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Ecotoxicological bioassays
Jakub Hofman & Klára Hilscherová
DaphniaMagna bacecoli lemna

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Introduction

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CHEMICAL ENTERS THE ENVIRONMENT
Bioavailable fraction
“EXPOSURE”
acute
chronic
Toxikokinetics
biotransformation
bioactivation
excretion / sequestration
Target site
“EFFECTS”
LEVELS, FATE, PROCESSES
CHEMICAL ENTERS THE ORGANISM
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Role of ecotoxicology in environmental protection
•FOCUS:
•Investigate relationships between organisms and contaminants
•
•ROLE:
•Scientific basis of env. protection; know-how; rational env. protection
•
•Activities:
Provide TOOLS for praxis:
-(Eco)toxicity testing = bioassays
-Chemical and pesticides
-Testing wastes, contaminated sites
-Environmental quality assessment
Research of:
–Principles …
–Mechanisms …
–Fate and bioavailability
–Mixture toxicity
–Biodiversity …

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Bioassays, ecotoxicity tests
•tools (methods) 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, dose-response …)
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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 !
Testing effects of chemicals and risk assessment
Most common in practice
MIXTURES OF
TOXICANTS

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•Chemical analyses only are not able to identify risks properly because:
1)
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?

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Bioassay development
•old bioassays – acute, ecologically irrelevant, testing pure chemicals, pesticides •new bioassays
– sublethal endpoints, ecological relevancy, chemical mixtures, miniaturization, simple to measure
endpoints
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•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
•
figure4 Eisenia_fetida scenedesmus_quadricauda_upr
Differenciation of bioassays
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1
Differenciation of bioassays
•size, complexity
•ecological relevancy
•controling the conditions
•old / fresh contamination
•no. of species
•…
cell lines
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auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict
Cu addition
Effect concentrations expressed in total/dissolved Cu
??? Safe concentrations ???
1 - Laboratory studies

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Expensive & time consuming (e.g. Pesticide testing)
Variable results (natural variability …)
Higher ecological relevancy
1 1
2 - Micro & Mesocosms
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Complex issue (chemistry, biology, geology, climate, pedology ..)
Ecotoxicology + Ecology
Comparing „contaminated“ to „control“ sites
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3 - Field assessment / biomonitoring

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Use of bioassays
•Testing chemicals
–Traditional / bioassays developed to assess individual chemicals
–Advantage: Standardized approaches
•OECD – Guideline methods - series „2“ Effects on biota
•ISO methods
–E.g. Fish tests - OECD 203 / ISO 7346
–E.g. D. magna - OECD 202 / ISO 6341
–Disadvantage: Limited ecological relevance
•often acute tests only
•„too standardized…“ (? Less representative ?)
•does not assess/consider bioavailability
•no consideration of mixture effects
•no consideration of specific modes of action
•no consideration of ecological situation
–
•
AcuteToxScheme_RM

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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
–
Example of light coloured springtail (Folsomia candida)
Èerná skládka
eluate 1:10
sdilution
IMG_4025 produce_ph05
Control soil
waste
Mixture at different ratios
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OECD guidelines
http://www.oecd.org/document/40/0,3746,en_2649_34377_37051368_1_1_1_1,00.html
Organisation for Economic Co-operation and Development
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

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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
Organisation for Economic Co-operation and Development
Organization for Economic Cooperation Development

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ISO guidelines
ISO Logo
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
ISO Logo
International Standardization Organization
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aColi_NIAID.jpg

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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
ISO Logo
International Standardization Organization
ISO guidelines
ISO Logo
International Standardization Organization
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ISO guidelines
ISO Logo
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
ISO Logo
International Standardization Organization
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ISO guidelines
ISO Logo
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 Hamilton-Buchanan (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 Hamilton-Buchanan (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 Hamilton-Buchanan (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-O-deethylase (EROD)
ISO/CD 23893-3
Water quality -- Biochemical and physiological measurements on fish -- Part 3: Determination of
vitellogenin
ISO guidelines
ISO Logo
International Standardization Organization
http://www.fishbase.org/images/species/Darer_m0.jpg

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ISO guidelines
ISO Logo
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
ISO Logo
International Standardization Organization

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ISO guidelines
ISO Logo
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
ISO Logo
International Standardization Organization
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ISO guidelines
ISO Logo
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
ISO Logo
International Standardization Organization
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http://upload.wikimedia.org/wikipedia/commons/thumb/e/e1/Illustration_Vicia_faba1.jpg/240px-Illustr
ation_Vicia_faba1.jpg
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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

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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

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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
•

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Testing strategy
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

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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
Schema testu Ps_putida

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Aquatic ecotoxicology bioassays

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Algal growth inhibition test (ISO 8692)
raphidocelis_subcapitata_upr scenedesmus_quadricauda_upr http://www.imp.mtu.edu/matchar/algal1.JPG
1

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Algal growth inhibition test (ISO 8692)
•Miniaturization
•
•
•
•
•
•
•
•
•
•Alternative: Algaltoxkit
•
•

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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
•
figure4

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Daphnia magna
C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg
Artemia salina
Ceriodaphnia dubia
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Gammarus
C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg
Chironomus riparius
Aquatic consumers - invertebrates
Potamopyrgus antipodarum
Tubifex tubifex
http://www.aquarium-kosmos.de/bilder/streifzuege/Lumbriculus%20variegatus_2.jpg
Lumbriculus variegatus
http://84.244.151.141/~persoone/images/slider/Thamnocephalus_platyurus.jpg
http://www.fcps.edu/islandcreekes/ecology/Arthropods/Scud/Hyaellallam1.jpg
Tamnocephalus platyurus
Hyalella azteca

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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 %

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Daphnia magna chronic test (ISO 10706)
daphnia
•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, pH
Mortality, survival
Reproduction
Juveniles
Behavior

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Daphnia magna acute vs chronic test
1

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Alternative: microbiotests


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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
•
IMG_0054 IMG_0061

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Guppy, Poecilia reticulata
figure4
Zebrafish, Danio rerio (syn. Brachydanio rerio)
Fathead minnow, Pimephales promelas (USA)
figure4
(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

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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.

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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

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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

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FETAX – Frog Embryo Teratogenicity Assay Xenopus
•Choriogonadotropin à 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
C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek
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FETAX
1

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Vibrio fisheri test (ISO 11348)
•5-30 min
•luminiscence inhibition
•
•
•
•
•
•problem with particles and colour
• è flash test
•
figure4 1

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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)
H295R

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Soil ecotoxicology bioassays

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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

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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

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LUFA soil (http://lufa-speyer.de/)
•Landwirtschaftliche Untersuchungs und Forschungsanstalt Speyer
•4 EUR / 1 kg
German

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Earthworm bioassays
CE8c mating LR CE8a-cocoon LR Lumdiap2 STUPD00Z
http://t3.gstatic.com/images?q=tbn:ANd9GcR7-3EeXeOV0fHO4s2TOGWEnFANnR74gi6jRAso6-WCvT_OWvmIZlw3FsoM
yA 11

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Earthworm acute toxicity test
•500 g soil + 10 adult Eisenia fetida
•14 days
•mortality and weight
38 62 71 019 012

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Earthworm reproduction test
•56 days
•500 g soil + 10 adult Eisenia fetida
•horse manure as food
•juveniles extracted using water bath
068
ISO 11268-1

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wahingwormsJPG
Soil preparation
WHC measurement
Water added
Soil weighted to jars
10 adults from culture
Washed
Weighting worms
10 adults to 1 jar
drysoils
Eisenia fetida reproduction test
ISO 11268-2

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Control of the jars, activity markers
Mortality assessment
Weighting the worms
trolleywithworms
Temperated room
E. fetida test – after 28 days
ISO 11268-2

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juvenileszoomedS juvpicking cocoonsievingS cocoonsearchontray cocoonsinthehand
After 20 min juveniles appear
Water bath, increasing temperature  40ºC - 60ºC
Sieving the soil
Collecting and counting juveniles
Counting
Hand sorting of cocoons
E. fetida – 8 weeks
ISO 11268-2

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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
ISO 17512-1

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Bioaccumulation test


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Enchytraeidae
Image14 grindal_worm IMG_3421_1 006

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Enchytraeidae
IMG_2981 IMG_2893 pokus IMG_2984
ISO 16387

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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

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Folsomia candida
005 008
016

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http://www.chaloupky.cz/fotografie/foto-150-640.jpg 012
Folsomia candida
023 024 028 031 NS4 P1010056 IMG_2850 pokus 3
ISO 11267

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Caenorhabditis elegans test
65
C. elegans life cycle C. elegans anatomy - cross section C. elegans anatomy

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ASTM: E2172-01 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode
Caenorhabditis elegans
ISO 10872
082
Caenorhabditis elegans test

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Mites
Hypoaspis
aculeifer
olog
prey
predator
cont. soil
0471971030

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Lactuca sativa root growth
Germination tests with plants – testing toxicity in the aquatic media (Sinapis alba) or in soil
(Lactuca sativa)
P1010052 pokus IMG_0215 IMG_0196 IMG_7021_al IMG_7034
ISO 11269-1
IMG_7022_al

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Higher plants chronic toxicity
IMG_0511 IMG_0499 IMG_0502 IMG_0819 IMG_0825 plant01.jpg (54.086 Byte)
ISO 22030

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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

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0010
Effects on microbial respiration
92

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Results of the bioassays and their use

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Data from ecotoxicity tests
Response

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Dose(concentration) - response relationship
AcuteToxScheme_RM
Note
No Observed Effect Concentration (NOEC)
Lowest Observed Effect Concentration (LOEC)
ECx ( x % effects concentration)
LCx ( x % lethal concentration)

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Bioassay vs real ecosystem
•Bioassay is only simplified ecosystem model
•species differs, matrix differs, single species vs multispecies, individuals vs populations
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64GCAZLE1HECAXIJ313CA3FNLEXCAC6L6XKCAZGB79LCAVIY0IY
OH9Z77CAI0ZPOSCADMHMQHCAY1N8ORCA4L1YM2CA782G0FCAMAU4PMCAKTGG8DCASQY6WXCAWEDONGCAYLV7Q9CATJ4B1CCA3EP
LAQCACYSSFWCA6IKG6BCAJVS9XJCAITWBCCCASYVSN1CAPXS9IZ
TRG2YLCAWVWGSOCAPGFRMYCA7XLGIMCAJZZOG8CAI0IV3MCAASHZH0CAEK0J3CCAY7SJM5CA85Q3DPCA48LM7SCA8GFBOYCA0VA
FDVCA71OTD9CAMEPKFUCAFHJ8Q4CARG9TF9CAGNPIJFCATVN1QM
XQ0C8LCAS8TXAKCAJBPMA3CAF2RV44CACFSL3QCABR6S93CAES5JJ1CA1G5UGZCAW92XUWCA5HR6OFCAFMNNS2CAQUPHKFCA6MP
0AUCAPKDV3DCAVUFCOACA8ZT1NACAMT8EFDCAXJ73WPCAK820Z6
BRB4R4CAN33GGICA8CL3TSCA3RLC5XCAWLCYIVCABTZL7WCA2O1G5WCAB4TUKICAA54GCDCAMKENOVCA8ZXJFOCASJY86TCAASZ
5JNCAOZTYHACA9MKBYRCA6AFGIDCAWHZ8QSCAGYVOESCAJWALFD IMG_4025
Pic1 fetida_adult_02
Eisenia fetida – compost worm !!
Aporrectodea caliginosa
caliginosa_adult_08 terrestris_adult_010
Lumbricus terrestris
vs

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Bioassay vs reality
•Objective = to protect real ecosystems, safe concentration
• è 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)
HC5 = 95% protection level
Extrapolace
PNEC (limits, EQS)

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Risk assessment with earthworms


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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

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  Thanks for your attention!!!!
Jakub Hofman
hofman@recetox.muni.cz
http://www.recetox.cz
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