chloé Bonnineau
(Irstea)</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>Fabrice Martin-Laurent
(INRA)</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Virginie Chapon
(CEA)</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>Jean-Michel Monier
(ENOVEO)</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>Jennifer Hellal
(BRGM)</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>Stéphane Pesce
(Irstea)</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>Corinne Leyval
(CNRS)</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>Thomas Pollet
(INRA)</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>Sylvie Nazaret
(CNRS)</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>Wilfried Sanchez
(FCS Rovaltain)</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>Agnès Richaume
(Univ. Lyon)</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>Stéphane Vuilleumier
(Univ. Strasbourg)</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>Sabine Stachowski-
Haberkorn
(IFREMER)</figcaption></figure>

**Local organizers**

Chloé Bonnineau, Irstea

Emilie Egea, FCS Rovaltain

Jean-Michel Monier, ENOVEO

Sylvie Nazaret, CNRS

Stéphane Pesce, Irstea

Agnès Richaume, University of Lyon

Wilfried Sanchez, FCS Rovaltain
<figure><img src="image_41.png" /></figure>
Frontiers Research Topic Publication

<figure><img src="image_42.png" /></figure>

frontiers

Frontiers, which is Gold Sponsor of ECOTOXICOMIC 2017, will edit a **Research Topic dedicated to Microbial Ecotoxicology** (Eds S. Pesce, F. Martin-Laurent, E. Topp, & JF. Ghiglione).

An exclusive special publishing discount will be offered to Conference delegates choosing to publish in the participating "Frontiers in" journals.

## Reduced Publishing Fees

Frontiers journals are Open Access and authors pay a publishing fee. For the general fee table, please visit the Frontiers website: http://www.frontiersin.org/about/PublishingFees.

Exclusively, for attendees of EcotoxicoMic 2017 Frontiers will offer a **50% discount** on the full publishing price for submissions to *Frontiers in Microbiology* and a **75% discount** to authors publishing in *Frontiers in Environmental Science*.

Authors who wish to contribute, but have not attended the conference will receive a 15% discount

## Abstract submission

Manuscript submission to the Research Topic is now opened.

Deadlines for the Research Topic are as follow:

- Abstract: 15 December 2017

- Manuscript: 15 April 2018

**The articles will be published as soon as they are accepted.**

Articles will be compiled into a free-of-charge and free-to-access *eBook*, which we can be distributed electronically.

Please do not hesitate to share this information within your networks.

More information on:

https://www.frontiersin.org/research-topics/6045/microbial-ecotoxicology
<figure><img src="image_43.png" /></figure>
## Sessions
<figure><img src="image_44.png" /></figure>
OPENING Session

Chair: Stéphane PESCE

Keynote conference: *Microbial ecotoxicology : Past, present, and future*, Dr. Edward TOPP, Agriculture and Agri-Food Canada, London Ontario, Canada.

<figure><img src="image_45.png" /></figure>

Dr. Ed Topp is a native of Montréal. He studied microbiology at McGill University, and he received his PhD in 1988 from the Department of Microbiology at the University of Minnesota. He is a Principal Research Scientist with Agriculture and Agri-Food Canada and also has adjunct appointments with the Department of Biology at Western University in London Ontario, and the Department of Soil and Water Sciences at the University of Florida in Gainesville. Dr. Topp has led several national studies that seek to better understand and to better manage the risks that food production practices pose for environmental quality and human health. This work has included terrestrial and aquatic exposure and fate assessments for numerous pharmaceuticals including various antibiotics, impacts of antibiotics on soil microorganisms, and fate of antibiotic resistance genes in soils receiving animal manures or sewage sludge. Dr. Topp is the national coordinator for the Genomics Research and Development Initiative project on antimicrobial resistance, a key component of the innovation pillar of the Canadian National AMR Action Plan. He is a former president (2011) of the Canadian Society of Microbiologists.
<figure><img src="image_46.png" /></figure>
Session BIODEGRADATION

The role of microbial communities in pollutant fate,
distribution and degradation

Chairs: Elizabeth A. EDWARDS and Corinne LEYVAL

A major challenge in microbial ecotoxicology concerns our ability to evaluate the role of microorganisms in the fate of the pollutants in the ecosystems (ecodynamic concept). Individual microorganisms were shown to degrade a large spectrum of pollutants but in the environment, the biodegradation is realized by a succession of microbial organisms. The relation between chemical evidence of biodegradation and the microbial 'degraders' is generally indirect and under-studied, which makes the prediction of the fate of pollutants in complex ecosystems difficult. Biotechnological approaches that are designed to carry out pollution remediation have received a great deal of attention in recent years, where the growing knowledge of bioremediation strategies proposed by microbial ecotoxicologists is of great interest. Biodisponibility concept is one of the challenge in biodegradation, exploring the accessibility of pollutants to biological barriers which separate microorganisms from their surrounding environment and limit their metabolization. In this session, original results facing this challenging field are welcome, and especially those at the interface between chemistry/microbiology disciplines.

**Keywords:** Bioremediation, Biodegradation, Biotransformation, Bioavailability, Metabolisation, Retroactive action, Ecological engineering

Keynote conference: *The role of microbial communities in pollutant fate, distribution and degradation*, Prof. Elizabeth A. EDWARDS, Toronto University, Canada.

<figure><img src="image_47.png" /></figure>

Dr. Elizabeth Edwards holds Bachelor's and Master's degrees in Chemical Engineering from McGill University, Montreal, and a PhD degree in Civil and Environmental Engineering from Stanford University. She is internationally known for her work on anaerobic bioremediation, the application of molecular biology and metagenomics to uncover novel microbial processes, and the transition of laboratory research into commercial practice to develop bioremediation and bioaugmentation strategies for groundwater pollutants. She is also the founding director of BioZone, a Centre for Applied Bioscience and Bioengineering Research at the University of Toronto and a Tier 1 Canada Research Chair in Anaerobic Biotechnology. In 2016, she was awarded the Canada Council of the Arts Killam Prize in recognition of outstanding career achievements.
<figure><img src="image_48.png" /></figure>
Session FUNCTION

Impact of pollutants on microbial functions

Chairs: Fernanda CASSIO and Ed TOPP

Microbial communities support a wide range of functions in ecosystems (e.g. primary production in waters, denitrification in soils and sediments...) and thus play a key role in biogeochemical cycles. Therefore the effects of pollutants observed at microbial level can have consequences at higher level of biological organization and alter ecosystem functions. The plasticity of microbial communities supports their adaptative response to the selective pressure exerted by pollutants. Functional indicators of adaptation (e.g. tolerance acquisition) have been used as indicators of contaminant effects and linked to changes in microbial structures. During the last decade, omics techniques (metatranscriptomics, metabolomics, metaproteomics) have been widely applied to complete the information given by classical indicators.

This session will then focus on the impact of contaminants on microbial functions and the consequences at different scales: from cellular biochemical pathway to biogeochemical cycles and ecosystem functions. To cover these different scales, we invite presentations of lab studies performed at different levels of biological organization (from single-species to communities) as well as field studies. Studies at community level, exploring the impact of contaminants on community and ecosystem functions are welcome as well as studies linking functional changes with structural ones (e.g. taxonomic diversity). In this session we also intend to show the wide range of techniques developed and validated to assess microbial functions in various ecosystems, in particular this session will explore the added value of omics tools and clarify how (meta)omics tools can be combined with more classical parameters to unravel the influence of contaminants on microbial functions, especially at community level.

**Keywords:** Ecosystem functions, Biogeochemical cycles, Functional diversity, Microbial plasticity / adaptation, Link between functions and taxonomic diversity, Metatranscriptomics, metabolomics, metaproteomics

Keynote conference: *How do aquatic microbes respond to emergent contaminants?* Dr. Fernanda CASSIO, Center of Molecular and Environmental Biology, Braga, Portugal.

<figure><img src="image_49.png" /></figure>

Dr. Fernanda Cássio is an Associate Professor with habilitation at the University of Minho, in Portugal. She got her PhD in Microbiology. She is the head of the Centre of Molecular and Environmental Biology, rated as Excellent by the Portuguese Foundation for Science and Technology. She has been studying the role of decomposers of organic matter in aquatic ecosystems, with particular emphasis on microbial diversity and activity, using traditional and molecular techniques.

She is also interested in ecotoxicological effects of emergent (pharmaceuticals and nanomaterials) and persistent contaminants (metals and PHAs) in freshwaters, studying effects from cells, to populations and communities levels.
<figure><img src="image_50.png" /></figure>
## Session MULTI-STRESS

# Response of microorganisms to pollutants under multi-stress conditions

Chairs: Mechthild SCHMITT-JANSEN and Stéphane VUILLEUMIER

A major challenge in environmental risk assessment of pollutants lies in the establishment of causal relationships between chemical exposure and the resulting community response within complex ecosystems which are generally subjected to multi-stress conditions due to multiple chemical and/or physical pressures. To face such a challenge, there is a need to develop microbial ecotoxicological approaches to better predict and evaluate the interactions between multiple pollutants and between pollutants and other environmental parameters, which can modulate both the exposure and the sensitivity of microorganisms to toxicants.

This session will welcome original presentations about experimental and in situ studies aiming at assessing, predicting or modeling the effects of pollutants on microorganisms (at various biological levels) in a context of multiple stresses (e.g. exposure to multi-pollutant mixtures, combined exposure to pollutants and physical disturbance...).

**Keywords:** Pollutants vs. other environmental parameters, Exposure (including bioavailability), Multivariate statistical analysis, Modelisation

Keynote conference: *Combined chemical and non-chemical stress: towards a mechanistic understanding of stressor interactions*, Dr. Mechthild SCHMITT-JANSEN, Helmholtz Centre for Environmental Research, Leipzig, Germany.

<figure><img src="image_51.png" /></figure>

Dr. Mechthild Schmitt-Jansen received her PhD in Hydrobiology at the Brandenburg University of Cottbus, Germany in the Department of Freshwater Conservation studying plankton input and dynamics in lowland streams. She continued her work as a postdoctoral researcher in the Department Chemical Ecotoxicology and the Department Effect Propagation at the Helmholtz Centre for Environmental Research – UFZ in the research areas of community ecotoxicology and site-specific toxicity assessment of aquatic systems. Since 2005 she has been working as a senior scientist at the Department Bioanalytical Ecotoxicology - UFZ leading the research group aquatic stress ecology. Since 2011 she has been holding a teaching assignment at the Technical University Bergakademie Freiberg with lectures related to aquatic ecology and ecotoxicology.

Dr Schmitt-Jansen's research interests concern community responses to multiple stressors and contaminants with special focus on biofilm ecology. Her work is based on ecological concepts like stress/pollution-induced community tolerance (SICT/PICT). In order to understand the underlying processes of stress responses, she investigates molecular responses like metabolomics and links them to phenotypic observations including physiologically-based (photosynthesis) and ecologically-based parameters (dynamics of species traits, community tolerance). Model systems she is working with range from algal cultures to periphyton communities.
<figure><img src="image_52.png" /></figure>
Session RISK ASSESSEMENT

Microorganisms as a tool for a priori and a posteriori
environmental risk assessment

Chairs: Dimitrios KARPOUZAS and Fabrice MARTIN-LAURENT

One of the primary objectives of ecotoxicology is to contribute to the development of innovative methods and tools for environmental risk assessment, both including predictive hazard and risk assessment as well as the in situ evaluation of environmental quality and ecotoxicological impacts in contaminated environments. Despite the recognized importance of microbial communities in supporting a range of ecosystemic functions and services and their ubiquity in all ecosystems, microorganisms are only rarely considered in the a priori environmental risk assessment of pollutants and they are typically not protected by any regulations or legislations. Moreover, whereas chemical analysis remains essential to assess the nature and extent of a contamination, the use of bioindicators can provide valuable complementary information on both the impact and fate of pollutants. The ubiquity of microorganisms in the environment and their capacity to adapt in many ways to a large range of pollutants make them good candidates as exposure and effects indicators, allowing them to contribute to the a posteriori environmental risk assessment. This session will focus on the development and/or application of microbial bioindicators/biomarkers/bioassays for environmental risk assessment including pollutant availability and transfer, evaluation of environmental quality and ecological health in various ecosystems. In this session we also intend to discuss about needs and possible strategies to promote the use of microorganisms (at different levels of biological organization) in a priori and a posteriori environmental risk assessment.

**Keywords:** Model organisms, sentinel species, Bioavailability, Bioindication, Bioassays, Biosensors, Environmental Legislation, Quality guidelines, Indices, Monitoring

Keynote conference: Assessing the soil microbial ecotoxicity of pesticides: Advances, limitations and new risk assessment schemes, Dr. Dimitrios KARPOUZAS, University of Thessaly, Volos, Greece.

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Dr. Dimitrios Karpouzas studied Crop Protection in the Aristotle University of Thessaly and then he moved to UK where he was awarded an MSc in Crop Protection from the University of Reading and a PhD on the microbial degradation of pesticides by the same University in collaboration with HRI-Wellesbourne. He is currently Associate Prof. in Environmental Microbiology and Head of the Lab of Plant and Environmental Biotechnology in the Department of Biochemistry and Biotechnology, University of Thessaly. His main research focus is on the interactions of pesticides with soil microorganisms which could lead either to biodegradation of the pesticides by soil microorganisms or to toxicity effects on the soil microbial community. On the former his focus is on the metabolic pathways involved and the genes/enzymes controlling each step of the process. In recent years within the frame of a series of EU-funded projects Dr. Karpouzas focused on the development of tools and procedures for the comprehensive assessment of the toxicity of pesticides on soil microorganisms which could be the basis for a forthcoming revision of the regulatory framework regarding pesticides soil microbial toxicity assessment.
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Session TROPHIC INTERACTIONS

Impact of pollutants on the interactions among micro - or
between micro- and macro-organisms

Chairs: Rick RELYEA and Jean-François GHIGLIONE

In many ecosystems, phototrophic organisms (including microorganisms) are at the base of "green" food webs supported by primary production while heterotrophic microorganisms play a pivotal function in detritus-based ("brown") food webs, whatever the kind of ecosystems. Generally, the brown and the green pathways are not separated and microbial communities are playing a key role in the between-pathways interactions, which are a major determinant of ecosystem functioning. Microorganisms are also part of living animals and plants (the 'holobiont' and 'hologenome' concept) which are no longer viewed as autonomous entities, a concept relatively new that challenged the general life science concepts. Moreover, the life of microorganisms themselves are closely linked to nutrient availability ("bottom-up control") and to viral lysis or predation by ciliates and flagellates ("top-down control"), which control their growth and activities. Pollutants are acting at different trophic levels, and the emerging discipline of microbial ecotoxicology is now facing the challenge of evaluating the role of microbial communities in ecosystem homeostasis by considering the complex array of interactions between micro- or micro-macro-organisms and their environment under pollution pressure.

This session welcome original presentations addressing both the indirect effects of pollutants on living
organisms and the alteration of interactions between organisms in all kinds of ecosystems (fresh or
marine water, soil...) . Indirect effects could arise from modification of consumer-prey balance (top-
down) or competition for resources (bottom-up) due to direct toxic pressure against one partner in
the frame of community ecology. Interactions such as mutualism, cooperation, and symbiosis can be
modified under pollution stress, either on one or several components of the interaction. Presentations
assessing ecological modeling to predict the effects of pollutants on the interactions among micro- and
between micro-macro-organisms are also fostered.

**Keywords:** Trophic interaction, Top-down (predation, lysis) vs. bottom-up (nutrients) controls, Host-microorganisms, Microbiota, Modelisation

**Keynote conference: The complexities of pollutants and ecological interactions: Lessons from freshwater ecosystems,** Dr. Rick RELYEA, Rensselaer Polytechnic Institute, Troy, NY, USA.

<figure><img src="image_55.png" /></figure>

Rick Relyea received his PhD from the University of Michigan in 1998 and is currently the David D. Darrin '40 Senior Endowed Chair in Biology at Rensselaer Polytechnic Institute (RPI). He has spent his career working to merge the fields of ecology, evolution, and ecotoxicology using a diversity of species and a wide range of experimental venues from the laboratory to outdoor field experiments. His research group has made a number of important discoveries regarding the synergistic effects of contaminants and natural stressors and how contaminant impacts on one species can have substantial impacts on the entire ecosystem due to the pervasive interconnectedness of food webs.
<figure><img src="image_56.png" /></figure>
Session ECOTOXICOMIC

EcotoxicoMic: towards an International Network on
Microbial Ecotoxicology?

Chairs: Philippe GARRIGUES, Jean-François GHIGLIONE, Stéphane PESCE, Fabrice MARTIN-LAURENT, Wilfried SANCHEZ

**Presentations and discussion regarding the creation of an international Network on Microbial Ecotoxicology.**

The general objective of the French EcotoxicoMic Multidisciplinary Thematic Network is to improve the visibility and federate the community of microbial ecotoxicologists, with a desire to promote cross-functional actions between the ecosystems studied and between researchers belonging to different research organizations.

**What about the extension of such objectives at an International Level?**

Keynote conference: **Ecotoxicomic: A team player part of the global game in Ecotoxicology**, Dr. Philippe GARRIGUES, National Center for Scientific Research, Bordeaux, France.

<figure><img src="image_57.png" /></figure>

Dr. Philippe Garrigues is a CNRS Research Director and currently at the University of Bordeaux. His research interests are the analytical aspects (chromatographies and spectroscopies) related to the detection of organic pollutants and the environmental fate and the toxicological effects of these compounds. He has been involved in the development of biochemical markers as early warning systems for the toxicological evaluation of marine ecosystems through the coordination of large research projects supported by the DG Research (EC, Brussels). His recent research interest deal with REACH regulation and how analytical chemistry is developed for its implementation.
Dr. Garrigues has authored about 180 publications. He has been member of the Executive Committee of SETAC-Europe (1997-1999) and Chair of the EuCheMS Division «Chemistry and the Environment» (2003-2008). He is presently Deputy-Chair of the French Chemical Society (SCF) and elected member of the Executive Board of EuCheMS (European Association of Chemical societies). He is/has been member of scientific committees in French institutions (CNRS, IFREMER, INERIS, Rovaltain Foundation). He has organized various Scientific Conferences dealing with environmental chemistry and toxicology, most of them related to scientific societies (SETAC, ISPAC, EuCheMS). He has also various editor's responsibilities (Polycyclic Aromatic Compounds, Environmental Science and Pollution Research, Analytical and Bioanalytical Chemistry).

Introduction to the Round Table: **Towards an international EcotoxicoMic network for an emerging discipline facing contemporary environmental threats worlwide**
Dr. Jean-François GHIGLIONE, National Center for Scientific Research, Banyuls sur mer, France.
<figure><img src="image_58.png" /></figure>
Communication Abstracts

TALKS
# Improving ecotoxicological assessment of chemicals using soil microbial communities

Kristian K. Brandt*¹

¹Department of Plant and Environmental Sciences, University of Copenhagen – Thorvaldsensvej 40
DK-1871 Frederiksberg, Denmark

## Abstract

Current environmental risk assessment (ERA) schemes aim to protect the biodiversity and ecological functioning of ecosystems, but ecosystem protection goals are only vaguely described in current ERA guidelines. Ongoing revisions of these guidelines focus on the needs for more specific ecosystem protection goals based on the ecosystem service concept. Consequently, key ecosystem services provided by microorganisms and their key drivers must be identified and experimentally assessed in order to establish improved microbial ecotoxicological assessment protocols useful for ERA. Based on recent and ongoing work performed in my research group in collaboration with other groups in Denmark, Sweden and France, I here propose a new approach for microbial community-based toxicity testing of chemicals (toxic metals, petroleum-derived hydrocarbons, antibiotics, biocides, and fungicides) in soil. The test scheme can be carried out in soil microcosms as outlined in current OECD protocols, but with a significantly expanded set of endpoints to evaluate effects on microbial community function, community structure, and pollution-induced community tolerance while at the same time accounting for chemical fate of the test compound(s). In essence, the proposed test scheme offers a tiered, polyphasic systems approach using sensitive techniques for measurement of bacterial and fungal *in situ* growth dynamics (1st tier) and comprehensive microbial community characterization (2nd tier) to account for effects on microbial diversity and resistance development. Our results reveal that different classes of toxicants, and sometimes even closely related compounds (e.g. isothiazolinones), can affect soil microbial communities in highly contrasting ways highlighting the need for the proposed polyphasic community approach.

**Keywords:** Antibiotics, Biocides, Copper, Zinc, Pesticides, Petroleum hydrocarbons, Soil microbial ecology, Molecular community analysis, Leucine incorporation, Acetate, in, ergosterol incorporation, PICT

<footnote>*Speaker</footnote>
# Dissipation of chloroacetanilides in agricultural soil microcosms: insights from stable isotope fractionation and bacterial community response

Ehssan Torabi<fnref n="1" />, Charline Wiegert<fnref n="2" />, Stéphane Vuilleumier*<fnref n="3" />, and Gwenaël Imfeld†<fnref n="4" />

## Abstract

Studying the degradation of pesticides and their effects on microbial populations in agricultural soils promises to provide insights into predicting the fate and ecotoxicity of agrochemicals. Dissipation and biodegradation of two widely applied chloroacetanilide herbicides butachlor and S-metolachlor were evaluated in two different agricultural soils (paddy and crop) using compound-specific isotope analysis (CSIA) and 16S rRNA gene based bacterial community analysis (Illumina MiSeq). CSIA results revealed that most dissipation (80-90%) and carbon fractionation of butachlor and S-metolachlor occurred within day 0-30 (bulk= -1.8 to -3.2) and 15-60 (bulk= -2.3 to -4.8) days following pesticide treatment, respectively, regardless of soil type or treatment dose (15 and 150 µg.g-1). Half-lives determined for the two pesticides at the two application rates of 15 µg g-1 and 150 µg.g-1 ranged between 7-17 and 12-42 days, respectively. Changes in bacterial community profiles were investigated by NDMS and analysis of similarities (ANOSIM). Soil type and incubation time were the main drivers of bacterial community composition, and no significant effect of pesticide type or spiking dose could be observed (p< 0.01). In paddy soil (flooded/anoxic conditions), the bacterial community was mainly dominated by anaerobic populations of Clostridiales and sulfate/sulfur reducers such as Desulfuromonadales and Syntrophobacterales. In the crop soil (20% moisture/oxic conditions), the majority of taxa belonged to Alphaproteobacteria, Actinobacteria and Bacillales. Obtained results provide new knowledge about the biodegradation of butachlor and S-metolachlor and corresponding changes in bacterial soil communities under different conditions and pesticide application regimes.

**Keywords:** Chloroacetanilide, CSIA, enrichment factor, bacterial communities, half, life

<footnote n="1">¹Department of Plant Protection, University College of Agricultural Natural Resources, University of Tehran, Karaj - Iran</footnote>

<footnote n="2">²Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS), UMR 7517 CNRS, and Department of Microbiology, Genomics and the Environment, UMR 7156 CNRS - université de Strasbourg - France</footnote>

<footnote n="3">³Department of Microbiology, Genomics and the Environment, UMR 7156 CNRS, Université de Strasbourg, France (UMR 7156 CNRS) - université de Strasbourg - 28 rue Goethe 67000 Strasbourg, France</footnote>

<footnote n="4">⁴Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS), UMR 7517 CNRS - université de Strasbourg - France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: imfeld@unistra.fr</footnote>
# Insights into flexibility of Rhodococci in transformation of PCBs

Ines Petric*†<fnref n="1" />, Vlasta Drevenkar<fnref n="2" />, and Fabrice Martin-Laurent<fnref n="3" />

## Abstract

Perhaps one of the most known POPs, polychlorinated biphenyls (PCBs), comprise a family of about 200 related compounds. Even though the manufacture and sale of PCBs were banned decades ago, they are continuously entering environment, mainly *via* point source pollution due to inadequately managed hazardous waste sites, by leaching from electrical equipment or during treatment of contaminated wastes. Microbial biodegradation of PCBs has been a subject of intensive research but until now, most of our knowledge on PCBs metabolism come from studies carried out on *Burkholderia xenovorans* LB400 and *Rhodococcus* sp. RHA1, two PCBs degraders whose genomes have been fully sequenced. Our knowledge on enzymes involved in PCB metabolism remains fragmentary and diversity of bacterial metabolisms in transforming PCBs is highly underestimated. In order to explore the metabolic pleiotropy of the *Rhodococci* we settled an experiment aiming at monitoring the transformation of structurally different PCB congeners (2,4,4'-trichlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl and 2,4,3'-trichlorobiphenyl) in four *Rhodococci* isolated from PCB-contaminated environments. These strains showed strong ability to transform various PCBs. Transformation products were detected by gas-chromatography and identified by mass spectrometry on aliquots of microbial cultures. We propose that bacteria belonging to the genus *Rhodococcus* do not only transform PCBs using the classical *bph* pathway, but also through a complex interplay of different catabolic pathway modules. Our study suggests that the PCB catabolic potential of *Rhodococci* is not yet fully understood.

**Keywords:** PCBs, biotransformation, catabolic pathway, intermediates, Rhodococcus, GC, MS

<footnote n="1">¹Rudjer Bosković Institute (RBI) – POB 180, 10002 Zagreb, Croatia</footnote>

<footnote n="2">²Institute for Medical Research and Occupational Health (IMI) – Ksaver street 2, Zagreb, Croatia</footnote>

<footnote n="3">³Agroécologie, AgroSup Dijon, INRA, Université de Bourgogne Franche-Comté (INRA Dijon) – Institut national de la recherche agronomique (INRA) – 17 Rue Sully, 21000 Dijon, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: ipetric@irb.hr</footnote>
# Capacity of leaf-associated microorganisms to biotransform pesticides in rivers

Louis Carles*<fnref n="1" />,<fnref n="2" />, Florent Rossi<fnref n="1" />, Muriel Joly<fnref n="1" />,<fnref n="2" />, Pascale Besse-Hoggan<fnref n="2" />, Isabelle Batisson<fnref n="1" />, and Joan Artigas<fnref n="1" />

## Abstract

Leaf-associated microbial communities, and especially fungi, possess a large panel of enzymes permitting the breakdown of leaf polymers as well as the transformation of xenobiotic compounds. This study aims (i) to assess the potential of leaf-associated microbial communities, exhibiting different exposure histories (upstream site, less polluted *versus* downstream site, more polluted), to biotransform three maize herbicides (mesotrione, S-metolachlor and nicosulfuron) in single and cocktail molecule exposures, and (ii) to identify the microorganisms involved in these biotransformations. The results showed a significant dissipation of nicosulfuron (29 % to 66 %, day 40) in both single and cocktail exposures, respectively, but not of mesotrione and S-metolachlor. The formation of nicosulfuron metabolites such as ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) confirmed the transformation of this molecule by leaf-associated microorganisms. In addition, the downstream communities showed a greater ability to transform nicosulfuron than the upstream communities suggesting that exposure history to pesticides can enhance the biotransformation potential of microorganisms. After 40-day single exposure to nicosulfuron, the downstream communities were also those experiencing the greatest shifts in fungal and bacterial community diversity. Specifically, the fungal strain (*Plectosphaerella cucumerina*) isolated from leaves was able to biotransform 100 % of nicosulfuron (25 mg L⁻<fnref n="1" />, in 14 days of incubation). Although leaf-associated fungal communities are known to mostly feed on litter constituents, the alternative use of xenobiotic compounds transported by flowing waters place fungi as good candidates for pesticide removal in polluted rivers.

**Keywords:** leaf, associated microorganisms, biodegradation, mesotrione, nicosulfuron, S, metolachlor, herbicide mixture

<footnote n="1">¹Laboratoire Microorganismes : génome et environnement (LMGE) – CNRS : UMR6023, Université Clermont Auvergne – 63000 Clermont-Ferrand, France</footnote>

<footnote n="2">²Institut de Chimie de Clermont-Ferrand (ICCF) – CNRS : UMR6296, Université Clermont Auvergne, SIGMA Clermont – 63000 Clermont-Ferrand, France</footnote>

<footnote>*Speaker</footnote>
# Micropollutant biotransformation potential of natural river biofilms

Werner Desiante*†¹, Bernadette Vogler¹, Cresten Mansfeldt¹, Stephanie Merbt¹, and Kathrin Fenner‡¹

¹Eawag – Überlandstrasse 133 8600 Dübendorf, Switzerland

## Abstract

Micropollutants, such as pesticides and pharmaceuticals, are released through several pathways into our environment and can pose non-negligible risks to aquatic organisms. Aquatic microbial communities exposed chronically to micropollutants can change significantly in terms of biodiversity and can gain an increase in community-tolerance. These findings raise the question what driving factors lead to such community changes. Since an improved ability to rid themselves of micropollutants could be a possible tolerance mechanism, in our research we investigate the potential of differently adapted natural river biofilms to biotransform a large mixture of environmentally relevant polar micropollutants. Biofilm has been cultivated in an indoor channel system, fed with river water from a sub-urban, treated wastewater-receiving stream in the Zurich area. Suspensions of this biofilm were used to seed a series of batch reactors and spiked with a mixture of 43 micropollutants (1 µg/L). Micropollutant concentrations were monitored over four days.

Seventeen of the 43 micropollutants showed decreasing concentrations over time, of which the abatement of eleven parent compounds could be attributed to biotransformation, whereas for the remaining six compounds, which all contained amine functional groups, concentration time patterns pointed towards intracellular ion-trapping.

The eleven chemicals showing potential biotransformation were found to belong to distinct chemical classes, including sulfonamide antibiotics, primary or secondary amide-containing compounds and substances containing carboxylic ester groups.
In an on-going study, the same approach is applied to investigate the biotransformation ability of biofilms grown up- and downstream of treated wastewater effluents, respectively, for two medium-sized rivers in central Switzerland.

**Keywords:** Biotransformation, Natural river biofilm, Periphyton, Micropollutants, Pollutant fate

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: werner.desiante@eawag.ch</footnote>

<footnote>‡Corresponding author: kathrin.fenner@eawag.ch</footnote>
# Hydrocarbon impact on microbial communities in marine coastal sediments

Robert Duran*¹, Cyril Noel, Cristiana Cravo-Laureau, and Christine Cagnon

¹Equipe Environnement et Microbiologie - IPREM UMR CNRS 5254 (EEM) – CNRS : UMR5254, Université de Pau et des Pays de l'Adour [UPPA] – IBEAS - UFR Sciences BP 1155 64013 PAU CEDEX, France

## Abstract

To date studies of hydrocarbon biodegradation have focused mainly on simplified systems (e.g. pure strains, individual molecules), which are not representative of natural environments. As a result, little is known about whole microbial communities in their actual context, and the impact of environmental fluctuations on pollutant degradation and microbial community structure is insufficiently investigated. Notably, little is known as yet about degradation within the transitional zone from oxic to anoxic conditions. It is thus important to determine the behavior of microbial communities in such oscillating conditions, especially the microbial populations involved on anaerobic metabolisms such as sulfate-reducing microorganisms. In order to decipher the mechanisms underlying microbial community organization after an oil spill, we develop experimental ecology approaches to manipulate the microbial communities. After an adaptive stage, the modification of the microbial community structure occurred, concomitant with the beginning of the degradation of hydrocarbon compounds, followed by a succession of bacterial community structures along the degradation process. Submitting sediments to different oxygenation regimes in bioreactors showed the influence of the oxygenation on hydrocarbonoclastic microbial-assemblages, which was favoured by oxygenation after a period of anoxia. Surprisingly, sulfate-reducers were not affected by oscillating conditions.

Applying different strategies of experimental ecology, our studies highlight the functional redundancy of microbial communities involved in hydrocarbons degradation. The description of the microbial communities functioning under different oxygenation regimes by omics approaches will allow to answer important ecological questions for understanding the mechanisms underpinning the bacterial communities assemblages.

**Keywords:** mesocosm, microbial ecology, oil biodegradation, intertidal sediments, experimental ecology, omic's approaches

<footnote>*Speaker</footnote>
Selective colonization processes of plastic-loving
bacteria in an abandoned landfill site

Edoardo Puglisi*†¹, Serena Galletti*¹, Francesco Romaniello*¹, Jie Feng², Alberto Frache², and Piersandro Cocconcelli‡¹

¹Istituto di Microbiologia, Università Cattolica del Sacro Cuore (UCSC) – Via Emilia Parmense 84,
29122 Piacenza, Italy, Italy

²Dipartimento Scienza Applicata e Tecnologia, Politecnico di Torino – Viale Teresa Michel 5,
Alessandria, Italy, Italy

Abstract

Polyethylene (PE), the most common petroleum-based plastic, is strongly recalcitrant to degradation. Despite this, recent evidences showed that a few microbial strains can partially degrade PE pre-oxidized by abiotic processes.

Here, we investigated plastic samples from an abandoned landfill, with the aim of elucidating the ecology of soil microorganisms able to grow on the plastics and to correlate them with plastic degradation levels.

Isolations were carried after detachment of the biofilm from the plastic surfaces and se-
rial dilutions on selective agar plates of mineral medium with paraffin as the only carbon
source. Microbial DNA derived from the plastic surfaces, the landfill soil and an uncontam-
inated neighboring agricultural soil was also analyzed by Illumina sequencing of 16S rRNA
amplicons.

A total of 49 strains growing on paraffin were identified: the most representative genus is *Bacillus* genus with 12 different species represented, followed by *Pseudomonas*, *Lysini-bacillus*, *Comanomonas* and *Acinetobacter*.

Differential scanning calorimetric analysis of the plastic samples showed an additional shoul-
der melting peak with various enthalpies besides the main melting peak, implying the plastic
exhibited different degrees of degradation. In parallel, Illumina 16S rRNA analyses showed
that the different plastics hosted different microbial communities, and that the bacterial
community derived from plastic sample with the highest degradation level was the most
similar to that of the soil derived from contaminated landfill. Results point to an interesting
correlation between the level of plastic degradation and the composition of the microbial
biofilm.

Keywords: polyethylene, biofilm, colonisation, Illumina

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: edoardo.puglisi@unicatt.it</footnote>

<footnote>‡Corresponding author: pier.cocconcelli@unicatt.it</footnote>
# Dynamics of bacterial communities in a field-scale
pilot treating As-rich acid mine drainage

Elia Laroche*<fnref n="1" />,<fnref n="2" />, Corinne Casiot<fnref n="1" />, Lidia Fernandez-Rojo<fnref n="1" />, Vincent Tardy<fnref n="1" />, Angélique Désoeuvre<fnref n="1" />, Gérard Grapin<fnref n="3" />, Julie Savignac<fnref n="3" />, Jolanda Boisson<fnref n="4" />, Fabienne Battaglia-Brunet<fnref n="2" />, Catherine Joulian<fnref n="2" />, and Marina Héry<fnref n="1" />

## Abstract

Arsenic rich AMDs (Acid Mine Drainages) represent a major source of pollution for aquatic ecosystems. Microbially driven iron (Fe) and arsenic (As) oxidation and precipitation represent a promising strategy to treat this pollution. A better understanding of the biogeochemical mechanisms involved is required prior any further exploitation of this microbial potential. A field-scale pilot was implemented at the Carnoulès mine (France) for the treatment of AMD. It is an ergonomic and passive aerobic system: five treatment units of 1.5 m2 are combined vertically in series and fed with the AMD water by gravitation flow. Biogenic precipitates (corresponding to Fe- and As-rich biofilms) covered the bottom of the units. Inlet water and biogenic precipitates were collected over a 7 months period. We determined the bacterial community structure in the precipitates by fingerprint (ARISA), metabarcoding (16S rRNA gene) and qPCR targeting arsenite oxidase gene *aioA*. Chemical and mineralogical analyses were conducted on the precipitates and on the feed water. The bacterial communities in the precipitates developed from the indigenous communities of the AMD used to feed the pilot. Our results showed an evolution of these communities over time associated with an increase of the potential genetic for As oxidation. The proportion of As(V) in the precipitates and arsenic removal efficiency fluctuated, with maximum levels of 99% and 97 % respectively. This work provided information about microbial dynamics and pollution removal efficiency in a treatment pilot under field conditions. It will serve for future design of a bioremediation system to treat As-rich AMD.

**Keywords:** bioremediation, arsenic, acid mine drainage, engineering, iron, arsenic, oxidizing bacteria

<footnote n="1">¹HydroSciences Montpellier (HSM) – CNRS : UMR5569, IRD, Université Montpellier II - Sciences et techniques – 163, rue Auguste Broussonet, 34090, Montpellier, France</footnote>

<footnote n="2">²Unité Biogéochimie environnementale et qualité de l'eau (BRGM) – Bureau de Recherches Géologiques et Minières (BRGM) – 3 avenue Claude-Guillemin BP 36009 45060 Orléans Cedex 2, France</footnote>

<footnote n="3">³Institut des Ressources Humaines Ingénieur Conseil – IRH – 427 rue Lavoisier - CS 50155, 54714, Ludres Cedex, France</footnote>

<footnote n="4">⁴Institut des Ressources Humaines Ingénieur Conseil – IRH – 197 avenue de Fronton, 31200, Toulouse, France</footnote>

<footnote>*Speaker</footnote>
# Insights into uranium tolerance of Microbacterium oleivorans A9 by proteogenomic analyses

Nicolas Gallois*†¹, Béatrice Alpha-Bazin², Philippe Ortet³, Mohamed Barakat³, Justine Long³, Laurie Piette¹, Séverine Zirah⁴, Adrienne Kish⁴, Jean Armengaud², Catherine Berthomieu¹, and Virginie Chapon¹

¹Laboratoire des Interactions Protéine-Métal (CEA/BIAM/LIPM) – CEA Cadarache (Saint Paul-lez-Durance) – France

²Laboratoire Innovations technologiques pour la Détection et le Diagnostic (Li2D) – Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA) - Grenoble – France

³Laboratoire d'écologie microbienne de la rhizosphère et d'environnements extrêmes (LEMiRE) – CEA Cadarache (Saint Paul-lez-Durance) – France

⁴Molécules de Communication et Adaptation des Micro-Organismes (MCAM) – CNRS : UMR7245, Musée National d'Histoire Naturelle - MNHN (France) – 57 rue Cuvier 75005 Paris, France

## Abstract

*Microbacterium oleivorans* A9 is a uranium-tolerant actinobacteria isolated from the trench T22 located near the Chernobyl nuclear power plant (Chapon *et al.* 2012). This site is contaminated with different radionuclides including uranium. Under uranium exposure, cells of *Microbacterium oleivorans* A9 exhibit three sequential mechanisms involved in uranium detoxification: a rapid metal removal within the first 30 minutes, then an active U(VI) release in the exposure medium and a final biomineralization step of uranium in autunite-like mineral phases (Theodorakopoulos *et al.* 2015). Using an innovative proteogenomic approach, we explored the molecular changes at the proteome level occurring in this strain upon uranyl exposure. We interpreted high-throughput proteomic data (1,221,282 spectra) against a six-reading frame ORF database deduced from the draft genome (Ortet *et al.* 2017), validated the identification of 1,532 proteins, identified these proteins and compared protein abundances from cells exposed or not to uranyl stress. These data show that a complex cellular response to uranium occurs in *Microbacterium oleivorans* A9. The abundance of 591 proteins was significantly different between conditions. In particular, the uranyl stress perturbed the phosphate and iron metabolisms pathways. A large ratio of proteins more abundant upon uranyl stress, are distant from functionally-annotated proteins, highlighting the lack of fundamental knowledge regarding numerous key molecular players from soil bacteria. Furthermore, several transporters have been identified to be specifically associated to uranyl stress, paving the way to the development of biotechnological tools for uranium decontamination.

**Keywords:** uranyl stress, Microbacterium, proteogenomic, radionuclides toxicity

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: nicolas.gallois@cea.fr</footnote>
Influence of plastic litters on marine microbial life

Claire Dussud*, Leila Meistertzheim, Matthieu Georges, Pascale Fabre, Maria-Luiza Pedrotti, Gaby Gorsky, and Jean-François Ghiglione*†1

¹Laboratoire d'Océanographie Microbienne (LOMIC) – CNRS : UMR7621, Université Pierre et Marie Curie (UPMC) - Paris VI – Observatoire Océanologique, Banyuls/mer, France

Abstract

The Mediterranean Sea is one of the most plastic-polluted zone and offer a unique op-
portunity to evaluate the ecotoxicity of plastic marine debris (PMD) on bacterioplankton
lifestyle. The large set of samples taken during the TARA expedition revealed for the first
time a clear niche partitioning between the PMD compared to the organic particle-attached
(PA) and the free-living (FL) bacteria. Bacterial counts in PMD presented higher enrich-
ment factors than generally observed in PA, as compared to the abundance of FL bacteria
in the surrounding waters. New generation sequencing showed that taxonomic diversity was
also higher in the PMD communities, where higher evenness indicated a favorable environ-
ment for a very large number of species. Cyanobacteria were particularly overrepresented
in PMD, together with essential functions for biofilm formation and maturation. The con-
sistent distinction of community structure and functional traits between the three lifestyles,
that exceeded the large-scale geographical variation in the Western Mediterranean basin,
support a new trade-off for the maintenance of a large biodiversity of bacterioplankton in
the Oceans. Bacterial production and ectoenzymes revealed also a higher activity on PMD,
thus suggesting that this community take significant part of the global biogeochemical cycles
in the Oceans.

**Keywords:** marine plastic pollution / microbial ecotoxicology / biofouling

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: ghiglione@obs-banyuls.fr</footnote>
Transfer and distribution of diuron in biofilms and
joint toxic effects

Betty Chaumet*<fnref n="1" />, Julie Neury-Ormanni<fnref n="1" />, Caroline Doose<fnref n="2" />, Brigitte Delest<fnref n="1" />, Joan Artigas<fnref n="3" />, Nicolas Mazzella<fnref n="1" />, and Soizic Morin<fnref n="1" />

Abstract

Biofilms are composed of microalgae and other microorganisms embedded in a polysac-
charidic matrix (EPS). We developed a new approach dedicated to the determination of
pesticide transfer and distribution into the matrix. This approach is based on the physi-
cal separation, then analysis of the different matrix compartments: pore water, EPS and
microorganisms. The aim of this study is to obtain a better understanding of mechanisms
taking part into pesticide bioaccumulation on biofilms. In parallel, toxicokinetic are coupled
with toxicodynamic studies, in order to investigate the relationship between accumulation
rate and joint effects of herbicides.

In this study, we determined the accumulation and distribution of diuron, a photosynthesis inhibitor. Simultaneously, we characterized biofilm biomass (dry weight, total organic carbon), protein and polysaccharide contents, coupled with the assessment of functional effects on enzymatic activities (β-Glucosidase and β-Glucosaminidase) and photosynthetic activity. After one month of colonization in Gazinet-Cestas pond (France) on glass slides, biofilms were exposed for 21 days in channels with three conditions: control, and 2 contaminated with 5 and 50 µg.L-1 of diuron, respectively.

Pesticide quantification in biofilm showed that diuron was quickly accumulated (i.e. after 15 minutes of exposure) in both EPS and cells, highlighting early distribution in the whole biofilm, followed by cell internalization. However, photosynthesis was clearly inhibited from 15 minutes until 21 days in the biofilms exposed to 50 µg.L-1 of diuron, as an expectable result of its mode of action. All these results provide an innovative basis to develop a pesticide toxicokinetic and toxicodynamic model for biofilms.

**Keywords:** Biofilm, Diuron, Bioaccumulation

<footnote n="1">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</footnote>

<footnote n="2">²Institut National de la Recherche Scientifique - INRS (CANADA) – Canada</footnote>

<footnote n="3">³Microorganismes : génome et environnement (LMGE) – Université Blaise Pascal - Clermont-Ferrand II – Université Blaise Pascal, Campus des Cézeaux, 24, avenue des Landais BP 80026 63 170 AUBIERE, France</footnote>

<footnote>*Speaker</footnote>
# Morphological plasticity in Chlamydomonas reinhardtii: an adaptive response to micropollutant stress

Giulia Cheloni*¹, Michel Goldschmidt-Clermont¹, and Vera Slaveykova¹

¹University of Geneva (UNIGE) – Switzerland

## Abstract

Phytoplankton are characterized by a great phenotypic plasticity and amazing morphological variability, both playing a primary role in the acclimation to changing environments. However only few data are available about morphological variability as adaptive responses to stress. The results that we present here highlight palmelloid colony formation of the green alga *Chlamydomonas reinhardtii* upon exposure to micropollutants.

*C. reinhardtii* was exposed to four micropollutants with different modes of action (copper, cadmium, PFOS and paraquat) for up to 96h. Effects on growth, cell traits (size, chlorophyll fluorescence), membrane integrity and oxidative stress were monitored daily via flow cytometry and fluorescence microscopy.

Colony formation was observed after 48h exposure to the four micropollutants. The number of cells forming the colony increased daily to reach up to 16 cells after 96 hours incubation. Microscopic observation showed the cells to be encased in a multilayer envelope, suggesting that the colony formation might be associated to the lack of hatching from the mother cell wall. When colonies were harvested, rinsed and inoculated in fresh medium without micropollutants *C. reinhardtii* cells reverted to their unicellular lifestyle. No effects on chlorophyll fluorescence, membrane integrity or oxidative stress were observed in cells forming colonies. In contrast such effects were observed at higher concentrations of the same micropollutants, suggesting that colony formation might be a stress response activated to face sub lethal micropollutant concentration.

From an ecotoxicological perspective, such influences on phytoplankton morphological plasticity might have important consequences on feeding dynamics at higher levels of the trophic chain.

**Keywords:** Phenotypic plasticity, adaptive stress response, palmelloid colonies, *C. reinhardtii*

<footnote>*Speaker</footnote>
# Genome-wide analyses of rare earth elements responsive genes in microbial model organisms

Nicolas Grosjean*¹,², Marie Le Jean², Elisabeth Gross², and Damien Blaudez¹

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 Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360, Université de Lorraine – Campus Bridoux - Rue du Général Delestraint, Université de Lorraine F-57070 METZ, France

## Abstract

The Rare Earth Elements (REEs) group gather 17 chemical elements covering 15 lanthanides (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu), Yttrium and Scandium. As part of the Technical Critical Elements (TCEs), they are of high importance in high-tech domains such as renewable energies, aeronautics, new communication technologies, and medicine. Although many investigations have been carried out to find innovative ways to supply the rising demand for these elements, the effects of the increasing release of these new emerging contaminants in the environment is poorly documented. Furthermore, the few existing studies on biota often mention controversial effects and also highlight the lack of knowledge on the impact of REEs at the cellular level. Among others, REE-mediated oxidative stress and modification of membrane permeability have been reported while almost nothing is known at the molecular level. Thus, we adopted high throughput strategies to study REE stress responses on several model organisms. These strategies consisted, on the one hand, of the screening of genome-wide mutant collections on several REEs, and on the other hand of transcriptional analyses (RNA-seq) under REE stress. These two large-scale methods allowed the identification of key genes, proteins or processes involved in a REE stress and/or their translocation in organisms (e.g. membrane transport). The results obtained from yeast and bacteria exposed to various REEs will be discussed, highlighting the potential impact of REEs on these organisms.

**Keywords:** Rare Earth Elements, Emerging contaminants, Bacteria, Yeast, RNA seq, Genomic phenotyping

<footnote>*Speaker</footnote>
# Novel nanopesticides introduce new challenges to microbes in agro-ecosystems and downstream wetlands

Marie Simonin*<fnref n="1" />, Steven Anderson<fnref n="1" />, Christina Bergemann<fnref n="1" />, Jennifer Rocca<fnref n="1" />, Benjamin Colman<fnref n="2" />, and Emily Bernhardt<fnref n="1" />

## Abstract

Widespread application of engineered nanoparticles for the purpose of crop protection – termed nanopesticides – are useful for agriculture, but little is known about the potential environmental impact on agro-ecosystems and downstream ecosystems, including wetlands. Here, we assessed the impact of a copper hydroxide nanopesticide on microbial community function and diversity in two large-scale mesocosm experiments. The first mesocosm experiment simulated the realistic application of the nanopesticide to an agro-ecosystem over a 6-month growing season. The second mesocosm study tested nanopesticide effects on wetlands with chronic weekly additions of low realistic concentrations over nine months. In these experiments, we tested the interactive effects of nanopesticides and nutrient enrichment treatment, as nanopesticide exposures will often co-occur with fertilizer enrichment. We hypothesized that resource availability will influence the resistance and resilience of microbial function to nanopesticides.

In the agro-ecosystem mesocosms, the eight measured microbial extracellular enzyme activities (EEAs) on soils were suppressed (-41 to -71%, *P* < 0.01) within 15 days under ambient nutrient conditions. In contrast, no negative effects were detected in fertilized mesocosms, but instead a stimulation of EEAs (+20 to +51%, *P* < 0.01) was observed after the third application at five months. In the wetland mesocosms, the microbial EEAs were decreased in the sediment and periphyton samples in both nutrient conditions after 6 months of exposure, but recovered after 9 months.

These findings suggest that nanopesticides could have more lasting effects in downstream ecosystems than in agro-ecosystems and that nutrient availability influence the ecosystem impacts of this emerging contaminant.

**Keywords:** Agro, ecosystem, nanomaterials, aquatic ecosystems, nutrient pollution, chronic exposure, Mesocosms

<footnote n="1">¹Duke university – Durham, NC 27708, United States</footnote>

<footnote n="2">²University of Montana – United States</footnote>

<footnote>*Speaker</footnote>
# Diversity and functions of microbial communities in historically multi-contaminated soils

Florian Lemmel*†¹, Florence Maunoury-Danger², Corinne Leyval¹, and Aurélie Cebron¹

¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360 – Faculté des Sciences et Technologies, BP70239, 54506 Vandoeuvre-lès-Nancy, France

²Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360, Université de Lorraine – Campus Bridoux, Avenue du général Delestraint, 57070 Metz, France

## Abstract

The intensive industrial activities of the 20th century in Lorraine Region (France), conducted to highly contaminated wasteland soils after coking plant dismantling. The organic and metallic multi-contamination may affect the microbial communities that are key actors of soil functioning and biogeochemical cycles. The aims of our study was to: i) evaluate microbial functional diversity in historically multi-contaminated soils, focusing on functions of the carbon cycle, and ii) determine how edaphic properties or pollutant concentrations contribute to shape microbial community. We hypothesized that high soil contamination would conduct to lower microbial diversity.

We worked on a range of 10 soils that were characterized for physicochemical (C, N and pollutant concentrations) and microbiological (microbial abundance using MPN and qPCR and microbial taxonomic diversity through Illumina MiSeq sequencing) properties. Microbial metabolic capacity, i.e. carbon substrate degradation rates using Biolog™ and MicroResp™ tools, were used as indicators of the functional diversity. Pollution induced community tolerance (PICT) was also tested after zinc input to soils.

Physicochemical properties discriminated the soils in 3 groups corresponding to their history (steel crusher; sludge ponds; old gravel pit and forest) while microbiological properties did not discriminate the soils in the same way. The functional diversity partly depends on the edaphic parameters such as the C/N ratio that was correlated with the basal mineralization and the substrate induced respiration yields. The pollution also shapes the microbial community because the highly polluted soils harboured the lowest metabolic diversity and were less sensitive to a metallic stress induced by zinc input.

**Keywords:** soils, wastelands, multipollution, microbial functions, biolog, microResp

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: florian.lemmel@univ-lorraine.fr</footnote>
Long-term effects of triclosan on community
function, community tolerance and metagenomic
composition in marine periphyton communities

K. Martin Eriksson*¹, Tobias österlund, Sanna Abrahamsson, Henrik Johansson,
Natàlia Corcoll, Kemal Sanli, Thomas Backhaus, Hans Blanck, and Erik Kristiansson

¹K. Martin Eriksson (K.M. Eriksson) – Chalmers University of Technology Dept. of Shipping and Marine Technology 41296 Gothenburg Sweden, Sweden

Abstract

This study presents data from a long-term flow-through microcosm experiment, describing the toxicity of triclosan (TCS) to natural marine periphyton communities. We measured the functional endpoints photosynthesis, bacterial carbon utilization, photosynthetic pigment content, as well as Pollution-Induced Community Tolerance (PICT) to TCS. In addition, we performed large-scale shotgun DNA sequencing (metagenomics), which resulted in detailed descriptions of TCS effects on taxonomic composition (16S and 18S marker genes) and composition of biological functions (protein-coding genes). These measurements enables us to compare how community functions, metabolic functions and taxonomic composition were affected by TCS. Results from functional endpoints showed that TCS stimulated photosynthesis and pigment content at concentrations of 31.6 nM, a change that co-occurred with an increase in PICT. TCS did not, however, affect the functional diversity or the composition of bacterial carbon utilization. In contrast to these results, the DNA sequencing results showed that both bacterial and eukaryote community composition was changed by TCS concentrations above 10 nM. These changes included *e.g.* increases in *Gammaproteobacteria* and decreases of other bacterial groups, as well as increases and decreases of specific diatom taxa. Also, changes in abundance of specific biological functions was detected at concentrations of 3.16 nM and higher. These included, for example, increases carbohydrate metabolism and transport genes and decreases photosynthesis genes. Overall, the study shows that the endpoints employed have different capabilities to detect effects from TCS. It also shows that functional redundancy and indirect effects from TCS are present in the complex biofilm communities studied here.

**Keywords:** metagenomics, shotgun sequencing, 16S, 18S, protein coding genes, PICT, pigment, triclosan, biofilm

<footnote>*Speaker</footnote>
Responses of microbial communities in mangrove
ecosystem under the influence of pretreated
wastewater discharges

Cécile Capdeville*†¹, Jonathan Gervaix², François Fromard¹, Jean-Luc Rols¹, Thomas Pommier², and Joséphine Leflaive¹

¹Laboratoire Ecologie Fonctionnelle et Environnement (EcoLab) – Université Paul Sabatier - Toulouse
III – France

²Ecologie microbienne – Université Claude Bernard - Lyon I – France

Abstract

Mangrove forests are coastal ecosystems continuously affected by various environmental
stresses but also under the influence of anthropogenic pressures. The aim of this study was
to evaluate the capabilities of microbial communities in sediments to respond to these an-
thropogenic pressures in function of the level of natural stress they are subjected to. We
used an *in situ* experimental system set up in Mayotte Island where mangrove is exposed
since 2007 to pretreated wastewater discharges (WD) to study its bio-epuration capability.
Six plots in two mangrove zones with different environmental constraints were exposed to
WD either for several years or since the beginning of the present study or not exposed. In two
additional plots WD have been stopped to assess the resilience capacities of microbial com-
munities. Sediments have been sampled after 1, 3, 8 and 12 months in 4 subplots per plot.
Since wastewaters were concentrated in ammonium, we focused on communities involved
in N-cycle. Microorganisms densities have been estimated by qPCR and the denitrification
activity (DEA) evaluated by potential activity measurements. The composition of microbial
photosynthetic communities at sediment surface was assessed by HPLC.
Long-term WD significantly impacted the composition of phototrophic microorganisms and
increased the density of microorganisms and DEA in both mangrove zones. The resilience
seemed to be faster on the less constrained mangrove zone. Within less than 8 months, the
DEA returned to the same level than control plots. These results confirm the high poten-
tial of mangrove for bio-epuration, associated with strong resilience properties of microbial
communities.

**Keywords:** Mangrove ecosystem, multiple disturbance, *in situ* experimental system, microbial community, N-cycle, resilience, wastewater discharge, natural environmental stress

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: cecile.capdeville1@univ-tlse3.fr</footnote>
# Do anthropogenic stressors impact the functional performance of leaf-degrading aquatic fungi? A case study using microcosms, fungicides, and modern biochemical tools

Patrick Baudy*¹, Nina Röder¹, Marco Konschak¹, Jochen Zubrod¹, Christiane Baschien², Ralf Schulz¹, and Mirco Bundschuh³

¹Institute for Environmental Sciences, University of Koblenz-Landau – Fortstraße 7, 76829 Landau, Germany

²Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH – Inhoffenstraße 7B, 38124 Braunschweig, Germany

³Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences – Lennart Hjelms väg 9, 75007 Uppsala, Sweden

## Abstract

Aquatic hyphomycetes – a polyphyletic group of true fungi – play a key role in leaf-litter breakdown as they contribute substantially to the leaf mass loss while increasing the nutritional value of leaf material as food source for detritivorous invertebrates. Despite their ecological significance, information about their communities’ functional composition is scarce, particularly under stress. In order to address this knowledge gap, we conducted a microcosm experiment with four common aquatic hyphomycete species (*Alatospora acuminata*, *Heliscella stellata*, *Neonectria lugdunensis*, and *Tetracladium marchalianum*) in every possible mono, binary, and quaternary combination. These combinations were exposed towards a model stressor, namely a five-component fungicide mixture over 14 days. Surprisingly, in monocultures, only *N. lugdunensis* and *T. marchalianum* were capable of degrading leaf material and they even maintained this capability up to high fungicide concentrations of 500 and 2,500 µg/L, respectively. Moreover, preliminary analyses of the decomposition data indicated non-additive species interactions in binary and quaternary culture combinations, which were influenced in presence of different fungicide-mixture concentrations. To gain a mechanistic understanding of the responses at the functional level, we are currently using modern biochemical tools including species-specific quantitative real-time polymerase chain reaction assays as well as enzyme activity assays (diverse hydrolases and phenol oxidase). Our preliminary results, nonetheless, point towards stressors’ potential to alter aquatic hyphomycete communities’ functional composition (due to an altered functional potential of single species and/or shifts in the structure of the communities). Thus, consequences for heterotrophic food webs due to their bottom-up directed regulation are likely.

**Keywords:** aquatic hyphomycetes, extracellular enzymes, fungicides, leaf decomposition, qPCR

<footnote>*Speaker</footnote>
# Proteomic responses to nanoparticulate and ionic silver in fungi from metal-polluted and non-polluted streams

Diana Barros*†¹, Arunava Pradhan¹, Cláudia Pascoal¹, and Fernanda Cássio¹

¹Centre of Molecular and Environmental Biology (CBMA) – Institute for Science and Innovation for Bio-sustainability (IB-S) University of Minho, 4710-057 Braga, Portugal

## Abstract

Enhanced production and usage of silver nanoparticles (AgNPs) raise concerns about their potential impacts in aquatic ecosystems. Effects of AgNPs and Ag+ were assessed based on the variations in the overall proteome and the activities of selected antioxidant enzymes in two fungal strains of *Articulospora tetracladia*, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61). For that, fungi were exposed to concentrations of AgNPs and Ag+ affecting 20% of growth (EC20). A total of 432 proteins were identified, of which 172 belonged to At72 and 260 to At61; 71 were expressed in both strains. At72 had 58% and 52% of the proteins induced by AgNPs and Ag+, respectively. For At61, the percentages were higher (~77%). The higher percentages of proteins suppressed by either Ag forms suggest higher stress in At72 than in At61, which is consistent with the background of this fungal strain. Major groups of proteins were related to carbohydrate metabolism, amino-acid and protein biosynthesis. Both Ag forms also induced stress-responsive proteins, including catalase and superoxide dismutase which remained consistent with the profile of their enzymatic activities. These results supported the ability of these fungi in initiating an efficient antioxidant response to cope with Ag-induced toxicity. Overall, the functional proteomic approach can be useful to get a mechanistic insight on the stress induced by AgNPs or Ag+ in aquatic fungi that play a key role in plant litter decomposition in streams.

**Keywords:** AgNPs and Ag+, stress response, functional proteomics, antioxidant enzymes, aquatic fungi.

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: dianamcbarros@gmail.com</footnote>
# The Microbiome Stress Project: A database for identifying stressor impacts on microbial community structure across multiple environments

Jennifer Rocca*†¹, Joanna Blaszczak¹, Firas Midani¹, Marie Simonin¹, and Alex
Washburne²

¹Duke university [Durham] – Durham, NC 27708, United States

²Montana State University – Bozeman, Montana 59717, United States

## Abstract

Understanding the responses of microbial communities to natural and anthropogenic stressors and their interactions is vital for determining ecosystem and host health. The impact of natural stressors (e.g. pH, temperature, storm, fire) and anthropogenic stressors (e.g. synthetic chemicals, land use) on microbial community structure have been extensively studied in various ecosystems. These studies highlight that microbial community composition is extremely sensitive to environmental change but despite an immense amount of data generated, cross-study identification of sensitive and/or tolerant microorganisms has not yet been performed. Therefore, we have constructed an extensive open-source database - The Microbiome Stress Project - to investigate how all types of stressors (natural and anthropogenic) impact microbial community structure across multiple environments (soil, biofilm, water, reactor, gut, sediment). This database is built with published/unpublished microbial data sets (using high-throughput sequencing only) and we are developing a computational pipeline using the R package 'Phylofactor' to identify microbial taxa specifically sensitive or resistant to these stressors. To test the full capacity of the Microbiome Stress Project, we preliminarily set up a smaller proof of concept dataset (8 studies). Specifically, we compared the influence of copper contamination on bacterial community structure in the soil, gut, and an activated-sludge reactor. Additionally, we examined the response of soil bacterial communities to a comprehensive set of stressors (Cu, Hg, antibiotics, PAHs, drought, nanoparticles, fire). This preliminary analysis tested if the relatedness of stressors holds true for their impact on microbial community structure and aimed at identifying indicator species responding ubiquitously to stress.

**Keywords:** Stress, Microbial Community, Microbiome, High, Throughput Sequencing, Diversity, Open Database

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: jennifer.rocca@duke.edu</footnote>
# Changes in bacterial communities in response to multicontamination phenomena and other anthropogenic forcing during different hydrological regimes in mediterranean watercourses.

Brice Reoyo-Prats*†<fnref n="1" />, Dominique Aubert<fnref n="1" />, Olivier Verneu<fnref n="1" />,<fnref n="2" />, and Carmen Palacios<fnref n="1" />

## Abstract

Mediterranean contrasted climate affects river dynamics and therefore the concentration of micropollutants encountered in freshwaters. Indeed, contaminants get concentrated in rivers watersheds during dry periods, while, at storm events, they get remobilized from soils and sediments or arrive from combined sewer overflows, leading to recurrent multipollution phenomena. Besides, microorganisms are capable of responding very rapidly to variations in the environment. In this study, we aim at understanding more particularly how aquatic bacteria attached to particles respond to changes in pollutants concentrations. To do so, a total of 60 samplings were carried out on a typically Mediterranean coastal watercourse, the Têt River, from upstream to coastal waters, in two complete drought-flood-drought hydrological cycles along 2013-2014 and 2015-2016. A high frequency monitoring at most impacted stations was also conducted during heavy rain periods, with a total of 27 samplings. Among physico-chemical parameters measurements, up to 350 contaminants have been analyzed including fecal indicators, nutrients, metals, pesticides and pharmaceuticals. A targeted metagenomics analysis with Illumina-based 16S rRNA gene sequencing was performed to study how the diversity of bacterial communities shifts with changes in contaminants concentrations. Multipollution phenomena occurring during storm events had a remarkable impact on bacterial alpha and beta diversity with an increment of the opportunistic phylum Proteobacteria among other changes. Thus, bacteria biofilms attached to suspended matter are able to quickly react to sudden changes in pollutants concentrations, being therefore potential good early signals of multipollution stress.

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

<footnote n="1">¹Centre de Formation et de Recherche sur les Environnements Méditérranéens - CEFREM (Perpignan, France) (CEFREM) – Université de Perpignan : VIADOMITIA - UPVD (FRANCE), Centre national de la recherche scientifique - CNRS (France) – France</footnote>

<footnote n="2">²Univ. for Environmental Sciences and Management – ZA-2520, Potchefstroom, South Africa</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: brice.reoyo@univ-perp.fr</footnote>
Interactive effects of pesticides and nutrients on
microbial communities responsible of litter
decomposition in streams

Florent Rossi*†<fnref n="1" />, Joan Artigas<fnref n="1" />, Clarisse Mallet<fnref n="1" />, Christelle Margoum<fnref n="2" />, Matthieu Masson<fnref n="2" />, and Stéphane Pesce<fnref n="2" />

Abstract

Streams contamination by pesticides and nutrients, resulting from human activities, is widespread. However, the interactive effect of these two stressors on heterotrophic microbial communities and their activity of organic matter decomposition is still poorly understood. In this context, a microcosm experiment was performed to evaluate the single and interactive effects of environmental concentrations of the herbicide S-metolachlor and the fungicide tebuconazole, on the decomposition ability of leaf-associated microbial communities, in either eutrophic or mesotrophic conditions. Eight treatments were tested in triplicate to assess, in laboratory channels, the resulting effects on natural microbial communities from a pristine forested stream (Couze d'Ardes, Centre France) developing on *Alnus glutinosa* leaf species. Every week for 40-days, water was renewed and samplings were performed to measure *Alnus* leaves' decomposition rates (K day), as well as the structure (biomass and diversity) and enzymatic activities of the leaf-associated microbial communities. Results showed a stronger influence of nutrients compared to that of pesticides. Indeed, while decomposition rates, microbial biomass, fungal diversity and ligninolytic activities (laccase, phenol oxidase) were higher in eutrophic channels compared to mesotrophic ones, only a three-fold increase of laccase activity by tebuconazole was observed in eutrophic condition. Surprisingly, such activity increase was not observed in eutrophic conditions when the fungicide and the herbicide were mixed. Overall, our study shows that the effect of realistic environmental concentrations of pesticides on leaf-associated microbial communities can be negligible compared to the effect of nutrients. However, the tested tebuconazole concentration was sufficient to trigger physiological response in fungal community.

**Keywords:** streams, microcosms, pesticides, nutrients interaction, leaf litter, microbial communities.

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

<footnote n="2">²Milieux aquatiques, écologie et pollutions (UR MALY) – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA – 5 rue de la Doua, BP 32108, 69616 Villeurbanne cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: florent.rossi@univ-bpclermont.fr</footnote>
# Monitoring the impact of multi-stress contamination on an environmental opportunistic pathogen, *Stenotrophomonas maltophilia*: application to antibiotics and metals

Armelle Crouzet*†, Géraldine Depret<fnref n="1" />, Justine Thiot<fnref n="2" />, David Lejon<fnref n="2" />, Alain Hartmann<fnref n="1" />, Lorenzo Spadini<fnref n="3" />, Sabine Favre-Bonté<fnref n="4" />, and Sylvie Nazaret<fnref n="4" />

## Abstract

*Stenotrophomonas maltophilia* is a ubiquitous bacterial species known to be a soil and rhizospheric inhabitant as well as a human opportunistic pathogen. Multi-drug resistant strains are frequently found in hospitals because of its ability to acquire resistance genes through horizontal gene transfer and its possession of efflux pumps. Recent data also showed the link between antibiotic resistance and metal resistance among both clinical and environmental strains. In this study, we evaluated over a 3 month study the impact of a cocktail of antibiotics (ciprofloxacin and sulfamethoxazole) and metals (Cu and Zn) on indigenous *S. maltophilia* in soil using an experimental design of soil columns exposed to manure containing various concentrations of the cocktail. In parallel, the toxicant adsorption, distribution, and migration in the soil matrix were measured to interpret the observed biological response. Data showed that regardless of the concentration of the cocktail i.e. from low (1X PEC) to high (40X PEC) doses, no changes in population abundance were observed after 7 and 100 days of exposure. Genetic diversity (REP-profile) analysis among more than 650 isolates showed that metals were the dominant stressors. Antibiotic resistance gene abundance (sulfonamide and quinolone) was unaltered and no resistance gene transfer was observed. Finally, no changes were observed on the phenotypic (antibiotic resistance) properties at low or high antibiotic doses. These data suggest the lack of a dose/response effect as well as the lack of synergic effects between metals and antibiotics on the genotypic and phenotypic evolution of *S. maltophilia* populations.

**Keywords:** Antibiotic resistance, human pathogen, bacteria, soil, multistress, dose/response

<footnote n="1">¹Agroécologie – Agrosup, Institut national de la recherche agronomique (INRA) : UMR1347, Université de Bourgogne – BP 86510, F-21000 Dijon, France</footnote>

<footnote n="2">²Rovaltain Research Company – Rovaltain Research Company – France</footnote>

<footnote n="3">³Institut des Géosciences de l'Environnement (IGE) – Institut de Recherche pour le Développement, Institut Polytechnique de Grenoble - Grenoble Institute of Technology, Centre National de la Recherche Scientifique, Université Grenoble Alpes – UGA - IGECS 4070038 058 Grenoble Cedex 9, France</footnote>

<footnote n="4">⁴Ecologie Micorbienne – Universite Claude Bernard Lyon1, CNRS : UMR5564 – Campus de la Doua 43 Bd du 11 Novembre 1918 69622 Villeurbanne, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: armellecrouzet@gmail.com</footnote>
Biofilm phosphorus uptake capacity responds to
chemical and environmental stressors in river
ecosystems.

Lorenzo Proia*¹, Anna Maria Romaní², and Sergi Sabater²,³

¹Ecology of Aquatic Systems - University of Bruxelles (ESA - ULB) – Belgium

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

³Catalan Institute for Water Research (ICRA) – Spain

Abstract

Biofilms are a key component in the nutrient removal from the water column. However, nutrient uptake by biofilms may be hampered by the occurrence of pollutants or other stressors. We investigated the responses of biofilm phosphorus uptake capacity (PUC) to different chemical and environmental stressors by using chamber experiments. PUC was calculated by measuring P temporal decay after performing controlled P-spikes in chambers with biofilm-colonized tiles. Four different experiments were conducted to evaluate the response of PUC to: (a) several river waters from increasing polluted sites; (b) the effect of the bactericide triclosan (TCS); (c) the combined effect of TCS and grazers; and (d) the effect of TCS after a drought episode that affected the biofilms. These experiments showed that biofilms decreased their PUC along the pollution gradient. The biofilm PUC was significantly reduced after receiving higher TCS concentrations, though lower TCS concentrations also affected the biofilm when this was submitted to grazing pressure. PUC decrease was induced by flow interruption which further enhanced the TCS negative effects. Overall, PUC was sensitive to the effects of pollutants like TCS as well as to the action of biological (grazing) and environmental (drought) factors. The study also showed that multiple stressors enhance the negative effects of pollutants on the PUC of biofilms. Our study values the use of biofilms' PUC as a sensitive ecological-based tool to assess the effects of chemicals on freshwater communities and their derived functioning in river ecosystems.

**Keywords:** Phosphorus, Uptake, Biofilm, River, Selfdepletion, Pollution

<footnote>*Speaker</footnote>
# Spatio-temporal dynamics of TPMT-harboring bacteria among urban deposits reveal significant shifts in Pseudomonas and Aeromonas populations matching pollutant concentrations

Emilie Bourgeois*¹, Romain Marti¹, Claire Bernardin¹, Céline Bécouze², Sébastien Ribun¹, Laurence Marjolet¹, Audrey Gleizal¹, Sylvie Barraud², Gislain Lipeme Kouyi², Laure Wiest², Didier Blaha¹, and Benoit Cournoyer¹

¹Ecologie Microbienne - Bacterial Opportunistic Pathogens and Environment - CNRS 5557, INRA
1418, Université Lyon 1, VetAgro Sup - France

²Laboratoire Déchets, Eaux, Environnement, Pollutions (DEEP) - INSA - Institut National des Sciences Appliquées - 34 avenue des Arts Bâtiment Bât. JCA Coulomb 69621 Villeurbanne cedex, France

## Abstract

Urban activities can generate surface deposits made, among others, of urban soils, fecal matter, and pyrogenic contaminants. These deposits harbor specific microbiota indicative of their origin and ecotoxicity. Here, the bacteriome of these deposits was investigated by an analysis of their TPMT harboring bacteria. The TPMT (thiopurine methyltransferase) can detoxify several toxic metalloid derivatives. It was found to have a distribution limited to 120 bacterial genera including major pathogens such as *Pseudomonas aeruginosa*, *Leionella pneumophila*, and *Vibrio cholerae*. A *tpm* (encoding TPMTs) meta-taxogenomics approach was developed and used to investigate shifts in TPMT-bacterial populations. Deposits were sampled over a 6 year-period from a detention basin. They had been washed off during rainfall events occurring over an industrial urban watershed in Lyon. The *tpm* genetic structures were compared between sampling years, and related to measured PAH and MTE concentrations. An important diversity of TPMT bacteria was observed (over 88 species). Bacterial structures were found impacted by the accumulation time-periods, with higher numbers of reads for opportunistic pathogens (e.g. *P. stutzeri*, *P. mendocina*, *Aeromonas caviae*, *A. hydrophila*) among recently trapped sediments. Reads of the phytopathogen *P. syringae* were enriched in old sediments, where significant plant developments had occurred. *P. aeruginosa* reads were found but were not enriched over the study period. TPMT bacterial structures were correlated to pollutant concentrations (e.g. fluorene, dibenzo(a,h)anthracene and indeno(1,2,3-cd)pyrene). The *tpm* meta-taxogenomics approach was found highly reliable to infer the incidence of pollutants on urban bacteriomes but also for performing a tracking of pathogenic bacterial species.

**Keywords:** urban ecosystem, bacterial opportunistic pathogens, Pseudomonas, Aeromonas, pollutant concentrations

<footnote>*Speaker</footnote>
# Ecotoxicological effects on the interactions of fullerenes and organic micro-contaminants in stream biofilms

Anna Freixa*†¹, Vicenç Acuña¹, Marina Gutierrez¹, Ferran Romero¹, Josep Sanchís², Lucia-Helena Santos¹, Sara Rodriguez-Mozaz¹, Damià Barceló¹,², Marinel·la Farré², and Sergi Sabater¹,³

¹Catalan Institute for Water Research (ICRA) – Spain

²Institute of Environmental Assessment and Water Research (IDAEA-CSIC) – Spain

³Institute of Aquatic Ecology - University of Girona (IEA - UdG) – Spain

## Abstract

An increased amount of carbon nanomaterials such as fullerenes (C60) is recently observed in rivers, which co-occur with multiple organic micro-contaminants, becoming a problem of unknown consequences for aquatic systems. Due to their high surface area, C60 make good compounds for the adsorption of organic micro-contaminants, their interaction potentially affecting the toxicity for stream biofilms. We designed several experiments with the main objective of defining the toxicity of fullerenes in the presence of 3 different organic micro-contaminants in biofilms.

We performed 3 different experiments with epilithic biofilm exposed to environmental relevant concentrations of C60 (1ppb), one micro-contaminant (triclosan; 10 ppb diuron 10 ppb or venlafaxine 100 ppb) and a mixture of each compound (C60 + micro-contaminant) during 72 h. Nutrients, micro-contaminants and C60 concentrations were analysed in water. Functional (extracellular enzyme activities, photosynthetic efficiency and respiration) and structural endpoints (extracellular polymeric substances (EPS) and chlorophyll-a content) were analysed in biofilm. Finally, quantitative RT-PCR was used for absolute quantification of 4 different genes (16S rRNA, 18S rRNA, *psaA* and *psbA*).

Our results showed a significant decrease on algal biomass fluorescence (F0) after the exposure of C60, but no significant responses in all other endpoints. The exposure of diuron and triclosan significant altered the photosynthetic efficiency, respiration and the EPS content in biofilms whereas no evidences were detected after the exposure of venlafaxine compound. Finally, when nanomaterials and contaminants were exposed together an antagonistic response (i.e. decrease) was produced to the toxicity of the micro-pollutants in biofilms after the interaction with fullerenes.

**Keywords:** Microcontaminants, carbon nanoparticles, biofilms, ecotoxicity, mixtures.

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: afreixa@icra.cat</footnote>
Influence of natural dissolved organic matter on
herbicide toxicity to two marine microalgae

Sabine Stachowski*<fnref n="1" />, Nathalie Coquillé†<fnref n="2" />,<fnref n="3" />,<fnref n="4" />, Dominique Ménard<fnref n="1" />, Julien Rouxel<fnref n="1" />,
Valentin Dupraz<fnref n="1" />, Mélissa Eon<fnref n="5" />, Hélène Budzinski<fnref n="6" />, Edith Parlanti<fnref n="6" />, and Soizic Morin<fnref n="2" />

Abstract

The continuum between ecosystems leads to herbicide transfer up to coastal areas, where they can affect microalgae. In addition, the dissolved organic matter (DOM) can also interact with herbicides and microalgae. In this study, the influence of natural DOM was assessed in controlled conditions, on the toxicity of three herbicides (diuron (D), ingarol (I) and S-metolachlor (S)) towards cultures of two marine microalgae, *Chaetoceros calcitrans* and *Tetraselmis suecica*. The microalgae were exposed during 6 days to each herbicide alone (D0.05 and D0.5 µg.L⁻<fnref n="1" />; I0.05 and I0.5 µg.L⁻<fnref n="1" />; S0.5 and S5 µg.L⁻<fnref n="1" />) and to mixtures (M1: I0.05+D0.05+S0.5 and M2: I0.5+D0.5+S5), with and without DOM. The effects were assessed on growth, photosynthetic efficiency and relative lipid content. The chemical environment (herbicides, nutrients, DOM quantity and quality) was also characterized.

Results showed that ingarol (I0.5) was the most toxic, without and with DOM. When M2 effects were similar to I0.5 effects in *C. calcitrans*, they were amplified in *T. suecica*. The addition of DOM to the cultures induced a decreased and increased toxicity in *C. calcitrans* and *T. suecica*, respectively. Chemical analyses in the cultures exposed to I0.5 and M2 showed a higher production of dissolved organic carbon, with more aromatic molecules and a higher molecular weight, than in control cultures.

The toxicity modulation with DOM could be explained by (i) a modification of herbicide bioavailability (complexation), (ii) a different composition of cell walls depending on species and/or (iii) a higher detoxification ability for *C. calcitrans* made possible by DOM utilization.

**Keywords:** Microalgae, Herbicides, Natural dissolved organic matter, Toxicity, Interactions, Mod-
ulation

<footnote n="1">¹Ifremer – Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Institut Français de Recherche pour l'Exploitation de la MER - IFREMER – France</footnote>

<footnote n="2">²Iirstea – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA (FRANCE) – France</footnote>

<footnote n="3">³Ifremer – Ifrer – France</footnote>

<footnote n="4">⁴EPOC-LPTC – EPOC-LPTC – France</footnote>

<footnote n="5">⁵IRSTEA (IRSTEA) – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture - IRSTEA (FRANCE) – France</footnote>

<footnote n="6">⁶EPOC-LPTC – CNRS – France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: nathalie.coquille@gmail.com</footnote>
# How climate change enhances nanoparticle toxicity towards freshwater biofilms – NanoTox project

Berta Bonet*†¹, Stefan Krause¹, and Iseult Lynch¹

¹The School of Geography, Earth and Environmental Sciences - The University of Birmingham (GEES - UoB) – School of Geography, Earth and Environmental Sciences University of Birmingham Edgbaston Birmingham B15 2TT United Kingdom, United Kingdom

## Abstract

Aquatic ecosystems are threatened by multiple environmental stressors. Release of toxics such as engineered nanoparticles (ENPs) into aquatic systems and their effects on these ecosystems remain poorly understood. This, combined with the acceleration of the global climate warming (ENP + ΔT°C), could have significant consequences for aquatic life. The NanoTox project focuses on responses of fluvial biofilms, as key points of ENP entry into aquatic food webs, and examines the effects of combined stressors at ecosystem level. Preliminary results from recirculating stream mesocosms measurements on how river warming, the presence of environmental concentrations of ENPs and the combination of both stresses may further stress biofilms at different levels (e.g. functional, community structural and ecosystem) will be presented. To achieve this aim, outdoor microcosm studies are being performed at different temperatures (i.e. 15°C, 20°C and 25°C) representing realistic warming scenarios (i.e. up to 4°C increase from current average UK summer temperatures). After biofilm colonization at different temperatures, ENP will be added obtaining 4 treatments: (i) control (only T°C), (ii) AgNP, (iii) Ag2S NP to represent the "environmentally aged" form of silver, known to have lower dissolution potential, and (iv) AgNO3 as a positive (ionic) control. Sampling will be at the beginning of ENP exposition (0h), after 6, 24 and 72 hours (addressing acute effects) and after 7, 14 and 30 days (addressing chronic effects). Endpoints assessed include: uptake/localisation of ENP in biofilms, changes in community structure and ecosystems services provision, quantification/identification of secreted biomolecules and resulting ENP-coronas, in an integrated approach.

**Keywords:** Climate change impact, engineered nanoparticles (ENPs), engineered nanoparticle toxicity, contamination, biofilms, microbial metabolic activity.

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: b.bonet@bham.ac.uk</footnote>
Real-time and in situ monitoring of aquatic
environments using indigenous microbial
community-based biosensors

Jean-Michel Monier*¹ and Olivier Sibourg¹

¹ENOVEO – ENOVEO – 7 place Antonin Poncet - 69002 Lyon, France

Abstract

Increasing contamination of aquatic environments by organic loads or toxic chemicals has resulted in the need for real-time monitoring tools to be used in pollution detection and risk assessment. While monitoring aquatic environments is critical to ensure their quality and sustainability, monitoring strategies often relies on the collection of individual spot samples. Such approach, usually costly and time-consuming, only provides a snapshot that may often fail to detect localized transient contamination events and describe the dynamic changes of the monitored environment. The objective of our work was to develop a cost-effective solution to monitor aquatic environment in real-time, inform targeted sampling by conventional means (*ie*, spot sampling) and act as a decision support tool. This novel biosensor uses microorganisms naturally occurring in the targeted environment as bioindicators and exploits their ability to convert chemical energy into an exploitable electrical signal. The electrical signal generated by the sensor is proportional to the organic load or affected by the presence of toxic compounds, as it directly reflects microbial activity at the surface of the colonized electrodes. Biosensors have been deployed at different sites in order to estimate either the organic load or BOD in real time (*eg*, groundwater, sewage networks...) or detect toxic compounds and assess their impact on their environmental sink (*eg*, industrial processes and wastewater treatment plants...). Through real case studies, the presentation will address the principle, information provided and advantages of such devices and how they can provide unique information to act as a decision support tool.

**Keywords:** biosensor, microbial communities, aquatic environments, in situ, real, time, toxicity, BOD, organic load

<footnote>*Speaker</footnote>
# Tolerance of stream biofilms to micropollutants indicates causality and reflects community recovery

Ahmed Tlili*†<fnref n="1" />, Natàlia Corcoll<fnref n="2" />, ãsa Arrhenius<fnref n="2" />, Thomas Backhaus<fnref n="2" />, Juliane Hollender<fnref n="1" />, Bettina Wagner<fnref n="1" />, and Renata Behra<fnref n="1" />

## Abstract

In Switzerland, wastewater treatment plants (WWTPs) are currently upgraded to reduce input of micropollutants into the receiving streams. This, offers the opportunity to study in real environments consequences of a reduction in micropollutants. Therefore, this study aims to examine tolerance to micropollutant mixtures of periphyton that has been sampled one year before and after the upgrade, in comparison to impacts on periphyton from non upgraded WWTPs. Results showed that one year after the upgrade, difference in micropol- lutant concentrations between up- and downstream of the effluent decreased. These results concurred with the tolerance measurements at this site, since no difference between up- and downstream periphyton was observed, unlike before the upgrade. Increased tolerance to the extracts, on the other hand, was detected at the non-upgraded sites in both sampling times. These results point to the strong relationship between microbial tolerance and the intensity of contamination by micropollutants. Furthermore, microbial diversity was consistent with tolerance measurements. This was the case at the non-upgraded sites in both sampling times, where bacterial and algal diversity of up- and downstream periphyton were different. On the contrary, differences at the upgraded site were less pronounced after than before the up- grade, indicating that micropollutants were a driver for the microbial diversity downstream of the WWTPs. Overall, our findings prove the relevance of using community tolerance for retrospective risk assessment of micropollutants, and to assess ecosystem recovery, while capturing the biological and chemical complexity in the environment.

**Keywords:** wastewater, periphyton, pollution, induced community tolerance, passive sampler

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

<footnote n="1">¹Swiss Federal Institute of Aquatic Science and Technology - EAWAG (SWITZERLAND) – Switzerland</footnote>

<footnote n="2">²Dept. Biological and Environmental Sciences, University of Gothenburg – Sweden</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: ahmed.tlili@eawag.ch</footnote>
# Unrecognized soil algal and cyanobacterial communities as a model, for herbicide risk assessment in agricultural soils.

Olivier Crouzet*<fnref n="1" />, Laurent Consentino<fnref n="1" />, Clarisse Mallet<fnref n="2" />, Jean-Pierre Aguer<fnref n="2" />, Pierre-Alain Maron<fnref n="3" />, and Annette Bérard<fnref n="4" />

## Abstract

Soil algae and cyanobacteria form microbiotic photosynthetic crusts, not only on desert soils, but also in temperate cropped soils. Despite a scarce literature about their ecology in agricultural soils, they could constitute an original model to improve the consideration of microbial component in the environmental risk assessment of herbicides. This work focused on the suitability of biochemical and molecular methods to characterize structural and functional responses of algae and cyanobacteria, to herbicides, in agricultural soils under different cropping systems, throughout laboratory and field approaches.

Cultural approaches are still helpful to isolate edaphic species for further ecotoxicological tests at the individual level. Photosynthetic pigments can provide biomass (chlorophyll a) or structural (pigment diversity) indicators. Based on chlorophyll a biomass, a modified pollution-induced community tolerance (PICT) approach was developed. Several genetic markers were successfully applied, to estimate their community composition and diversity. Toxic effects on algal and cyanobacterial biomasses and diversities have been evidenced at doses sometimes below recommended field rates, in microcosm experiment. Overall, edaphic algal and cyanobacterial communities showed a higher sensitivity to herbicides, compared to commonly studied soil bacterial and fungal community, in field monitoring. The PICT approach highlighted a higher tolerance to phenyl urea herbicides of the photosynthetic microbial communities in conventional versus organic soils. Characterization of the diversity of the algal and cyanobacterial communities, unravel links between some taxa and shift in tolerance levels. The structural stability of soil surface aggregates, considered as a functional output of algae and cyanobacteria crusts, were disturbed by herbicide treatments.

**Keywords:** soil, pesticides, algae, communities, functions, diversity, tolerance

<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">²UMR 6023 LMGE - Centre National de la Recherche Scientifique - CNRS, Université Clermont</footnote>

<footnote>Auvergne - Campus universitaire des Cézeaux Avenue des Landais 63622 AUBIERE cedex, France</footnote>

<footnote n="3">³UMR 1347 Agroecologie - Institut national de la recherche agronomique (INRA) : UMR1347, Agrosup Dijon, Université de Bourgogne - 21000 Dijon, France</footnote>

<footnote n="4">⁴UMR 1114 EMMAH - Institut National de la Recherche Agronomique - INRA - 84914 Avignon, France</footnote>

<footnote>*Speaker</footnote>
Towards the implementation of Pollution Induced
Community Tolerance (PICT) approaches in water
regulatory frameworks: Developing standard
procedures for the sediment compartment

Stéphane Pesce*†<fnref n="1" />, Anaïs Charton<fnref n="1" />, Aymeric Dabrin<fnref n="1" />, Emilie Lyautey<fnref n="2" />, Ayanleh Mahamoud Ahmed<fnref n="2" />, Emmanuel Naffrechoux<fnref n="3" />, Olivier Perceval<fnref n="4" />, and Chloé Bonnineau<fnref n="1" />

Abstract

Assessing the ecological effects of pollutants is an important aspect of regulations dealing with the sustainable management of water resources. In this context, scientists and regulators face the challenge of going beyond the estimation of pollutant concentrations to take into account the ecotoxicological effects on exposed aquatic communities. Among microbial ecotoxicological approaches, the pollution induced community tolerance (PICT) concept has been proven to be a suitable biomonitoring tool for *in situ* assessment of either the response of aquatic microbial communities to toxicant exposure [1] or of their recovery following the improvement of water chemical quality [2,3]. The PICT is a powerful microbial metric to better link the assessment of ecological and chemical status of ecosystems by providing causal analysis in effect-based monitoring of impacted field sites [4]. However, prior its implementation in regulatory frameworks, further work is required to standardize PICT measurement and define baseline tolerance levels at large geographical scales. In this context, we will present the first results of the application of PICT approaches in the sediment compartment (with copper as model compound). We will specifically focus on both the development of standardized protocols (from sampling to dose-response modeling) and on the evaluation and control of potential confounding factors to make measurements comparable in space and time by considering robust reference conditions. [1] Tlili et al., 2017. *Water Res* 111; [2] Larras et al., 2016. *ESPR* 23; [3] Pesce et al., 2016. *Agr Ecosyst Environ* 221; [4] Tlili et al., 2016. *Freshwater Biol* 61

**Keywords:** Benthic communities, microbial ecotoxicology, PICT, sediments, copper, standardization

<footnote n="1">¹UR MALY – Irstea – 5 rue de la Doua, BP32108, 69616 Villeurbanne Cedex, France</footnote>

<footnote n="2">²Centre Alpin de Recherche sur les Réseaux Trophiques des Ecosystèmes Limniques (CARRTEL) – Université de Savoie, Institut national de la recherche agronomique (INRA) – Domaine Universitaire, 73376 Le Bourget-du-Lac Cedex, 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 n="4">⁴Agence Française pour la Biodiversité – AFB, France – 5 Square Félix Nadar, 94300 Vincennes, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: stephane.pesce@irstea.fr</footnote>
# Abiotic, biotic (microflora and fauna) and functional parameters of derelict soils - towards a potential use of derelict soils as a resource.

Quentin Vincent*†²,¹, Apolline Auclerc², Thierry Beguiristain¹, and Corinne Leyval¹

²Laboratoire Sols et Environnements (LSE) – Institut national de la recherche agronomique (INRA) : UMR1120, Université de Lorraine – 2 avenue de la Forêt de Haye, 54500 Vandoeuvre-lès-Nancy, France

¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360, Université de Lorraine – Faculté des Sciences et Technologies, 54500 Vandoeuvre-lès-Nancy, France

## Abstract

The intensification and closure of industrial activities during the last century led to large surfaces of derelict lands. The derelict soils have a low fertility and might be contaminated, and remain unused. However, with the increasing demand of soil surface, they might be considered as a resource for non-food biomass production. The study of their biodiversity and biological activity may be an indication for a potential re-use of derelict soils. The objective of our study was to investigate the quality of six derelict soils, considering abiotic, biotic and functional approaches. Soil bacteria, fungi, plant, meso- and macro-fauna communities were studied in a set of six different derelict soils (coking plant, settling pond, constructed, inert waste storage soils) as well as the decomposition process, considering the decomposer trophic network, enzyme activity, mineralization activity and organic pollutant degradation. Biodiversity levels in the derelict soils were rather high but depended on the considered biotic component. Multivariate analysis showed that biotic parameters co-varied more with fertility proxies than with soil contamination parameters. Similarly, functional parameters significantly co-varied with abiotic parameters. Co-inertia analysis showed that several structural and functional components of biota were useful to discriminate the derelict soils and evaluate their quality. We found that the compost amended constructed soil had the highest quality while the settling pond soil and the contaminated constructed soil had the lowest one. Although differences between the derelict soils were observed, this study showed that derelict soils may provide a biodiversity ecosystem service and are functional for decomposition.

**Keywords:** Derelict soil, Microflora, Fauna, Pollution, Mineralization, Function, Soil quality

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: quentin.vincent@univ-lorraine.fr</footnote>
# Are fungicides a threat for heterotrophic aquatic food webs?

Jochen Zubrod*<fnref n="1" />, Ralf Schulz<fnref n="1" />, and Mirco Bundschuh<fnref n="2" />

## Abstract

Fungicides can affect heterotrophic organisms and the functions they provide via multiple pathways. Here we discuss the impact of fungicides on leaf-associated biofilms (i.e., aquatic fungi and bacteria) and how these modifications modulate the nutritional quality of leaf material for leaf-shredding invertebrates. When leaves were exposed to the inorganic fungicide copper during microbial conditioning, an increased palatability of the leaves for the amphipod shredder *Gammarus fossarum* was observed. This preference was attributed to an increased fungal biomass accrual and a shift in fungal community composition favoring species considered as more palatable. When, however, fed with these preferred leaves over multiple weeks, *Gammarus* showed impairments in growth and energy reserves. Organic fungicides, in contrast, favored leaf-associated fungal species considered less palatable, and thus induced a rejection of this leaf material, while feeding on these leaves for several weeks again induced impairments in gammarids' growth. These insights, thus, suggest that the food choice of selectively feeding detritivores may not be predictive for indirect effects on their physiological level. Nonetheless, it was shown that fungicides can modify the physiological fitness of detritivores via bottom-up directed effect pathways indicating long-term impacts in the population development of these species at field relevant levels.

**Keywords:** fungicides, leaf, associated microorganisms, shredders, trophic interactions

<footnote n="1">¹University of Koblenz-Landau – Germany</footnote>

<footnote n="2">²Swedish University of Agricultural Sciences – Sweden</footnote>

<footnote>*Speaker</footnote>
# Evaluation of the ecotoxicological impact of municipal wastewaters on wildlife: a case study in Tuscany

Federica Spina*¹, Laura Palli, Mario Aragno, A Bonari, A Bossi, I Pompilio, S Dugheri, D Santianni, S Caffaz, M Vincenzi, Riccardo Gori, and Giovanna Cristina Varese

¹Mycotheca Universitatis Taurinensis, University of Turin – Turin, Italy

## Abstract

As effect of human activities, persistent organic pollutants are discharged in the environment and can be found in surface waters. They could ultimately accumulate in wastewaters treatment plants (WWTPs) which unfortunately are rarely designed to face this problem. The aim of this study was to evaluate the quality of waters in Tuscany region, in terms of pharmaceutical active compounds (PhAC) concentration and environmental risk assessment. Three WWTPs managed by Publiacqua SpA located in Pistoia, Figline Valdarno and Lastra a Signa were taken into consideration. The process efficiency was studied, evaluating also the effect of seasonal variability: samples were collected from both the influents and the effluents in spring, summer and winter. The chemical characterization revealed the presence of nine PhAC including three non-steroidal anti-inflammatory drugs (diclofenac, ketoprofen, paracetamol), three antibiotics (amoxicillin, clarithromycin, doxycycline), one β-blocker (atenolol), one antiepileptic drug (carbamazepine) and one hormone (β-estradiol). As expected, their concentration was strongly affected by the season, with high antibiotics concentration detected during cold periods. As regards the wastewaters ecotoxicity, several bioassays were performed based on three seeds (watercress, sorghum and cucumber), algae, crustaceous and luminescent bacteria. *Raphidocelis subcapitata* was often the most sensitive organism, but data confirmed the importance of a battery of tests with model organisms representative of different trophic levels of the aquatic ecosystem. The WWTPs did not always reduce the toxicity indicating that the waters discharged in the receiving basin still had relevant effects on the ecosystem. Ecotoxicological index will be performed to better define the actual environmental risk.

**Keywords:** ecotoxicological bioassays, micropollutants, fungi

<footnote>*Speaker</footnote>
# Changes in the interactions between periphytic microalgae and micromeiofauna induced by environmental exposure to diuron and/or imidacloprid

Julie Neury-Ormanni*¹, Jacky Vedrenne¹, Gwilhern Jan¹, Betty Chaumet¹, Mélissa Eon¹, and Soizic Morin†¹

¹Iirstea-Bordeaux – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture – 50 Avenue de Verdun 33612 Gazinet Cestas, France

## Abstract

Only a little part of agricultural pesticides reach their target organisms; the remaining is transferred by phenomena like leaching to aquatic ecosystems, where they are found in mixtures. Chemical stressors induce direct and indirect changes on the fauna and/or the flora, thus complicating pesticide risk assessment.

We exposed mature biofilms for 14 days in aquaria to four treatments: control, the herbicide diuron (priority substance in the Water Framework Directive) and the insecticide imidacloprid (included in the Watchlist), singly and in mixture, at environmental concentrations (5µg/L). Our aim was to understand biofilm community responses when exposed to pesticides, based on changes in structure (species composition, diversity and density) and community tolerance, within a PICT (Pollution-Induced Community Tolerance) framework. We quantified weekly microalgal and micromeiofaunal composition, biofilm dry weight, chlorophyll a concentration and exopolysaccharide content.

In the controls, biofilm dry weight increased over time; whereas in the three pesticide treatments the biomass did not increase after 7 days of exposure. PICT approaches based on photosynthesis inhibition revealed pre-exposure to diuron, while imidacloprid didn't affect directly the microalgae. Contrastingly, the autotrophy index increased with imidacloprid pressure, in agreement with its known target action on animals. These impacts were confirmed by taxonomy analyses. The binary mixture had the same effects on microalgae as diuron, decreasing photosynthetic efficiency and chlorophyll concentration.

Altogether the results of this study highlighted changes in the proportions of microalgae and micromeiofauna reflecting their interactions and exposure history. They also demonstrate that micromeiofauna should not be overlooked in microbial ecotoxicology.

**Keywords:** biofilm, micromeiofauna, microalgae, diuron, imidaclopride, community response, PICT

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: soizic.morin@irstea.fr</footnote>
# Impacts of silver nanoparticles on aquatic detritus-based food webs

Claudia Pascoal*¹, Daniela Batista¹, and Fernanda Cassio¹

¹CBMA, University of Minho – Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, Braga, Portugal

## Abstract

The production and use of silver nanoparticles (AgNPs) has significantly grown over the last decade, increasing the chances of these NPs entering in freshwaters. In these ecosystems, AgNPs and ionic silver released from dissolution of NPs can have toxic effects on aquatic species and can compromise important ecosystem processes. We used a microcosm approach, to assess the impacts of environmentally-realistic concentrations of AgNPs and ionic Ag across a freshwater detrital food web comprising plant litter, microbes and invertebrate detritivore species. Exposure to increasing concentrations of AgNPs and ionic Ag led to a decrease in fungal diversity and reproduction and inhibited plant litter decomposition. The toxicity of AgNP to microbial decomposers may be related to AgNP absorption on leaves, where the fungi are growing, preventing the release of spores, limiting fungal growth and inhibiting the activity of microbial decomposers. Moreover, AgNPs and ionic Ag were able to trigger oxidative and neuronal stress in invertebrate detritivores. The consumption of leaves by detritivores was lower in treatments with leaves pre-exposed to nano and ionic Ag compared to water exposure treatments, which can be related to a food avoidance behavior with impacts to detrital-based food webs.

**Keywords:** Nanoparticles, Silver, Detritus foodwebs, Freshwaters

<footnote>*Speaker</footnote>
Dysbiosis in plant-endophyte partnership: repeated
short exposures of sulfametho- xazole &
trimethoprim at microconcen- trations can disturb
the microbial community in soft rush, Juncus effusus

Muhammad Arslan*<fnref n="1" />, Marcello Santoni, Arndt Wiessner<fnref n="1" />, Thomas Neu<fnref n="2" />, Dietmar Pieper<fnref n="3" />, and Jochen Müller<fnref n="1" />

Abstract

Plants harbour complex endophytic communities that play beneficial role for growth of
their host. A disturbance of these interactions can be detrimental for the plant. In this
study, our objectives were to query whether certain organic micropollutants might interfere
with the plant-bacteria partnership and, if so, how the endophytic community responds.
Towards these aims, a wetland plant *Juncus effusus* was repeatedly exposed to low aque-
ous concentrations of sulfamethaxazole and trimethoprim, antibiotics frequently found in
the environment. Plant status was assessed based on evapotransporation rates as indica-
tor of plant fitness and by visual inspection. Changes of endophytic communities in roots
and shoots of exposed and unexposed plants were tracked via culture-dependent (MPN),
and culture-independent (qPCR, FISH, Illumina 16S rRNA sequencing) approaches. The
*in planta* physiological response was evaluated via detection of reactive species (ROS/RNS)
using confocal laser scanning microscopy (CLSM). During the first dose of antibiotics, evap-
otransporation rates decreased, and plants became infested with insects. After omitting the
antibiotics, evapotransporation recovered to some extent, but after repeated exposures it
was almost zero. Eventually, plant tissue turned necrotic and CLSM analysis showed in-
creased generation of ROS/RNS in stressed plants, i.e., evidence for infection. Endophytic
fungi were never observed microscopically. Comparative analyses of bacterial endophytes
revealed that post-treated roots, but not shoots, had significantly increased abundances of
*Gammaproteobacteria*, *Firmicutes*, and *Actinobacteria*. Nevertheless, community profiles of
post-treated roots and shoots differed substantially from their non-treated counterparts. In
conclusion, study provides evidence that antibiotics at environmental concentrations can
trigger a dysbiosis in a wetland plant.

**Keywords:** plant, endophyte partnership, dose antimicrobials, microbial ecology, organic micropol-
lutants

*Speaker

<footnote n="1">¹Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology (UFZ) –
Permoserstr. 15, Leipzig, Germany</footnote>

<footnote n="2">²Helmholtz Centre for Environmental Research, Department of River Ecology – Brückstr. 3A, 39114 Magdeburg, Germany</footnote>

<footnote n="3">³Helmholtz Center for Infection Research – Microbial Interactions and Processes Research Group (HZI) – 38124 Braunschweig, Germany</footnote>
RECOTOX: a network of research sites to implement
multiple and multi-scale approaches for a posteriori
environmental risk assessment in agricultural systems

Christian Mougin*¹, Véronique Gouy†², and Vincent Bretagnolle‡³

¹Ecologie fonctionnelle et écotoxicologie des agroécosystèmes (ECOSYS) – Institut National de la Recherche Agronomique : UMR1402 – Pôle Ecotoxicologie, Route de St-Cyr, 78016 Versailles, France

²Milieux Aquatiques, Écologie et Pollutions (MAEP) – Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture – 5 rue de la Doua, 69626, Villeurbanne, France

³Centre d'Etudes Biologiques de Chizé (CEBC) – CNRS : UMR7372 – 405 Route de La Canauderie, 79360, Villiers-en-Bois, France

Abstract

One of the greatest current challenges is the ever increasing environmental pollution with its concomitant environmental and human impacts. For many years, the national scientific community has pointed out the lack of long-term field and large-scale monitoring of the effects of chemicals on natural media such as water, soil and air. In that context, the network RECOTOX aims at promoting transversal, spatially explicit and integrated research to meet the scientific challenges of understanding and anticipating the environmental and health impacts of pesticides and biocides (organics, biopesticides, metals). RECOTOX includes 15 sites in metropolitan France and French West Indies, which are mostly already endorsed by national organizations (AllEnvi, INRA, CNRS, IRSTEA, universities) and instrumented networks (SOERE RBV, network of catchment area; SOERE RZA, network of long term ecological research observatories; long-term cropping system trials...). These sites allow *in natura* observation and experimentation in ecotoxicology in offering various and complementary agro-pedo-climatic situations. The network aims at: promoting transversal and integrated research, analyzing the whole chain "pressures-exposures-impacts" of pesticides, sharing between its sites a common culture of ecotoxicology. RECOTOX encourages the implementation of multiple and large geographical scale approaches for a *posteriori* environmental risk assessment in agricultural systems. Through its scientific project and its transverse activities, it will contribute to promote the use of bio-indicators as, microorganisms, for environmental risk assessment by ensuring the validity of biological tools and their operational potential. https://www6.inra.fr/ecotox/Animation-nationale/Infrastructures/RECOTOX

**Keywords:** network, sites, in natura, pesticides, ecotoxicology, pressures, fate, exposure, effects, risk
assessment, microbial communities, invertebrates, vertebrates, spatial and temporal scales, landscape

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: veronique.gouy@irstea.fr</footnote>

<footnote>‡Corresponding author: Vincent.BRETAGNOLLE@cebc.cnrs.fr</footnote>
Communication Abstracts

POSTER CORNERS
# Bioremediation of an aged dioxins and furans contaminated soil by saprotrophic telluric fungi and a mild chemical oxidation

Isabelle Delsarte*¹, Catherine Rafin*¹, Yann Landkocz¹, 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

An innovative method for the bioremediation of contaminated soils by dioxins and furans (PCDD/F) by telluric saprotrophic fungi is developed. The degradation of PCDD/F has two main obstacles to efficient biological treatments by fungi: their low bioavailability and the difficulty of initiating the oxidation of so stable molecules. To overcome these technological obstacles, we propose to couple a mild chemical oxidation to a biological one by saprotrophic telluric fungi.

A collection of 33 fungal strains is built up using a PCDD/F historically contaminated soil originating from an experimental site (Halluin) in the North of France. A simplified study system in mineral medium under axenic conditions allowed us to determine the best parameters i.e. the fungal strains (*Penicillium brasilianum* and *Fusarium solani*), the type and the dose of chemical oxidant (the Fenton's reagent) for the degradation lab experiment conducted in historically PCDD/F contaminated soil microcosms.

Twelve treatments (biological and/or chemical) are realized in triplicate for only two months of incubation and the concentrations of 17 PCDD/F congeners are then analysed. The inoculation of the endogen strain *P. brasilianum* in presence of cardboard chips as a bulking agent led to a decrease in PCDD/F toxicity of 40.5 % in comparison with the control.

This work draws new perspectives for remediation techniques of contaminated soils by PCDD/F that are cost-effective and environmentally friendly.

Acknowledgement: Financial support for this research was provided by Region Hauts de France, Pôle Métropolitain Côte d'Opale (PMCO) and the European Funds for Regional Development (ERDF).

**Keywords:** Bioremediation, Dioxins, Fenton's reagent, Furans, Saprotrophic fungi, Soil

<footnote>*Speaker</footnote>
# On-farm bioremediation of leftover pesticide solutions using biobed

Talaat Elsebai*¹

¹National Research Centre (NRC) – National Research Centre, 12622 Dokki, Giza, Egypt., Egypt

## Abstract

Emissions of pesticides from agricultural soils to adjacent environmental compartments are deriving from direct (point source) and indirect (non-point source) losses. Direct losses of pesticides from point sources are of different origins among which (leftover pesticide solution, spillages during filling and cleaning of the spraying equipment, leakages of spraying equipment, etc.). In Egypt, leftover pesticide solution represents the main source of pesticides emissions due to the small size of Egyptian farm. Biobed is a smart system designed to receive and dissipate pesticide contained in wastewaters, it is of a prime interest. The biomixture (mix of straw, compost or peatmoss and top soil at a volumetric proportion of 2:1:1 respectively) is the key component of biobed. This study aimed to remediate a complex of leftover pesticide mixtures (methomyl, pirimiphos methyl, diniconazole, pyridaben, hexythiozox, hymexazole and abamectin) at recommendation rate. Four biobeds were prepared; (i) pesticide solution was replaced by water (control), (ii), for insecticides remediation (iii), for fungicides remediation (iv) for remediation of complex of insecti- and fungi-cide, the moisture content was maintained around 60% of WHC. Samples of the biobed material at different depths were withdrawn at the end of experiment for pesticide residues analysis (6 months after the last addition). Our data indicated that more than 90% of all tested pesticides were dissipated. In addition, dehydrogenase activity of all treatment was comparable of this of the control treatment. It could be suggested from our findings that biobeds are an efficient technique to limit pesticide release in the environment.

**Keywords:** pesticide pollutions, point source, biobed, bioremediation

<footnote>*Speaker</footnote>
# Dichloromethane degradation in multi-contaminated groundwater: coupling biomolecular and compound-specific isotope analyses

Louis Hermon*<fnref n="1" />,<fnref n="2" />, Jérémie Denonfoux<fnref n="3" />, Jennifer Hellal<fnref n="1" />, Catherine Joulian<fnref n="1" />, Stéphanie Ferreira<fnref n="3" />, Stéphane Vuilleumier<fnref n="2" />, and Gwenaël Imfeld†<fnref n="4" />

## Abstract

*In situ* and *in vitro* biodegradation of DCM were investigated in groundwater from a former solvent-processing industrial site where DCM is a major contaminant together with chlorinated ethenes and ethanes. Compound-specific isotope analysis (CSIA) of DCM enabled to evaluate and quantify *in situ* biodegradation at the field scale. Applying DCM-specific isotopic enrichment factors (εC) from reference strains, biodegradation extent (B%) varied across the plume and through time from 4 to 50%. Carbon isotope enrichment factors of 21.8 ± 3 and -31.0 ± 3 for aerobic and anaerobic degradation of DCM by *Hyphomicrobium* sp. GJ21 grown in contaminated site groundwater and in synthetic medium were obtained. Under aerobic conditions, significant differences in εc were observed for contaminated site groundwater and culture mineral medium, suggesting an effect of field physico-chemical conditions on the degradation mechanism of DCM. High-throughput sequencing of the 16S rrnA gene amplified from groundwater samples along the contamination plume revealed the occurrence of several genus-level taxa previously associated with DCM biodegradation under both aerobic and under anaerobic conditions. The abundance of dehalogenase genes associated with DCM biodegradation was quantified by qPCR of samples along the contamination plume, totalling 4102-1107 copies/L depending on the location. Highest abundances of dehalogenase genes were detected in the source zone where the highest co-contaminant concentrations were measured. Obtained results highlight the potential for on-site DCM biodegradation. The proposed integrative approach coupling biomolecular and CSIA may help identify microorganisms, microbial pathways and suitable redox conditions associated for bioremediation of the investigated site.

**Keywords:** Bioremediation, contaminated groundwater, dichloromethane, degradation, CSIA, biomarkers

<footnote n="1">¹Département Eau Environnement Ecotechnologies (D3E/BGE) – Bureau de Recherches Géologiques et Minières (BRGM) – France</footnote>

<footnote n="2">²Department of Microbiology, Genomics and the Environment – université de Strasbourg, CNRS : UMR7156 – 28 Rue Goethe 67000 Strasbourg, France</footnote>

<footnote n="3">³Communautés microbiennes – genoscreen – 1 rue du Professeur Calmette, Lille, France</footnote>

<footnote n="4">⁴Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS) – université de Strasbourg, CNRS : UMR7517 – 1 rue Blessig, 67084 Strasbourg Cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: imfeld@unistra.fr</footnote>
# Microbiology of Acid Mine Drainage: from natural attenuation to bioremediation

Marina Héry*¹, Odile Bruneel¹, and Corinne Casiot¹

¹Hydrosciences Montpellier (HSM) – Institut de recherche pour le développement [IRD], CNRS : UMR5569, Université Montpellier II - Sciences et techniques - CC57, 163, rue Auguste Broussonet, 34090, Montpellier, France, France

## Abstract

Acid mine drainages (AMD) resulting from the oxidation of sulfides contained in mining wastes lead to the release of toxic elements into the aquatic environment. They represent a severe treat for the ecosystems and public health. Arsenic (As) is one of the most toxic elements commonly associated with AMD. In the abandoned Carnoulès mine (Gard, France), the formation of an AMD containing hundred of mg.L-1 of As coincides with the spring of the Reigous stream. HydroSciences Montpellier lab has conducted for almost 20 years interdisciplinary work aiming at deciphering the biogeochemical processes controlling the fate of As in the Reigous waters. Along this stream, natural attenuation leads to the removal of up to 90% of the dissolved As from the water. This natural process is based on microbially driven oxydation of iron and arsenic followed by their co-precipitation in the streambed. Such capacities offer a promising strategy for the development of passive biological treatments of As-rich AMD. The IngECOST-DMA ANR project proposed a multidisciplinary approach associating microbiologists, process engineers, geochemists, mineralogists, materials engineers, to develop an integrated strategy for the management of As-rich AMD. Biotreatment options were first studied on a pilot-scale system and then tested in situ with in a field-scale pilot implemented at the Carnoulès former mine. This presentation will provide a review of (i) the results obtained on the biogeochemistry of the Carnoulès AMD and of (ii) the recent work conducted on innovative biotreatment options of this extreme pollution.

**Keywords:** acid mine drainage, natural attenuation, iron and arsenic oxidation, bacterial communities, bioremediation

<footnote>*Speaker</footnote>
# Microbial resources management for bioremediation

Olivier Sibourg<sup>†1</sup>, Cedric Malandain<sup>1</sup>, Celine Baguelin<sup>1</sup>, Sandra Entresangles<sup>1</sup>, and
Jean-Michel Monier*<sup>1</sup>

<sup>1</sup>ENOVEO – ENOVEO – 7 place Antonin Poncet - 69002 Lyon, France

## Abstract

Successful site remediation through microbial degradation requires a thorough understanding of the microbial mechanisms implicated in the biodegradation of the pollutant in question. Regardless of the nature of the pollutant or environmental matrix, the success of a bioremediation project hinges on the consideration of two essential components: the presence of a microbial community which can be stimulated in order to promote biodegradation activity and the bioavailability of the pollutant (often influenced by the chemical and physical characteristics of the site). Understanding microbial degradation processes is therefore a major contribution to the success of such projects. ENOVEO's biotechnological solutions include the characterization of the microbial degradation potential of a site and the evolution of the activity of indigenous bacteria. On-going technological advancements in the fields of metagenomics, metatranscriptomics and metaproteomics continue to bring new perspectives to our understanding of microbial mechanisms. Our presentation will illustrate how different molecular biology tools can be utilized to characterize and manage microbial resources, from the development of the remedial action plan to site treatment and closure, to identify any potential difficulties associated with up-scaling and to enable the optimization of depollution strategies.

**Keywords:** bioremediation, polluted sites, microorganisms, molecular biology tools

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: o.sibourg@enoveo.com</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 Cantarel¹, Jonathan Gervaix¹, Jean Martins², and Agnès Richaume*†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

²Institute Geosciences Environment (IGE) – CNRS : UMR5001, Université Grenoble Alpes – Université Grenoble Alpes CS 40 700. F-38058 Grenoble cedex, France

## 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>*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 David<sup>1,2</sup>, Celine Roosevelt-Amsaleg<sup>2</sup>, Fabrice Alliot<sup>2</sup>, Marjolaine Bourdat-Deschamps<sup>1</sup>, and Olivier Crouzet*†1

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

<sup>2</sup>Milieux 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>
Impact of pesticides on denitrification in
groundwaters

Caroline Michel*¹, Nicole Baran¹, Laurent Andre¹, and Catherine Joulian¹

¹BRGM – Bureau de Recherches Géologiques et Minières (BRGM) – 3 avenue Claude Guillemin, B.P. 36009, Orléans cedex 2, France

Abstract

Denitrification processes are involved in the decrease of nitrate (NO3-) concentrations in groundwaters impacted by human activities. Non-target/side effects of pesticides on soil microbial denitrification have been demonstrated. It is however now well established that groundwater is an ecosystem in itself with specific microbial communities different from soils' ones. If the impact of pesticides on groundwater bacterial communities has already been studied, the impact of pesticides on the denitrification activity in groundwater has still to be investigated. Groundwater samples are collected from several wells on a catchment submitted to agricultural practices with fertilizers and pesticides uses. Groundwaters contain nitrate (lower than limit of quantification up to 80 mg/l), pesticides and some of their metabolites in low concentrations. The abundance of genes involved in denitrification (*narG*, *napA*, *nirS*) shows the occurrence of bacteria able to perform, at least, the two first steps of denitrification. Three groundwaters distributed within the catchment and exhibiting different denitrification signatures based on genes identification and physic-chemical properties are selected. They are used in laboratory to perform batch experiments in conditions boosting microbial denitrification (addition of KNO3 and acetate, anaerobic conditions, 25°C). The microbial ecotoxicology study is conducted in the presence or absence of pesticides spiked as single or cocktail pesticides. Several parameters (pH, concentrations of acetate, NO3, NO2, N2O, biomass) are recorded over time to quantify denitrification rate according to experimental conditions. This experimental work constitutes a first step in the understanding of the inhibitor/catalyzer role of pesticides on denitrification process.

**Keywords:** denitrification, groundwater microbial community, pesticides, side effects

<footnote>*Speaker</footnote>
Long Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Bacteria Occupying Nodules of Soybeans (Glycine max)

Cécile Revellin<fnref n="1" />, Alain Hartmann*<fnref n="1" />, Edward Topp<fnref n="2" />, and Sébastien Solanas<fnref n="1" />

**Abstract**

Antibiotics can be entrained onto agricultural land through the application of organic wastes or wastewater for irrigation. In order to evaluate the impacts on soil microorganisms of exposure to antibiotics (ATB), replicated plots treated each year with a mixture of sulfamethazine, chlortetracycline and tylosin at concentrations ranging from 0.1 to 10 mg/kg, were initiated in 1999 at the AAFC research farm in London ON. Plots were seeded with soybeans. Soybean nodules were collected in 2012, after 14 annual ATB treatments. The bradyrhizobia strains were characterized by serology and molecular methods. The sensitivity of isolates to the 3 ATB was also evaluated. Based on molecular characterization the collection of 278 bradyrhizobia was very diverse. The distribution of isolates in RSα fingerprint groups was significantly different in the 3 soils treated with antibiotics compared to control soil, with an increase in proportion of strains belonging to RSα types a, b, c and q. This result confirms those based on serotyping: strains belonging to *B. liaoningense* serogroup 135 are more abundant in soils having received antibiotics (compared to control). Using RSα fingerprinting results, Shannon diversity indexes were computed, T-tests revealed that Shannon diversity indexes were significantly different between control soil and soil having received the lower dose of antibiotics. Overall, the present study indicates that long term treatment with veterinary antibiotics i.e. tylosin, chlortetracycline and sulfamethazine alters the composition of Bradyrhizobial populations occupying soybean nodules. The sensitivity of bradyrhizobia to the three antibiotics was not associated with the treatment from which they were recovered.

**Keywords:** Bradyrhizobium, antibiotics, soil, molecular characterization, serology

<footnote n="1">¹Agroécologie [Dijon] – Université de Bourgogne, Institut National de la Recherche Agronomique : UMRINRA 1347, Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Université Bourgogne Franche-Comté – Université de Bourgogne - 17 rue Sully - BP 86510 - 21000 Dijon, France</footnote>

<footnote n="2">²London Research and Development Centre (AAFC) – 1391 Sandford St London, Ontario N5V 4T3, Canada</footnote>

<footnote>*Speaker</footnote>
# SYNAQUA: A French-Swiss program to modernize the environmental biomonitoring of Lake Geneva and transboundary rivers

Agnès Bouchez*†¹ and Jan Pawlowski

¹UMR CARRTEL – Institut National de la Recherche Agronomique - INRA (FRANCE) – 75 av. de Corzent, 74203 Thonon-les-Bains, France

## Abstract

Rivers and lakes play a major role in the environment by providing not only habitat for fauna and flora, but also improving the landscape and therefore the quality of life. The effectiveness of environmental protection measures rests on the early and precise identification of pressure points, which should be facilitated by environmental genomics. To implement measures to preserve or restore aquatic areas, the SYNAQUA project proposes to use the genetic tools based on the recognition of bio-indicator organisms present in the aquatic environment directly from their DNA. The method of environmental DNA analysis, is tested by Swiss and French scientists in collaboration with public and private stakeholders on diatoms, a bioindicator commonly used for water quality assessment.

The aim is to bring together scientists, environment managers as well as public and private partners in Switzerland and France to develop and apply this DNA-based broadband approach to biomonitoring of regional aquatic ecosystems. Collaboration between public and private stakeholders will help to develop a robust and reliable tool that is tailored to their needs and will strengthen the current practice of environmental diagnosis. The innovative tools that will be developed in synergy in France and Switzerland should make it possible to respond to the need for better monitoring and protection of the regional heritage of aquatic environments in a rapidly changing area subject to multiple pressures.

**Keywords:** aquatic ecosystem, biomonitoring, eco, genomic tools, High Throughput Sequencing, DNA, barcoding

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: agnes.bouchez@inra.fr</footnote>
Environmental persistence of pharmaceutical residues
from hospital and urban effluents and their impacts
on environmental aquatic microbial communities.

Agnès Bouchez*†¹, Teofana Chonova, Benoit Cournoyer, Leslie Mondamert, and
Jérôme Labanowski

¹INRA CARRTEL – Institut National de la Recherche Agronomique - INRA (FRANCE) – 75 bis av. de Corzent, 74200 Thonon-les-Bains, France

Abstract

Pharmaceutical residues coming from wastewater treatment plant (WWTP) discharges are regularly observed in natural waters. In this respect, the one-health issues associated with this contamination are currently being examined with considerable attention. The lack of knowledge and tools to monitor environmental exposure make their risk assessments hard to perform. The French SIPIBEL pilot site, monitored from 2011, includes a WWTP where urban and hospital wastewaters are treated separately. Treated effluents are delivered into a connected river. On this unique pilot site, the reliability of natural aquatic biofilms for monitoring the ecological impact of pharmaceuticals was investigated. The relations between field parameters, pharmaceutical concentrations, and organization of microbial communities were investigated. Microbiological and chemical tools were used to i) monitor the exposure of aquatic biofilms to pharmaceutical residues (analysis of different matrices: water, passive samplers, biofilms), and ii) assess changes in biofilm communities related to pharmaceuticals (community structure assessed through molecular fingerprinting or High Throughput Sequencing - HTS). Aquatic biofilms were grown from urban and hospital treated wastewaters, and from river surface waters located up- and downstream the WWTP discharge area. The finer the evaluation tools used to investigate the biofilm exposure and changes, the more reliable were the relations observed between pharmaceutical concentrations and community genetic patterns. Thus, the impact of low-level pharmaceutical concentrations could be investigated in situ. The combination of these monitoring tools highlighted environmental changes among the investigated aquatic ecosystem that might have impacted its functioning and ecological quality status.

**Keywords:** environmental monitoring, pharmaceutical residues, monitoring tools, biofilm

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: agnes.bouchez@inra.fr</footnote>
Shifts in planktonic and biofilm prokaryotic
community composition along a chemical
multicontamination gradient in a north-western
coastal mediterranean environment (Toulon bay,
France)

Coclet Clément*¹, Jean-François Briand², Gaël Durrieu¹, Christophe Le Poupon¹,
Sébastien D'onofrio¹, Lyria Berdjeb¹, Dario Omanović³, Cédric Garnier¹, and Benjamin
Misson¹

¹PROTEE – Université de Toulon : EA3819 – 83041 Toulon, France

²MAPIEM – Université de Toulon : EA4323 – 83041 Toulon, France

³Ruer Bošković Institute – Bijenička 54, Zagreb, Croatia

Abstract

Mediterranean coastal environments are currently subjected to various anthropogenic in-
puts, resulting in multiple chemical contaminations. Microbial communities play an essential
role in ecosystem functioning, however their response to such chemical multicontaminations
remains poorly characterized. Samples were collected from June 1st to June 29th, 2015 at
two depths (top and bottom of the water column) in five chemically contrasted sites along
a gradient of multicontamination (in several trace metals and nutrients) in Toulon Bay.
By combining flow cytometry and 16S rRNA gene sequencing (Illumina MiSeq), we assessed
spatio-temporal variability of planktonic and biofilm microbial abundance and diversity in re-
lation to natural and human-influenced environmental variables (physico-chemical variables,
nutrients and trace metals (TM) concentrations). Strong spatial gradients of TM concentra-
tions were observed in both surface and bottom seawater, with up to 115-fold enrichments.
On the one hand, samples collected at the most contaminated sites exhibited a significantly
higher planktonic prokaryote abundance than those from less contaminated sites, while no
difference was observed in biofilms. On the other hand, alpha diversity (richness, Shannon
and Chao1) was the lowest at the most contaminated sites. Moreover, microbial assemblages
appeared to differ strongly all along the contamination gradient (45 to 57% dissimilarity,
unweighted Unifrac distances). The main taxonomic groups identified in contaminated sites
were Bacteroidetes (Flavobacteriia) and Proteobacteria (Alpha- and Gammaproteobacteria)
while in the less contaminated site Proteobacteria (Alphaproteobacteria) and Cyanobacte-
ria (Synechococcophycideae) were found dominant. Respective contributions of natural and
anthropic environmental gradients to biological spatio-temporal shifts remain to be precise.

**Keywords:** Prokaryotic community, Biofilm, Environmental factors, Mediterranean Sea, High throughput sequencing

<footnote>*Speaker</footnote>
Combination of high-throughput sequencing and geochemistry methods for evaluating the impact of single and multi-metal pollutions in soil at the aggregate scale.

Jean Martins*¹, Frédéric Lehembre, Emilie François, Aline Navel, Erwann Vince, and Lorenzo Spadini

¹Institut des Géosciences de l'Environnement (IGE) – Institut National Polytechnique de Grenoble (INPG), INSU, OSUG, CNRS : UMR5001, Institut de recherche pour le développement [IRD] : UR252, Université Grenoble Alpes – IGE - HyDRIMZ – Université Grenoble Alpes CS 40 700. F-38058 Grenoble cedex 9, France

**Abstract**

J.M.F. Martins, F. Lehembre, E. François, A. Navel, E. Vince and L. Spadini.
IGE UMR5001, UGA-CNRS-GINP-IRD, France

In soils toxic effects of metals, especially in mixtures, disturb and modify competitive balance between species of microorganisms, involving in return a deterioration of the fundamental bio-geochemical cycles (C, N, P...). The sensitivity of microorganisms responses to environmental stresses make them relevant ecotoxicological indicators. Soils are spatially heterogeneous environments structured in aggregates of different sizes. Similarly to microorganisms, metals present heterogeneous and specific distribution patterns among these micro-aggregates. The objective of this work was to study the restructuring action of a copper, chromium and cadmium mixture on the microbial communities of a structured-soil at the microaggregate-scale. A grassland-soil contaminated with equitoxic-concentrations of Cu-Cd-Cr, alone or mixed. The contaminated soils were gently physically fractionated during a 2 months period. Each soil size-fraction (N> 250µm, 250-63µm, 63-20µm and N< 20µm) and the bulk soil were analyzed for mass-balance, determination of free-soluble-total metals contents and intrinsic properties (pH-Eh and OC-N contents). Microbial communities were characterized by estimating biomass and bacterial density/diversity were estimated by 16SrDNA rtPCR and high-throughput sequencing (MiSeq-Illumina). The results provide better understanding of the spatial impact of heavy-metal mixtures on soil microbial-communities, according to their respective distribution within the different soil-microhabitats, and the temporal evolution of microbial communities. A new vision of the microstructure of soil bacterial community is presented, which also opens the way for the detection and identification of rare-taxa that may be new bio-indicators of soil health

**Keywords:** Metals, mixtures, soil, spatialized impact, aggregates, microhabitats, microbial community, MiSeq, distribution

<footnote>*Speaker</footnote>
Quick metaproteomics for environmental risk
assessment

Jean Armengaud*¹, Sophie Ayrault, Guylaine Miotello, Emilie Avazeri, Gérard Steinmetz, Jean-Charles Gaillard, Charlotte Mappa, Karim Hayoun, Béatrice Alpha-Bazin, and Olivier Pible

¹Innovative technologies for Detection and Diagnostics (Li2D) – CEA Marcoule – BP17171, F-30207 BAGNOLS-SUR-CEZE, France

Abstract

Metaproteomics allows in-depth exploration of microbiota through i) establishing an in-
ventory of organisms present in a sample, even for a complex sample, and ii) documenting
new insights into functional pathways and interactions between microorganisms. We propose
to simplify metaproteomics data acquisition and data treatment for being able to explore any
microbiota in a few hours even without sequencing data. This allows quick metaproteomics
of water or soil samples become a reality. We designed a new bioinformatics pipeline for
quick analysis of samples and a lab-assembled microbiota reference comprising 24 bacterial
species covering 20 genera and 5 phyla for evaluating data processing procedures for metapro-
teomics. We exemplified the possible applications of this procedure for environmental risk
assessment on soil samples from the Seine River basin downstream of Paris Megacity. Major
changes in biodiversity may be evaluated from a 60 min tandem mass spectrometry run with
a broad vision as the method is able to assess bacteria, archaea, fungi, and microalgae at
once. Results on diverse environmental samples of interest for monitoring anthropogenic
impacts will be commented.

References:

Armengaud J (2016) Next-generation proteomics faces new challenges in environmental biotechnology. *Curr Opin Biotechnol.* 38:174-82;

Pible O & Armengaud J (2015) Improving the quality of genome, protein sequence, and taxonomy databases: a prerequisite for microbiome meta-omics 2.0. *Proteomics* 15:3418-23

Jagtap P, Griffin T, Armengaud J (2015) Microbiomes - embracing complexity. *Proteomics*. 15:3405-6.

Armengaud J. (2013) Microbiology and proteomics, getting the best of both worlds! *Environ Microbiol.* 15:12-23.

**Keywords:** Microbiota, Metaproteomics, Tandem mass spectrometry, Bacteria, Archaea, Fungi, Soil, Water

<footnote>*Speaker</footnote>
# From the screening of soil eukaryotic metatranscriptomes to the functional characterization of new metallothioneins

Laurence Fraissinet-Tachet*<fnref n="1" />, Antoine Ziller<fnref n="1" />, Rajiv Yadav<fnref n="1" />, Frédéric Lehembre<fnref n="1" />, Laurent Vallon<fnref n="1" />, Jan Colpaert<fnref n="2" />, and Roland Marmeisse<fnref n="1" />

## Abstract

Metal-polluted soils are colonised by microbial communities which have developed different adaptations that allow them to resist. Knowledge of eukaryotic resistance mechanisms to pollutants often results from studies on a limited number of model species. The objective of this study was to isolate and characterize new environmental genes expressed directly in soils by eukaryotic microorganisms and involved in metal resistance mechanisms. For this purpose, we have implemented an environmental genomics approach, the functional metatranscriptomics, to isolate resistance genes expressed in soils.

Metatranscriptomics is based on the direct extraction of eukaryotic polyadenylated mRNAs from soil samples followed by their conversion into cDNAs which are cloned in a yeast expression vector. These environmental cDNA libraries were used to complement a Cd-sensitive yeast mutant and resistant genes were recovered and analysed. Fifty percent of these genes were completely new, without homologs in databases. Some of these genes encode cysteine-rich proteins that we have called CRPs. These protein sequences present specific features that relate them to metallothioneins (MTs). They, however, also display unusual features: an "unusual" length (110-132 aa), the presence of a CCC cluster, two conserved His residues and a cysteine-free linker dividing the protein sequence in two parts. After CRP expression in E. coli and purifications, ESI-TOF-MS assays revealed that CRPs are able to chelate Cd, Zn and Cu and belong to a completely new MT family.

These results highlight the power of the functional metatranscriptomic approach which allows the discovery of new metal resistance genes.

**Keywords:** Microbial ecotoxicology. Heavy metals. Functional metatranscriptomics. Metal resistance genes. Metallothioneins. Protein purification and biochemical characterization

<footnote n="1">¹Ecologie microbienne (EM) – Institut national de la recherche agronomique (INRA) : UR1193, CNRS : UMR5557, Université Claude Bernard - Lyon I (UCBL), Ecole Nationale Vétérinaire de Lyon – BAT GREGOR MENDEL (ex 741)-4 et 43 Bvd du 11 Novembre 1918 69622 VILLEURBANNE CEDEX, France</footnote>

<footnote n="2">²University of Hasselt - Centrum voor Milieukunde – Belgium</footnote>

<footnote>*Speaker</footnote>
Volatolomics in microbial ecotoxicology, a pathway
for detecting metabolic signatures of exposure to
xenobiotics

Kevin Hidalgo*<fnref n="1" />, Erwan Engel<fnref n="1" />, Jean Louis Bonnet<fnref n="2" />, and Philippe Bouchard†<fnref n="2" />

Abstract

Monitoring the presence of xenobiotics in ecosystems is an important environmental,
health and economic challenge. Development of alternative analytical methodologies based
on "omics" technologies makes possible to consider an exposure biomarker approach to xeno-
biotics. It is therefore no longer a matter of quantifying residues of phytosanitary products,
but of measuring the level of exposure of sentinel organisms in various compartments of the
environment.

The stress tolerance of tissue and/or cell involves response processes whose metabolic sig-
natures are highly volatile metabolites. The objective of this work was to evaluate the
feasibility of a novel rapid approach based on a non-targeted analysis of volatile compounds
in microorganisms to detect previous exposure to different types of xenobiotic. We seek
to detect metabolic fingerprints specifically indicative of an exposure to metabolic response
libraries obtained under treated *versus* control conditions of contamination.

Our analytical process is based on the *in situ* concentration of such volatile molecules using solid phase micro-extraction techniques (SPME) and gas chromatography coupled to a mass spectrometer (GC-MS). We highlight that low-dose exposure (NOEC) of two pyrethroïds (deltamethrin and cypermethrin) and a tricetone (sulcotrione) in soil and water sentinel micro-organisms (*Bacillus megaterium*, *Pseudomonas fluorescens* and the ciliate *Paramecium*) induces specific metabolomic fingerprints, indicative of the exposure.

The toxicogenomics objective is now to feed a database of metabolic biomarkers revealing exposure to phytosanitary agents in micro-organisms then in animal models, consequent of a continuum in food production (e.g. bees, fish and poultry).

Keywords: Volatolomic, Metabolomic, Biomarker, Toxicogenomic

<footnote n="1">¹UR 370 Qualité des Produits Animaux, Equipe Micro-contaminants Arôme et Sciences Séparatives (MASS) (QuaPA, MASS) – Institut National de la Recherche Agronomique - INRA (FRANCE) – INRA Centre de recherches Auvergne-Rhône-Alpes Theix 63122 Saint-Genès-Champanelle, France</footnote>

<footnote n="2">²Microorganismes : génome et environnement (LMGE) – CNRS : UMR6023, Université Clermont Auvergne – Laboratoire Micro-organismes Génome et Environnement, Campus Universitaire des Cézeaux 1 impasse Amélie Murat, TSA 60026, CSA 60026 63178 Aubière Cedex, France</footnote>

<footnote>*Speaker</footnote>

<footnote>† Corresponding author: philippe.bouchard@uca.fr</footnote>
# Development of a standardized aquatic hyphomycete laboratory assay for fungicide risk assessment

Patrick Baudy*<fnref n="1" />, Jochen Zubrod<fnref n="1" />, Ralf Schulz<fnref n="1" />, and Mirco Bundschuh<fnref n="2" />

## Abstract

During the aquatic environmental risk assessment (ERA) of herbicides and insecticides, the testing of the presumably most sensitive taxa (i.e., aquatic plants and arthropods, respectively) is required. During fungicide ERA, in contrast, no aquatic fungi are tested. The protectiveness of a priori ERA for aquatic fungi is thus largely unknown, while recent studies indicate effects on these organisms at field-relevant fungicide concentrations. Furthermore, a posteriori ERA is mostly based on algae as benchmark microorganisms, while the correlation between the sensitivity of algae and aquatic fungi was never investigated. To address this fundamental knowledge gap, we are currently developing a suggestion for a standardized laboratory assay using aquatic hyphomycetes – an ecologically important group of fungi. This involves the assessment of the fungi’s reproduction (i.e., sporulation) over 4 days as well as their growth on fungicide-spiked agar over 56 days. In preliminary experiments (n=3), we exposed three aquatic hyphomycete strains towards eight concentrations of either of the fungicides dimethomorph or tebuconazole (0 to 10,000 µg/L). Sporulation was three orders of magnitude more sensitive towards fungicide exposure than growth. Respective EC50 values were partly in the low µg/L-range, which, however, requires further verification due to high variation. Based on these first insights, we conducted a power analysis to optimize the assay’s replication and thus statistical performance. Using this adjusted design, we are currently screening a range of fungicides frequently detected in surface waters to check for the protectiveness of fungicide ERA and the correlation of fungal and algal sensitivities towards these pesticides.

**Keywords:** aquatic hyphomycetes, fungicides, growth, reproduction, risk assessment

<footnote n="1">¹Institute for Environmental Sciences, University of Koblenz-Landau – Fortstraße 7, 76829 Landau, Germany</footnote>

<footnote n="2">²Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences – Lennart Hjelms väg 9, 75007 Uppsala, Sweden</footnote>

<footnote>*Speaker</footnote>
# Integrated biomarker and ecological response (IBER) index

Helena Guasch*<fnref n="1" />,<fnref n="2" />, Berta Bonet, Chloé Bonnineau, and Laura Barral

## Abstract

Biomarkers, defined in ecotoxicology as functional measures of exposure to chemicals, may not be informative on the consequences of exposure at the scale of interest, which is the entire ecosystem. This drawback is because links and interactions existing between these measures and the biological system at a larger scale are not always sufficiently known. In this presentation, we propose the application of an integrative multi-metric approach providing unequivocal information concerning exposure, early effects, adaptation and ecosystem damage caused by metal pollution. The IBER index is based on biomarkers of effect and/or exposure (e.g. antioxidant enzyme activities, community structure and resistance genes), together with other chemical, biogeochemical and ecological metrics related to microbial metabolism (e.g. bioaccumulation, chemical speciation, organic matter decomposition or nutrient cycling).

**Keywords:** biomarker, integrated response, index, metal pollution, fluvial ecosystem

<footnote n="1">¹Universitat de Girona – Spain</footnote>

<footnote n="2">²Institute of Aquatic Ecology (University of Girona, Spain) (IEA) – Faculty of Sciences, C Aurelia Capmany, Spain</footnote>

<footnote>*Speaker</footnote>
Microbial molecular and isotopic biomarkers for
evaluating natural attenuation of chlorinated
hydrocarbons in multi-contaminated sites

Jennifer Hellal*¹, Jérémie Denonfoux², Catherine Joulian¹, Stéphane Vuilleumier³,
Stéphanie Ferreira², Louis Hermon¹,³, and Gwenaël Imfeld⁴

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

²Genoscreen [Lille] – Institut Pasteur de Lille – Campus de l'Institut Pasteur, 1 rue du Professeur Calmette, Lille 59000, France

³Génétique moléculaire, génomique, microbiologie (GMGM) – université de Strasbourg, Centre National de la Recherche Scientifique : UMR7156 – 28 Rue Goethe 67083 STRASBOURG CEDEX, France

⁴Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS), UMR 7517 CNRS – université de Strasbourg – France

Abstract

Groundwater contamination by aliphatic chlorinated hydrocarbons (e.g. tetrachloroethene (PCE) and its degradation products) and dichloromethane (DCM) in the environment is high due to their extensive use as solvents and their persistence in the environment. Their efficient remediation mainly relies on prior plume characterization. Although physico-chemical assessment is indispensable, it can be complemented with an evaluation of bacterial diversity and activity on-site, using molecular biomarkers to evaluate *in situ* natural attenuation. The aim of this collaborative research project funded by the French Environment and Energy Management Agency (ADEME) is to evaluate the occurrence of PCE, TCE, DCE and CV degradation in contaminated groundwater, and how it can be linked to specific genes to evaluate the added value of molecular biomarkers and compound-specific isotope analysis (CSIA) for enhanced diagnosis and monitoring of multi-contaminated groundwater. Groundwater was sampled biannually in 12 piezometers at the source, in the plume and its fringes at a contaminated site (France). Four comparative approaches were used to analyze the samples: i) groundwater physical-chemical parameters, ii) CSIA of the pollutants (δ13C), iii) functional gene abundance, and iv) new generation sequencing of microbial diversity (Illumina MiSeq). In addition to on-site monitoring, the degradation potentials of PCE, DCE, VC and DCM were assessed in different redox conditions in batch assays. These complementary experiments coupling all or several of the 4 comparative approaches contributed to interpreting the onsite data. The expected impact of this project is to validate a set of tools to help define remediation and site management strategies.

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

<footnote>*Speaker</footnote>
High throughput microbial array for complex
environmental sample assessment

Anna Hua*¹, Mickael Cregut¹, Sullivan Jouanneau¹, Christophe Cordella², Gérald Thouand†¹, and Marie-José Durand‡¹

¹Laboratoire de génie des procédés - environnement - agroalimentaire (GEPEA) – CNRS : UMR6144,
Université de Nantes : UMR6144 – France

²Inra, AgroParisTech – AgroParisTech – France

Abstract

Ecotoxicological assessment using different bioassays for environmental sample are largely
used this last 20 years. Among them, recombinant bioluminescent or fluorescent bacterial
bioassays have been developed to determine the presence and the toxic effect of pollutants.
Chemical analyzes are sensitive and specific to detect pollutant, but they failed in the de-
termination of bioaccessibility and biodisponibility of these contaminants. To overcome this
problem, recombinant bioluminescent bacteria have been developed over the past years.
These recombinant bacteria carry reporter genes for bioluminescence or fluorescence fused
with gene promoters involved in metal resistance mechanisms or with genes involved in the
degradation of organic compounds. These bioassays are generally not specific for one com-
pound, in particular those dedicated to Metal Trace Element (MTE). Because of the lack of
specificity of these recombinant bacteria, applications for environmental samples are limited.
To deal with this problem, we used a set of bioluminescent recombinant bacteria sensitive
to MTE, which bioluminescence increases in the presence of MTE, and a collection of 1896
fluorescent recombinant bacteria covering the majority of gene promoters of *Escherichia coli*
strain. Results obtained on complex environmental samples (wood-waste leachates, soil)
showed variation of bacterial response according to the contamination allowing an interest-
ing method of screening. Because improvement of ecotoxicological assessment needs a large
panel of bioassays, we propose a mixture of targeted and non-targeted high throughput ar-
rays of bacteria to describe more realistically complex samples toxicity, combined with data
mining interpretation.

**Keywords:** Bioassays, recombinant bacteria, assessment, bioaccessibility

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: gerald.thouand@univ-nantes.fr</footnote>

<footnote>‡Corresponding author: durand-thouand-mj@univ-nantes.fr</footnote>
Communication Abstracts

POSTERS
# Reduction of pesticide contamination in agricultural soils: a bioprophylactic approach.

Louis Carles<fnref n="1" />,<fnref n="2" />, Pascale Besse-Hoggan<fnref n="2" />, and Isabelle Batisson*†<fnref n="1" />

## Abstract

The major problem with pesticides is linked to the more or less substantial proportion of the active ingredient quantity spread that does not reach its intended target, and contam- inate environmental compartments. We developed a bioprophylactic process based on the simultaneous application of pesticide and pesticide-degrading microorganisms, providing op- timal conditions for microbial degradation of pesticides after their specific action and before their transport to environmental compartments. Up to now, studies related to bioprophy- laxis rather focused on non-agricultural uses of pesticides [1-2]. The aim of our study was to determine the suitability of this concept in agriculture, with a model of herbicide (2,4-D, 2,4-dichlorophenoxyacetic acid) and one of its bacterial degrading-strain (*Cupriavidus neca- tor* JMP134).

The biodegradation of 2,4-D was studied in soil microcosms planted with sensitive and non- sensitive plants. Simultaneous application of 2,4-D at agricultural recommended doses and inoculation with 105 cells.g-1 dw of soil of JMP134 strain were carried out. The quantifica- tion of residual 2,4-D in soil, the survival rate of inoculated bacteria and the assessment of the herbicidal effect on sensitive plants were monitored. In soil inoculated with C. necator JMP134, the herbicidal effect was conserved and the 2,4-D concentration was reduced by 97% within seven days, while only 55% reduction was observed in the non-inoculated con- trol.

By using realistic conditions as close as possible to those of the field (i.e. recommended field dose of pesticide, commercial formulation), our study constitutes first bases for the development of bioprophylaxis as a good means to reduce environmental contamination by agricultural pesticides.

**Keywords:** Pesticide, 2, 4, dichlorophenoxyacetic acid, Bioremediation, Cupriavidus necator

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

<footnote n="2">²Institut 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</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: isabelle.batisson@uca.fr</footnote>
# Natural petroleum seepages shapes the microbial community diversity and activities of a forest ecosystem

Aurélie Cebron*†<fnref n="1" />, Thierry Beguiristain<fnref n="1" />, Kifayatou Rabiou Cholabomi<fnref n="1" />,<fnref n="2" />, Delphine Catteloin<fnref n="2" />, Catherine Lorgeoux<fnref n="2" />, and Pierre Faure<fnref n="1" />

## Abstract

Rapid selection of microbial populations and biodegradation functions after accidental petroleum contaminated is well studied. However, the long-term adaptation of microbial diversity on chronically polluted environment is less documented. The aim of our study was to evaluate how petroleum contamination shapes microbial communities and how it selects for specific microbial taxa or modulates microbial functions. We worked on a unique site (forest of Haguenau, Pechelbronn, France) contaminated for hundreds of years by petroleum seepages inherited from a tertiary source rock. Two seepages presenting different pedological characteristics were studied. We sampled soils at five points (in 3 replicates) along a 20 m gradient from the seepage source to the forest control soil. Hydrocarbon content was quantified (GC-FID) and qualified (GC-MS), microbial enzymatic activities related to C, N and P cycles were measured and microbial abundance and diversity were evaluated. On both sites, results were roughly similar. Hydrocarbon indexes showed lateral gradients of contamination, i.e. petroleum was unaltered at the seepage and biodegradation evidences appeared with increasing distance from the seepage. Moreover, microbial mineralization activities, some enzymatic activities, microbial abundance and occurrence of some hydrocarbon-degrading bacteria were low at the source point, became higher at the closest points (2-3 m from the petroleum source) and then decreased toward the less contaminated area, indicating modifications of the bacterial and fungal community structures.

**Keywords:** petroleum, natural seepages, forest soil, biodegradation, enzymatic activities, microbial diversity

<footnote n="1">¹Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC) – CNRS : UMR7360 – Faculté des Sciences et Technologies, BP70239, 54506 Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="2">²GeoRessources – CNRS : UMR7359, Université de Lorraine – Faculté des Sciences et Technologies, BP70239, 54506 Vandoeuvre-lès-Nancy, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author: aurelie.cebron@univ-lorraine.fr</footnote>
# Dissipation of chlorpyrifos, pendimethalin, and thiophanate-methyl pesticides in compost based-versus peatmoss based-biomixtures of biobeds

Talaat El Sebai*†<fnref n="1" />, Wafaa Zidan<fnref n="1" />, Wafaaabd El Rehim<fnref n="1" />, Alaa Marzouk<fnref n="2" />, Marion Divers<fnref n="3" />, and Fabrice Martin-Laurent<fnref n="3" />

## Abstract

Biobeds is a simple and a cost effective way to treat point source pesticide's contamination. In this study, we replaced peat-moss (P) with compost (C) which is locally available to compare the effectiveness of both bio-mixtures (biomix) P and C on pesticide dissipation. Chlorpyrifos, pendimethalin, and thiophanate methyl were added separately (at three concentrations 25, 50, and 75 mg/kg of both biomix P and C). Our results showed that biomix composition influenced the dissipation of pesticides. For chlorpyrifos, the highest dissipation rate was recorded in biomix P. This result was confirmed by the mineralization kinetics, since, 25% of 14C-chlorpyrifos initially added to biomix evolved to 14CO2 in biomix P while only 14% evolved to 14CO2 in biomix C. In addition, chlorpyrifos dissipation rate was found to be influenced by the initial concentration applied to biomix P and C. In contrary, highest rate of dissipation of pendimethalin was observed in biomix C. More than 76% of pendimethalin was dissipated in biomix C while only 67% were dissipated in biomix P. The efficiency of both biomix (C and P) was similar in the dissipation of thiophanate-methyl but less efficient compared to other tested pesticides. The addition of the three tested pesticides as mixture at 25 mg/kg of biomix C had a positive effect on both chlorpyrifos and thiophanate-methyl dissipation, while pendimethalin dissipation was similar when applied separately or mixed. In both biomixtures, dehydrogenase activity of microorganisms was stimulated by the addition of pesticides.

**Keywords:** biobeds, pesticid, contamination, point source, bioremediation

<footnote n="1">¹Agricultural Microbiology Dept. National Research Centre – Cairo, Egypt</footnote>

<footnote n="2">²Laboratory of pesticide analysis, Agriculture Research Centre – Cairo, Egypt</footnote>

<footnote n="3">³Agroécologie – Institut National de la Recherche Agronomique : UMR1347, Université de Bourgogne, Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement – Université de Bourgogne - 17 rue Sully - BP 86510 - 21000 Dijon, France</footnote>

<footnote>*Speaker</footnote>

<footnote>†Corresponding author:</footnote>