Background - What is bioavailability? Comparison of leaching methods for the estimation of bioavailability in two CCAcontaminated

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1 Comparison of leaching methods for the estimation of bioavailability in two CCAcontaminated soils (1,2), Anders Lättström (3), Mats Tysklind (2), Lars Lövgren (), Solomon Tesfalidet (3) Per Leffler (1), 1) Division of NBC Defence, Swedish DefenceResearch Agency (FOI),Umeå,Sweden 2) Department of chemistry, Environmental chemistry, Umeå University, Sweden 3) Department of chemistry, Analytical chemistry, Umeå University, Sweden ) Department of chemistry, Analytical chemistry, Umeå University, Sweden FOI NBC-defence Dept. Of Threat Assessment North Sweden Soil Remediation Center, MCN, Background - Risk Assessment of polluted sites Total concentrations of contaminants compared to permissible levels (e.g. PNECS, guideline limit values from national EPA s) estimation of risk Lack of site-specific information, soil conditions, combination effects, and bioavailability may lead to over- and underestimation of risk Other tools to better estimate risk is necessary Environmental Chemistry, Umeå University Background - Risk Assessment of polluted sites Combination of toxicological methods and chemical analysis is beneficial Background - What is bioavailability? Leaching/extraction methods Simple bioassays specific bioassays Standardised bioassays ( USEPA, ASTM, OECD,etc.) have been proposed Plants, soil living organisms e.g. earthworms, springtails, plants etc. Ecologically relevant, but often laborious high costs!! Spatial distribution is necessary at large contaminated sites Fast och cheap surrogate methods for screening purposes are needed Lanno et al,

2 Aims of the study Leaching/extraction methods for estimation of bioavailability Compare the relative bioavailability of arsenic and arsenic species between methods Leaching/extraction efficiency yield of metals/metalloid in the leachates Cytotoxicity and genotoxicity in the leachates ASE - Accelerated Solvent Extraction ( or Pressurized liquid extraction (PLE)) MPa and 15 C, C, 5 C Batch leaching EN , L/S=, 2h. Batch leaching 1 M CaCl 2, L/S, 2h (Houba et al. 199). IVG anaerobic, 37 C, 2 x 1h (Rodriguez et al., 1999) ASE - Accelerated Solvent Extraction Bioassays Environmental sample in High through put, ca 2 min/sample High temp. & pressure Solvent is water L-929-cytotoxicity: General toxicity, disturbed basal cellular functions affecting growth. Genotoxicity using CHO-cells: mutagenic properties e.g. metal/metalloid (Helleday et al.21) AA8 UV AA8:Wild-type UV: Deficient in DNA-repair H2O Biocompatible Extract out Medium effective extraction Worst case scenario of mobile and water soluble fraction of contaminants 9 9 UV5 9 EM9 9 5µl DMEM + µl µl µl µl leachate UV5: Deficient in DNA-repair EM9: Deficient in DNA-repair 2

3 Test soils Wood impregnation sites using CCA-based preservatives Results Concentration of metals in leachates Arsenic Cadmium Chromium Cobalt Copper Lead Nickel Zinc TOC Forsmo mg/kg % Robertsfors mg/kg % log µg/l Cr Cu As FASE RASE Principal component analysis (PCA) Metal yield Cr Cu As Chemical characteristics RCaCl:3 RCaCl:2 RCaCl:1 Cd Yield Cond. % FASE RASE. t[2] 15% 2-2 FCaCl:1 FCaCl:2 FCaCl:3 :3 RASE:3 :1 :2 :1 :3 :1 :2 :3 RASE:1 :2 :1 :3 RASE:2 FASE:2 :2 FASE:3 FASE:1 RB:1 RB:2 RB:3 FB:1 FB:2 FB: t[1] 3% RIVG:3 RIVG:1 RIVG:2 FIVG:3 Leaching capacity FIVG:2 FIVG p[2] ph - Co Yield Zn Yield Ni L/S Yield Pb Yield Cu Yi Cr Yi As5+ As3Y DOC p[1] 3

4 ASE5 leachate Forsmo soil ASE5 leachate Robertsfors soil UV Mean UV Mean Batch LS leachate Forsmo Soil Batch LS leachate Robertsfors Soil UV Mean UV Mean Leachate toxicity Leachate Genotoxicity Growth inhibition ( % ) Basal cytotoxicity L-929 & AA8 L-929 AA8 FASE RASE Growth inhibition ( % ) 8 2 Genotoxicity in CHO-cells from soil leachates FASE RASE AA8 UV UV5 EM9 ASE5 Batch Forsmo leachates ( % ) FASE Extraction of total arsenic (%) RASE Growth inhibition ( % ) 8 2 Genotoxicity in CHO-cells from soil leachates FASE RASE AA8 UV UV5 EM9 Robertsfors leachates

5 Partial least squares to latent structures (PLS). 5.. Ratio As3+/As5+ FASE RASE u[1] (1%) FCaCl:1 RB:1 RB:3 FCaCl:3 FASE:3 FB:1RB:2 RCaCl:2 FCaCl:2 FB:3 FASE:1 FB:2 :3 :2 RCaCl:1 RCaCl:3 :1 Genotoxicity :3 :1 RASE:1 RASE:2 RASE:3 :2 :2 :1 FASE:2 :3 Exposure vs Toxicity t[1] (%) Basal cytotoxicity RASE w*c[2] - - ph L/S X Cd(l) Cond. As3+/As5+ Cu(l) w *c[1] Y UV5 Co(l) EM9 Ni(l) AA8 L-929 UV As(l) Cr(l) Zn(l) DOC Pb(l) Risk associated to oral intake according to IVG Conclusion 1 % 5 3 As yield (%) Ratio As3+/As5+ Higher yield and toxicity in PLE/ASE at 15 C suitable for screening purposes to assess the toxic potential in soil samples 2 IVG Forsmo IVG Robertsfors 5 IVG Forsmo IVG Robertsfors Comparable results from ASE at lower temperatures ( C & 5 C) and the batch leaching methods, EN and 1M CaCl2 Higher Yield in Forsmo = Higher bioavailability Similar ratio of As 3+ / As 5+ 5

6 Conclusion 2 Conclusion 3 Speciation of Arsenic is essential for risk assessment of CCA - contaminated sites Drink Forsmo soil leachates - Eat Robertsfors soil! Both analysis of chemical speciation and bioassays for genotoxicity reveal these differences compared to total concentrations! Thank you for your attention!!