Ecotoxicological bioassays Jakub Hofman & Klára Hilscherová Introduction Protection of environment / nature • Is and must be primary aim of sustainably developing society • why? – discussion (ecosystem services, human, ethics ...) How to protect ? • Policy • Legislation • Research • Education Ecotoxicology – offers knowledge and tools useful for the effective and reasonable environmental protection tools = ecotoxicological bioassays Ecotoxicology molecules organelle populations contaminant environmental fate environmental chemistry living system ecology (environmental) toxicology toxicology cells organs, organ complexes organisms community ecosystem Discipline on the border of ecology and toxicology studying and evaluating direct and indirect effects of man-made or natural harmful chemicals or other stressors on animals (except human), plants and microorganisms at all levels of biological organization Ecotoxicity bioassay, ecotoxicity test • a tool (method, procedure ...) for ecotoxicological research and praxis – for environmental legislation and protection • biota (tissue, organism, population, ecosystem …) is exposed to chemicals (and/or other factors), in the lab (controlled conditions) or in the field (less controlled) and effects are evaluated and related to exposure • WHY? To understand the cause-effects relationships (causality, doseresponse …) Chemical analyses only 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) Analytical methods are limited vs. wide spectrum of possibly toxic chemicals Why bioassays? Bioassays useful for: • prospective ecological risk assessment – using bioassays for chemical compounds, pesticides – using bioassays for materials, mixtures – before they enter the environment ecotoxicity tests concentration effects control application dose PROSPECTIVE APPROACH NOEC ECx LCx controlled field experiments experiments microcosms mesocosms § Bioassays useful for: • retrospective ecological risk assessment – using bioassays for real environmental samples – searching the causalities between pollution and effects ecotoxicity tests of samples soil properties TRIAD control soil tested soil RETROSPECTIVE APPROACH observation, description, analysis bioindication (biomonitoring) contamination (monitoring) kauzalita INDIVIDUAL TOXICANTS MIXTURES OF CHEMICALS CONTAMINATED ENVIRONMENT RETROSPECTIVE PROSPECTIVE Bioassessment Field assessment Monitoring Bioassessment Field assessment Monitoring Lab studies Lab studies Simulated small ecosystems DISASTERS PREDICTIONS for future Time: NOW ! Bioassays useful for: MIXTURES OF TOXICANTS Bioassays useful for: ecotoxicity tests of samples soil properties TRIAD control soil tested soil ecotoxicity tests concentration effects control application dose RETROSPECTIVE APPROACH PROSPECTIVE APPROACH operability controllability NOEC ECx LCx controlled field experiments reality ecologicalrelevancy variability unknownfactors inaccuracy artificiality extrapolationsneed practical feasibility price duration observation, description, analysis experiments microcosms mesocosms bioindication (biomonitoring) contamination (monitoring) § kauzalita Bioassay development • old bioassays – acute, ecologically irrelevant, testing pure chemicals, pesticides • new bioassays – sublethal endpoints, ecological relevancy, chemical mixtures, miniaturization, simple to measure endpoints • major trophic levels – producers – consumers – destruents • aquatic / soil • single / multiple species • acute / chronic effects • contact bioassays / eluate bioassays / TIE • legislative / standardized (practical) / experimental (research) • toxicity / bioaccumulation / biodegradation tests Differenciation of bioassays Differenciation of bioassays size, complexity ecological relevancy controling the conditions old / fresh contamination no. of species … cell lines Differenciation of bioassays ecological relevancy complexity of the system test on filter paper soil ecotoxicity bioassay microcosm mesocosm managed field experiment more species and interactions real species real soils real conditions sublethal effects relevancy ? prolongation of the tests Differenciation of bioassays • limit test / comparison test • concentration – response tests – preliminary, final test organisms control/reference soil tested chemical or chemical mixture tested material – complex environmental mixture (soil from contaminated site, sewage sludge, sediment, manure, fertilizer, waste ...) relationship concentration – effect test comparison test concentration effect concentration effect Use of bioassays • Testing toxicity of natural contaminated matrices – Rather new in ecotoxicology – many open challenges • Whole effluent toxicity testing (WET) • Contact soil toxicity assays – More complex and more complicated • „cause-effects“ often not clear – Natural variability in matrices – Algal tests - nutrients (Nitrogen, Phosporus) >> Toxic compounds eluate 1:10 Control soil waste Mixture at different ratios OECD guidelines http://www.oecd.org/document/40/0,3746,en_2649_34377_37051368_1_1_1_1,00.html Test No. 201: Alga, Growth Inhibition Test 11 July 2006 Test No. 221: Lemna sp. Growth Inhabition Test 11 July 2006 Test No. 202: Daphnia sp. Acute Immobilisation Test 23 Nov 2004 Test No. 211: Daphnia magna Reproduction Test 16 Oct 2008 Test No. 203: Fish, Acute Toxicity Test 17 July 1992 Test No. 204: Fish, Prolonged Toxicity Test: 14-Day Study 04 Apr 1984 Test No. 210: Fish, Early-Life Stage Toxicity Test 17 July 1992 Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages 21 Sep 1998 Test No. 215: Fish, Juvenile Growth Test 21 Jan 2000 Test No. 229: Fish Short Term Reproduction Assay 08 Sep 2009 Test No. 230: 21-day Fish Assay 08 Sep 2009 Test No. 231: Amphibian Metamorphosis Assay 08 Sep 2009 Aquatic organisms Test No. 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment 23 Nov 2004 Test No. 219: Sediment-Water Chironomid Toxicity Using Spiked Water 23 Nov 2004 Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment 23 July 2010 Test No. 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment 15 Oct 2007 Sediment organisms Organization for Economic Cooperation Development Soil organisms Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test 17 Aug 2006 Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test 17 Aug 2006 Test No. 207: Earthworm, Acute Toxicity Tests 04 Apr 1984 Test No. 220: Enchytraeid Reproduction Test 23 Nov 2004 Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 23 Nov 2004 Test No. 228: Determination of Developmental Toxicity of a Test Chemical to Dipteran Dung Flies(Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) 16 Oct 2008 Test No. 232: Collembolan Reproduction Test in Soil 08 Sep 2009 Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil 16 Oct 2008 Test No. 216: Soil Microorganisms: Nitrogen Transformation Test 21 Jan 2000 Test No. 217: Soil Microorganisms: Carbon Transformation Test 21 Jan 2000 Test No. 213: Honeybees, Acute Oral Toxicity Test 21 Sep 1998 Test No. 214: Honeybees, Acute Contact Toxicity Test 21 Sep 1998 Test No. 205: Avian Dietary Toxicity Test 04 Apr 1984 Test No. 206: Avian Reproduction Test 04 Apr 1984 Test No. 223: Avian Acute Oral Toxicity Test 23 July 2010 Other tests OECD guidelines http://www.oecd.org/document/40/0,3746,en_2649_34377_37051368_1_1_1_1,00.html Organization for Economic Cooperation Development ISO guidelines Aquatic microorganisms ISO 10712:1995 Water quality -- Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test) ISO 11348-1:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 1: Method using freshly prepared bacteria ISO 11348-2:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 2: Method using liquid-dried bacteria ISO 11348-3:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 3: Method using freeze-dried bacteria ISO 13641-1:2003 Water quality -- Determination of inhibition of gas production of anaerobic bacteria -- Part 1: General test ISO 13641-2:2003 Water quality -- Determination of inhibition of gas production of anaerobic bacteria -- Part 2: Test for low biomass concentrations ISO 13829:2000 Water quality -- Determination of the genotoxicity of water and waste water using the umu-test ISO 16240:2005 Water quality -- Determination of the genotoxicity of water and waste water -- Salmonella/microsome test (Ames test) ISO/DIS 11350 Water quality -- Determination of the genotoxicity of water and waste water -- Salmonella/microsome fluctuation test (Ames fluctuation test) ISO 15522:1999 Water quality -- Determination of the inhibitory effect of water constituents on the growth of activated sludge microorganisms ISO 21338:2010 Water quality -- Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples on the light emission of Vibrio fischeri (kinetic luminescent bacteria test) ISO 8192:2007 Water quality -- Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation ISO 9509:2006 Water quality -- Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms International Standardization Organization ISO guidelines International Standardization Organization Aquatic plants ISO 20079:2005 Water quality -- Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) -- Duckweed growth inhibition test ISO 8692:2004 Water quality -- Freshwater algal growth inhibition test with unicellular green algae ISO/CD 16191 Water quality - Determination of the toxic effect of sediment and soil on the growth behaviour of Myriophyllum aquaticum Myriophyllum test ISO 10253:2006 Water quality -- Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum ISO 10710:2010 Water quality -- Growth inhibition test with the marine and brackish water macroalga Ceramium tenuicorne ISO 14442:2006 Water quality -- Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water ISO/DIS 13308 Water quality -- Toxicity test based on reproduction inhibition of the green macroalga Ulva pertusa ISO/TR 11044:2008 Water quality -- Scientific and technical aspects of batch algae growth inhibition tests ISO guidelines International Standardization Organization ISO guidelines International Standardization Organization ISO guidelines Aquatic invertebrates ISO 6341:1996 Water quality -- Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) -- Acute toxicity test ISO 10706:2000 Water quality -- Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea) ISO/DIS 14380 Water quality -- Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca) ISO/CD 16303 Water quality -- Determination of toxicity of fresh water sediments using Hyalella azteca ISO 10872:2010 Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) ISO 16712:2005 Water quality -- Determination of acute toxicity of marine or estuarine sediment to amphipods ISO 20665:2008 Water quality -- Determination of chronic toxicity to Ceriodaphnia dubia ISO 20666:2008 Water quality -- Determination of the chronic toxicity to Brachionus calyciflorus in 48 h ISO 14669:1999 Water quality -- Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) ISO/DIS 14371 Water quality -- Determination of freshwater-sediment subchronic toxicity to Heterocypris incongruens (Crustacea, Ostracoda) ISO 7828:1985 Water quality -- Methods of biological sampling -- Guidance on handnet sampling of aquatic benthic macro-invertebrates ISO 8265:1988 Water quality -- Design and use of quantitative samplers for benthic macro-invertebrates on stony substrata in shallow freshwaters ISO 8689-1:2000 Water quality -- Biological classification of rivers -- Part 1: Guidance on the interpretation of biological quality data from surveys of benthic macroinvertebrates ISO 8689-2:2000 Water quality -- Biological classification of rivers -- Part 2: Guidance on the presentation of biological quality data from surveys of benthic macroinvertebrates ISO/DIS 10870 Water quality -- Guidelines for the selection of sampling methods and devices for benthic macroinvertebrates in fresh waters ISO/WD 16778 Water quality -- Calanoid copepod development test with Acartia tonsa ISO guidelines International Standardization Organization ISO guidelines Aquatic vertebrates ISO 15088:2007 Water quality -- Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio) ISO 7346-1:1996 Water quality -- Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio HamiltonBuchanan (Teleostei, Cyprinidae)] -- Part 1: Static method ISO 7346-2:1996 Water quality -- Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio HamiltonBuchanan (Teleostei, Cyprinidae)] -- Part 2: Semi-static method ISO 7346-3:1996 Water quality -- Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio HamiltonBuchanan (Teleostei, Cyprinidae)] -- Part 3: Flow-through method ISO 10229:1994 Water quality -- Determination of the prolonged toxicity of substances to freshwater fish -- Method for evaluating the effects of substances on the growth rate of rainbow trout (Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae)) ISO 12890:1999 Water quality -- Determination of toxicity to embryos and larvae of freshwater fish -- Semi-static method ISO 21427-1:2006 Water quality -- Evaluation of genotoxicity by measurement of the induction of micronuclei -- Part 1: Evaluation of genotoxicity using amphibian larvae ISO 21427-2:2006 Water quality -- Evaluation of genotoxicity by measurement of the induction of micronuclei -- Part 2: Mixed population method using the cell line V79 ISO 23893-1:2007 Water quality -- Biochemical and physiological measurements on fish -- Part 1: Sampling of fish, handling and preservation of samples ISO/TS 23893-2:2007 Water quality -- Biochemical and physiological measurements on fish -- Part 2: Determination of ethoxyresorufin-Odeethylase (EROD) ISO/CD 23893-3 Water quality -- Biochemical and physiological measurements on fish -- Part 3: Determination of vitellogenin ISO guidelines International Standardization Organization ISO guidelines Soil microorganisms ISO 10381-6:2009 Soil quality -- Sampling -- Part 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory ISO 14240-1:1997 Soil quality -- Determination of soil microbial biomass -- Part 1: Substrate-induced respiration method ISO 14240-2:1997 Soil quality -- Determination of soil microbial biomass -- Part 2: Fumigation-extraction method ISO 16072:2002 Soil quality -- Laboratory methods for determination of microbial soil respiration ISO 17155:2002 Soil quality -- Determination of abundance and activity of soil microflora using respiration curves ISO 15685:2004 Soil quality -- Determination of potential nitrification and inhibition of nitrification -- Rapid test by ammonium oxidation ISO 14238:1997 Soil quality -- Biological methods -- Determination of nitrogen mineralization and nitrification in soils and the influence of chemicals on these processes ISO 23753-1:2005 Soil quality -- Determination of dehydrogenase activity in soils -- Part 1: Method using triphenyltetrazolium chloride (TTC) ISO 23753-2:2005 Soil quality -- Determination of dehydrogenase activity in soils -- Part 2: Method using iodotetrazolium chloride (INT) ISO/DIS 11063 Soil quality -- Method to directly extract DNA from soil samples ISO/TS 29843-1:2010 Soil quality -- Determination of soil microbial diversity -- Part 1: Method by phospholipid fatty acid analysis (PLFA) and phospholipid ether lipids (PLEL) analysis ISO/PRF TS 29843-2 Soil quality -- Determination of soil microbial diversity -- Part 2: Method by phospholipid fatty acid analysis (PLFA) using the simple PLFA extraction method ISO/TS 10832:2009 Soil quality -- Effects of pollutants on mycorrhizal fungi -- Spore germination test ISO/TS 22939:2010 Soil quality -- Measurement of enzyme activity patterns in soil samples using fluorogenic substrates in micro-well plates ISO 11266:1994 Soil quality -- Guidance on laboratory testing for biodegradation of organic chemicals in soil under aerobic conditions ISO 15473:2002 Soil quality -- Guidance on laboratory testing for biodegradation of organic chemicals in soil under anaerobic conditions ISO 14239:1997 Soil quality -- Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions ISO guidelines International Standardization Organization ISO guidelines Soil invertebrates ISO 11268-1:1993 Soil quality -- Effects of pollutants on earthworms (Eisenia fetida) -- Part 1: Determination of acute toxicity using artificial soil substrate ISO 11268-2:1998 Soil quality -- Effects of pollutants on earthworms (Eisenia fetida) -- Part 2: Determination of effects on reproduction ISO 11268-3:1999 Soil quality -- Effects of pollutants on earthworms -- Part 3: Guidance on the determination of effects in field situations ISO 11267:1999 Soil quality -- Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants ISO 16387:2004 Soil quality -- Effects of pollutants on Enchytraeidae (Enchytraeus sp.) -- Determination of effects on reproduction and survival ISO 15952:2006 Soil quality -- Effects of pollutants on juvenile land snails (Helicidae) -- Determination of the effects on growth by soil contamination ISO 20963:2005 Soil quality -- Effects of pollutants on insect larvae (Oxythyrea funesta) -- Determination of acute toxicity ISO 17512-1:2008 Soil quality -- Avoidance test for determining the quality of soils and effects of chemicals on behaviour-- Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei) ISO/DIS 17512-2 Soil quality -- Avoidance test for determining the quality of soils and effects of chemicals on behaviour-- Part 2: Test with collembolans (Folsomia candida) ISO 23611-1:2006 Soil quality -- Sampling of soil invertebrates -- Part 1: Hand-sorting and formalin extraction of earthworms ISO 23611-2:2006 Soil quality -- Sampling of soil invertebrates -- Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina) ISO 23611-3:2007 Soil quality -- Sampling of soil invertebrates -- Part 3: Sampling and soil extraction of enchytraeids ISO 23611-4:2007 Soil quality -- Sampling of soil invertebrates -- Part 4: Sampling, extraction and identification of soil-inhabiting nematodes ISO/DIS 23611-5 Soil quality -- Sampling of soil invertebrates -- Part 5: Sampling and extraction of soil macro-invertebrates ISO/DIS 23611-6 Soil quality -- Sampling of soil invertebrates -- Part 6: Guidance for the design of sampling programmes with soil invertebrates ISO guidelines International Standardization Organization ISO guidelines Plants ISO 11269-1:1993 Soil quality -- Determination of the effects of pollutants on soil flora -- Part 1: Method for the measurement of inhibition of root growth ISO 11269-2:2005 Soil quality -- Determination of the effects of pollutants on soil flora -- Part 2: Effects of chemicals on the emergence and growth of higher plants ISO 17126:2005 Soil quality -- Determination of the effects of pollutants on soil flora -- Screening test for emergence of lettuce seedlings (Lactuca sativa L.) ISO 22030:2005 Soil quality -- Biological methods -- Chronic toxicity in higher plants ISO/CD 29200 Soil quality -- Assessment of genotoxic effects on higher plants -- Micronucleus test on Vicia faba ISO guidelines International Standardization Organization US EPA http://www.epa.gov/ocspp/pubs/frs/home/draftguidelines.htm Aquatic 850.1010 - Aquatic Invetebrate Acute Toxicity, Test, Freshwater Daphnids (PDF) (10 pp, 36K) 850.1020 - Gammarid Acute Toxicity Test (PDF) (11 pp, 36K) 850.1025 - Oyster Acute Toxicity Test (Shell Deposition) (PDF) (9 pp, 32K) 850.1035 - Mysid Acute Toxicity Test (PDF) (10 pp, 34K) 850.1045 - Penaeid Acute Toxicity Test (PDF) (9 pp, 32K) 850.1055 - Bivalve Acute Toxicity Test (Embryo Larval) (PDF) (7 pp, 27K) 850.1075 - Fish Acute Toxicity Test, Freshwater And Marine (PDF) (13 pp, 45K) 850.1085 - Fish Acute Toxicity Mitigated By Humic Acid (PDF) (10 pp, 35K) 850.1300 - Daphnid Chronic Toxicity Test (PDF) (12 pp, 42K) 850.1350 - Mysid Chronic Toxicity Test (PDF) (10 pp, 36K) 850.1400 - Fish Early-Life Stage Toxicity Test (PDF) (15 pp, 66K) 850.1500 - Fish Life Cycle Toxicity (PDF) (4 pp, 16K) 850.1710 - Oyster BCF (PDF) (14 pp, 50K) 850.1730 - Fish BCF (PDF) (25 pp, 74K) 850.1735 - Whole Sediment Acute Toxicity Invertebrates, Freshwater (PDF) (19 pp, 65K) 850.1740 - Whole Sediment Acute Toxicity Invertebrates, Marine (PDF) (14 pp, 50K) 850.1790 - Chironomid Sediment Toxicity Test (PDF) (16 pp, 57K) 850.1800 - Tadpole/Sediment Subchronic Toxicity Test (PDF) (15 pp, 49K) 850.1850 - Aquatic Food Chain Transfer (PDF) (4 pp, 16K) 850.1900 - Generic Freshwater Microcosm Test, Laboratory (PDF) (28 pp, 76K) 850.1925 - Site-Specific Aquatic Microcosm Test, Laboratory (PDF) (21 pp, 91K) 850.1950 - Field Testing For Aquatic Organisms (PDF) (7 pp, 21K) 850.4400 - Aquatic Plant Toxicity Test Using Lemna Spp., Tiers I and II (PDF) (10 pp, 36K) 850.4450 - Aquatic Plants Field Study, Tier III (PDF) (9 pp, 30K) 850.5400 - Algal Toxicity, Tiers I and II (PDF) (11 pp, 42K) 850.6800 - Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals (PDF) (9 pp, 37K) US Environmental Protection Agency US EPA http://www.epa.gov/ocspp/pubs/frs/home/draftguidelines.htm US Environmental Protection Agency Soil 850.2450 - Terrestrial (Soil-Core) Microcosm Test (PDF) (19 pp, 123K) 850.4000 - Background-Nontarget Plant Testing (PDF) (15 pp, 50K) 850.4025 - Target Area Phytotoxicity (PDF) (15 pp, 51K) 850.4100 - Terrestrial Plant Toxicity, Tier I (Seedling Emergence) (PDF) (8 pp, 29K) 850.4150 - Terrestrial Plant Toxicity, Tier I (Vegetative Vigor) (PDF) (8 pp, 28K) 850.4200 - Seed Germination/Root Elongation Toxicity Test (PDF) (8 pp, 29K) 850.4225 - Seedling Emergence, Tier II (PDF) (10 pp, 36K) 850.4230 - Early Seedling Growth Toxicity Test (PDF) (9 pp, 33K) 850.4250 - Vegetative Vigor, Tier II (PDF) (10 pp, 35K) 850.4300 - Terrestrial Plants Field Study, Tier III (PDF) (8 pp, 27K) 850.4600 - Rhizobium-Legume Toxicity (PDF) (14 pp, 73K) 850.4800 - Plant Uptake and Translocation Test (PDF) (13 pp, 35K) 850.5100 - Soil Microbial Community Toxicity Test (PDF) (11 pp, 46K) 850.6200 - Earthworm Subchronic Toxicity Test (PDF) (13 pp, 43K) 850.2100 - Avian Acute Oral Toxicity Test (PDF) (11 pp, 38K) 850.2200 - Avian Dietary Toxicity Test (PDF) (12 pp, 42K) 850.2300 - Avian Reproduction Test (PDF) (16 pp, 53K) 850.2400 - Wild Mammal Acute Toxicity (PDF) (5 pp, 18K) 850.2500 - Field Testing For Terrestrial Wildlife (PDF) (43 pp, 115K) 850.3020 - Honey Bee Acute Contact Toxicity (PDF) (8 pp, 27K) 850.3030 - Honey Bee Toxicity of Residues on Foliage (PDF) (6 pp, 23K) 850.3040 - Field Testing for Pollinators (PDF) (5 pp, 18K) Other Testing strategy • Battery of assays • Fast screening tests (Vibrio fisheri bioluminiscence, 30 min toxicity) • Standardized acute toxicity tests • Further studies with chronic assays • Combine trophic levels! Combine exposure routes! • Various purposes -> guidelines and recommendations • REACH (EU - Registration, Evaluation and Authorisation of Chemicals) • Plant protection products + biocides • Veterinary and human pharmaceuticals • Waste materials … • The most common set ups • algae / D. magna / fish for aquatic environment • earthworm (enchytraeid/springtail) / plant for soil environment Testing strategy Retention function – Biotests with eluates Ecotoxic contents Genotoxic contents Habitat function - Biotests with solids Luminescent bacteria test Algal inhibition test Site inherent test organisms Added test organisms Respiration test Nitrification test Bacteria contact test Earthworm avoidance test Plant test Earthworm test Collembolan test Umu-test 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 1) Prepare the organism Culture media, standardized numbers, age, etc. 2) Prepare the sample Dilution series water/culture media – direct organism exposure Include BLANK (medium only) solvent for organic compounds – minimum to be added Include SOLVENT CONTROL 3) Expose organisms … for appropriate time, number of repetitions, under specified conditions 4) Evaluate and report results measure the endpoint / count organisms validity criteria statistical evaluation (means, ANOVA, dose-response …) General scheme of bioassay Aquatic ecotoxicology bioassays Algal growth inhibition test (ISO 8692) Algal growth inhibition test (ISO 8692) • Miniaturization • Alternative: Algaltoxkit Duckweed bioassay (ISO 20079) • Lemna minor • 10 leaves per 1 beaker • pH 6.5; 10 000 lx; 24°C • 96 hours • growth, biomass, no. of leaves • image analysis possible • validity: – 8x increase in control – IC50 for K2Cr2O7 10-60 mg/L Daphnia magna Artemia salina Ceriodaphnia dubia Gammarus Chironomus riparius Aquatic consumers - invertebrates Potamopyrgus antipodarum Tubifex tubifex Lumbriculus variegatus Tamnocephalus platyurus Hyalella azteca Daphnia magna test (ISO 6341) • 5 individuals per replicate (min 2 ml) • no food • 20°C; dark or 16h light / 8h dark • 24h, 48h • medium • CaCl2 · 2H2O 11,76 g/l • MgSO4 · 7H2O 4,93 g/l • NaHCO3 2,59 g/l • KCl 0,23 g/l • 25 ml each to 1 L • pH 7.8 • aeration • no. of immobilized individuals • validity: – O2 > 80 % (2 mg/l) – mortality in control 10 % Daphnia magna chronic test (ISO 10706) • 10 juveniles (24h old) per replicate • 50ml medium • 3 times per week medium change • 21 days • 20 ± 2°C • pH 7-9 • disolved O2 > 3mg/l •16 h light / 8 h dark • food - algae • week controls: O2, T, pHMortality, survival Reproduction Juveniles Behavior koncentrace 135 mg/l 0 2 4 6 8 10 12 14 16 18 20 22 čas (dny) 0 20 40 60 80 100 přežívání(%) contorl BIOMASS AQ.EXTRACT C18 PERMEATE C18 ELUATE koncentrace 15 mg/l 10 12 14 16 18 20 22 čas (dny) 0 20 40 60 80 100 120 140 160 180 200 220 240 početnarozenýchjedinců control BIOMASS AQ.EXTRACT C18 PERMEATE C18 ELUATE SPINACH Daphnia magna acute vs chronic test Alternative: microbiotests Chironomus riparius test (OECD 218) • 10 larvae (cca 10d old) per beaker • OECD sediment • 100 ml sediment / 175 ml water • 20 ± 2°C; food, aeration • 16h light / 8h dark; controled pH, O2 • 10 d • survival and growth Guppy, Poecilia reticulata Zebrafish, Danio rerio (syn. Brachydanio rerio) Fathead minnow, Pimephales promelas (USA) (Rainbow) trout (Onchorhynchus sp.) Medaka, Oryzias latipes Nile tilapia, Oreochromis niloticus acute tests (96 h) prolonged tests embryolarval tests chronic tests - reproduction - growth Specific endpoints – genotoxicity, endocrine disruption Fish bioassays Fish bioassay acute (ISO 7346 1-3) • Brachydanio rerio, … • 26°C • medium: – pH 7,8 ± 0,2 – CaCl2.2H2O, MgSO4.7H2O, NaHCO3, KCl – disolved O2 > 90% • 24, 48, 72, 96 h • validity: mortality in control < 10%, O2 etc. Prolonged and chronic fish tests Prolonged • OECD 204 – 14d, ISO 10229 - 21 d • semistatic or flow through • food; controled pH, O2 • endpoints: breathing, gills, behavior, orientation, mortality • 14-21d LC50, NOEC, LOEC Chronic • OECD 210; US EPA OPPTS 850.1500 • 7 - 200 days • starting with eggs or embryos or juveniles • endpoints: survival, behavior, feeding, lenght, growth, weight, biochemical parameters, bioaccumulation Embryolarval test (ISO 12890, OECD 210, 212) • 100 embryos per replicate • 6-7 days • no food • T, pH, O2 • Endpoints: hatching, survival, morphology, bahavior, weight, lenght, anomalies FETAX – Frog Embryo Teratogenicity Assay Xenopus • Choriogonadotropin  eggs after 9-12h • 25 embryos per Petri dish, 10 ml test solution • 24°C; pH 6.5-9 • 96h • validity: – 6-aminonicotinamid LC50 2,23 mg/ml, EC50 0,005 mg/ml • mortality, growth, abnormalities • Atlas of Abnormalities – John A. Bantle FETAX Vibrio fisheri test (ISO 11348) • 5-30 min • luminiscence inhibition • problem with particles and colour  flash test Specific mechanisms of toxicity • Cell lines (H4IIE.luc – diox.; MVLN, T47D.Luc – estrog.) • Nuclear receptors (AhR, ER, AR, RAR/RXR) • AhR/ER – luciferase reporter gene • CALUX (Chemical Assisted LUciferase eXpression) LightLight Luciferase Nuclear Factors 1 P + AhR HSP90 HSP90 Src HSP90 HSP90 Adapted from Blankenship (1994) DRE-Luc AhR ARNT Modulation of Gene Expression ARNT Src “Activated” 2 P P Cytosolic Proteins Membrane Proteins IncreasedProtein Phosphorylation Ligand (TCDD) LightLight Luciferase Nuclear Factors 1 P + AhR HSP90 HSP90 SrcSrc HSP90 HSP90 Adapted from Blankenship (1994) DRE-Luc AhR ARNT Modulation of Gene Expression ARNT Src “Activated” 2 P P Cytosolic Proteins Membrane Proteins IncreasedProtein Phosphorylation Soil ecotoxicology bioassays Exposure methods • Tested chemical mixed with soil – Artificial soil (OECD, ISO) – Real soil (LUFA 2.2 …) • Topic applications, injections, forced feeding … not so relevant 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. LUFA soil (http://lufa-speyer.de/) • Landwirtschaftliche Untersuchungs und Forschungsanstalt Speyer • 4 EUR / 1 kg LUFA 2.1 LUFA 2.2 LUFA 2.3 LUFA 5M LUFA 6S organic carbon (%) 0.81 ± 0.21 2.16 ± 0.40 0.98 ± 0.05 1.29 ± 0.20 1.75 ± 0.11 particles < 0.02 mm (%) 8.2 ± 0.9 13.9 ± 1.1 22.7 ± 1.1 25.3 ± 1.8 65.1 ± 2.7 pH (0.01M CaCl2) 5.1± 0.4 5.4 ± 0.1 6.4 ± 0.6 7.2 ± 0.1 7.2 ± 0.1 cation exchange capacity (meq/100g) 4± 1 10 ± 1 8 ± 2 15 ± 3 22 ± 6 water holding capacity (g/100g) 33.2 ± 1 48.2 ± 5 34.4 ± 2 42.1 ± 4 40.7 ± 5 weight per volume (g/1000ml) 1404 ± 46 1197 ± 60 1291 ± 30 1212 ± 56 1264 ± 90 <0.002 3.0 ± 0.9 6.4 ± 0.9 9.4 ± 0.9 10.8 ± 1.3 42.1 ± 1.8 0.002 - 0.006 2.2 ± 0.7 3.5 ± 0.7 4.2 ± 0.8 5.4 ± 0.3 10.8 ± 0.7 0.006 - 0.02 2.9 ± 0.7 3.8 ± 0.7 9.1 ± 0.5 9.1 ± 0.5 12.1 ± 1.3 0.02 - 0.063 5.3 ± 1.8 5.4 ± 1.2 18.6 ± 2.3 19.5 ± 1.3 14.1 ± 2.5 0.063 - 0.2 27.0 ± 3.1 35.4 ± 2.3 29.3 ± 3.4 38.9 ± 1.0 8.7 ± 0.9 0.2 - 0.63 57.2 ± 4.3 44.8 ± 2.7 26.9 ± 0.7 14.9 ± 1.0 9.0 ± 0.3 0.63 - 2.0 2.4 ± 0.6 0.7 ± 0.1 2.5 ± 0.8 1.4 ± 0.1 3.2 ± 0.7 soil type sand (S) loamy sand (lS) loamy sand (lS) silty sand (uS) clayey loam (tL) <0.002 3.0 ± 0.9 6.4 ± 0.9 9.4 ± 0.9 10.8 ± 1.3 42.1 ± 1.8 0.002 - 0.05 8.8 ± 1.8 12.2 ± 0.6 29.8 ± 3.0 27.5 ± 2.2 36.0 ±2.3 0.05 - 2.0 88.2 ± 1.2 81.4 ± 1.2 60.8 ± 2.6 61.7 ± 3.2 21.9 ± 1.6 soil type sand loamy sand sandy loam sandy loam clay Particle size (mm) distribution according to German DIN (in %): Particle size (mm) distribution according to USDA (in %) Earthworm bioassays Earthworm acute toxicity test • 500 g soil + 10 adult Eisenia fetida • 14 days • mortality and weight Earthworm reproduction test • 56 days • 500 g soil + 10 adult Eisenia fetida • horse manure as food • juveniles extracted using water bath ISO 11268-1 Soil preparation WHC measurement Water added Soil weighted to jars 10 adults from culture Washed Weighting worms10 adults to 1 jar Eisenia fetida reproduction test ISO 11268-2 Control of the jars, activity markers Mortality assessmentWeighting the worms Temperated room E. fetida test – after 28 days ISO 11268-2 After 20 min juveniles appear Water bath, increasing temperature 40ºC - 60ºC Sieving the soil Collecting and counting juveniles CountingHand sorting of cocoons E. fetida – 8 weeks ISO 11268-2 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 ISO 17512-1 Bioaccumulation test Enchytraeidae Enchytraeidae ISO 16387 Avoidance test with E. albidus 24 hrs 1 2 3 Ø 8,4 cm 4 5 10 adults 10 x 2 x 10 g Counting of adults in halves waste Artificial soil Folsomia candida Folsomia candida ISO 11267 Caenorhabditis elegans test ASTM: E2172-01 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode Caenorhabditis elegans ISO 10872 Caenorhabditis elegans test Mites Hypoaspis aculeifer prey predator cont. soil Lactuca sativa root growth Germination tests with plants – testing toxicity in the aquatic media (Sinapis alba) or in soil (Lactuca sativa) ISO 11269-1 Higher plants chronic toxicity ISO 22030 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% 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 Effects on microbial respiration Expensive & time consuming (e.g. Pesticide testing) Variable results (natural variability …) Higher ecological relevancy Micro & Mesocosms Results of the bioassays and their use Data from ecotoxicity tests tested stressor observed response freqvency qualitative nominal limit test qualitative nominal comparison test qualitative ordinal comparison test quantitative qualitative binary qualitative ordinal quantitative continuous 0 100 C S freqvency 0 100 C S1 frequency 0 100 A B frequency 0 100 C Vz 0 C S response S2 frequency 0 100 C S1S2 C S1S2 C frequency 0 100 A B C A B C frequency 0 100 0 concentration 0 concentration 0 concentration frequency 0 100 0 response 0 response 0 response Dose(concentration) - response relationship Note No Observed Effect Concentration (NOEC) Lowest Observed Effect Concentration (LOEC) ECx ( x % effects concentration) LCx ( x % lethal concentration) Bioassay vs real ecosystem • Bioassay is only simplified ecosystem model • species differs, matrix differs, single species vs multispecies, individuals vs populations Eisenia fetida – compost worm !! Aporrectodea caliginosa Lumbricus terrestris vs Bioassay vs reality Objective = to protect real ecosystems, safe concentration  need to extrapolate bioassay results to be valid for ecosystems • how much information we have? • uncertainty factors, 1, 10, 100, 1000 Species sensitivity distribution (SSD) C. riparius S. fontanilis O. kisuth O. reinhardtii P. fluviatilis P. partenog. P. notatus H. azteca N. barablutus P. promelas D. pulex C. dubia I. punctatus D. magna O. mykiss G. pulex C. magnifica C. decisum 0 20 40 60 80 100 1 10 100 1000 dissolved copper (µg/l) cumulativedistributionfunction Hazen plotting Loglogistic fitting HC5 = 95% protection level Extrapolace PNEC (limits, EQS) Data Assessment factor L(E)C50 short-term toxicity tests NOEC for1 long-term toxicity test NOEC foradditionallong-term toxicity tests of 2 trophic levels NOEC foradditionallong-term toxicity tests of 3 species of 3 trophic levels 1000 100 50 10 Risk assessment with earthworms Summary - Take home message • Bioassays are necessary addition to chemical analyses • Lot of standardized/experimental bioassays to choose from • Necessary know-how (lab tricks, weakpoints, when to use, interpretation, data evaluation …) • No single test gives all information - battery of bioassays Thanks for your attention!!!! Jakub Hofman hofman@recetox.muni.cz http://www.recetox.cz