Role of Higher Tier Data in the Derivation of the Ni EQS Chris Schlekat, Ph.D., DABT NiPERA Durham, NC USA Expert Sub-group Discussion March 15th 2012 Brussels

Overview Review of discussion at Feb 11, 2011 Expert-sub Group meeting SCHER Opinion Focus of subsequent discussions Statistical analysis of snail endpoint from Fraunhofer indoor mesocosm experiment Overall Weight of Evidence

Review of discussion at Feb 11, 2011 Expert-sub Group meeting Higher tier data were introduced WCA Environment field-based analysis Fraunhofer Institute indoor mesocosm experiment Expert Sub-group agreed that these data should be sent to the SCHER for their opinion on the Ni EQS Consistent with WFD and EQS TGD Annex V of WFD: Proposed EQSs should be compared with any evidence from field studies

Field-based analyses Approach Followed methods outlined in Crane et al. (2007) Crane et al. (2007) specifically referred to in Section 2.9.2 of EQS TGD as an appropriate approach for comparison with EQS Paired data from UK surface waters Detailed water chemistry (pH, DOC, hardness, Ni concentrations) Occurrence of benthic macroinvertebrates (including snails) Kind of data used to determine compliance with WFD EQS Relationship between benthic macroinvertebrate abundance and bioavailable Ni were calculated

Field-based analyses Results Results indicated that bioavailable Ni concentrations need to be greater than 10.3 µg Ni/L for ecological status to change from Good to Moderate 3.9 µg Ni/L before snail abundance cannot be distinguished from reference condition TGD Section 2.9.2: Given that field exposures include other stressors (metals, organic chemicals, non-chemical stressors), results should be considered as conservative estimates of effects attributable to substance under evaluation Furthermore, “the maximum concentration that still permits good biological quality… will be a conservative estimate” Suggests that value of 10.3 µgbioavailable Ni/L is appropriate to compare with EQS Commission proposal of 31 January 2012 proposed an EQS of 4 µgbioavailable/L Proposed EQS is well below maximum concentration that still permits good biological quality

Indoor Mesocosm Study Relevance Mesocosm data are widely referred to in EQS TGD For example, Section 3.3.1.3 states that mesocosm data “may be used to help select the size of AF applied to the HC5” for substances where an SSD is used Fits the situation for Ni Section 3.3.1.3 contains criteria to judge acceptance of mesocosm studies Indoor mesocosm study was designed with these criteria in mind More detail in presentation by U. Hommen In general, intent of EQS TGD is for mesocosm data to serve as a broad means of comparison to the HC5 from the SSD (e.g., to evaluate the level of agreement between lab and field evidence) Field/mesocosm data = supporting data, not critical data Section 2.9.2: “Given the variability in field data (and indeed in laboratory ecotoxicity data), small differences between a laboratory-based QS and field data should not be given undue weight.”

Indoor Mesocosm Study Approach, Methods, Results, Further evaluation: To be covered by U. Hommen (Fraunhofer Institute) in detail Very briefly, the primary conclusions from the Fraunhofer Institute study are as follows: Using standard statistical methods (as identified by OECD Guidance Document 53), the study-wide NOEC is 24 µg Ni/L Using detailed statistics focused on the most sensitive endpoint (relative growth rate of snails), a NOEC of 12 µg Ni/L can be considered

SCHER Opinion SCHER Opinion was delivered on May 25, 2011 Opinion affirmed: That the EQS has been correctly and appropriately derived That the most critical endpoints (bioavailable Ni) were identified That higher tier data (field/mesocosm) should be used in a Weight of Evidence to determine the Ni EQS Opinion noted: “that the HC5 derived in the EU RAR and used in the EQS derivation is lower than NOECs from laboratory studies (SSD), the mesocosm study and the effect levels established in the extensive field study. This is also true when a NOEC value of 6 μg/L is used derived from the mesocosm experiment.” Opinion recommended: In-depth statistical analysis on mesocosm data by independent statisticians

Follow-up on SCHER Opinion NiPERA asked Dr. T. Brock (Alterra, the NL) to perform a broad review of the Fraunhofer Institute mesocosm study Dr. Brock has extensive experience in the development, application, and interpretation of statistical analyses of ecological endpoints in natural and semi-natural systems for regulatory purposes1 Dr. Brock’s review was submitted to the Commission on July 19th, 2011 Briefly, the review suggested the following: The Fraunhofer Institute study satisfies all criteria identified in Section 3.3.1.3 of EQS TGD for acceptance of mesocosm results for risk assessment purposes; The statistical analysis performed by the Fraunhofer Institute was in general “state-of-the-art”, and that in particular the statistical analysis performed for the snail endpoints was appropriate; The overall NOECmicrocosm could be either 24 µg Ni/L or 12 µg Ni/L, depending on the weight that is placed on the snail endpoint. 1: http://www.wewur.wur.nl/popups/vcard.aspx?id=BROCK002&lang=uk

Follow-up on SCHER Opinion DEPA asked Dr. B. Ersbøll of the Danish Technical University (DTU) to perform a review: Details of the review will be discussed in the presentation of U. Hommen We briefly note the following: DTU review focuses on use of William’s test on snail endpoint, and does not categorically reject this analysis DTU analysis supports regression analysis, which was used by Fraunhofer Institute in addition to William’s test DTU analysis does not offer support for DEPA’s statistical analysis and its conclusion that the NOEC is 6 µg Ni/L

Summary of relevance of field/mesocosm data Do field and mesocosm data fulfill the role identified in the EQS TGD? TGD Section 3.3.1.3 identifies several key concerns that field/mesocosm data should address: Proper characterization of exposure, and analysis of effects in the context of this exposure Bioavailable [Ni] calculated for both studies Coverage of relevant water systems: Lentic (mesocosm) and lotic (field analysis) systems covered Completely different ecological communities covered Inclusion of sensitive species: Guidance: “taxa that are expected to be sensitive” should be included Mesocosm: Single most sensitive species (Lymnaea stagnalis) Two most sensitive taxa (snails, cladocerans) Four out of the ten most sensitive species in the SSD (L. stagnalis, Paracantha truncata, Daphnia longispina, and D. magna) Many, many previously untested species Field study: Snails EPT

Direct comparisons? Keep in mind that the mesocosm water chemistry was reflective of high bioavailability conditions Lymnaea stagnalis: Most sensitive EC10/NOEC from laboratory studies: EC10 = 1.3 µg Ni/L, NOEC = 3 µg Ni/L Mesocosm study: NOEC = 12 µg Ni/L Paracantha truncata (cladoceran): NOEC = 2.5 µg Ni/L No effects observed as high as 96 µg Ni/L (highest test concentration) Same pattern for Daphnia longispina (lowest lab NOEC = 26.6 µg Ni/L) and D. magna (lowest lab EC10 = 16 µg Ni/L) No effects observed at highest test concentration Only example where high sensitivity is shown in both laboratory and higher level data: Lymnaea stagnalis All other examples: Laboratory data more sensitive than field/mesocosm data

Overall Weight of Evidence Analysis for Ni EQS Guidance for determining AF? Section 3.3.1.2: Extrapolation using SSDs: Issues considered in determining Assessment Factor for aquatic compartment Availability for Ni? The overall quality of the database and the endpoints covered, e.g., if all the data are generated from “true” chronic studies (e.g., covering all sensitive life stages) True chronic data available Data cover more than10th – 90th percentiles of physicochemical characteristics of EU surface waters The diversity and representativity of the taxonomic groups covered by the database, and the extent to which differences in the life forms, feeding strategies and trophic levels of the organisms are represented 31 species Taxonomic diversity allows use of SSD Knowledge on presumed mode of action of the chemical (covering also long-term exposure). Ionoregulatory toxicant 4 BLMs available Explicit “spot-check” performed to validate extrapolation of BLMs to non-BLM species When more than 1 BLM is available, more stringent model is always used Statistical uncertainties around the HC5 estimate, e.g., reflected in the goodness of fit or the size of confidence interval around the 5th percentile, and consideration of different levels of confidence (e.g. by a comparison between the median estimate of the HC5 with the lower estimate (90% confidence interval) of the HC5) Log-normal distribution always used, which yields more precautionary outcome than use of best-fitting model Comparisons between field and mesocosm studies and the 5th percentile and mesocosm/field studies to evaluate the laboratory to field extrapolation Mesocosm and field data available, which show that bioavailability-normalized HC5 is protective of higher-tier ecological effects : Available during EU RA, and used to determine AF of 2 : Available after EU RA

Summary of Ni IND Position An AF of 2 was concluded during the EU RA HC5 determination included many precautionary steps Only missing information was field/mesocosm data Field and mesocosm data have been generated since close of EU RA These data were developed in line with guidance offered by EQS TGD These data have been interpreted following guidance offered by EQS TGD SCHER opinion supports their consideration in EQS determination One endpoint (from > 90) from Fraunhofer indoor mesocosm study was questioned in subsequent discussions Independent review by Alterra supports Fraunhofer conclusions: Study wide NOEC can be no lower than 12 µg Ni/L Field and indoor mesocosm data show that the bioavailability normalized HC5 is protective of higher-tier ecological effects All areas of uncertainty identified in Section 3.3.1.2 for evaluating the magnitude of the AF have been addressed