Implementation of the Water Framework Directive: Lessons Learned and Future Perspectives for an Ecologically Meaningful Classification Based on Phytoplankton of the Status of Greek Lakes, Mediterranean Region.

The enactment of the Water Framework Directive (WFD) initiated scientific efforts to develop reliable methods for comparing prevailing lake conditions against reference (or nonimpaired) states, using the state of a set biological elements. Drawing a distinction between impaired and natural conditions can be a challenging exercise. Another important aspect is to ensure that water quality assessment is comparable among the different Member States. In this context, the present paper offers a constructive critique of the practices followed during the WFD implementation in Greece by pinpointing methodological weaknesses and knowledge gaps that undermine our ability to classify the ecological quality of Greek lakes. One of the pillars of WDF is a valid lake typology that sets ecological standards transcending geographic regions and national boundaries. The national typology of Greek lakes has failed to take into account essential components. WFD compliance assessments based on the descriptions of phytoplankton communities are oversimplified and as such should be revisited. Exclusion of most chroococcal species from the analysis of cyanobacteria biovolume in Greek lakes/reservoirs and most reservoirs in Spain, Portugal, and Cyprus is not consistent with the distribution of those taxa in lakes. Similarly, the total biovolume reference values and the indices used in classification schemes reflect misunderstandings of WFD core principles. This hampers the comparability of ecological status across Europe and leads to quality standards that are too relaxed to provide an efficient target for the protection of Greek/transboundary lakes such as the ancient Lake Megali Prespa.

Plankton microorganisms coinciding with two consecutive mass fish kills in a newly reconstructed lake.

Lake Karla, Greece, was dried up in 1962 and its refilling started in 2009. We examined the Cyanobacteria and unicellular eukaryotes found during two fish kill incidents, in March and April 2010, in order to detect possible causative agents. Both microscopic and molecular (16S/18S rRNA gene diversity) identification were applied. Potentially toxic Cyanobacteria included representatives of the Planktothrix and Anabaena groups. Known toxic eukaryotes or parasites related to fish kill events were Prymnesium parvum and Pfiesteria cf. piscicida, the latter being reported in an inland lake for the second time. Other potentially harmful microorganisms, for fish and other aquatic life, included representatives of Fungi, Mesomycetozoa, Alveolata, and Heterokontophyta (stramenopiles). In addition, Euglenophyta, Chlorophyta, and diatoms were represented by species indicative of hypertrophic conditions. The pioneers of L. Karla's plankton during the first months of its water refilling process included species that could cause the two observed fish kill events.

A catastrophic change in a european protected wetland: From harmful phytoplankton blooms to fish and bird kill.

Understanding the processes that underlay an ecological disaster represents a major scientific challenge. Here, we investigated phytoplankton and zooplankton community changes before and during a fauna mass kill in a European protected wetland. Evidence on gradual development and collapse of harmful phytoplankton blooms, allowed us to delineate the biotic and abiotic interactions that led to this ecological disaster. Before the mass fauna kill, mixed blooms of known harmful cyanobacteria and the killer alga Prymnesium parvum altered biomass flow and minimized zooplankton resource use efficiency. These blooms collapsed under high nutrient concentrations and inhibitory ammonia levels, with low phytoplankton biomass leading to a dramatic drop in photosynthetic oxygenation and a shift to a heterotrophic ecosystem phase. Along with the phytoplankton collapse, extremely high numbers of red planktonic crustaceans-Daphnia magna, visible through satellite images, indicated low oxygen conditions as well as a decrease or absence of fish predation pressure. Our findings provide clear evidence that the mass episode of fish and birds kill resulted through severe changes in phytoplankton and zooplankton dynamics, and the alternation on key abiotic conditions. Our study highlights that plankton-related ecosystem functions mirror the accumulated heavy anthropogenic impacts on freshwaters and could reflect a failure in conservation and restoration measures.

Phytoplankton and water quality in a Mediterranean drinking-water reservoir (Marathonas Reservoir, Greece).

Phytoplankton and water quality of Marathonas drinking-water Reservoir were examined for the first time. During the study period (July-September 2007), phytoplankton composition was indicative of eutrophic conditions although phytoplankton biovolume was low (max. 2.7 mm³ l⁻¹). Phytoplankton was dominated by cyanobacteria and diatoms, whereas desmids and dinoflagellates contributed with lower biovolume values. Changing flushing rate in the reservoir (up to 0.7% of reservoir's water volume per day) driven by water withdrawal and occurring in pulses for a period of 15-25 days was associated with phytoplankton dynamics. Under flushing pulses: (1) biovolume was low and (2) both 'good' quality species and the tolerant to flushing 'nuisance' cyanobacterium Microcystis aeruginosa dominated. According to the Water Framework Directive, the metrics of phytoplankton biovolume and cyanobacterial percentage (%) contribution indicated a moderate ecological water quality. In addition, the total biovolume of cyanobacteria as well as the dominance of the known toxin-producing M. aeruginosa in the reservoir's phytoplankton indicated a potential hazard for human health according to the World Health Organization.

Occurrence and diversity of cyanotoxins in Greek lakes.

Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm3MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754 ng mg-1 dw, followed by MC-LR (458 ng mg-1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria).

Molecular diversity of bacteria in commercially available "Spirulina" food supplements.

The cyanobacterium Arthrospira is among the most well-known food supplements worldwide known as "Spirulina." While it is a widely recognized health-promoting natural product, there are no reports on the molecular diversity of commercially available brands of "Spirulina" supplements and the occurrence of other cyanobacterial and heterotrophic bacterial microorganisms in these products. In this study, 454-pyrosequencing analysis of the total bacterial occurrence in 31 brands of "Spirulina" dietary supplements from the Greek market was applied for the first time. In all samples, operational taxonomic units (OTUs) of Arthrospira platensis were the predominant cyanobacteria. Some products contained additional cyanobacterial OTUs including a few known potentially toxic taxa. Moreover, 469 OTUs were detected in all 31 products collectively, with most of them being related to the Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria and Verrucomicrobia. All samples included heterotrophic bacterial OTUs, ranging from 9-157 per product. Among the most common OTUs were ones closely related to taxa known for causing health issues (i.e., Pseudomonas, Flavobacterium, Vibrio, Aeromonas, Clostridium, Bacillus, Fusobacterium, Enterococcus). The observed high cyanobacterial and heterotrophic bacterial OTUs richness in the final product is a point for further research on the growth and processing of Arthrospira biomass for commercial purposes.