Fanny Desbiolles

Laetitia De Jong

Xavier Moreau

Isabelle Laffont-Schwob

Laure Malleret

Christophe Tiliacos

Nicolas Tiliacos

Pascal Wong-Wah-Chung

Non-destructive biological and chemical traits to track down long term effects of environmentally relevant low concentrations of pharmaceutical mixtures using two freshwater species

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For OxCBZ, a slight metabolic stress was observed latter whereas neither CBZ nor the mixture at low concentrations impacted this index.

 Nitrogen balance index:

OxCBZ and CBZ at the highest concentrations increased the nitrogen balance index after 11 days.

It shows a modification of allocation of nitrogen and carbon that may impact photosynthesis in of duckweed.

Chlorophyll index: This primary metabolite was stable for all the compounds and their mixture. In exposed polyps, slowing down and decrease of budding rate were observed (Figs A-B) that could be related to morphological changes. Indeed, as the decrease of budding rate occurred from the d6 after exposure, i.e. the time required to the Hydra polyps to restore their normal morphology, trade-offs between the survival of polyps and the reproductive success of the population could be hypothesized. As polyps exposed to CBZ and mixture never reached the maximal budding rate observed in control, these compounds have negative effect on the reproductive success.

Whatever the exposure conditions, all Hydra polyps were still able to catch and ingest prey : no alteration of feeding behaviour was observed.

 Both models enable to study non-destructive biological pertinent endpoints.  The macrophyta, Lemna minor, showed deleterious effects only at high concentrations. This specie does not seem highly sensitive to these compounds under the tested conditions.  Bioassays using Hydra attenuata showed that the selected compounds at environmental concentrations induced deleterious effects at early (morphological changes) and later stages (budding rates). Such negative effects raise the question of the impact on natural populations that are chronically exposed to pollutants.  These preliminary results could help to develop new tools for water quality monitoring and for chronic toxicity assessment at realistic environmental concentrations.

  -Marseille Université, CNRS, Laboratoire de Chimie de l'Environnement, UMR 7376, 13545, Aix-en-Provence, France 2 -Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale, UMR CNRS 7263 / IRD 237, Aix-Marseille Université, Avignon Université, 3 Place Victor Hugo, 13331 Marseille, France 3 -SEAKALIA, Héliopolis -Technopôle de Château Gombert -3 allée des Mairaîchers, 13013 Marseille, France fanny.desbiolles@univ-amu.fr Context & Objectives Acknowledgement Lemna minor L. Hydra attenuata Pall.

  Light : 9h/day, Moisture : 60%, Temperature : 15°C, Hoagland media (10%) Growth conditions: Single strain, Light : 12h/day, Temperature : 19.5°C, TES* buffer, ETDA, CaCl 2 * 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid References 1 Cleuvers, M. 2003. Aquatic Ecotoxicity of Pharmaceuticals Including the Assessment of Combination Effects. Toxicology Letters 142 (3): 185-94. 2 Brian Q., Gagné F., Blaise C. 2008. An Investigation into the Acute and Chronic Toxicity of Eleven Pharmaceuticals (and Their Solvents) Found in Wastewater Effluent on the Cnidarian, Hydra Attenuata. Science of The Total Environment 389 (2-3): 306-14. 3 De Jong L., Pech N. , de Aragão Umbuzeiro G. , Moreau X. 2016. Multi-scale biomarker evaluation of the toxicity of a commercial azo dye (Disperse Red 1) in an animal model, the freshwater cnidarian Hydra attenuata. Water Research 96 : 62-73. Phenolic index of compounds and control during a 17 day-long exposure  Phenolic index: The highest concentration of 9-CAA induced an early metabolic stress in Lemna minor L. as shown by the increase of the phenolic index probably due to a defence mechanism.

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  Anthocyanin and flavonol indexes were below detection limits. SETAC 26 th annual meeting, Nantes, 22 -26 May 2016 not removed in wastewater treatment plants Carbamazepine (CBZ), OxCarbazepine (OxCBZ) and one of their metabolite Acridine-9-carboxylic acid (9-CAA)  Morphological changes, reflecting progressive intoxication, were observed after only one day of exposure : 21% of polyps exposed to CBZ, OxCBZ or 9-CAA showed first signs of intoxication and 16,6% of polyps exposed to the mixture showed more pronounced signs of intoxication. Interestingly, after 6 days of exposure morphological signs of intoxication tended to disappear and polyps exhibited normal morphological stage until the end of exposure (21 days). These observations suggest that H. attenuata possesses defence mechanisms to face intoxication. A-B : Budding rate (Ktot) of exposed polyps and controls followed during 21 days. K tot values were calculated 3 with the following formula K tot = (ln(n tot )-ln(n))/d n: number of polyps at the beginning of the experiment (d0); n tot the sum of attached buds plus the number of polyps (i.e. detached buds and initial polyps) after x days of exposure (d3 to d21). *:P< 0.05, **:P< 0.01,***:P< 0.001, significantly different from control (n= 24 in triplicate; Kruskall-Wallis followed by Dunn test).

  Hydra attenuata Pall., EC 50 : 15.52 mg.L -1 , 96h (chronic toxicity)LC 50 : 29.4 mg.L -1 , 96h (acute toxicity) Lemna minor L., EC 50 : 25.5 mg.L -1 , growth inhibition, 7d, (chronic toxicity)