Workplan

How and when are we going to do it?

PEPCY's workplan is structured into 5 workpackages Networking between them will be intensive both with respect to results, deliverables and tools produced by one and used in other WPs, as well as with respect to their input for risk management.

WP 1

provides new molecular genetic and mass spectrometric tools for characterising genotypes containing cyanopeptides. This WP is large in terms of person-months, as it has an important service function for the others by producing mutants with impaired peptide production and export, which is very labor-intensive. Objectives WP 1The molecular biology of toxin production by cyanobacteria is only at its very beginning, although molecular and genetic tools are of great importance to determine the potential risk emanating from Cyanobacterial "chemotypes", i.e. those containing specific substances. Knowledge of the genes encoding for the production of these substances enables their identification as specific "genotypes" with molecular tools. Objectives of this WP are therefore:

identify new potentially toxic cyanopeptides from the cyanobacterial taxa most frequently present in European waterbodies;
assess the genetic potential of these strains to produce such compounds; i.e. to match "genotype" and "chemotype"
generate mutants lacking such compounds as tools for assessment of toxicity and to elucidate their role in mass development of cyanobacteria in WPs 2-4
develop and use genetic tools for rapid screening and detection of toxin-producing cyanobacterial genotypes, as a contribution to exposure assessment and risk assessment (WP 5).

WP 2

elucidates cyanopeptide occurrence in cyanobacterial populations to which humans are potentially exposed. With respect to person-months input, WP 2 is the largest, as it comprises three areas of particularly labor-intensive work: (i) field investigations for assessing peptide occurrence in nature and isolating strains producing potentially hazardous peptides; (ii) laboratory experiments for assessing whether these peptides are cellular constituents which are always produced or whether environmental factors trigger their production, and (iii) development of invertebrate biomonitoring systems for toxic peptides.Objectives WP2

Quantify continuity/discontinuity in production of bioactive cyanopeptide content in cells
Provide quantitative data on variability of occurrence of different cyanopeptide genotypes in relation to environmental factors in the field; database on genotype/chemotype occurrence in the field
Develop rapid, easy-to-use cyanopeptide biomonitoring systems, based on microscopical analyses of zooplankton symptoms, including single and combinatorial exposures

WP 3

is in part a "service WP", focusing on producing sufficient amounts of candidate peptide toxins for toxicity testing and hazard characterisation in WP 4. Its further deliverables are physicochemical and immunobiological methods for quantitative cyanopeptide analyses. Objectives: WP3Purified cyanobacterial peptides methods for their detection, identification and quantification are central requirements for PEPCY WP1, WP2 and WP4 objectives to be achieved. The WP 3 objectives are also essential to provide quantitative data for hazard characterization, exposure assessment and risk assessment in WP5. The objectives are:

Peptide purification from laboratory cultures of cyanobacteria and environmental samples of cyanobacterial mass populations (blooms, scums) from European freshwater resources
Establishment of quantitative physicochemical analytical methods for cyanobacterial peptides (MALDI-TOF MS; HPLC)
Establishment of quantitative procedures for peptide extraction from cyanobacterial cells and other matrices
Establishment of quantitative immunoassay-based methods for cyanobacterial peptides

WP 4

characterises toxicity of field samples, crude extracts and purified peptides, investigates toxicokinetics and toxicodynamics, and produces hazard characterisation for the key toxic cyanopeptides, thus providing key factors for decision trees and risk management.Objectives WP4

Provision of dose-response data on potentially toxic cyanopeptides
Description of modes of action and type(s) of effects
Identification of most likely exposure routes and consequently hazard assessment of potential human health effects

WP 5

Finally in WP 5 the exposure scenarios from WP 2 and hazard characterisation from WP 4 will be integrated for developing a risk management framework developed in WP 5. A further important element of this will be protocols for monitoring and surveillance, using the user-friendly analytical methods provided by WP 3. The proposed approaches will be discussed with practitioners at an international conference held in year 3 and then communicated in dissemination packages for different user groups.Objectives WP 5

assess the risk of exposure from data on occurrence of cyanobacteria containing hazardous peptides
develop an Alert Levels Framework with decision trees for exposure to cyanopeptides via drinking, recreation and occupational exposure
develop risk communication strategies and materials for cyanopeptide hazards, exposure risks, and their avoidance/reduction for the public and professional groups
develop a risk management approach for cyanopeptides from WP4 hazard characterisation and objectives 1-4 in collaboration with international (WHO), European and national institutions, public health authorities and environment agencies