Biocapteurs pour le contrôle de la toxicité des eaux : application des bioélectrodes algales

L'amélioration de la qualité de l'environnement passe par la réalisation de contrôles de toxicité in situ et en continu des sources de pollution ou des milieux contaminés, à l'aide de systèmes automatisés à réponse rapide. Les systèmes donnant une réponse en temps réel permettent d'intervenir immédiatement à la source, d'interrompre le rejet d'un flux toxique et de prévenir ainsi les accidents de pollution. Ce type de stratégie ne peut être développé qu'au moyen de biocapteurs : les méthodes d'essais conventionnelles n'autorisent que des contrôles de toxicité épisodiques, en laboratoire, effectués dans des conditions statiques quelque peu éloignées des conditions dynamiques.Nous nous sommes intéressés à la mesure de l'activité photosynthétique d'algues unicellulaires immobilisées. La photosynthèse induite par des stimuli lumineux est en effet un processus dont la réponse est immédiate et aisément mesurable à l'aide de transducteurs électrochimiques. Il apparaît donc intéressant d'utiliser ces réactions photosynthétiques pour la détection des polluants.Deux dispositifs mesurant l'activité photosynthétique d'algues unicellulaires ont été testés. Le premier dispositif mesure le transfert d'électrons le long de la chaîne photosynthétique lors d'une illumination des micro-organismes. Le second système permet de quantifier la production d'oxygène résultant de cette excitation lumineuse.La mesure du transfert d'électrons photosynthétiques nécessite l'addition d'une substance oxydo-réductible (médiateur) dans le milieu pour capter ces électrons. De la série de médiateurs testés, seuls les dérivés à caractère lipophile (2,6-diméthylbenzoquinone et p-benzoquinone) ont permis de mesurer un transfert d'électrons. Toutefois la durée de vie de ce biocapteur s'est révélée limitée à moins de 24 heures, ce qui exclut toute utilisation en continu.Le second dispositif développé présente en revanche une longévité d'une semaine, ce qui le rend intéressant en vue d'une utilisation in situ. Les performances de ce capteur à oxygène ont été testées sur des produits de type herbicides, cyanures, métaux et comparées aux valeurs obtenues à l'aide de tests algues classiques ou de méthodes de détection rapide de la toxicité.Environmental monitoring of pollutants with automatic systems, applied on-line and allowing rapid response constitutes one of the most successful ways to improve the quality of the environment. Real time analysis offers the advantage of detecting rapidly sources of pollution and preventing any accidental release of pollutants. Such a strategy is possible only by means of biosensors : current methods, commonly used far toxicity testing are usually carried out in Laboratory in static conditions, making real lime analysis impractical.Two types of amperometric environmental sensor incorporating eukaryotic algae were investigated for use in monitoring industrial pollution of aquatic systems. Both sensors allowed the monitoring of photosynthetic events.The first sensor follows photosynthetic electron chain events within the cell resulting in the reduction of mediator acting as terminal electron accepter. Reoxidation of the mediator at the biosensor electrode surface rues in a flow of current, the magnitude of which is proportional to the level of photosynthetic activity of the microalgae.In the second approach photosynthetic oxygen evolution by the illuminated biocatalyst is measured by reduction at a cathodic electrode. Enzymic systems associated with the water splitting and oxygen evolution are amongst the most fragile components of the photosynthetic apparatus, and the monitoring of algal oxygen production is therefore a useful approach to early detection of toxic environmental pollutants.Several species of unicellular algae were used for these experiments : Chlorella vulgaris, Scenedesmus subspicatsus and Selenastrum capricomutum. Algal cultures were harvested in the exponential growth phase and diluted to 0.5 O.D. (655 nm); then 1 ml aliquots were centrifuged at 900xg for 3 min. After centrifugation, cells were resuspended in growth medium, LEFEBVRE and CZARDA (LC), and immobilized by aspiration onto a filter disc. This filter disc was placed onto the carbon working electrode surface. Filters were held in place by a fine nylon mesh.This biosensor is a two electrode system comprising a carbon working electrode and Ag/AgCl reference/counter electrode. Solution was continuously flowed through the electrochemical cell at a flow rate of 2 ml min-1. Illumination of the algal biocatalyst was supplied by light emitting diodes with a peak wavelength of 635 nm and a light intensity of 125 millicandellas. Periodicity of illumination was chosen in order to obtain a stable photosynthetic response.Biosensors exploiting direct electron transfer from a biocatalyst to an electrical system are not feasible. Indeed, the tell wall of the biocatalyst act as a barrier to the exchange of electrons between the electrode and the redox intermediates oft the cell. Electroactive compounds (mediators) must be used to shuttle electrons from the photosynthetic electron transfer chain to the electrode. Mediators were added to the flowing solution of LC medium, and a potential of 550 mV applied at the working electrode to reoxidize mediator reduced by the biocatalyst. The mediator must be lipophilic to access the chloroplast electron transport chain of eukaryotic algae. We tested a wide range of mediators but only p-benzoquinone (p-BQ) and 2,6-dimethylbenzoquinone gave measurable responses.A concentration of 0.2 mM p-BQ (21.5 mg/l) was employed to measure photosynthetic activity. Experiments showed that 15 minutes light period followed by a 15 minutes dark period gave a steady photosynthetic response. However, this high concentration of mediator was toxic for the cells. Static algal tests using Chlorella vulgaris have shown that growth is totally inhibited after 72 hours at a concentration of 5.4 mg/l. The working life of this sensor was therefore very short, less than 24 hours : after 16 hours of continuous monitoring, the recorded photosynthetic current was less than 20 % of initial response. Sensor life was not increased when the probe was used alternately with recovery periods in nutrient medium (4 hours of working period/4 hours of recovery period).The same apparatus was used for the oxygen electrode based biosensor. The working electrode was coated with a Teflon gas permeable membrane to protect the sensor against poisoning by electrochemically active compounds. Separation of the working and reference/counter electrode requires addition of electrolyte in the flowing solution. With such a semi-protected oxygen electrode, mass transport controlled oxygen reduction currents were obtained when the Teflon covered cathode was poised at -700 mV.The oxygen biosensor responded more rapidly than the mediated biosensor to changes in the light regime, and alternating light and dark periods of 1 min of light followed by 4 min of dark were used. The sensor also showed good long term stability, with a working life of up to seven days using Chlorella vulgaris or Scenedesmus subspicalus as biocatalysts.The sensitivity of this oxygen electrode based biosensor was tested on herbicides (isoproturon, propanil and atrazine), cyanide and heavy metals (copper and mercury). Results were compared with chose obtained with three toxicity tests : a standard algal growth inhibition test, the inhibition of photosynthetic activity in spinach leaves and the alga Chlamydomonas reinhardii, and the Microtox test using the luminescent bacterium Photobacterium phosphoreum.IC50 obtained for isoproturon and atrazine were very similar for the growth inhibition and the oxygen sensor tests. The inhibition of oxygen production by spinach leaves was less sensitive to atrazine; no toxic affect could be detected with the Microtox test. The oxygen sensor was also very sensitive to cyanide but the response of the probe was quite different if Selenastrum capricornutum or Chlorella vulgaris was used.The sensor allowed metals detection but this detection of toxicity was slow compared to that of herbicides or cyanide. Inhibition growth tests and Microtax test were more sensitive than the algal sensor for copper and mercury

Infections dues aux hantavirus : cas particulier de la France

L'auteur propose une synthèse bibliographique de la Fièvre Hémorragique avec Syndrome Rénal ou Hantavirose, zoonose d'origine virale, transmise par les rongeurs de la famille des Muridae. Les Hantavirus, membres de la famille des Bunyaviridae sont des virus enveloppés à ARN négatif. L'hantavirose est largement répandue dans le monde : les flux migratoires des rongeurs au cours des siècles ont en effet contribué à la dissémination des virus sur les différents continents. L'auteur centre son étude sur l'Hantavirose en France. Il présente ainsi les données épidémiologiques et les données cliniques de la maladie. Il aborde également les méthodes diagnostiques, les possibilités de traitement et enfin les mesures prophylactiques

Applicability of the Caenorhabditis elegans survival, growth and reproduction test to assess the effects of biosolids used in agriculture

Modern high productivity agriculture has led to an impoverishment of nutrients and organic carbon in field soils, and as a consequence plants nutrition complements like fertilisers or wastes have to be used to keep high yield. These complex materials can also be a source of toxicants, depending on their origin, so there is a necessity to assess their environmental effects before field use. In this study, biosolids used in agriculture were tested as a mix with standard soil at different field dose through ecotoxicity test on the nematode Caenorhabditis elegans, endpoints for this organism being survival, growth and reproduction. In order to run these tests we first followed the standardized protocol for sediment and soil testing with C. elegans (ISO 10872, 2010). These results showed that the mixtures could be significantly different according to the type of soil (e.g. two phases system with overlaying water). Indeed, soil moistening in the standardized protocol is based on soil dry weight which means that the same amount of water is added for every soil, regardless of the water holding capacity (WHC) of the samples. Moreover, some biosolids can have a high WHC what increase humidity's differences among soil and soil mixed with biosolids when moistened as recommended by this protocol. To prevent this, improvements of the standardized protocol were developed, in which water addition is based on soil WHC instead of soil dry weight. Moreover, food volume added to the samples was lowered in order to be able to include it in the volume for soil moistening. In this context and in order to validate this new method, comparisons between standardized and optimized protocols were carried out for endpoints responses in five soils with different textures. This optimized protocol was used to assess the effect of different biosolids (limed sludge, manure ...) mixed with standard soil at different field rates and an example for a limed sludge is given. Regarding these results, improvements of the standardized protocol are adapted to assess the effects of these materials on C. elegans

Contribution of ecotoxicological tests in the evaluation of soil bioremediation efficiency

Clean-up of contaminated soils became a high priority only recently. Several techniques have been developed forthis purpose such as chemical, physical, thermic or microbiological methods. Efficiency of the remediation can be estimated using two approaches : a chemical specific approach and a toxicity-based approach. So far, the efficiency of the decontamination process was based essentially on chemical analyses which does not integrale the toxicity of all the soil contaminants and does not give a response on effects caused by the bioavailable fraction of these contaminants äs the toxicity-based approach. In the present study, bioremediation efficiency of a soil contaminated by 4-chlorobiphenyl was evaluated using chemical and biological analyses. Experiments were carried out in microcosms contaminated at a rate of 1 g/kg. Control microcosms without specific degrader were performed simultaneously. Acute toxicity to earthworms and inhibition of growth of barley roots were selected, from previous work, äs relevant ecotoxicological test

Effect of non-ageing and ageing ceria nanoparticles suspensions on fresh water micro-algae

When assessing the hazards properties of nanomaterials in the environment, the main research challenges are numerous. Firstly, determining if nanomaterials are more or less toxic than the bulk forms of the same materials and the extent to which toxicity is governed or influenced by the physico-chemicals properties of the nanoparticles. Secondly, it appears necessary to study the effect of nanomaterials and nanoparticles throughout their life cycle including both initial forms and physico-chemically modified form (i.e. aggregated or agglomerated forms) resulting from an ageing process. Our work focused on the effect of commercial ceria nanoparticle (nCeO2) suspensions, towards freshwater micro-algae assessing the effect nCeO2 suspensions with different agglomeration/aggregation state obtained by using an artificial ageing process. Both ageing and non-ageing nCeO2 suspensions were fully characterized using dynamic light scattering (ZetaSizer, Malvern Instruments) or laser diffraction (MasterSizer, Malvern Instruments) and transmission electron microscopy (TEM). In addition, the interaction between NPs and algae were investigated using flow-cytometry and environmental scanning electron microscope technique (E-SEM).The results obtained showed that the algae growth inhibition was similar after exposure to non-ageing or ageing nCeO2 suspensions. The results obtained from flow-cytometry and E-SEM proved that the ceria NPs are able to tightly entrap the algae cells, which could in part contribute to the effect recorded. Those results also support the fact that aggregation or agglomeration has a few influences when focusing on the standardized algae ecotoxicity test. Moreover by comparison to our previous studies performed with other ceria suspensions, it was shown that the primary particle size and consequently the particle surface area is a relevant parameter in assessing the ecotoxicity of nanoparticles

Evaluation of an in vitro hsp 70 induction test for toxicity assessment of complex mixtures : comparaison with chemical analyses and ecotoxicity tests

International audienceThe aim of this study was to assess the potential of a human cell line containing the hsp70 promoter linked to the chloramphenicol acetyltransferase reporter gene in evaluating the toxic potential of complex mixtures. Cells were exposed to eluates of industrial wastes and the cellular responses were compared with the metal contents of the samples and with standardized aquatic (microalgal growth inhibition, daphnia Immobilization, bacterial luminescence inhibition, Ceriodaphnia dubia reproduction inhibition) and terrestrial (earthworm lethality, plant growth inhibition) tests. The hsp70 promoter was significantly induced by 11 of 14 samples, with different dose-response patterns. Significant correlations of in vitro induction potency with aquatic ecotoxicity, especially with chronic tests, and with the metal contents of the samples were observed. Our study provides new information on the relevance of hsp70 gene induction as a criterion of toxicity and suggests its usefulness for the detection of toxicity associated with metallic pollution in complex mixtures

Physico-chemical and biological characterization of an aquifer polluted with ETBE

International audiencePetroleum compounds and among them, gasoline, is the most massively used chemicals worldwide and, as a consequence gasoline derives compounds are the most frequently found contaminants in groundwate

Adapting oecd aquatic toxicity tests for use with manufactured nanomaterials: key issues and consensus recommendations

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values