La contamination du Léman par les micropolluants – revue de 40 ans d’études

Lake Geneva pollution by micropollutants – a review of 40 years of study. – Lake Geneva drains a watershed of about 8000 km2, subject to anthropogenic pressures due to a large population, agriculture, and industries. Contaminants released by these activities may eventually end up in the lake water, sediments and biota. For more than 40 years, the International Commission for Lake Geneva Protection (Commission Internationale pour la Protection des Eaux du Léman), and research institutes located around the lake, look after these contaminants by following the evolution of analytical technics. This review particularly focuses on the evolution of the lake contamination by mercury, PCBs, pesticides, drugs and some emerging pollutants. Mercury and PCBs were the first contaminants detected in sediments in the 1970s. These contaminants were particularly targeted because of their propensity to bioaccumulate in organisms and biomagnify in the food chain, and because of their high ecotoxicity. If the contamination of fish by mercury seems controlled with values ~10 times lower than the legal limits, PCBs still pose problems for fatty fish such as Arctic char (Salvinus alpinus). Improved techniques for analyz- ing organic products revealed the presence in the waters of more than one hundred pesticides and thirty pharmaceuticals. In addition, contamination of Foramsulfuron in 2005, from industry, has almost reached the legal limit for drinking water. This review shows that the contamination of the lake over the last 40 years has evolved with the growing presence of many contaminants, either by the release of new substances or by improving analytical techniques. Monitoring of water quality is essential to act early enough to reduce inputs in the lake. And special vigilance must be maintained for both ‘old’ pollutants (Hg, PCBs) and emerging contaminants such as pharmaceuticals, microplastics, and products of industrial synthesis.

Sources, Spreading and Fate of Antibiotic Resistance Genes and Resistant Bacteria in Vidy Bay, Lake Geneva, Switzerland

Nadine Czekalski

Increasing numbers of multiresistant bacterial pathogens cause severe health problems on a global scale. Large quantities of bacteria and their resistance genes are discharged into natural environments via human and animal waste. Not much is known so far about the fate and further spread of resistance determinants in the environment. There is evidence that aquatic environments may function as important compartments for preserving and exchanging antibiotic resistance genes (ARG) from sewage and drinking water. Lake Geneva is the largest fresh water reservoir of Western Europe and supplies the surrounding cities. At the same time these cities discharge sewage into the lake. Lausanne is the largest city in the lake’s catchment and discharges the highest amount of treated, as well as in case of heavy rain events, partly untreated waste waters. Also two rivers, Chambering and Flown, transport untreated sewage (bypass), in case of storm events. Only 3.4 km from the sewage discharge point lake water is pumped for drinking water preparation for Lausanne. Many studies have reported on contamination of the bay with heavy metals, inorganic nutrients, organic carbon and fecal indicator bacteria due to the sewage discharge. The present thesis aimed on investigating the sources, spreading and fate of antibiotic resistant bacteria and antibiotic resistance genes in the Vida bay, Lake Geneva. In the first part of this dissertation, a screening for antibiotic- and specifically multi-resistant bacteria was carried out as one of the first studies in a Swiss waste water stream and the receiving fresh water lake. For this purpose, a combined set of culture based (viable bacterial counts on nutrient media supplemented with antibiotics) and molecular tools (detection and quantitation of antibiotic resistance genes from environmental DNA-extracts using quantitative real-time PCR) was applied. Raw hospital and communal sewage, as well as partly treated/mixed sewage, which is discharged into the lake via an effluent pipe 700 m off shore, were taken into account. Furthermore lake water samples nearby the end of the waste water discharge pipe and the drinking water pump as well as close to the two river mouths were analyzed. Finally, the role of Lausannes WTP was evaluated for its potential to remove resistant and multiresistant bacteria, as well ARG. Results show highest levels of multiresistant bacteria and ARG in both hospital and communal sewage. Incidence was found that the WTP is a selective environment for multiresistant bacteria and resistance genes. Moreover, lake water and sediments nearby the end of the WTP discharge pipe and the two river mouths exhibited significantly higher levels of resistant determinants as compared to samples from the drinking water uptake. If the level of antibiotic resistance close to the drinking water uptake represents the natural background level of the lake or whether it is impacted by the WTP plume remained open during this first survey. The spatial distribution and persistence of ARG discharged via waste water into lake systems has been evaluated for the first time in the second part of this thesis. 22 sediment cores were sampled along two major transects (leading either into the deep lake or along the south-western shore line to the drinking water uptake) and in the vicinity of the WTP discharge pipe in the Vidy bay. A subset of samples was also analyzed for contamination with a “conventional” pollutant of sewage (mercury), for which a similar transport behavior (particle bound) as for bacteria and their resistance genes is assumed. The microbial community composition in all sediment as well as in waste water samples was analyzed using ARISA. Strong pollution with both ARG and mercury was detected in close proximity of the WTP discharge pipe and both contaminants significantly decreased along the transect into the deep lake. In general however, the spatial contamination of mercury and ARG was only moderately correlated. While the mercury contaminated sediment surface area tended to have a pollution tail in south eastern direction, ARG showed only a slow decrease along the south western transect. These findings imply differences in transport and fate of mercury and ARG in the bay, or additional sources of ARG contamination. Analysis of the microbial diversity in the samples support these hypotheses. This study revealed feasibility of qPCR as a tool to semi-quantitatively track spatial ARG contamination in fresh water lake sediments. Not much is known yet on the antibiotic resistance background level of fresh water lakes and how this may be influenced by anthropogenic activities in the surrounding catchment. Water samples from 21 Swiss lakes have been collected and were analyzed for the prevalence of different ARG (2 sulfonamide resistance genes sul1 & sul2, 3 tetracycline resistance genes tetB, tetM, tetW and one plasmid mediated fluoroquinolone resistance gene qnrA). In order to document the abundance of these genes in the bacterial populations, they were normalized to bacterial 16S rRNA gene copy numbers. The lakes differ in size, geographical position, land use pattern, and trophical status. The composition of the microbial community of all lakes was analyzed using ARISA. Sulfonamide resistance genes were detected in all investigated lakes (0.2-4%, sul1 and 0.01-0.3%, sul2), with highest levels in Lake Baldegger (21%). Lake Baldegger also contained all of the three tetracycline resistance genes, which were found in only 4 other lakes. None of the lakes was positive for qnrA. Important anthropogenic catchment activities linked to sul gene prevalence could not be identified based on our data and may require further analysis and an additional variables to complete our data set. However, our study demonstrates contamination of Swiss lakes with sulfonamide resistance genes and the potential of fresh water lakes preserve these genes, even remote from direct sources of pollution.

EPFL2013

Development of a hyphenated analytical methodology for the fractionation and characterization of environmental colloids

Enrica Alasonati

Natural colloids are defined as macromolecules and nanoparticles with sizes ranging between 1 nm and 1 µm. Colloidal pool is complex "ill-defined" mixture of humic substances, exopolymeric substances (EPS), inorganic oxides, clays, etc. Aquatic colloids have been recognized as key mediators in the removal of trace elements, facilitating contaminant transport, organic carbon cycling and micronutrient bioavailability. The residence times of the colloidal pool (and associate contaminants) can be order of magnitude different from those of the particulate fraction, but unlike the "truly" dissolved fraction, can be subjected to different coagulation and sedimentation processes. Colloids may also influence metal bioavailability by affecting free metal ion concentrations, constraining diffusion and affecting the dissociation kinetics of biological processes. The important environmental role of natural colloids is largely influenced by their surface area and closely related colloidal size. Nonetheless, it is currently unclear to what extend colloidal size can influence their behaviour and contaminant (e.g. toxic metals) binding properties. In our view this is partially related to the difficulties in determination of the colloidal size distributions and their impact in metal behaviour, mainly due to the inherent heterogeneity of the environmental colloids and their low concentrations in the environment. Furthermore, in addition to average parameters, there is a lack of appropriate sample collection and processing procedures, as well as analytical techniques to measure simultaneously the size (or related parameter) distributions of the colloid associated metal. The objective of the present work was therefore to develop sensitive analytical procedures for the fractionation of natural colloids and to apply them to improve the current understanding on the fate of the colloids and associated toxic metals in the freshwaters. To achieve these goals, the capabilities of asymmetric flow-field flow fractionation (aFlFFF) coupled to several detectors was explored. aFlFFF is a versatile technique separating colloids according to their diffusion coefficient. The system was hyphenated with multi-angle laser light scattering (MALS), differential refractive index (DRI), UV detector and, when necessary, high resolution inductively coupled plasma mass spectrometry (ICP-MS). Correct interpretation of the experimental results and extrapolation of meaningful molecular parameters by using an analytical tool with such a level of complexity requires improvement of the knowledge of colloids behaviour in the aFlFFF channel and careful optimization of the separation conditions. Given the very complex and dynamic nature of environmental colloids, different experimental procedures was first optimized and validate under lab conditions by using "standardized" colloids (e.g. alginate and EPS isolate from lab bacterial cultures), then applied to characterize colloidal organic matter (COM) isolated from natural water systems. The influence of critical operating parameters, such as crossflow rate, carrier composition and concentration, and sample load, on the alginate retention was carefully evaluated. Combined information obtained simultaneously by DRI and MALS detectors, allowed to set the appropriate combination of optimal parameters for the characterization of alginate, as a model COM. The metal distributions of metal-alginate complexes were probed by aFlFFF - ICP-MS: average values and continuous distributions of molar masses, radius of gyration and hydrodynamic radius, critical for understanding their role as carriers of metal pollutants, were evaluated in presence of lead or cadmium and compared to those in metal-free solutions of alginate. Furthermore, the molecular characteristic of exopolymeric substances (EPS) secreted by bacterium Sinorhizobium meliloti wild type and exoY strains, which is deficient in the production of the succinoglycan, were studied. EPS were collected in the extracellular environment or desorbed from the cell surface using different extraction protocols. EPS fractions with different chemical characteristic and molar mass were found, depending on the strain and extraction protocol. Molecular parameters as the distributions and average values of molar mass and gyration radius, and the polydispersity index have been measured and compared with data obtained by the chemical characterization. Size exclusion chromatography coupled to UV detection was also employed to provide additional information on the small class of size. Moreover, the effect of the cadmium, lead and calcium addition to exopolymeric substances excreted in the extracellular environment was investigated, with the aim to study how does EPS composition, size and mass distributions, can affect the trace metal binding properties of EPS and how the metal addition affects the EPS conformation. Obtained results revealed that cadmium and calcium bound preferentially on the low-size species population and the amount of the bound metal increased with the increasing of the concentration of added metal. In contrast, lead bound on preferentially larger classes of EPS size. The aFlFFF separation parameters were also optimized for the fractionation and characterization of humic substances, as other COM component, and applied to probe the temporal and spatial variability in the size and mass characteristics of COM samples collected in the Amazon River basin. COM characterization demonstrated the existence of a clear decrease of size of the colloidal material when passing from small to large watercourses (spatial variation). No significant changes were found in size distributions of samples collected in different period of the year, during the low and high flow, respectively (temporal variation). The analytical methodology, adapted to determine not only mean characteristics, but the population distributions, developed in the present work, allowed to obtain new information about the colloidal organic matter size distribution, which play utmost role in colloids behaviour and fate in the environment, in particular as toxic metal carriers.

EPFL2009

Assessment of microbial community changes and limiting factors during bioremediation of hydrocarbon-polluted soil with new miniaturized physiological methods

Karin Kaufmann

Due to human activities, organic pollutants are spilled to the environment where they threaten public health, often as contaminants of soil or groundwater. Living organisms are able to transform or mineralize many organic pollutants, and bioremediation techniques have been developed to remove pollutants from a contaminated site. However, fast and easy methods to document both the efficacy of bioremediation and the changes in soil microbial communities during bioremediation are not well developed. The major aim of this thesis was to develop miniaturized methods targeting the physiology of microbial communities during pollutant degradation in soil and to assess pollution-induced community changes. Furthermore, the methods should identify factors limiting efficient pollutant biodegradation. Petroleum hydrocarbons have been chosen as pollutants because these compounds are frequently spilled, readily water-soluble, partly toxic and carcinogenic, and therefore undesirable in soils and drinking water supplies. The influence of petroleum hydrocarbons on a microbial community in the vadose zone was assessed under field conditions. An artificial hydrocarbon mixture consisting of volatile and semi-volatile compounds similar to jet-fuel was emplaced in a previously uncontaminated vadose zone in nutrient-poor glacial melt water sand. The experiment included monitoring of microbial parameters and CO2 concentrations in soil gas over 3 months, in- and outside the hydrocarbon vapor plume that formed around the buried petroleum. Microbial and chemical analyses of vadose zone samples were performed on material from 9 cores drilled on 3 days at 3 distances from the buried petroleum mass to 3.3 m depth. Significantly elevated CO2 concentrations were observed after contamination. Total cell numbers as determined by fluorescence microscopy were strongly correlated with soil organic carbon and nitrogen content but varied little with contamination. Redundancy analysis (RDA) allowed direct analysis of effects of selected environmental variables or the artificial contamination on microbiological parameters. Variation in biomass and CO2 production was to 46 % explained by soil parameters and to 39.8 % by the duration of contamination. The microbial community structure was assessed by community-level physiological profile analysis using BiologTM EcoPlates. Only 35.9 % of variation in BiologTM data could be attributed to soil parameters and contamination, however, the samples with greatest exposure to hydrocarbons grouped together on RDA plots. It is concluded that at this nutrient-poor site, the microbial community was dominated by the natural heterogeneity and that the influence of petroleum hydrocarbon vapor was weak. BiologTM Eco-Plates combined with RDA were able to distinguish between samples with high and low hydrocarbon exposure. However, the method was not sensitive enough to produce consistent patterns in three replicates when extracts from subsurface soils with low cell numbers were inoculated. A more sensitive, MPN-based system was therefore developed and its ability to provide information about specific pollutant degraders was tested in alluvial sand. Eight dilutions of sand extracts were inoculated on medium- and substrate-containing polypropylene deep-well plates. To keep the concentrations of the volatile substrates constant over several days of incubation without intoxicating the soil community, a substrate-containing organic carrier phase was applied to each vial. The biochemically inert 2,2,4,4,6,8,8-heptamethylnonane (HMN) was appropriate for this purpose. Thus, the volatile substrates partition from HMN into the microorganism-containing soil water where they achieve relatively low concentrations. The sensitive watersoluble tetrazolium dye (WST-1) was added to each well after 7 days incubation to detect dehydrogenase activity. A dark yellow signal was developed for the substrates toluene, n-octane, n-dodecane, 1,2,4-trimethylbenzene and methylcyclohexane but not for isooctane, being strongest in n-alkane amended wells. With this method, more hydrocarbon degrading bacteria were detected in an alluvial sand exposed to kerosene for 72 hours compared to the community in the pristine sand. Both communities were mainly composed of n-alkane degraders. Although encouraging results were achieved, we noticed that the tetrazolium reduction was inhibited in some cases. Wells with high cell numbers, in which obvious growth occurred, did not produce a signal. In a next step, the two-liquid phase system with HMN was applied to modify a miniaturized respiration detection system using whole soil samples instead of soil extracts (Campbell et al., AEM 69, p. 3593). Soil was incubated in deep-well plates in presence of single volatile organic hydrocarbons. The carrier phase was added to the interstitial space in a bead layer on the bottom of the well (below the soil sample), and the hydrocarbons diffused into the soil-water. The soil activity was determined by means of an agar plate containing a pH indicator, which changes color as a function of the produced CO2. Physiological profiles specific to petroleum hydrocarbons in pristine and contaminated soils were assessed. The substrate concentrations, which induced highest microbial activity, as well as concentrations causing inhibition, were determined with effect-concentration curves. Community-level physiological profiles based on hydrocarbon degradation were obtained by applying multivariate analysis on the CO2 yields. The first measurement period of 0-6 hours after addition of relatively high hydrocarbon concentrations to the system yields values, which best separated soil types as well as pristine and contaminated soils. If low concentrations were applied, we obtained only significant separations after 24 hours incubation, probably after a growth step. The same micro-respiration system equipped with a 14CO2 detection plate was applied to study N and P limitation of aerobic mineralization of 14C-toluene in a soil previously exposed to petroleum. Significant nutrient limitation during short-term tests (24-48 hours) was identified and the optimized combination of nutrient addition was determined. Conclusively, miniaturized physiological methods have been proven to detect soil microbial community changes in petroleum hydrocarbon-contaminated soils. Adding an organic carrier phase to the miniaturized systems allowed assuring a volatile organic hydrocarbon supply in non-toxic concentrations in the soil water or the medium for several days. Thus simple, cost-effective systems to investigate the soil community during bioremediation and to address questions of nutrient shortage in soils have been provided with this thesis.

EPFL2004