Eutrophication is one of the most common causes of water quality impairment of inland and marine waters. Its best-known manifestations are toxic cyanobacteria blooms in lakes and waterways and proliferations of green macro algae in coastal areas. The term eutrophication is used by both the scientific community and public policy-makers, and therefore has a myriad of definitions. The introduction by the public authorities of regulations to limit eutrophication is a source of tension and debate on the activities identified as contributing or having contributed decisively to these phenomena. Debates on the identification of the driving factors and risk levels of eutrophication, seeking to guide public policies, have led the ministries in charge of the environment and agriculture to ask for a joint scientific appraisal to be conducted on the subject. Four French research institutes were mandated to produce a critical scientific analysis on the latest knowledge of the causes, mechanisms, consequences and predictability of eutrophication phenomena. This paper provides the methodology and the main findings of this two years exercise involving 40 scientific experts.

Abstract.A better understanding of the factors controlling N 2 fixation is a pre-requisite for improving our knowledge on the contribution of N 2 fixation process in the nitrogen cycling. Trace-metal clean nutrient/dust addition bioassays (+P, +PFe, +dust) were performed at three stations located in the western, central and eastern Mediterranean Sea, in summer 2008 as part of the BOUM cruise. The main goals were (1) to investigate the nutrient factor(s) limiting N 2 fixation (uptake of 15 N 2 ) and (2) to evaluate the potential impact of a Saharan dust event on this biological process during the stratification period. Initially, surface waters at the three stations were DIP-depleted (<10 nM) while the DFe concentrations were relatively high (from 1.2 to 2.3 nM) most likely due to atmospheric iron accumulation in the surface mixed layer. At all stations, Saharan dust input relieved the ambient nutrient limitation of the diazotrophic activity as demonstrated by the strong stimulation of N 2 fixation (from 130 % to 430 %). The highest dust stimulation of N 2 fixation was recorded at the station located in the eastern basin. The response of diazotrophic activity to nutrient additions was variable between the sampled stations suggesting a spatial variability of the factor controlling N 2 fixation over the whole basin. At allCorrespondence to: C. Ridame (celine.ridame@upmc.fr) stations, N 2 fixation was not limited by Fe nor co-limited by P and Fe. At the western station, N 2 fixation was DIP limited while at the eastern one, N 2 fixation was first DIP limited, then was limited by one or several chemical element(s) released by dust. Our results demonstrated that a Saharan dust input was able to relieve these successive on going limitations. Very interestingly, at the station located in the central basin, N 2 fixation was not limited by the availability of P yet it was strongly stimulated by dust addition (x3.1). A chemical element or a combination of several, released by the added dust may have been responsible for the observed stimulations of N 2 fixation. These results indicated that Saharan dust pulses to the surface Mediterranean waters, in addition to P and Fe, could be a source of chemical(s) element(s) that are necessary for metabolic processes and therefore influence rates of N 2 fixation.

Unicellular diazotrophic cyanobacteria (UCYN 2 -Fix lineage) were detected using whole-cell hybridization of specific Nitro821 oligonucleotide probe at the coastal and oligotrophic station SOMLIT off Marseilles (France). This station was sampled monthly, for a year and a half (June 2006-November 2007. The UCYN 2 -Fix community was dominated at 99.9% by picoplankters (0.7-1.5 mm) mainly as free living in the 0.2-3-mm size fraction. They were present all the year long with a mean density of 4.6 cell mL 21 , except in summer 2006, when concentrations reached 1.9 3 10 3 cell mL 21 and 5.3 3 10 3 cell mL 21 in June and July, respectively. During this bloom, picoplanktonic UCYN 2 -Fix represented 10.7% of the picocyanobacterial community. The larger size fractions (3-10 mm and .10 mm) were also dominated (98.9%) by picoplanktonic UCYN 2 -Fix cells, associated with inert particles, or dinoflagellates. However, hardly any nanoplanktonic UCYN 2 -Fix were detected (0-0.12 cell mL 21 ), and Trichodesmium sp. was observed only once in summer at low concentration (0.03 trichome mL 21 ). We hypothesize that a combination of abiotic parameters, such as elevated temperature, absence of nitrate, presence of phosphate, and an exceptionally high urban pollution event, explain the large bloom of potentially diazotrophic picocyanobacteria. Further studies are needed to confirm the identity of these small cells and their role in nitrogen cycle and marine productivity, especially since some effects of climate change (e.g., increased surface warming and upper-water-column stratification) may increase their importance in a near future.

Abstract. The Mediterranean Sea is one of the most oligotrophic marine areas on earth where nitrogen fixation has formally believed to play an important role in carbon and nitrogen fluxes. Although this view is under debate, the diazotrophs responsible for this activity have still not been investigated in the open sea. In this study, we characterised the surface distribution and species richness of unicellular and filamentous diazotrophs across the Mediterranean Sea by combining microscopic counts with size fractionated in situ hybridization (TSA-FISH), and 16S rDNA and nifH genes phylogenies. These genetic analyses were possible owing to the development of a new PCR protocol adapted to scarce microorganisms that can detect as few as 1 cell ml −1 in cultures. Low concentrations of diazotrophic cyanobacteria were detected and this community was dominated at 99.9% by picoplankton hybridized to the Nitro821 probe, specific for unicellular diazotrophic cyanobacteria (UCYN). Among filamentous cyanobacteria only 0.02 filament ml −1 of Richelia were detected in the eastern basin, while small (0.7-1.5 µm) and large (2.5-3.2 µm) Nitro821-targeted cells were recovered at all stations with a mean concentration of 3.5 cell ml −1 . The affiliation of the small Nitro821-targeted cells to UCYN-A was confirmed by 16S and nifH phylogenies in the western Mediterranean Sea. In the central and the eastern Mediterranean Sea no 16S rDNA and nifH sequence from UCYN was obtained as cells concentration were close to, or below PCR detection limit. Bradyrhizobium sequences dominated nifH clone libraries from picoplanktonic size fractions. A few sequences of γ -proteobacteria were also detected in the central Mediterranean Sea. While low phosphate and iron concentrations could explain the absence of Trichodesmium sp., the factors that prevent the developCorrespondence to: I. C. Biegala (biegala@univmed.fr) ment of UCYN-B and C remain unknown. We also propose that the dominating picoplankters probably developed specific strategies, such as associations with protists or particles, and/or photosynthetic activity, to acquire carbon for sustaining diazotrophy.

A better understanding of the factors controlling N₂ fixation is a pre-requisite for improving our knowledge on the contribution of N₂ fixation in the nitrogen cycling in the Mediterranean Sea. Trace-metal clean nutrient/dust additions bioassays (+P, +PFe, +dust) were performed at three stations located in the western, central and eastern Mediterranean Sea, in summer 2008 as part of the BOUM cruise. The main goals were to investigate the nutrient factor(s) limiting N₂ fixation (uptake of ¹⁵N₂) and to evaluate the potential impact of a Saharan dust event on this biological process during the stratification period. Initially, surface waters at the three stations were DIP-depleted (<10 nM) while the DFe concentrations were relatively high (from 1.2 to 2.3 nM) most likely due to atmospheric iron accumulation in the surface mixed layer. At all stations, Saharan dust input relieved the ambient nutrient limitation of diazotrophic activity as demonstrated by the strong stimulation of N₂ fixation (from x2.3 to x5.3). The highest dust stimulation of N₂ fixation was recorded at the station located in the eastern basin (x5.3). The responses of diazotrophic activity to nutrients addition were contrasted at the sampled stations suggesting a spatial variability of the factor controlling N₂ fixation over the whole basin. At all stations, N₂ fixation was not limited by Fe nor co-limited by P and Fe. At the western station, N₂ fixation was DIP limited while at the eastern one, N₂ fixation was first DIP limited then was limited by one or several chemical element(s) released by dust. Our results demonstrated that a Saharan dust input was able to relieve the successive on-going N₂ fixation limitations. Very interestingly, at the station located in the central basin, N₂ fixation was not limited by the availability of P yet it was strongly stimulated by dust additions (up to x3.1). A chemical element or a combination of several, released by the added dust may have been responsible for the observed stimulations of N₂ fixation. These results indicated that Saharan dust pulses to the surface Mediterranean waters, in addition to P and Fe, could be a source of chemical(s) element(s) that are necessary for metabolic processes and therefore influence rates of N₂ fixation

Cyanobacterial blooms in eutrophic freshwater is a global threat to the functioning of ecosystems, human health and the economy. Parties responsible for the ecosystems and human health increasingly demand reliable predictions of cyanobacterial development to support necessary decisions. Long-term data series help with identifying environmental drivers of cyanobacterial developments in the context of climatic and anthropogenic pressure. Here, we analyzed 13 years of eutrophication and climatic data of a shallow temperate reservoir showing a high interannual variability of cyanobacterial development and composition, which is a less occurring and/or less described phenomenon compared to recurrant monospecific blooms. While between 2007–2012 Planktothrix agardhii dominated the cyanobacterial community, it shifted towards Microcystis sp. and then Dolichospermum sp. afterwards (2013–2019). The shift to Microcystis sp. dominance was mainly influenced by generally calmer and warmer conditions. The later shift to Dolichospermum sp. was driven by droughts influencing, amongst others, the N-load, as P remained unchanged over the time period. Both, climatic pressure and N-limitation contributed to the high variability of cyanobacterial blooms and may lead to a new equilibrium. The further reduction of P-load in parallel to the decreasing N-load is important to suppress cyanobacterial blooms and ameliorate ecosystem health.

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