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