Standardisation – fundament for change

The use of effect-based methods (EBMs) is currently being discussed in connection with the further development of the European Water Framework Directive [1]. This is also reflected, for example, in the extended definition of the environmental quality standard (EQS) proposed by the EU Commission [2]. In addition to the usual substance-based threshold values for assessing the chemical status of surface waters, this also allows the definition of effect-based threshold values – for example for the maximum effect potential that leads to the activation of the estrogen receptor by endocrine disruptors. The implementation of EBMs in water body regulation follows an extended assessment approach with the aim of a more comprehensive assessment of the water body status. The further development of planar bioassay methods, which are based on a direct coupling of thin-layer chromatography and bioassay, allow both a substance-related and an effect-based assessment of the chemical status of a surface water sample and thus represent a bridge between the two assessment approaches [3]. An essential prerequisite for the regulatory implementation of measurement methods is their harmonisation and standardisation as well as the derivation of effect-based threshold values for the assessment of EBMs. In this sense, standardisation is the prerequisite for change, as this contributes to the acceptance of a procedure, creates transparency and enables the generation of court-proof data that can be used for test procedures in a regulatory context. To demonstrate sufficient sensitivity of the planar yeast estrogen screen (p-YES) for analysing estrogenic compounds/effects, a surface water sample from the Rhine as well as aliquots with various concentrations of the compounds estrone (E1), 17α ethinylestradiol (EE2), 17β estradiol (E2) and estriol (E3) were enriched 1000-fold using solid phase extraction (HLB). 50 µL of each sample was applied to a HPTLC-plate (HPTLC plates silica gel 60 F 254) and separated by thin-layer chromatography (Chloroform/Aceton/Petroleum fraction, 75/10/15) after a focusing step with methanol. Following the separation, a yeast estrogen screen was performed directly on the plate surface in accordance with ISO 19040–1 [4, 5] by spraying the yeast cells onto the plate. After a three-hour exposure at 30°C in a saturated water vapor atmosphere, the activity of the reporter gene lacZ was quantified by the formation of a fluorescent dye as a measure of the agonistic activity on the estrogen receptor. Figure 1 demonstrates the sensitivity of the p-YES to detect a spike-level of EE2 of 10 pg/L. No signal corresponding to EE2 is detectable in the extract of the unspiked water from river Rhine (a). In the sample spiked with 10 pg/L EE2 (b) a respective signal emerges that is above the limit of quantification and baseline-separated from the adjacent E2-Signal. Thus, a quantification of EE2 below its discussed environmental quality standard of 17 pg/L is feasible. Due to the lower separation power of HPTLC compared to HPLC false positive assessments regarding the presence of EE2 might occur if estrogenic compounds with a similar mobility on the HPTLC-plate are present in the sample. However, a reliable falsification of the presence of an estrogenic compound such as EE2 is possible which allows the application of this approach as a screening method. In addition to the compound specific evaluation the overall estrogenicity of a sample can be determined and assessed against the proposed effect- based trigger value for the p-YES of 0.5 ng E2-Equivalents per liter [6]. Figure 1 shows in fact the presence of further estrogenic compounds besides the discussed priority compounds E1, E2 and EE2 in the investigated surface water, but these compounds add to the overall estrogenicity only to a minor extend. Currently, a technical specification for the p-YES is under development in a DIN-working group, a DIN EN ISO standard for the derivation of biological equivalence concentrations from bioassays [7] and the series DIN EN ISO 19040 [4] for the quantification of the overall estrogenicity of water samples is available. In sum, all relevant elements for the use of EBMs for the analysis of estrogenicity in surface waters in line with the EU-WFD (Water Framework Directive) are available – the implementation of these EBMs in the WFD would open the stage for further EBMs and would be a first step towards a paradigm-change towards the use of more holistic methods in the WFD for the assessment of the chemical status of surface waters. The authors thank the German Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection for support via the BfG-BMUV action 109 and Marina Ohlig (BfG) for excellent technical assistance.