The ENDIS-RISKS project can be divided into five different phases. Four of these phases concern research activities, the fifth phase is a communication and valorisation phase.

• Phase I: Distribution of endocrine disrupting substances in the Scheldt estuary (chemical analyses, analyses on biota, in vitro analyses)

• Phase II: Evaluation of the exposure of biota from the Scheldt estuary to endocrine disrupting substances

• Phase III: Ecotoxicological evaluation of the effects of endocrine disrupting substances occurring in the Scheldt estuary on resident mysid shrimp populations (laboratory and field studies)

• Phase IV: Risk assessment

• Phase V: Communication and valorisation

The project runs over a period of four years, from 1 Feb 2002 until 30 Apr 2006. The way the planning of the tasks is spread over the four years for each of the partners, is described in the tasks-sceme.

Phase I: Distribution of endocrine disrupting substances in the Scheldt estuary (chemical analyses, analyses on biota, in vitro analyses)

The Scheldt estuary will be sampled three times a year (spring, summer and winter) for the duration of the project. On each sampling date, sediment, water, suspended solid and biota (mysid shrimp and gobies) will be collected. Based on the distribution of the measured contaminants during the first three sampling campaigns (7 different sampling stations, situated along a salinity gradient from Vlissingen near the mouth of the estuary up to Antwerp) a possible reduction in sampling locations could be suggested. The different stations were selected from the sampling stations mentioned by Mees et al. (1993), the orientation study for the presence of endocrine active substances in aquatic systems in the Netherlands (LOES-project; RIZA rapport 99.007, RIKZ Rapport 99.024), the homogenous measuring network of the International Commission for the Protection of the Scheldt (ICBS) and the sampling stations used by the Waterways and Maritime Affairs Administration(AWZ). Sampling and sample preservation will be done according to the protocols and recommendations described in the orientation study for the presence of endocrine active substances in water systems in the Netherlands (LOES-project). Samples will be taken with the available research ships (e.g. Belgica, Zeeleeuw, Ter Strepe or Luctor). Simultaneously, temperature, salinity, conductivity, dissolved oxygen, pH and Secchi depth will be measured. From the sampled biota, density, diversity, population composition (sex and developmental stage) and fecundity will be determined.

The different water, sediment and suspended solid extracts will be tested in vitro to assess their potential to bind with the (human) estrogen and androgen receptor. For this purpose, a yeast assay will be used in which the respective receptor is cloned in such a way that the estrogen or androgen binding potential of the extract can be quantitatively determined. The results of these analyses will allow an identification of the occurrence and distribution of chemicals - in different environmental matrices of the Scheldt estuary - with the ability to bind to the estrogen and androgen receptor; i.e. chemicals which are potential endocrine disruptors.

Phase II: Evaluation of the exposure of biota from the Scheldt estuary to endocrine disrupting substances

Based on the results of the first sampling campaigns (after 3 sampling campaigns) priority substances will be selected. Chemicals are indicated as priority substances when they are found in the sampled biota or if they are present in a sufficiently high concentration in the other sampled matrices (indicating a potential risk). The concentrations will therefore be compared to literature data on the effect concentrations of these substances. For this purpose, the available database on endocrine disruptors developed at the Laboratory of Environmental Toxicology and Aquatic Ecology (OSTC-project EDNorth) will be used. These priority endocrine disrupting chemicals will be used in laboratory experiments with mysid shrimp to asses acute and chronic effects (Phase III).

Phase III: Ecotoxicological evaluation of the effects of endocrine disrupting substances occurring in the Scheldt estuary on resident mysid shrimp populations (laboratory and field studies)

Acute and chronic laboratory tests with mysid shrimps

To evaluate the possible effects of the pollutants (retained in phase II) on the mysid shrimps, acute and chronic test will be performed in the laboratory. The biomarkers that will be used to evaluate potential endocrine disruption in mysid shrimp are: energy and (ecdy)steroid metabolism, DNA damage and specific protein expression. Next to the biomarkers for endocrine disruption, also growth, molting and population parameters will be assessed (e.g. sex-ratio, mortality, …). These parameters will allow an evaluation of the interference of the tested substances with the growth regulation, the ecdysteroid metabolism and sexual differentiation of the test organisms. Mysid shrimp will also be exposed in multi-generation experiments during two generations (until the first generation of juveniles produces juveniles again) to assess the effects of endocrine disruptors during prolonged exposure. The same endpoints will be measured as in the shorter experiments (in addition to reproductive endpoints).

Population study of the native mysid shrimp in the Scheldt estuary

The mysid shrimp population of the Scheldt estuary will be sampled and studied during one year. The density, biomass, population structure and brood size will be measured according to previously published studies by Mees et al. (1993) and Mees et al. (1994). In this way, the present population dynamics can be compared with the available historical data and possible changes can be detected. In addition, a more in-depth field study on the possible endocrine disruption effects will be conducted based on the results of the laboratory toxicity experiments. Sensitive endpoints identified in the laboratory experiments will be validated in situ on the resident mysid shrimp population in the Scheldt estuary. The laboratory and field studies will evaluate potential endocrine disruption in mysid shrimp populations in the Scheldt estuary. This may possibly result in an ecotoxicological surveillance system aimed at the detection of endocrine disruptive effects.

Phase IV: Risk assessment

In the last phase, the results of the three previous phases will be incorporated into an integrated risk assessment of the selected (Phase II) endocrine disrupting substances (for the Scheldt estuary). Using the results of the laboratory toxicity tests, the in situ field study and the ecological data obtained in the present study and from historical studies, Predicted No Effect Concentrations will be calculated for the tested compounds and the studied environmental compartments. The most recent (probabilistic) risk assessment techniques will be applied. Moreover, an overall risk assessment for the Scheldt estuary will be conducted with these data in correlation with environmental concentrations of the tested endocrine disruptors. Recommendations for a sustainable management and priority substances will be developed.

Phase V: Communication and valorisation

In order to ensure (rapid) dissemination and valorisation of the obtained results, the following actions will be undertaken:

• All results will be published in peer-reviewed journals.
• Transfer (and discussion) of the obtained knowledge and insights to national and international representatives of policy-making and policy-supporting governmental services. This discussion will offer an essential contribution to the international contractual engagements of Belgium.
• Diffusion of data and results through scientific databases
• Interactive communication forum during the project (project website)
• The results of the project will be brought together in a book which will include the datasets on CD-rom and the main research results. The diffusion and valorisation of the information as described above will be developed and maintained by the Flanders Marine Institute (VLIZ) (subcontracted) under supervision of the coordinator.

Supported by the Belgian Federal Science Policy Office

General coordination: Colin Janssen, An Ghekiere Web site and databases hosted by VLIZ