Bi2003 Ecotoxicology
Ecotoxicological bioassays
Jakub Hofman
1

Content
§Introduction – what, why, how, concept
§Types of bioassays
§Ecotoxicological bioassays’ design and results
§Aquatic bioassays - examples
§Soil bioassays – examples
§Use of bioassays in praxis
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Introduction – what, why, how, concept
3


Protection of environment / nature
§Is and must be primary aim of sustainably developing society
§why?
How to protect ?
§Policy
§Legislation
§Research
§Education
§
Ecotoxicology – offers knowledge and tools useful for the effective and reasonable environmental
protection (these tools = ecotoxicological bioassays)
http://s3.amazonaws.com/dfc_attachments/images/3154905/iStock_000004307050Small_web.jpg
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Ecotoxicology
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
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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 stress
factors), in the lab (controlled conditions) or in the field (less controlled) and effects are
evaluated and related to exposure – its experiment (!)
§WHY? To understand the cause-effects relationships (causality, dose-response …)
obut sometimes also e.g. accumulation, biodegradation ...
http://www.teara.govt.nz/files/p15500pc.jpg http://www.ebpi.ca/Rototox%20Kit%20M%20Kit.bmp
http://www.ees.ufl.edu/homepp/bitton/images/metpad_metplate.jpg
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Ecotoxicity bioassay, ecotoxicity test
toxicity test versus ecotoxicity test
à see toxicology versus ecotoxicology
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Why ecotoxicological tests ?
CHEMICAL ANALYSES ALONE CANNOT show real risk to living organisms:
(1)
(1)real exposure varies according to bioavailability of toxic substances
(2)in real environment, there is always mixture of toxicants that acts differently from individual
compounds
(3)negative effects of matrix itself, regardless of toxicant content, on organisms or interaction
of matrix with effects of toxicants
(4)spectrum of analytical methods (i.e. limit values) is limited and un-analysed significantly
toxic substances may be present in the sample
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ADVANTAGES of chemical analyses
- Reproducibility, standard-ability
- Exact numerical outputs understandable to all people: use in the law

Bioassays useful for:
§prospective ecological risk assessment
ousing bioassays for chemical compounds, pesticides
ousing bioassays for materials, mixtures
obefore they enter the environment
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Bioassays useful for:
Objectives of the prospective approach:
§evaluation of hazards of contaminants (individual and mixtures) and other stressors
§analysis of relationships between concentration and effect ("dose-response relationship")
§hazard quantification, risk assessment (including legislatively required assessment) and
prediction of negative effects of real environmental samples
§setting limit values for (legislative) regulation of chemical substances, pesticides and materials
that may come in contact with the environment (waste, sludge, fertilizers…)
§knowledge of processes and mechanisms related to the effects of contaminants (or other stressors)
on biota, fate and bioavailability of contaminants in the environment and exposure of organisms
§understanding causes of harmful effects of contaminants on organisms
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Bioassays useful for:
§retrospective ecological risk assessment
ousing bioassays for real environmental samples
osearching the causalities between pollution and effects
ISO 19204:2017 Soil quality — Procedure for site-specific ecological risk assessment of soil
contamination (soil quality TRIAD approach)
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Bioassays useful for:
Objectives of the retrospective approach:
§knowledge of the links (causality) between the occurrence and fate of contaminants (stressors) and
the state of the biota
§knowledge of past events and their regularities allows to estimate the development for the future
in similar situations (prediction)
§evaluation of interventions on real components of the environment in real ecosystems (evaluation
of fertilization, remediation, assessment of contaminated sites…)
§knowledge of processes and mechanisms related to the effects of contaminants (or other stressors)
on the biota, the fate and bioavailability of contaminants in the environment and the exposure of
organisms
§understanding the consequences of the harmful effects of contaminants, especially at higher levels
of the biological organization
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Bioassays useful for:
§
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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 !
MIXTURES OF
TOXICANTS

Bioassays useful for:
§Each methodological approach has its limitations and can be interpreted only with regard to its
information content and focus
§
§It is optimal to combine both approaches !!!
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Types of 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
http://www.ebpi.ca/thamnocephalus%20platyurus%20toxicity%20test%20kit.jpg
http://www.ebpi.ca/Rototox%20Kit%20M%20Kit.bmp http://www.ebpi.ca/daphtoxkit%20toxicity%20pulex.jpg
http://www.teara.govt.nz/files/p15500pc.jpg
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Mikrobiotests
§they use some dormant stages of testing organisms
§practical = everything in one box
§cheap and easy, however, often not very relevant for real situations
§http://ebpi.ca/index.php?option=com_content&view=article&id=20&Itemid=50
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Obsah obrázku fotka, různé, pták Popis byl vytvořen automaticky

Differenciation of bioassays
§According to the trophic level of test organisms:
otests with producers, consumers, destruents
§Depending on the duration of exposure and the nature of the effects:
oacute, semiacute (semichronic), sub-acute, chronic
-the specific length depends on the generation time of the organism (bacteria <<< trout), the
classification is not completely uniform; Division usually into:
acute = 24, 48 to 96 hours, usually assessment of lethality
chronic - days, weeks to months, evaluation of non-lethal effects
§According to the number of species involved:
osingle species, two species, multi-species
o
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figure4 Eisenia_fetida scenedesmus_quadricauda_upr
http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/210px-Escherichi
aColi_NIAID.jpg

Differenciation of bioassays
§According to the level of the biological system (and complexity):
oenzymes, bioprobes, in vitro cell and tissue cultures, intact living organism, population, micro /
mesocosm, field experiments
o
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C:\Documents and Settings\Ludek Blaha\Plocha\1.gif

Differenciation of bioassays
§According to the type of sample:
ochemical substance, mixture of substances, natural sample from the environment
§According to the tested matrix:
owater, soil, air, sediment, waste, chemical
§According to the sample modification:
oleachate (organic solvent, DMSO, water…), contact (Solid Phase Tests), direct (Direct tests, Whole
effluent test), TIE - toxicity identification evaluation
§According to the evaluated effect:
omortality tests, reproduction tests, escape tests, growth tests, teratogenicity tests,
carcinogenicity, xenoestrogenicity, etc.
§According to implementation:
oin situ and in vitro
§+ process bioassays: bioaccumulation, bioconcentration, biodegration
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Ekological relevance of the bioassays
§the tested species should represent the relevant functional group
§the test should respect the ecology of the organism
§monitored responses should be ecologically relevant and indicate the state and function of the
organism (survival, growth, reproduction, food intake and mobility)
§when monitoring reproduction, the exposure should cover most of the life cycle
§abiotic and biotic factors in the test should be similar to those in the habitat
§exposure paths should mimic real exposures
§the bioavailability of the contaminant should be similar to that in reality
§concentrations should be environmentally realistic
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Ekological relevance of test species
§play a key role in the functioning of the ecosystem
§they occur in a number of ecosystems in higher abundance
§easy to use in field and laboratory conditions
§they come into contact with pollutants
§they are sensitive enough to stress
§
The problem of ecotoxicology in general:
I will use organisms A in the tests (for a number of reasons), but the target organisms in the
system are B à what is the relationship of the results for A and B?
Example: Eisenia fetida - the most famous soil test
§
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Earthworm species
Pic1
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Differenciation of bioassays
§limit test / comparison test
§concentration – response tests – preliminary, final
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Standard-ability, legislation …
§Law given tests
oVery little, especially for new chemicals, pesticides, waste
oThe big boom in the use of bioassays in recent years - ecological criteria for environmental
quality
§Standardized, standardized
oMany tests
oStandardization ≠ duty, binding
oEconomic reasons - accreditation of laboratories
§Experimental
oA series of tests
oSpace for efforts to achieve ecological realism
oApplication of new knowledge about mechanisms and effects
oEcological studies
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Norms, standards, guidelines
Objective: to reduce interlaboratory variability
Over time, standard procedures have been developed for evaluating effects in laboratory tests up to
in situ bioindication methods
Advantages:
oguaranteeing uniformity and repeatability of results
ocomparability of results from different laboratories following the procedure
ovalidated results suitable for decision making
olittle need for optimization
Disadvantages:
overy specific and limited informative value ("acute lethality for Daphnia crustaceans")
ousually suitable only for classification of substances (more - moderately - less toxic ...)
olimited number of standardized procedures, usually simple (acute) effects
odifficult to apply to other situations or to answer other questions
oonly on a few model species - the question of transferability of results
oused in inappropriate situations (research, evaluation of cause and effect)
oit may not be applicable to a real environment
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OECD guidelines – water 1
§
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Organisation for Economic Co-operation and Development
https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef
fects-on-biotic-systems_20745761
Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
Test No. 221: Lemna sp. Growth Inhibition Test
2006
Test No. 238: Sediment-Free Myriophyllum Spicatum Toxicity Test
2014
Test No. 239: Water-Sediment Myriophyllum Spicatum Toxicity Test
2014
Test No. 202: Daphnia sp. Acute Immobilisation Test
2004
Test No. 211: Daphnia magna Reproduction Test
2012
Test No. 231: Amphibian Metamorphosis Assay
2009
Test No. 242: Potamopyrgus antipodarum Reproduction Test
2016
Test No. 243: Lymnaea stagnalis Reproduction Test
2016
Test No. 235: Chironomus sp., Acute Immobilisation Test
2011
Test No. 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment
2004
Test No. 219: Sediment-Water Chironomid Toxicity Using Spiked Water
2004
Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked
Sediment
2010
Test No. 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment
2007
Test No. 224: Determination of the Inhibition of the Activity of Anaerobic Bacteria
2007
Test No. 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)
2010
Test No. 244: Protozoan Activated Sludge Inhibition Test
2017

OECD guidelines – water 2
§
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Organisation for Economic Co-operation and Development
https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef
fects-on-biotic-systems_20745761
Test No. 210: Fish, Early-life Stage Toxicity Test
2013
Test No. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages
1998
Test No. 215: Fish, Juvenile Growth Test
2000
Test No. 234: Fish Sexual Development Test
2011
Test No. 236: Fish Embryo Acute Toxicity (FET) Test
2013
Test No. 203: Fish, Acute Toxicity Test
2019
Test No. 229: Fish Short Term Reproduction Assay
2012
Test No. 204: Fish, Prolonged Toxicity Test: 14-Day Study
1984
Test No. 230: 21-day Fish Assay
2009
Test No. 240: Medaka Extended One Generation Reproduction Test (MEOGRT)
2015
Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA)
2015
Test No. 248: Xenopus Eleutheroembryonic Thyroid Assay (XETA)
2019

OECD guidelines - soil
§
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Organisation for Economic Co-operation and Development
https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef
fects-on-biotic-systems_20745761
Test No. 216: Soil Microorganisms: Nitrogen Transformation Test
2000
Test No. 217: Soil Microorganisms: Carbon Transformation Test
2000
Test No. 207: Earthworm, Acute Toxicity Tests
1984
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
Test No. 232: Collembolan Reproduction Test in Soil
2016
Test No. 220: Enchytraeid Reproduction Test
2016
Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil
2016
Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies(Scathophaga
stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))
2016
Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test
2006
Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test
2006
http://upload.wikimedia.org/wikipedia/commons/thumb/0/0b/Redwiggler1.jpg/240px-Redwiggler1.jpg
http://www.falw.vu.nl/en/Images/Folsomia%20candida_tcm24-30644.jpg
http://www.zuova.cz/sluzby/kontaktni-testy-toxicity2.jpg
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http://beetlespace.wz.cz/druhy/fotky/Oxythyrea_funesta_01.jpg

OECD guidelines - other
§
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Organisation for Economic Co-operation and Development
https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-2-ef
fects-on-biotic-systems_20745761
Test No. 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure
2013
Test No. 213: Honeybees, Acute Oral Toxicity Test
1998
Test No. 214: Honeybees, Acute Contact Toxicity Test
1998
Test No. 245: Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding)
2017
Test No. 246: Bumblebee, Acute Contact Toxicity Test
2017
Test No. 247: Bumblebee, Acute Oral Toxicity Test
2017
Test No. 228: Determination of Developmental Toxicity to Dipteran Dung Flies(Scathophaga
stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae))
2016
Test No. 223: Avian Acute Oral Toxicity Test
2016
Test No. 205: Avian Dietary Toxicity Test
1984
Test No. 206: Avian Reproduction Test
1984

ISO standards – aquatic plants
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ISO Logo
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO 14442:2006
Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials,
volatile compounds, metals and waste water
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 20227:2017
Water quality — Determination of the growth inhibition effects of waste waters, natural waters and
chemicals on the duckweed Spirodela polyrhiza — Method using a stock culture independent
microbiotest
ISO 16191:2013
Water quality — Determination of the toxic effect of sediment on the growth behaviour of
Myriophyllum aquaticum
ISO 10253:2016
Water quality — Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum
tricornutum
ISO 10710:2010
Water quality — Growth inhibition test with the marine and brackish water macroalga Ceramium
tenuicorne
http://www.clean-flo.com/wp-content/uploads/pic-1.jpg
http://www.microscopy-uk.org.uk/mag/imgoct05/Scenedesmus-quadricauda.jpg
http://www.algaebase.org/_mediafiles/algaebase/5B7BE95A076ca2C19Dsxv2CAFF8E/k857fWdJXeJD.jpg
http://rostlinna-akvaria.cz/eshop/obrazky/366-Myriophyllum-matogrossense.jpg

ISO standards – aquatic invertebrates
§
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ISO Logo http://pcwww.liv.ac.uk/~stewp123/Daphnia%20magna.jpg
http://84.244.151.141/~persoone/images/slider/Thamnocephalus_platyurus.jpg
http://www.fcps.edu/islandcreekes/ecology/Arthropods/Scud/Hyaellallam1.jpg
http://caramelosblog.es/wp-content/gallery/fauna/caenorhabditis_elegans_worm.jpg
http://cfb.unh.edu/CFBKey/html/Organisms/PRotifera/GBrachionus/brachionus_calyciflorus/brachionusca
lyciflorus1large.jpg http://www.dr-ralf-wagner.de/Bilder/Heterocypris_incongruens-50x_17.jpg
ISO 6341:2012
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 10872:2020
Water and soil quality — Determination of the toxic effect of sediment and soil samples on growth,
fertility and reproduction of Caenorhabditis elegans (Nematoda)
ISO 14371:2012
Water quality — Determination of fresh water sediment toxicity to Heterocypris incongruens
(Crustacea, Ostracoda)
ISO 14380:2011
Water quality — Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea,
Anostraca)
ISO 14669:1999
Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)
ISO 20665:2008
Water quality — Determination of chronic toxicity to Ceriodaphnia dubia
ISO/TS 18220:2016
Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes
ISO 16303:2013
Water quality — Determination of toxicity of fresh water sediments using Hyalella azteca
ISO 16778:2015
Water quality — Calanoid copepod early-life stage test with Acartia tonsa
ISO 17244:2015
Water quality — Determination of the toxicity of water samples on the embryo-larval development of
Japanese oyster (Crassostrea gigas) and mussel (Mytilus edulis or Mytilus galloprovincialis)
ISO 20666:2008
Water quality — Determination of the chronic toxicity to Brachionus calyciflorus in 48 h
ISO 19820:2016
Water quality — Determination of the acute toxicity to the marine rotifer Brachionus plicatilis
ISO 19827:2016
Water quality — Determination of the acute toxicity to the freshwater rotifer Brachionus
calyciflorus

ISO standards – aquatic vertebrates
§
33
ISO Logo
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 12890:1999
Water quality — Determination of toxicity to embryos and larvae of freshwater fish — Semi-static
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 15088:2007
Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)
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 23893-3:2013
Water quality — Biochemical and physiological measurements on fish — Part 3: Determination of
vitellogenin
ISO 21115:2019
Water quality — Determination of acute toxicity of water samples and chemicals to a fish gill cell
line (RTgill-W1)
http://www.fishbase.org/images/species/Darer_m0.jpg

ISO standards – aquatic microorganisms
§
34
ISO Logo
ISO 11348-1:2007/Amd 1:2018
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 —
Amendment 1
ISO 11348-2:2007/Amd 1:2018
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 —
Amendment 1
ISO 11348-3:2007/Amd 1:2018
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 —
Amendment 1
ISO 10712:1995
Water quality — Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication
inhibition test)
ISO 15522:1999
Water quality — Determination of the inhibitory effect of water constituents on the growth of
activated sludge microorganisms
ISO 11350:2012
Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome
fluctuation test (Ames fluctuation test)
ISO 16240:2005
Water quality — Determination of the genotoxicity of water and waste water — Salmonella/microsome
test (Ames test)
ISO 13829:2000
Water quality — Determination of the genotoxicity of water and waste water using the umu-test
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 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
http://enfo.agt.bme.hu/drupal/sites/default/files/Pseudomonas%20putida_0.jpg
http://www.ou.edu/cas/botany-micro/faculty/pictures/vibrio.jpg
http://4.bp.blogspot.com/-TN0HBeFL1Pk/T3NrRmy44sI/AAAAAAAABI8/uWCONXl_q18/s1600/salmonella.jpg
http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/250px-Escherichi
aColi_NIAID.jpg

ISO standards – in vitro tests
§
35
ISO Logo
ISO 19040-1:2018
Water quality — Determination of the estrogenic potential of water and waste water — Part 1: Yeast
estrogen screen (Saccharomyces cerevisiae)
ISO 19040-2:2018
Water quality — Determination of the estrogenic potential of water and waste water — Part 2: Yeast
estrogen screen (A-YES, Arxula adeninivorans)
ISO 19040-3:2018
Water quality — Determination of the estrogenic potential of water and waste water — Part 3: In
vitro human cell-based reporter gene assay
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/Cor 1:2009
Water quality — Evaluation of genotoxicity by measurement of the induction of micronuclei — Part 2:
Mixed population method using the cell line V79 — Technical Corrigendum 1
ISO/CD 24295
Water quality — Determination of the dioxin-like potential of water and wastewater — Method using
in vitro mammalian cell-based reporter gene assay

ISO standards – biodegradation
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36
ISO Logo
ISO 11733:2004
Water quality — Determination of the elimination and biodegradability of organic compounds in an
aqueous medium — Activated sludge simulation test
ISO 10707:1994
Water quality — Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of
organic compounds — Method by analysis of biochemical oxygen demand (closed bottle test)
ISO 7827:2010
Water quality — Evaluation of the "ready", "ultimate" aerobic biodegradability of organic compounds
in an aqueous medium — Method by analysis of dissolved organic carbon (DOC)
ISO 10708:1997
Water quality — Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic
compounds — Determination of biochemical oxygen demand in a two-phase closed bottle test
ISO 11734:1995
Water quality — Evaluation of the "ultimate" anaerobic biodegradability of organic compounds in
digested sludge — Method by measurement of the biogas production
ISO 14592-1:2002
Water quality — Evaluation of the aerobic biodegradability of organic compounds at low
concentrations — Part 1: Shake-flask batch test with surface water or surface water/sediment
suspensions
ISO 14592-2:2002
Water quality — Evaluation of the aerobic biodegradability of organic compounds at low
concentrations — Part 2: Continuous flow river model with attached biomass
ISO 14593:1999
Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium — Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)
ISO 16221:2001
Water quality — Guidance for determination of biodegradability in the marine environment
ISO 9408:1999
Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium by determination of oxygen demand in a closed respirometer
ISO 9439:1999
Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium — Carbon dioxide evolution test
ISO 9887:1992
Water quality — Evaluation of the aerobic biodegradability of organic compounds in an aqueous
medium — Semi-continuous activated sludge method (SCAS)
ISO 9888:1999
Water quality — Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium — Static test (Zahn-Wellens method)

ISO standards – terrestrial plants
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37
ISO 11269-1:2012
Soil quality — Determination of the effects of pollutants on soil flora — Part 1: Method for the
measurement of inhibition of root growth
ISO 11269-2:2012
Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
contaminated soil on the emergence and early 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 18763:2016
Soil quality — Determination of the toxic effects of pollutants on germination and early growth of
higher plants
ISO 22030:2005
Soil quality — Biological methods — Chronic toxicity in higher plants
ISO 29200:2013
Soil quality — Assessment of genotoxic effects on higher plants — Vicia faba micronucleus test
ISO 21479:2019
Soil quality — Determination of the effects of pollutants on soil flora — Leaf fatty acid
composition of plants used to assess soil quality
ISO Logo http://media-2.web.britannica.com/eb-media/69/10269-004-EEF044DB.jpg
http://upload.wikimedia.org/wikipedia/commons/thumb/e/e1/Illustration_Vicia_faba1.jpg/240px-Illustr
ation_Vicia_faba1.jpg
http://lh3.google.com/luirig/R5yaRJTetsI/AAAAAAAAOL8/WO5-p8bmIQE/s800/sinapis_alba_2.jpg

ISO standards – soil invertebrates
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38
ISO 11268-1:2012
Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei
ISO 11268-3:2014
Soil quality — Effects of pollutants on earthworms — Part 3: Guidance on the determination of
effects in field situations
ISO 11267:2014
Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants
ISO 16387:2014
Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of
effects on reproduction
ISO 21285:2019
Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil
contaminants
ISO 23266:2020
Soil quality — Test for measuring the inhibition of reproduction in oribatid mites (Oppia nitens)
exposed to contaminants in soil
ISO 15952:2018
Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of the
effects on growth by soil contamination
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 E. andrei)
ISO 17512-2:2011
Soil quality — Avoidance test for determining the quality of soils and effects of chemicals on
behaviour — Part 2: Test with collembolans (Folsomia candida)
ISO 20963:2005
Soil quality — Effects of pollutants on insect larvae (Oxythyrea funesta) — Determination of acute
toxicity
ISO 18311:2016
Soil quality — Method for testing effects of soil contaminants on the feeding activity of soil
dwelling organisms — Bait-lamina test
ISO/DIS 24032
Soil quality — In situ caging of snails to assess bioaccumulation of contaminants
ISO 23611-1:2018
Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and 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 23611-3:2019
Soil quality — Sampling of soil invertebrates — Part 3: Sampling and extraction of enchytraeids
ISO 23611-4:2007
Soil quality — Sampling of soil invertebrates — Part 4: Sampling, extraction and identification of
soil-inhabiting nematodes
ISO 23611-5:2011
Soil quality — Sampling of soil invertebrates — Part 5: Sampling and extraction of soil
macro-invertebrates
ISO 23611-6:2012
Soil quality — Sampling of soil invertebrates — Part 6: Guidance for the design of sampling
programmes with soil invertebrates
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ISO standards – soil microorganisms
§
39
ISO 14238:2012
Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in
soils and the influence of chemicals on these processes
ISO 15685:2012
Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid
test by ammonium oxidation
ISO 18187:2016
Soil quality — Contact test for solid samples using the dehydrogenase activity of Arthrobacter
globiformis
ISO 17155:2012
Soil quality — Determination of abundance and activity of soil microflora using respiration curves
ISO/TS 10832:2009
Soil quality — Effects of pollutants on mycorrhizal fungi — Spore germination test
ISO/CD 23265
Soil quality — Test for estimating organic matter decomposition in contaminated soil
ISO 16072:2002
Soil quality — Laboratory methods for determination of microbial soil respiration
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 23753-1:2019
Soil quality — Determination of dehydrogenases activity in soils — Part 1: Method using
triphenyltetrazolium chloride (TTC)
ISO 23753-2:2019
Soil quality — Determination of dehydrogenases activity in soils — Part 2: Method using
iodotetrazolium chloride (INT)
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/TS 29843-2:2011
Soil quality — Determination of soil microbial diversity — Part 2: Method by phospholipid fatty
acid analysis (PLFA) using the simple PLFA extraction method
ISO 11063:2020
Soil quality — Direct extraction of soil DNA
ISO 17601:2016
Soil quality — Estimation of abundance of selected microbial gene sequences by quantitative PCR
from DNA directly extracted from soil
ISO 20130:2018
Soil quality — Measurement of enzyme activity patterns in soil samples using colorimetric
substrates in micro-well plates
ISO/TS 20131-1:2018
Soil quality — Easy laboratory assessments of soil denitrification, a process source of N2O
emissions — Part 1: Soil denitrifying enzymes activities
ISO/TS 20131-2:2018
Soil quality — Easy laboratory assessments of soil denitrification, a process source of N2O
emissions — Part 2: Assessment of the capacity of soils to reduce N2O
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 14239:2017
Soil quality — Laboratory incubation systems for measuring the mineralization of organic chemicals
in soil under aerobic conditions
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US EPA - US Environmental Protection Agency
OPPTS - Office of Prevention, Pesticides & Toxic Substances
Group A - Aquatic and Sediment-dwelling Fauna and Aquatic Microcosms
40
https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t
est-guidelines
850.1010 - Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids (December 2016)
850.1300 - Daphnid Chronic Toxicity Test (December 2016)
850.1020 - Gammarid Amphipod Acute Toxicity Test (December 2016)
850.1025 - Oyster Acute Toxicity Test (Shell Deposition) (December 2016)
850.1035 - Mysid Acute Toxicity Test (December 2016)
850.1045 - Penaeid Acute Toxicity Test (December 2016)
850.1055 - Bivalve Acute Toxicity Test (Embryo-Larval) (December 2016)
850.1710 - Oyster Bioconcentration Factor (BCF) (December 2016)
850.1075 - Freshwater and Saltwater Fish Acute Toxicity Test (December 2016)
850.1400 - Fish Early Life Stage Toxicity Test (December 2016)
850.1730 - Fish Bioconcentration Factor (BCF) (December 2016)
850.1735 - Spiked Whole Sediment 10-Day Toxicity Test , Freshwater Invertebrates (December 2016)
850.1740 - Spiked Whole Sediment 10-Day Toxicity Test, Saltwater Invertebrates (December 2016)

US EPA - US Environmental Protection Agency
OPPTS - Office of Prevention, Pesticides & Toxic Substances
Group B – Terrestrial Wildlife
41
https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t
est-guidelines
850.2100 - Avian Acute Oral Toxicity Test (June 2012)
850.2200 - Avian Dietary Toxicity Test (June 2012)
850.2300 - Avian Reproduction Test (June 2012)
850.2400 - Wild Mammal Toxicity Testing (June 2012)
850.2500 - Field Testing for Terrestrial Wildlife (June 2012)

US EPA - US Environmental Protection Agency
OPPTS - Office of Prevention, Pesticides & Toxic Substances
Group C – Terrestrial Beneficial Insects, Invertebrates, and Soil and Wastewater Microorganisms
42
https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t
est-guidelines
850.3020 - Honey Bee Acute Contact Toxicity Test (June 2012)
850.3030 - Honey Bee Toxicity of Residues on Foliage (June 2012)
850.3040 - Field Testing for Pollinators (June 2012)
850.3100 - Earthworm Subchronic Toxicity Test (June 2012)
850.3200 - Soil Microbial Community Toxicity Test (June 2012)
850.3300 - Modified Activated Sludge, Respiration Inhibition Test (June 2012)

US EPA - US Environmental Protection Agency
OPPTS - Office of Prevention, Pesticides & Toxic Substances
Group D – Terrestrial and Aquatic Plants, Cyanobacteria, and Terrestrial Soil Core Microcosm
43
https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-t
est-guidelines
850.4230 - Early Seedling Growth Toxicity Test (June 2012)
850.4100 - Seedling Emergence and Seedling Growth (June 2012)
850.4150 - Vegetative Vigor (June 2012)
850.4800 - Plant Uptake and Translocation Test (June 2012)
850.4300 - Terrestrial Plants Field Study (June 2012)
850.4500 - Algal Toxicity (June 2012)
850.4550 - Cyanobacteria (Anabaena flos-aquae) Toxicity (June 2012)
850.4400 - Aquatic Plant Toxicity Test Using Lemna spp. (June 2012)
850.4450 - Aquatic Plants Field Study (June 2012)
850.4600 - Rhizobium-Legume Toxicity (June 2012)
850.4900 - Terrestrial Soil-Core Microcosm Test (June 2012)