1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212570_28446780.jpg logo_mu_cerne.gif trojlogo.gif Soil ecotoxicology: soil bioassays Dr. Jakub Hofman hofman@recetox.muni.cz 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif What is soil? Soil Science Glossary (Soil Science Society of America). •The unconsolidated mineral or organic matter on the surface of the earth that has been subjected to and shows effects of genetic and environmental factors of: climate (including water and temperature effects), and macro- and microorganisms, conditioned by relief, acting on parent material over a period of time. A product-soil differs from the material from which it is derived in many physical, chemical, biological, and morphological properties and characteristics. Soil Taxonomy, second edition. •Soil is a natural body comprised of solids (minerals and organic matter), liquid, and gases that occurs on the land surface, occupies space, and is characterized by one or both of the following: horizons, or layers, that are distinguishable from the initial material as a result of additions, losses, transfers, and transformations of energy and matter or the ability to support rooted plants in a natural environment. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Chernozem2 •important part of nature •non-renewable source •non-replaceable functions of ter. ecosystems •base for plant growth •nutrient storage - fertility – production •start and end of food chains •biogeochemical cycles •decomposition of organic matter, humification •filtration, immobilization and degradation of pollutants •water cycling •biodiversity treasure • • •WE MUST PROTECT SOIL QUALITY Why soil does matter? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 190 soil = biotic + abiotic = complex •biota is important for •formation of soil, for soil structure •soil fertility •organic residues decomposition, release nutrients •element cycles •air and water regime bacteria protozoa fungi algae nematoda rotifera annelida arthropodes collembola mollusca ….. twobugsb 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Soil biota 029 029 142 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Soils have problems •EU Thematic Strategy for Soil Protection (COM/2006/231) defines main threats for soils (sealing, erosion, compaction, salinisation, OM loss, contamination …) •3,5 mil. contaminated sites in EU •0.5 mil. are seriously contaminated and need remediation •Costs related to contaminated sites in EU: 2-17 bil. € (Impact assessment (SEC/2006/620)) • impermeabilizz_small logo2 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Contamination of soils csi015-fig02june07 csi015_fig03 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif FOCUS: • soil as environmental compartment • soil organisms • exposure in solid medium THE ENVIRONMENT (SOIL), WHERE RELATIONSHIP BETWEEN ORGANISM AND THE CHEMICAL EXISTS, MUST BE STUDIED TOO !! Soil ecotoxicology 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Very different from aquatic ecotoxicology •Solid matrices are heterogenous •Soil contains all three phases SOLID, LIQUID (pore water) and GAS (soil air) •Solid phase especially influences strongly FATE and BEHAVIOUR of chemicals •Depending on soil and chemical properties and depending on TIME, chemical is DISTRIBUTED in soil, chemical SPECIATION occurs •SORPTION is the crucial process and leads to changes of BIOAVAILABILITY – the key factor of soil ecotoxicology •All this changes final TOXICITY and RISKS •All this hampers EXTRAPOLATION possibilities Soil environment is very different from aquatic 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif CHEMICAL entry ORGANISM Chemical in organism Metabolism, elimination, effects CHEMICAL in soil spatially distributed; chemical speciation EXPOSURE depends on: Fate of chemical in soil + Organism properties (morphology, physiology, ecology ...) Exposure in soil matrix 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Anorganic pollutant 0.1 nm 100 nm Fungi 2-10 um Organic pollutant 1 nm Bacteria 1-2 um 1 nm 10 um 1 um 10 nm 0.1 nm 100 um Clay particle or humic acid 2-0.2 um Stationery folsom01 1 mm Springtail 1 – 2 mm Stationery Stationery Stationery Stationery Stationery folsom01 Interaction of organisms and chemicals Tkaná rohož Stationery Tkaná rohož 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif From Bradham et al. (2003) Why to bother with bioavailability ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Factors affecting bioavailability •Soil properties –Soil composition, organic matter, texture, pH, CEC, moisture, temperature, structure - pores •Chemical properties –Chemical structure, Kow, Sw, Koc, pKa, MW, H, pv •Organisms properties –physiology (uptake, metabolism, elimination), morphology, ecology •Time effects –Aging, sequestration •Other chemicals (např. NAPL) and interactions 139 triangle fetida_adult_02 caliginosa_adult_08 rubellus_adult_08 Go to fullsize image Závisí na látce, půdních vlastnostech, okolních podmínkách, čase a vlastnostech organismu. Klíčové vlastnosti jsou u nepolárních polutantů jejich hydrofobicita a u kovů jejich speciace v půdě. Z půdních vlastností se jako klíčové opakovaně ukazují obsah a vlastnosti půdní organické hmoty, pH a kationtová výměnná kapacita, protože všechny tyto faktory stěžejně ovlivňují sorpci kontaminantů. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Why to bother with bioavailability ? •For correct risk assessment: –Soil animals (individuals, communities) –Organisms eating soil (e.g. children) –Plants •Prediction of biodegradation and remediation efficiencies •Legislative framework –Not the total concentrations for limits! •Extrapolation possibilities: –Between different soils –From aquatic to soil tests –From lab experiments to field situation 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif How to measure bioavailability ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bio-indication – (bio)monitoring Ecotoxicity bioassays BD05168_[1] Meadow4 ? [USEMAP] IN00647_[1] IN00647_[1] IN00647_[1] Dose Response Goal Define and describe relationship between biota conditions and contamination Goal Define safe concentration, describe risks WORM4_1.JPG (50221 bytes) 359 Potworms (Enchytraeids doerjesi) Example of light coloured springtail (Folsomia candida) C. Elegans, coming soon VBPmites_LR tullgren Approaches of soil ecotoxicology ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Role of soil ecotoxicology in soil protection •FOCUS: Investigate relationships between soil organisms and contaminants • •ROLE: Scientific basis of soil protection • •Activities: Provide tools - bioassays for routine praxis: -Chemical and pesticides testing -Testing wastes, sludge, contaminated sites -Soil quality assessment Research of: –Fate and bioavailability –Mixture toxicity –Biodiversity … 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •Chemical analyses are not able to identify risks properly because: 1)Real exposure is different – bioavailability in particular situation 2)Pollutant mixture - always in real ecosystems 3)Matrix itself has effects or interacts with effects of contaminants 4)Anylytical methods are limited vs. Wide spectrum of possibly toxic chemicals Why bioassays? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Example of light coloured springtail (Folsomia candida) •Eluate tests are not able to predict solid phase exposure Èerná skládka eluate 1:10 sdilution IMG_4025 produce_ph05 Control soil waste Mixture at different ratios WHY ? à real bioavailability à effect of matrix involved Why soil bioassays? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif EU: COMMISSION DIRECTIVE 2001/59/EC of 6 August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances; Annex V. (earthworms, plants) USA: TSCA; OPPTS (The Office of Prevention, Pesticides and Toxic Substances) (earthworms, microbes, plants) OECD: Guidelines for the testing of chemicals (many ……) ISO: many …….. Are soil bioassays used now ? EU: COUNCIL DIRECTIVE 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market; Annex II. USA: TSCA - OPPTS (The Office of Prevention, Pesticides and Toxic Substances) OECD: Guidelines for the testing of chemicals ISO + EPPO, IOBC, SETAC, BBA Chemicals Plant protection products 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bioassay standards - OECD Valid standards 207 Earthworm, Acute Toxicity Tests (4th April 1984) 208 Terrestrial Plants, Growth Test (19th July 2006) 216 Soil Microorganisms, Nitrogen Transformation Test (21st January 2000) 217 Soil Microorganisms, Carbon Transformation Test (21st January 2000) 220 Enchytraeid Reproduction Test (13th April 2004) 222 Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) (13th April 2004) 227 Terrestrial Plant Test: Vegetitive Vigour Test (19th July 2006) Drafts Predatory Mite Reproduction Test in Soil (Hypoaspis (Geolaelaps) Aculeifer) Determination of Developmental Toxicity of a Test Chemical to Dipteran Dung Flies (Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) In preparation: Folsomia sp. test a bioaccumulation test 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bioassay standards - ISO ISO Logo ISO 15799 2003 Guidance on the ecotoxicological characterization of soils and soil materials ISO 11268-1 1993 Effects of pollutants on earthworms (Eisenia fetida) - Part 1: Determination of acute toxicity using artificial soil substrate ISO 11268-2 1998 Effects of pollutants on earthworms (Eisenia fetida) - Part 2: Determination of effects on reproduction ISO 11267 1999 Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants ISO 16387 2004 Effects of pollutants on Enchytraeidae (Enchytraeus sp.) - Determination of effects on reproduction and survival ISO 20963 2005 Effects of pollutants on insect larvae (Oxythyrea funesta) - Determination of acute toxicity ISO 15952 2006 Effects of pollutants on juvenile land snails (Helicidae) - Determination of the effects on growth by soil contamination ISO/DIS 17512-1 Avoidance test for testing the quality of soils and effects of chemicals on behaviour - Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei) ISO 11269-1 1993 Determination of the effects of pollutants on soil flora - Part 1: Method for the measurement of inhibition of root growth ISO 11269-2 2005 Determination of the effects of pollutants on soil flora - Part 2: Effects of chemicals on the emergence and growth of higher plants ISO 22030 2005 Chronic toxicity in higher plants ISO 14238 1997 Determination of nitrogen mineralization and nitrification in soils and the influence of chemicals on these processes ISO 14240-1 1997 Determination of soil microbial biomass - Part 1: Substrate-induced respiration method ISO 14240-2 1997 Determination of soil microbial biomass - Part 2: Fumigation-extraction method ISO 16072 2002 Laboratory methods for determination of microbial soil respiration ISO 17155 2002 Determination of abundance and activity of soil microflora using respiration curves ISO 15685 2004 Determination of potential nitrification and inhibition of nitrification - Rapid test by ammonium oxidation ISO 23753-1 2005 Determination of dehydrogenase activity in soils - Part 1: Method using triphenyltetrazolium chloride (TTC) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bioassay standards – US EPA 850.2450 Terrestrial (soil-core) microcosm test 850.4000 Background-Nontarget plant testing 850.4100 Terrestrial plant toxicity, Tier I (seedling emergence) 850.4150 Terrestrial plant toxicity, Tier I (vegetative vigor) 850.4200 Seed germination/root elongation toxicity test 850.4225 Seedling emergence, Tier II 850.4230 Early seedling growth toxicity test 850.4250 Vegetative vigor, Tier II 850.4300 Terrestrial plants field study, Tier III 850.4600 Rhizobium-legume toxicity 850.4800 Plant uptake and translocation test 850.5100 Soil microbial community toxicity test 850.6200 Earthworm subchronic toxicity test 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Use of soil bioassays in soil protection •Sofar, mostly for assessment of hazard of chemicals and pesticides • •Increase of use for evaluation of hazard of complex mixtures like wastes, sewage sludge, sediments, composts, fertilizers … • •Great potential in the future for assessment of soil quality e.g. Before and after the remediation, contaminated sites assessment etc. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Solid material toxicity testing logs Abbruch_reliktische_Bausubstanz-3 Halde-2 produce_ph05 Go to fullsize image ISO 15799 (2003): Guidance on the ecotoxicological characterization of soils and soil materials ISO 17616 (2008): Guidance on the choice and evaluation of bioassays for ecotoxicological characterization of soils and soil materials Grasbrook 010 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif EU – test battery for wastes 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Exposure methods •Tested chemical mixed with soil –Artificial soil (OECD, ISO) –Real soil (LUFA 2.2 …) • • • • • •Topic applications, injections, forced feeding … not so relevant Implanting a VIE tag into an earthworm kunstgrond IMG_1085 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif What is artificial soil ? •Is standard medium for many soil bioassays … •Is much more relevant than solution, agar, filter paper … •Should solve problem of high variability of natural soils … •Should resemble natural loamy soil … •Should enable the toxicity extrapolation to natural soils … OECD 1984. Guideline for testing chemicals 207. Earthworm acute toxicity test. IMG_1085 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Soil microbial assays •EPA (1996): OPPTS 850.5100 Soil microbial community toxicity test. Ecological effects test guidelines. United States Environmental Agency. • •EPPO (1994): Decision making scheme for the environmental risk assessment of plant protection products. EPPO Bulletin 24, Chapter 7, Soil Microflora. • •Lynch, M.R. (1995): Procedures for assessing the environmental fate and ecotoxicity of pesticides. SETAC, Brussels, Belgium. • •OECD (1999): Proposal for a new guideline 217. Soil microorganisms: Carbon transformation test. OECD guideline for the testing of chemicals. OECD. • •OECD (1999): Proposal for a new guideline 216. Soil microorganisms: Nitrogen transformation test. OECD guideline for the testing of chemicals. OECD. • •ISO 14238 (1997): Soil quality - Determination of nitrogen mineralization and nitrification in soil and the influence of chemicals on these processes. International Organization for Standardization. Geneve, Switzerland. • 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Real uncontaminated agricultural soil with indigenous microflora: pHKCl = 7 – 7.5 Cbio = 400 – 700 µg.gd.w.-1 Corg = 1.5% BR = 0.5 – 0.7 µg CO2-C.h-1.gd.w.-1 sand = 70% Lokalita_04_05b Soil sampling Storage Pre-incubation (7 days) START Substance application Negative control Positive control 7th day 14th day 21st day 28th day Microbial parameters 10 g per replicate aerobic conditions 60% WHC; 22°C; dark Soil microbial assay according to OECD, ISO 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 0010 Effects on microbial respiration 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Respirometry 92 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Nematodes C. elegans anatomy C. elegans anatomy - cross section 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif C. elegans life cycle Caenorhabditis elegans L4-young-egl-20x L1-40x-b L2-20x L3-10x-d L4-10x 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif ASTM: E2172-01 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode Caenorhabditis elegans 082 Caenorhabditis elegans 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Enchytraeidae Image14 grindal_worm IMG_3421_1 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 019 024 027 032 031 Enchytraeidae 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Avoidance test with E. albidus 24 hrs 1 2 3 Ø 8,4 cm 4 5 WORM0_3.JPG (27190 bytes) 10 adults 10 x 2 x 10 g Counting of adults in halves waste Artificial soil 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Bioaccumulation experiments with enchytraeids 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Earthworms CE8c mating LR CE8a-cocoon LR Lumdiap2 image31 STUPD00Z 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 068 Earthworm acute toxicity test •14 days •500 g soil + 10 adult Eisenia fetida •mortality and weight 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Earthworm reproduction test •56 days •500 g soil + 10 adult Eisenia fetida •horse manure as food •juveniles extracted using water bath 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif WHC vesselsready controlwithworms wormsweighing wahingwormsJPG Příprava půd Měření WHC půd Ovlhčená AS rozvážená do testovacích nádob Výběr 10 reprezentativních adultů z chovu a jejich omytí dH20 Zvážení jedinců Přídavek 10 adultů do nádoby na test drysoils Eisenia fetida reproduction test 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif wormssearching wormsweighing bottomofvessel Prohlídka nádob (známky aktivity) Zhodnocení mortality Zvážení žížal trolleywithworms Nádoby během testu v kontrolované místnosti E. fetida test – po 28 dnech 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif waterbathS juvenileszoomedS juvpicking cocoonsievingS cocoonsearchontray cocoonsinthehand Po cca 20 min juvenilové na povrchu Vodní lázeň s narůstající teplotou 40ºC až 60ºC Přesátí půdy Sbírání a počítání Počítání Ruční třídění kokonů E. fetida – po 8 týdnech 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Avoidance test Guideline: ISO/DIS 17512 (draft) Species: E. andrei Substrate: LUFA St. 2.2 standard soil Duration: 1 - 2 days Parameter: Behaviour of the worms Test vessels: Dual chamber DSC07739 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Risk assessment with earthworms 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Folsomia candida 005 008 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 021 Folsomia candida 023 024 028 031 NS4 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Mites Hypoaspis aculeifer 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif olog prey predator cont. soil 0471971030 Mites 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Plants IMG_0511 IMG_0499 IMG_0502 IMG_0819 IMG_0825 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Lactuca sativa root growth IMG_0196 IMG_7022_al IMG_7021_al IMG_7034 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Plant protection products risk assessment 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 121 tme01_b.jpg (66.851 Byte) Microcosms - TME 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif tme03_b.jpg (52.421 Byte) tme04_b.jpg (57.399 Byte) Microcosms - TME