Sensitive vs. tolerant Nitzschia palea (Kützing) W. Smith strains to atrazine: a biochemical perspective.

Organic contaminants, and herbicides in particular, represent a risk for aquatic ecosystems. The primary target of herbicides are producers, the base of food webs, but frequently they end up far from the application point affecting non-target species. Its presence can work as sub-lethal stimulus, which sort the genetic and phenotypic differences within a species. Intraspecific variation allows adaptation to changes in the environment but also to new niches due to variations in species' sensitivity and biochemical response to a certain chemical. A better understanding of these variations can lead to the development of improved strategies for ecosystem protection. This research aimed to compare a sensitive and a tolerant strain of the freshwater diatom Nitzschia palea to atrazine. Strains were exposed to three concentrations within their tolerance range, during 96 h. The activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione-S-transferases and glutathione peroxidases was determined. In addition, chlorophylls a and c, carotenoids, reduced glutathione, proteins and lipid peroxidation were quantified. Both strains displayed different strategies to deal with atrazine toxicity: while the sensitive strain decreased the oxidative stress, increasing the activity of antioxidant enzymes such as superoxide dismutase, the tolerant strain invested in conjugation pathways and carotenoids' maintenance.

Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms.

The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.

Typology of diatom communities and the influence of hydro-ecoregions: a study on the French hydrosystem scale.

By comparing diatom communities in natural and disturbed sites, indicators for different types and levels of anthropogenic disturbance can be found. As a first step, this study aims to describe the different natural and disturbed community types found throughout the French hydrosystem. 836 diatom samples were analysed with an unsupervised neural network, the self-organising-map, a well accepted method for community ordination. 11 different communities were identified, 5 corresponding to non-impacted or slightly impacted conditions and representing the diatom natural variability of our dataset. These 5 communities corresponded to 5 different hydro-ecoregions, i.e. 5 river types with similar geological context and range in altitude. The 6 other communities were typical of rivers under anthropogenic pressure. The influence of natural conditions within the hydro-ecoregions was overwhelmed by the nature and the intensity of the pollution at the sampling stations. This work was done in the context of the application and enforcement of the Water Framework Directive.

Procedures for determining the pesticide sensitivity of indigenous soil algae: a possible bioindicator of soil contamination?

Soil algae are present in all kinds of soils, both arable and virgin, in large amounts and in great diversity. These photosynthetic microorganisms, which are concentrated in the top few centimeters of the soil profile, are organized in a community structure that varies depending on soil type, farming method and pesticide application. We first tested several extraction and conservation methods for soil algae, and a 14C incorporation procedure to evaluate the photosynthetic activity of these micro-organisms. In a second study, we assessed the sensitivity to atrazine of soil microalgae from two corn fields managed by different agricultural practices (conventional vs. organic). Changes in indigenous diatom communities were monitored, together with photosynthetic tests performed on the whole algal community. Comparison of the data for the treated field with those for the reference, untreated organic cornfield showed that previous atrazine application in the conventional cornfield had changed the species composition of the soil diatom communities. Short-term ecotoxicological tests, using photosynthetic activity as endpoint, also showed that the communities that had developed under pesticide stress were more tolerant to further atrazine application than the control communities. The stress caused by major environmental disturbances, such as ground dressing, was not sufficient to mask the difference between the two crops, suggesting that telluric microalgae could be used as indicators of xenobiotic contamination in soils. These initial findings about using soil microalgal communities as bioindicators are promising. In addition, their photosynthetic activity, which reflects their sensitivity to xenobiotic compounds, seems to be a relevant bioindicator of soil contamination.

Effects of atrazine and nicosulfuron on freshwater microalgae.

Growth modifications caused by various concentrations of atrazine and nicosulfuron were monitored in closed and continuous culture of Chlorella vulgaris (chlorophyta), Navicula accommoda (diatomophyta), and Oscillatoria limnetica (cyanophyta). The concentration at which algal growth rate was reduced twofold (EC50) was determined in the three species for both herbicides. Comparatively, the two toxicants were applied at 10 microg/l level in microcosms inoculated with natural phytoplankton from Lake Geneva. The relative abundances of major phytoplanktonic species were measured by algal cell count at the beginning and at the end of each experiment. Atrazine and nicosulfuron have different targets in plant metabolism, respectively, photosystem II (PSII) and acetolactate synthase (ALS), and the expected effects were different. Generally, the cultured phytoplankton exhibited various sensitivities, depending on species or herbicide. In the microcosms, the major taxa of natural phytoplanktonic samples exhibited various patterns, from acute toxicity to growth enhancement. For example, the diatoms inside the community were not affected by atrazine and nicosulfuron, except for Stephanodiscus minutulus that was sensitive to both, and Asterionella/formosa that was sensitive only to nicosulfuron. The specific physiology and the relationships among the phytoplanktonic communities have to be carefully considered when one would try to predict the extent of herbicide action on natural phytoplankton using in vitro tests. There is a need to test the toxic effect on various cultured strains, representative of most of the taxonomic composition of natural communities, to take into account the wide range of sensitivities and reaction to herbicide contamination. But this is not enough to give a solid frame when transposing the results to the field, and the use of more ecologically relevant systems is recommended.

Effects of atrazine and nicosulfuron on phytoplankton in systems of increasing complexity.

We have tested the sensitivity of phytoplankton to the herbicides atrazine and nicosulfuron in experiments conduced in increasingly complex systems, from single strain phytoplankton cultures (microplates) to mesocosms mimicking whole ecosystems. The endpoints used to assess sensitivity to atrazine and nicosulfuron were total biomass increase, photosynthetic efficiency, and community diversity, depending on the system considered. Nicosulfuron appeared to be very much less toxic to phytoplankton than atrazine, in accord with the planned changes in agricultural practices to reduce the effects of surface water contamination on aquatic biota. Nevertheless, nicosulfuron had significant effects in some systems (principally microcosms), whereas the single monocultures were almost insensitive to it. This points out the inaccuracy of standardized toxicity test on phytoplanktonic algae alone for predicting the effects of xenobiotics on natural communities and the need for tests in microcosms and mesocosms to obtain reliable evidence about the toxicity of a given chemical on freshwater aquatic ecosystems.