1. 1 Incorporation of bioavailability Patrick Van Sprang – ARCHE OECD Workshop on Metals Specificities in Environmental Hazard Assessment, Paris, 7-8 september 2011

2. 2 Metals are found in different forms in the environment These are referred to as metal “species” Changing in the environment is called “ (chemical) speciation” or “transformation” Kinetics and chemical speciation under environmentally relevant conditions crucial for PNEC derivation & read-across Important point: Not all metal species are toxic Introduction

3. 3 – with inorganic ligands (OH -, CO 3 2-, HCO 3 -,..) – with dissolved NOM 2 (measured as DOC 3 : humic and fulvic acids)  each of these processes may reduce metal bioavailability/toxicity Dissolved Metal Complexes Metals exist in the environment… – Free-ion forms tend to bind to biological ligands, e.g.physiologically active sites at the gill  these species mainly causes metal toxicity Dissolved Free Metal – adsorbed to suspended solids (POC 1 or mineral surfaces)  each of these processes may reduce metal bioavailability/toxicity Particulate Metal 1 POC: Particulate Organic Carbon 2 NOM: Natural Organic Matter 3 DOC: Dissolved Organic Carbon

4. 4 For terrestrial and sediment systems, the concentration of a metal that is determined after destruction of the mineral matrix. For aqueous systems: the total amount of metal present, including the fraction sorbed to particles and to dissolved organic matter and the fraction in the mineral matrix; = particulate (sorbed + precipitated) + dissolved (inorganic complexes + organic complexes + free ionic forms) Total Metal Concentration Metal toxicity can be expressed as… Dissolved Metal Concentration* most often, the dissolved fraction in ecotoxicity tests refers to the fraction that passes through a filter of 0.45 µm. = inorganic complexes + organic complexes + free ionic forms * It should be noted, however, that this definition may not necessarily refer to the metals in solution. In the range of 0.01- 0.45 µm colloid inert particles containing metal ions that remain suspended, may still exist;

5. 5 - the degree to which a metal species is available to interact with the biotic ligand (e.g. physiologically active sites at the gill) to exert its effect. = free ionic forms (mainly) Bioavailable Metal Concentration - Biotic Ligand* Model (BLM): assumes that both the free metal ion activity and the interaction of the available cationic forms with the organism reflect the toxicity. - Free Ion Activity Model (FIAM): assumes that the free metal ion activity reflects the chemical reactivity and toxicity of the metal Metal toxicity can be expressed as… * A "biotic ligand" is a biochemical receptor that is metal-binding and is treated similarly to other ligands in the exposure water, except that it is on the organism. An example of a biotic ligand is a fish gill.

6. 6 Why incorporate bioavailability in CSR of metals? NOEC/EC10 in laboratory test media which often maximizes bioavailability (e.g. low DOC in water; low OC in soil)  may not reflect ‘real environment’ (rivers may have different DOC, pH) ! Database often contains NOEC/EC10 obtained in test media with widely varying chemistry (= very different bioavailability)  which toxicity values to select (species mean ?, lowest NOEC/EC10 ?) ? Generic/uncorrected SSD does not represent ‘intrinsic sensitivity’ alone but rather a mix of ‘intrinsic sensitivity’ + bioavailability effects  toxicity values should therefore be normalized towards similar physico-chemical conditions !

7. 7 Why inorporate bioavailability in CSR of metals? Incorporation bioavailability Concentration (µg/l) Cumulative Distribution Function (%) Generic PNEC Concentration (µg/l) Cumulative Distribution Function (%) Normalized PNEC Bioavailability models ‘remove’ the variability in sensitivity due to differences in physico-chemistry

8. 8 Does bioavailability matter in EU waters ? Acute effects (LC50 in µg/l) of copper to Daphnia magna, tested in 11 different EU surface waters Factor 30 difference in acute Cu-toxicity across EU surface waters !! De Schamphelaere et al., 2002 Sampling location

9. 9 Does bioavailability matters in EU soils ? Chronic effects of nickel (NOEC/EC10 in mg/kg) to soil organisms/processes tested in 16 different EU surface soils Factor between 10-45 difference in chronic Ni-toxicity across EU soils !!

10. 10 Approaches for bioavailability ?

11. 11 1. Transformation from total to soluble fraction - approach No conversion needed - Assumption: Dissolved metal concentrations more closely approximate the biologically available fraction than does total metal concentrations = dissolved metal concentrations (e.g. Ni, Cu, Zn) Total metal concentrations ≠ dissolved metal concentrations (e.g. Pb) Conversion needed - Speciation model (e.g. Minteq: requires phys-chem characterisation of medium (pH, Hardness,..)) - Analytical measurements testing (filtration, then e.g. ICPMS/AAS)

12. 12 2. Use of speciation models - approach - Assumption: chemical species (mainly free metal ion activity) is able to explain the observed toxicity  FIAM Dissolved metal concentrations - Speciation model (e.g. Minteq/WHAM: requires phys- chem characterisation of medium (pH, Hardness,..)) - Analytical measurements testing (filtration, then e.g. Ion Selective Electrode (ISE), Donnan membrane technique (DMT))

13. 13 3. Toxicity related bioavailability models: approach - Chronic BLM’s have been developed & validated for several metals (e.g. Ni, Zn, Co, Cu in freshwater)/ Acute BLM’s also exist for other metals (e.g. Ag, Cd) - Assumption: chemical species (mainly free metal ion activity) and the interaction of the available cationic forms with the organism reflect the toxicity  BLM Dissolved metal concentrations - FIAM - speciation model (e.g. Minteq/WHAM) - Gill Surface Interaction Model (e.g. CHESS) The BLM requires a description of water chemical parameters that can influence metal toxicity: - pH - DOC (a convenient measure of NOM) - Major ions: Calcium, Magnesium - Others: e.g. Sodium (Cu)

14. 14 MeOH + MeCO 3 Me-DOC pH [Me] on ‘biotic ligand’ Toxic effect WaterOrganism H+H+ pH Me 2+ Ca 2+ Na + Mg 2+ ‘biotic ligand’ e.g. gill, cell surface Speciation (WHAM) Intrinsic sensitivity Competition (log K’s) Log K CaBL Log K MgBL Log K NaBL Log K HBL

15. 15 De Schamphelaere & Janssen, 2002 Acute Chronic CaMg BLM: development (1) 3. Toxicity related bioavailability models

16. 16 De Schamphelaere et al., 2002 Sampling of waters Chemical analyses (pH, DOC, Ca, Na,…) Adding ≠ concentrations Determine toxicity Test BLM Factor 2 BLM: validation (1) 3. Toxicity related bioavailability models

17. 17 Factor 10 to 30 variability in toxicity… reduced to factor 2 in > 90% of the cases..for invertebrates and fish BLM: validation (2) 3. Toxicity related bioavailability models

18. 18..for algae Factor 10 to 30 variability in toxicity… reduced to factor 2 in > 90% of the cases BLM: validation (3) 3. Toxicity related bioavailability models

19. 19 BLM: similar response across metals ? (1) Invertebrates Algae Fish Invertebrates, fish & algae NOEC (µg/l Zn) algae pH DOC invertebrates fish pH NOEC (µg/l Zn) Invertebrates, fish & algae NOEC (µg/l Zn) Hardness Ca > Mg Invertebrates, fish & algae NOEC (µg/l Ni) Invertebrates, fish, algae pH DOC Mg > Ca Invertebrates, fish & algae NOEC (µg/l Ni) Hardness - Zn - Ni 3. Toxicity related bioavailability models

20. 20 Invertebrates, fish & algae NOEC (µg/l Cu) algae pH DOC invertebrates fish pH NOEC (µg/l Cu) Hardness does not significantly affect chronic toxicity - Cu - Toxicity response = f(organism; phys-chem parameter) - Toxicity response = pH: similar for algae; different for invertebrates & fish = DOC:similar for all organisms = H: ± similar for all organism (> Ca for Zn; > Mg for Ni; less significant for Cu) BLM: similar response across metals ? (2) 3. Toxicity related bioavailability models

21. 21 BLM: applicability domain BLMs developed & validated within 90 th % of phys.-chem from EU waters and should therefore only be applied within such boundaries !! Specific conditions outside boundaries need special attention (e.g. model extrapolation, additional specific testing…. 3. Toxicity related bioavailability models

22. 22 BLM developed for limited number of species: – P. subcapitata (green alga) – D. magna/C. dubia (cladoceran, invetebrate), – O. mykiss/P. promelas (fish) Ecotoxicity database contains NOEC/EC 10 for other species/taxonomic groups (e.g. molluscs, rotifers, insects) Given that individual development for all existing aquatic species is impossible, can a BLM developed for one species be used for another species?... Extrapolation assumes similar mechanism of actions (e.g. similar stability constants between the cations (Ca, Mg, H) and the biotic ligands, similar site of action) BLM: extrapolation across other species ? (1) 3. Toxicity related bioavailability models

23. 23 Read across? BLM model fish (rainbow trout) BLM model algae (Raphidocelis) BLM model water flea (Daphnids) BLM: extrapolation across other species ? (2) 3. Toxicity related bioavailability models

24. 24 How ?: perform ‘spot checking’ of the BLMs for species for which no validation had been undertaken. BLM: extrapolation across other species ? (3) Literature toxicity data (e.g. Cu) Toxicity testing (e.g. Ni) - Insect: Chironomus tentans - Rotifer: Brachionus calyciflorus - Molluscs: Lymnaea stagnalis - Higher plant: Lemna minor BLM predictions were within a factor ± 3 3. Toxicity related bioavailability models

25. 25 BLM: Implementation in risk assessment (1) Full BLM normalization across all species 3 BLM’s (alga, invertebrate, fish) available ? ‘Spot checks’ available for at least 3 other species ? Partial BLM normalization or BioF approach Partial BLM normalization allowed Yes No 3. Toxicity related bioavailability models

26. 26 Full BLM normalization across all species (e.g. Ni, Cu-RA) Partial BLM normalization or BioF approach (e.g. Zn RA) 2. Calculate the bioavailability factors (BioF) for the BLM species PEC bioavailable =PEC * BioF water,X 1. Normalise the NOEC for the BLM species towards site specific conditions (NOEC x ) and towards EU reference water chemistry conditions (NOEC ref ) 3. Select the highest BioF for the BLM species 4. - Calculate the bioavailable PEC concentration - Use D. magna/C. dubia BLM to normalise all other invertebrates (e.g. molluscs, rotifers,..) - Use O. mykiss/P. promelas to normalize all fish/amphibians - Use R. subcapitata to normalize all other algae BLM: Implementation in risk assessment (2) 3. Toxicity related bioavailability models - Or calculate the bioavailable PNEC concentration PNEC bioavailable =PNEC generic /BioF water,X

27. 27 - Coastal/open sea waters are characterised by… -high pH (typically between 7.8–8.3), high salinity (35‰), high ionic strength. -DOC levels may vary considerably between marine waterbodies -Freshwater and marine organisms face very different iono- and osmo-regulatory issues related to living in either a very dilute or concentrated salt environment. freshwater BLMs can NOT directly be used for marine environments - Me-DOC binding freshwater different then marine waters = Speciation modelling to be modified with the ionic strength DOC normalization if applicable = bioavailability correction = not species-specific 27 4. Bioavailability models in marine waters

28. 28 4. Bioavailability models in marine waters Bioavailability correction (DOC): derivation of normalized PNEC value – e.g. Cu Model accuracy - Bioavailability prediction within a factor of 2 Toxicity - DOC regressions for 6 marine species