New Concepts in the Evaluation of Biodegradation/Persistence of Chemical Substances Using a Microbial Inoculum

The European REACH Regulation (Registration, Evaluation, Authorization of CHemical substances) implies, among other things, the evaluation of the biodegradability of chemical substances produced by industry. A large set of test methods is available including detailed information on the appropriate conditions for testing. However, the inoculum used for these tests constitutes a “black box.” If biodegradation is achievable from the growth of a small group of specific microbial species with the substance as the only carbon source, the result of the test depends largely on the cell density of this group at “time zero.” If these species are relatively rare in an inoculum that is normally used, the likelihood of inoculating a test with sufficient specific cells becomes a matter of probability. Normally this probability increases with total cell density and with the diversity of species in the inoculum. Furthermore the history of the inoculum, e.g., a possible pre-exposure to the test substance or similar substances will have a significant influence on the probability. A high probability can be expected for substances that are widely used and regularly released into the environment, whereas a low probability can be expected for new xenobiotic substances that have not yet been released into the environment. Be that as it may, once the inoculum sample contains sufficient specific degraders, the performance of the biodegradation will follow a typical S shaped growth curve which depends on the specific growth rate under laboratory conditions, the so called F/M ratio (ratio between food and biomass) and the more or less toxic recalcitrant, but possible, metabolites. Normally regulators require the evaluation of the growth curve using a simple approach such as half-time. Unfortunately probability and biodegradation half-time are very often confused. As the half-time values reflect laboratory conditions which are quite different from environmental conditions (after a substance is released), these values should not be used to quantify and predict environmental behavior. The probability value could be of much greater benefit for predictions under realistic conditions. The main issue in the evaluation of probability is that the result is not based on a single inoculum from an environmental sample, but on a variety of samples. These samples can be representative of regional or local areas, climate regions, water types, and history, e.g., pristine or polluted. The above concept has provided us with a new approach, namely “Probabio.” With this approach, persistence is not only regarded as a simple intrinsic property of a substance, but also as the capability of various environmental samples to degrade a substance under realistic exposure conditions and F/M ratio

Bacterial Bioluminescent Biosensor Characterisation for On-line Monitoring of Heavy Metals Pollutions in Waste Water Treatment Plant Effluents

International audienc

7-alkylguanines et leurs dérivés fluorescents comme biomarqueurs d'exposition aux agents alkylants

Biological fluids, such as gastric juice, often contain alkylating genotoxic substances which may paly a role in the aetiology of human cancer. The major adducts formed in DNA by most alkylating agents are 7-alkyldeoxyguanosines. These addcuts are labile and 7-alkylguanines are released by heating or enzyme repair. We have explored the possibility of using a probe DNA method - calf thymus DNA is incubated with nitrite-treated gastric juice, recovered and heated to induce depurination. The 7-alkylguanines are detected by direct fluorescence after an immunoaffinity purification, but with low sensitivity. 7-alkylguanines react with phenylmalondialdehyde to give derivatives with good HPLC-fluorescence properties. This method increases the detection sensitivity of 7-alkylguanineLes fluides biologiques comme les sucs gastriques peuvent contenir des substances génotoxiques ou leurs précurseurs qui joueraient un rôle dans le développement des cancers. Parmi ces composés se trouvent de nombreux agents alkylants. Les adduits majeurs produits par ces agents sont sur la position n7 de la désoxyguanosine de l'ADN. Ces adduits sont instables, l'ADN peut être facilement dépuriné pour donner les n7-alkylguanines correspondantes. De l'ADN de thymus de veau est incubé avec des sucs gastriques nitrosés chimiquement. Les 7-alkylguanines contenues dans le surnageant de dépurination de l'ADN sont détectées par mesure de la fluorescence après purification sur colonne d'immunoaffinité. La sensibilité de cette méthode étant insuffisante pour les applications biologiques. La dérivation des 7-alkylguanines par la phénylmalondialdéhyde a permis d'accroître leur fluorescence et de faciliter leur détection par spectrofluorimétri

Développement de bioessais à l'aide de bactéries génétiquement modifiées pour la détection de polluants environnementaux

La Décision Européenne n2455/2001/CE a publié en 2001 une liste de 33 substances à détecter dans l eau. Nous avons abordé, avec des méthodes biologiques innovantes, le problème de la détection de deux de ces substances, le tributylétain (TBT), un biocide couramment utilisé dans les peintures antisalissures et interdit dans de nombreux pays, et l atrazine, un herbicide. La bactérie Escherichia coli TBT3, qui a été obtenue par mutagenèse aléatoire, est sensible aux organoétains mais son mécanisme d induction est inconnu. L étude au niveau moléculaire de cette bactérie a permis d aboutir à un mécanisme vraisemblable de la réponse au TBT, grâce à la localisation d un promoteur et d un nouveau régulateur transcriptionnel, responsables de l induction de la bioluminescence. Un dispositif innovant a été fabriqué pour distinguer les peintures interdites contenant du TBT de celle n en contenant pas. Un bioessai simple est proposé comme alternative aux analyses chimiques. En parallèle, une nouvelle bactérie, dont la bioluminescence est inductible par l atrazine, a été développée en détournant le système de dégradation de l atrazine provenant de la bactérie environnementale Pseudomonas sp. ADP. Cette nouvelle bactérie bioluminescente permettra une détection simple et rapide de l atrazine dans l eau et le sol.The European decision n2455/2001/EC published in 2001 a list of 33 priority substances to be detected in water. We addressed the problem of the detection with innovative biological methods for two substances, the tributyltin (TBT) a biocide broadly used in antifouling paint and now banned for many countries and the herbicide atrazine. The bacterium E. coli TBT3, which was made by random mutagenesis, is sensitive to organotins but its induction mechanism remained unknown. A comprehensive molecular study of this bacterium led to a plausible mechanism of TBT response, thanks to the localization of a promoter and a new transcriptional regulator YgaV involved in the induction of bioluminescence. An innovative device was made to distinguish prohibited paints containing TBT from those without TBT. A simple alternative bioassay is now proposed to the conventional analyses. In a second step, a new bacterium, whose bioluminescence was activated by atrazine, was also developed by using the biodegradation system of atrazine from an environmental bacterium Pseudomonas sp. ADP. This new bioluminescent bacteria will allow a simple and rapid way to detect atrazine both in water and in soil.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Coupling traditional biodegradation assessments with spectroscopic tools for plastic wastes: case of Polyurethane wastes assessment.

International audienc

Online Detection of Metals in Environmental Samples: Comparing Two Concepts of Bioluminescent Bacterial Biosensors

International audienc

A new bioassay for the inspection and identification of TBT-containing antifouling paint

International audienc

Design of a toxicity biosensor based on Aliivibrio fischeri entrapped in a disposable card

International audienceThe degradation of the marine environment is a subject of concern for the European authorities primarily because of its contamination by hydrocarbons. The traditional methods (ISO 11348 standard) of general toxicity assessment are unsuitable in a context of in situ monitoring, such as seaports or bathing zones. Consequently, to address this issue, bacterial biosensors appear to be pertinent tools. This article presents the design of an innovative bioluminescent biosensor dedicated to in situ toxicity monitoring. This biosensor is based on the entrapment of the wild marine bioluminescent bacterial strain Aliivibrio fischeri ATCC® 49387™ in an agarose matrix within a disposable card. A pre-study was needed to select the most biological parameters. In particular, the regenerating medium’s composition and the hydrogel concentration needed for the bacterial entrapment (mechanical resistance) were optimized. Based on these data, the ability of the bacterial reporter to assess the sample toxicity was demonstrated using naphthalene as a chemical model. The biosensor’s results show a lower sensitivity to naphthalene (EC50 = 95 mg/L) compared with the results obtained using the reference method (EC50 = 43 mg/L). With this architecture, the biosensor is an interesting compromise among low maintenance, ease of use, appropriate sensitivity, relatively low cost and the ability to control online toxicity

Applying Raman spectroscopy to the assessment of the biodegradation of industrial polyurethanes wastes

International audienc

From laboratory to environmental conditions: a new approach for chemical's biodegradability assessment

International audienceWith thousands of organic chemicals released every day into our environment, Europe and other continents are confronted with increased risk of health and environmental problems. Even if a strict regulation such as REgistration, Authorization and restriction of CHemicals (REACH) is imposed and followed by industry to ensure that they prove the harmlessness of their substances, not all testing procedures are designed to cope with the complexity of the environment. This is especially true for the evaluation of persistence through biodegradability assessment guidelines. Our new approach has been to adapt “in the lab” biodegradability assessment to the environmental conditions and model the probability for a biodegradation test to be positive in the form of a logistic function of both the temperature and the viable cell density. Here, a proof of this new concept is proposed with the establishment of tri-dimensional biodegradability profiles of six chemicals (sodium benzoate, 4-nitrophenol, diethylene glycol, 2,4,5-trichlorophenol, atrazine, and glyphosate) between 4 to 30 °C and 104 to 108 cells ml−1 as can be found in environmental compartments in time and space. The results show a significant increase of the predictive power of existing screening lab-scale tests designed for soluble substances. This strategy can be complementary to those current testing strategies with the creation of new indicators to quantify environmental persistence using lab-scale tests