Bi2003 Ecotoxicology
Ecotoxicological bioassays
Jakub Hofman
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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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Aquatic bioassays - examples
3


Aquatic bioassays
§huge number of the tests (aquatic ecotoxicology had been for a long time the only one
ecotoxicology)
§today, standardized bioassays cover the whole range of levels:
osuborganismal level
-laboratory experiments: study of toxicity mechanisms, in vitro biomarkers, specific types of
toxicity (dioxin like toxicity, xenoestrogenicity ...)
oindividual species, individuals
-laboratory experiments: traditional ecotoxicological bioassays with individual species, comparison
of susceptibility of different species ...
opopulation effects
-laboratory tests - longer-term experiments - lifelong toxicity tests, tests with early
developmental stages, plants - reproduction, germination ..., invertebrates - vertebrates -
reproductive toxicity tests
4

Aquatic bioassays
§today, standardized bioassays cover the whole range of levels (cont):
oeffects in communities
-laboratory microcosms - artificially established communities of organisms of various species under
defined conditions (producers - consumers - destruents)
-field multispecies manipulated in situ studies - field studies, aquatic mesocosms (flowing,
standing, littoral – coastal
ofield manipulated studies (in situ)
-studies with individual species - growing plants on contaminated and control areas, cage
experiments in aquatic environments (molluscs, fish)
oecosystem effects (these are, in fact, not „bioassays“ but „bioindication“)
-field observation - evaluation of effects in populations and communities in a real natural
situation
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Aquatic bioassays - producers
§cyanobacteria – photosynthetic, nitrogen-fixing eubacteria; colonies, filaments, single cells
§unicellular algae – eucaryotic; cells, filamentous, colonies; freshwater or marine
§both usually evaluated by the change in the number of cells (growth) – measured often as green
color
§
§vascular plants – aquatic plants
§size, length – growth, mortality
§
§other endpoints: concentration of pigments (eg chlorophyll a), physiological activity (nutrient
intake) and metabolic activity (photosynthetic activities, enzymatic activities)
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Aquatic bioassays – producers - algae
§unicellular algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum),
Desmodesmus subspicatus (formerly Scenedesmus subspicatus), diatoms (Navicula pelliculosa) or
cyanobacteria Anabaena flos-aquae, Synechococcus leopoliensis
§cultures or lyophylised stock may be used
§equipment needed with high light intensity
about 60-120 μE m-2 s-1 (~4500-9000 lux)
at 400-700 nm
§inicial density of the culture:
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Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO Logo Organisation for Economic Co-operation and Development

Aquatic bioassays – producers - algae
§defined medium Ã
§72 h exposure at light and 20-24°C
§algal biomass determination: cell counts (electronic or microscope or flow cytometer) or
fluorescence or color (fluorimeter, spectrophotometer)
§
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Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO Logo Organisation for Economic Co-operation and Development

Aquatic bioassays – producers - algae
validity
§controls:
oexponential growth by factor of > 16 over 72 h
ocoefficient of variation (relative standard deviation, i.e. standard deviation divided by mean) <
35% (each day evaluation) and for average specific growth rate < 7%
§reference substance:
oe.g. 3,5-dichlorophenol or potassium dichromate should be tested at least twice a year
oeffect (EC50) must be within the prescribed range
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Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO Logo Organisation for Economic Co-operation and Development

Aquatic bioassays – producers - algae
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Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO Logo Organisation for Economic Co-operation and Development

Aquatic bioassays – producers - algae
miniaturization
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Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test
2011
ISO 8692:2012
Water quality — Fresh water algal growth inhibition test with unicellular green algae
ISO Logo Organisation for Economic Co-operation and Development
http://ebpi.ca/_slideshows_Algaltoxkit%20F%20slide%20show.pdf

Aquatic bioassays – producers - plants
§„duckweed“, Lemna minor or gibba
§start with 10 leaves (fronds) per 1 beaker
§several recommended special media
§96 h; 6500 - 10000 lux; pH 6.5; 24°C
§growth: biomass – weight and no. of fronds (image analysis possible)
validity:
§control: doubling time < 2.5 days
§reference compound: 3,5-dichlorophenol or K2Cr2O7 (EC50: 10-60 mg/L)
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ISO Logo Organisation for Economic Co-operation and Development
Test No. 221: Lemna sp. Growth Inhibition Test
2006
ISO 20079:2005
Water quality — Determination of the toxic effect of water constituents and waste water on duckweed
(Lemna minor) — Duckweed growth inhibition test
figure4

Aquatic bioassays – producers - plants
§miniaturization with Spirodela
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ISO Logo
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
https://www.microbiotests.com/wp-content/uploads/2019/07/duckweed-toxicity-test_duckweed-toxkit-f_s
tandard-operating-procedure.pdf

Aquatic bioassays – producers - plants
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ISO Logo Organisation for Economic Co-operation and Development
ISO 16191:2013
Water quality — Determination of the toxic effect of sediment on the growth behaviour of
Myriophyllum aquaticum
ISO 10710:2010
Water quality — Growth inhibition test with the marine and brackish water macroalga Ceramium
tenuicorne
Test No. 238: Sediment-Free Myriophyllum Spicatum Toxicity Test
2014
Test No. 239: Water-Sediment Myriophyllum Spicatum Toxicity Test
2014

Aquatic bioassays – consumers - invertebrates
§are very very common - sometimes ecotoxicology is confused with "Daphnia bioassays„
§standard layouts:
obeakers/vessels, acute tests 1-few days, extended tests 21 d
oevaluation of lethality, growth ... short-term - usually static
oevaluation of reproduction etc. ... longer exposures, need for food, well controlled supply of
oxygen ...
§aquatic planktonic crustaceans - the most common
oDaphnia magna, Ceriodaphnia dubia, Artemia salina (marine)
§other invertebrates
obenthic - Gammarus, Hyallela azteca
ooligochaetic worms - Tubifex, Lumbriculus
osnails – sand snail
oinsects - midges (Chironomus), mayflies ...
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C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg

Aquatic bioassays – consumers - invertebrates
§also many of them developed to microbiotests
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https://www.microbiotests.com
http://ebpi.ca/_slideshows_Daphtoxkit%20F%20magna%20slide%20show.pdf

Aquatic bioassays – consumers - invertebrates
Daphnia magna acute test
§5 juvenile daphnids per replicate (min 2 ml)
§medium = so called reconstituted water
1.CaCl2 · 2H2O 11,76 g/l
2.MgSO4 · 7H2O 4,93 g/l
3.NaHCO3 2,59 g/l
4.KCl 0,23 g/l
mix 25 ml of each to 1 L, pH 7.8, aeration
§24h, 48h; dark or 16h light / 8h dark;
O2 > 80 % (2 mg/l); 20°C; no food
§mortality = immobilization
validity:
§control: mortality< 10 %;
§reference compound: K2Cr2O7 (LC50: mg/L)
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ISO Logo Organisation for Economic Co-operation and Development
Test No. 202: Daphnia sp. Acute Immobilisation Test
2004
ISO 6341:2012
Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera,
Crustacea) — Acute toxicity test
C:\DOCUME~1\MARTIN~1\LOCALS~1\Temp\\msotw9_temp0.bmp
C:\DOCUME~1\MARTIN~1\LOCALS~1\Temp\\msotw9_temp0.bmp

Aquatic bioassays – consumers - invertebrates
Daphnia magna acute test
§
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ISO Logo Organisation for Economic Co-operation and Development
Test No. 202: Daphnia sp. Acute Immobilisation Test
2004
ISO 6341:2012
Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera,
Crustacea) — Acute toxicity test
other videos:
https://www.youtube.com/watch?v=ElENqCeGNSA  https://www.youtube.com/watch?v=3AxO36DLjsU

Aquatic bioassays – consumers - invertebrates
Daphnia magna reproduction test
§24 h juveniles, 10 per replicate, 50 ml or more
§medium reconstituted water or M4/M7
§21 days; 16/8 light/dark; O2 > 3 mg/l);
20°C; food (algae)
§week check, aeration or medium change
§mortality + number of juveniles
+ other parameters (behavior, malformations ...)
validity:
§control: mortality< 10 %;
§reference compound: K2Cr2O7 (LC50: mg/L)
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ISO Logo Organisation for Economic Co-operation and Development
Test No. 211: Daphnia magna Reproduction Test
2012
ISO 10706:2000
Water quality — Determination of long term toxicity of substances to Daphnia magna Straus
(Cladocera, Crustacea)

Aquatic bioassays – consumers - invertebrates
other invertebrates used in the bioassays
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ISO Logo Organisation for Economic Co-operation and Development
Test No. 242: Potamopyrgus antipodarum Reproduction Test
2016
Test No. 243: Lymnaea stagnalis Reproduction Test
2016
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 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 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)
Obsah obrázku jídlo Popis byl vytvořen automaticky

Aquatic bioassays – consumers - fish
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...

Aquatic bioassays – consumers - fish
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§fish cultures require specific equipment – culture labs with very controlled conditions
§need for special approval to work experiments with vertebrates !!!

Aquatic bioassays – consumers - fish
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§fish cultures require specific equipment – culture labs with very controlled conditions

Aquatic bioassays – consumers - fish
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§standard layout:
oaquaria smaller or larger, aeration needed
ovarious arrangements (static, flow-through ...)
oacute tests - 96 h, prolonged tests - days to months
olethality, growth, reproduction
olot of sublethal endpoints possible: behavior, spasms, food intake, breathing, health, histology,
bioaccumulation, teratogenicity, carcinogenicity, xenoestrogenicity è è è

Aquatic bioassays – consumers - fish
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ISO Logo Organisation for Economic Co-operation and Development
Acute toxicity test
§juvenile fish, acclimatized
§medium: reconstituted water, groundwater or clean water
§24-96 h
§conditions depend on species, e.g. pH, temp, photoperiod
§disolved O2 >60% saturation
§no feeding
§mortality, size, weight
validity
§controls: mortality < 10%, O2 etc.
§dissolved O2 ≥ 60% of air saturation
§analytical measurement of test concentrations is compulsory
Test No. 203: Fish, Acute Toxicity Test
2019
ISO 7346-1:1996
Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish
(Brachydanio rerio ...) - Part 1: Static method
ISO 7346-2:1996
Part 2: Semi-static method
ISO 7346-3:1996
Part 3: Flow-through method

Aquatic bioassays – consumers - fish
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ISO Logo Organisation for Economic Co-operation and Development
Prolonged, chronic toxicity tests
§prolonged = longer exposure with mortality endpoint, sublethal endpoints also recorded
§from 14 to 28 d
§chronic = also other endpoints like reproduction
§7-200 d
Test No. 210: Fish, Early-life Stage Toxicity Test
2013
Test No. 215: Fish, Juvenile Growth Test
2000
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
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))

Aquatic bioassays – consumers - fish
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ISO Logo Organisation for Economic Co-operation and Development
Embryonal, embryolarval tests
§fertilized eggs are exposed to chemicals for several days
§effects on development = teratogenicity
§prolonged to hatching and larvae development
§endpoints: hatching, survival, development, behavior, size
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. 236: Fish Embryo Acute Toxicity (FET) Test
2013
ISO 15088:2007
Water quality — Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)

1
Aquatic bioassays – consumers - frog
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FETAX – Frog Embryo Teratogenicity Assay Xenopus
§fertilized eggs are exposed to chemicals for several days
§effects on development = teratogenicity
frog_pict C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek
Blaha\Plocha\1.gif

Aquatic bioassays – destruents - bacteria
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ISO Logo
Vibrio fisheri luminescece test
§marine bacteria - bad for samples with high minerals or organic matter - stimulation
§very quick - 5-30 min
§luminiscence inhibition
§problem with particles and colour è flash test
§
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
Part 2: Method using liquid-dried bacteria — Amendment 1
ISO 11348-3:2007/Amd 1:2018
Part 3: Method using freeze-dried bacteria — Amendment 1
figure4 1

Aquatic bioassays – destruents - bacteria
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ISO Logo
Pseudomonas putida growth inhibition test
§...
ISO 10712:1995
Water quality — Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication
inhibition test)

Aquatic bioassays – destruents - bacteria
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ISO Logo
Mutation and genotoxicity tests
§with bacteria, but used also for general testing of mutagenicity and genotoxicity
§with liver fraction S9 simulate activation of xenobiotics within de-toxification in vertebrates
Salmonella sp. – Ames test
§mutants cannot live in medium without histidine
§in case of mutagenic chemical the reverse mutation is induces – they grow
Umu-C
§transgenic bacteria with luciferase gene introduced to operon for DNA reparation
§genotoxicity causes activation of reparation and thus luciferase and light
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

Aquatic bioassays – destruents - bacteria
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Testing the effects on activated sludge
§important because of biotechnologies – water treatment plants
§complex microbial community and its activities: respiration, nitrification
§in erlenmeyer flasks etc.
§measurements of biological oxygen demand (BOD) or respirometry
ISO Logo Organisation for Economic Co-operation and Development
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
ISO 15522:1999
Water quality — Determination of the inhibitory effect of water constituents on the growth of
activated sludge microorganisms
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

Aquatic bioassays – sediments
§sediment = aquatic equivalent of the soil è very heterogenic matrix, contains solid phase (mineral
particles and organic matter) and pore water
§sediment is often potential long-term reservoir / source of contaminants
§distribution of contaminatns between water and sediment (sorption) – sediment organisms are
exposed to contaminants in solid and/or liquid components of sediment
§benthic organisms
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Aquatic bioassays – sediments
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Chironomus tentans
Chironomus riparius
C:\Documents and Settings\Ludek Blaha\Plocha\figure4.jpg
Tubifex tubifex
http://www.aquarium-kosmos.de/bilder/streifzuege/Lumbriculus%20variegatus_2.jpg
Lumbriculus variegatus
Heterocypris incongruens
Hyalela aztecaincongruens

Aquatic bioassays – consumers - invertebrates
Tests with chironomids
OECD 235
§same as Daphnia acute test, just with Chironomus larvae
OECD 218+219
§10 larvae (cca 10d old) per beaker
§OECD artificial sediment
§100 ml sediment / 175 ml water
§21-28 d; 16/8 h light/dark; controled pH, O2
§food
§survival and growth
OECD 233
§reproduction = development to midges
§
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Organisation for Economic Co-operation and Development
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
IMG_0054 IMG_0061 IMG_0059

Soil bioassays - examples
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Soil bioassays – producents - plants
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§large number of different tests
various endpoints
§seed germination and root elongation
§emergence
§seedling growth
§biomass production
§life cycle (changes in weight, size, number of flowers, seeds ..)
§physiological tests (photosynthesis, respiration)
§enzymatic tests
§symbiosis – N fixation, mycorhiza

Soil bioassays – producents - plants
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species
§usually needed at least monocolyledenous  + dicolyledenous
§Sinapis alba, Lactuca sativa, Lepidium sativum, Hordeum vulgare, Zea mays
§
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 Hordeum
vulgare - Barley - THE WORLDWIDE VEGETABLES botany, corn (Zea mays), field, Germany,
Additional-Rights-Clearance-Info-Not-Available Stock Photo - Alamy

Soil bioassays – producents - plants
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Root elongation inhibition
§preparation of seeds to 2 mm
§15 seeds to 200-300 g soil
§3-5 d; soil pH; dark; 24°C
§root length
ISO Logo
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
P1010052 pokus IMG_0215 IMG_0196 IMG_7021_al IMG_7034 IMG_7022_al

Soil bioassays – producents - plants
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Emergence, early growth, growth, chronic
ISO Logo Organisation for Economic Co-operation and Development IMG_0511 IMG_0502 IMG_0819 IMG_0825
plant01.jpg (54.086 Byte)
Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test
2006
Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test
2006
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

Soil bioassays – producents - plants
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§
https://www.microbiotests.com
http://ebpi.ca/_slideshows_Phytotoxkit%20slide%20show.pdf

Soil bioassays – consumers - invertebrates
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Earthworms
§earthworms are the most and longest time used representative of soil organisms in ecotoxicology –
the oldest test OECD from 1984
§Benefits and reasons:
othe whole development cycle takes place in the soil - a typical geobiont
oconsume large amounts of soil (high food exposure and accumulation of contaminants)
ohave very close physical contact with soil (skin exposure + oral)
othey have significant bioaccumulation and bioconcentration character – macroconcentrators
overy significant role in soil formation, decomposition processes, soil fertility
okey role in the transfer of pollutants in food chains
ooccurrence in almost all soils in high numbers and weights
owell introduced in laboratory tests (easy breeding)
oeasily identified in real samples (thanks to size)...
STUPD00Z Lumdiap2

Soil bioassays – consumers - invertebrates
43
Earthworms - acute
§Eisenia fetida / Eisenia andrei
§culcure in mixture manure/soil/garden-substrate/peat
§7,14 days
§500 g soil; 50-60 % water holding capacity
§artificial soil or LUFA 2.2 or other
§
ISO Logo Organisation for Economic Co-operation and Development
Test No. 207: Earthworm, Acute Toxicity Tests
1984
ISO 11268-1:2012
Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
Eisenia Cycle CE8a-cocoon LR
http://t3.gstatic.com/images?q=tbn:ANd9GcR7-3EeXeOV0fHO4s2TOGWEnFANnR74gi6jRAso6-WCvT_OWvmIZlw3FsoM
yA 11

Soil bioassays – consumers - invertebrates
44
Earthworms - acute
§
ISO Logo Organisation for Economic Co-operation and Development
Test No. 207: Earthworm, Acute Toxicity Tests
1984
ISO 11268-1:2012
Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
62 71 019 012 019 012

Soil bioassays – consumers - invertebrates
45
Earthworms - reproduction
§2 months: after 1 month adults removed, after 2nd month juveniles counted
§soil and conditions same as for acute test but food added (dung)
§various techniques how to extract juveniles from soil (usually heat)
ISO Logo Organisation for Economic Co-operation and Development
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei
068

Soil bioassays – consumers - invertebrates
46
Earthworms - reproduction
ISO Logo Organisation for Economic Co-operation and Development
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei
WHC vesselsready controlwithworms wormsweighing wahingwormsJPG
Soil preparation
WHC measurement
Water added
Soil weighted to jars
10 adults from culture
Washed
Weighting worms
10 adults to 1 jar
drysoils

Soil bioassays – consumers - invertebrates
47
Earthworms - reproduction
ISO Logo Organisation for Economic Co-operation and Development
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei
wormssearching wormsweighing bottomofvessel
Control of the jars, activity markers
Mortality assessment
Weighting the worms
trolleywithworms
Temperated room

Soil bioassays – consumers - invertebrates
48
Earthworms - reproduction
ISO Logo Organisation for Economic Co-operation and Development
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei
waterbathS
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

Soil bioassays – consumers - invertebrates
49
Earthworms - reproduction
§...
ISO Logo Organisation for Economic Co-operation and Development
Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)
2016
ISO 11268-2:2012
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei

Soil bioassays – consumers - invertebrates
50
Enchytraeids
§Enchytraeus albidus or Enchytraeus crypticus
§28 d; 20-30 g soil; 50-60% water holding capacity
§artificial soil or LUFA 2.2 or other
ISO Logo Organisation for Economic Co-operation and Development
Test No. 220: Enchytraeid Reproduction Test
2016
ISO 16387:2014
Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of
effects on reproduction
grindal_worm IMG_3421_1 006 Image14

Soil bioassays – consumers - invertebrates
51
Enchytraeids
ISO Logo Organisation for Economic Co-operation and Development
Test No. 220: Enchytraeid Reproduction Test
2016
ISO 16387:2014
Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of
effects on reproduction
IMG_3360 019 024 027 032 IMG_2981 IMG_2893 pokus IMG_2984 031

Soil bioassays – consumers - invertebrates
52
Enchytraeids
ISO Logo Organisation for Economic Co-operation and Development
Test No. 220: Enchytraeid Reproduction Test
2016
ISO 16387:2014
Soil quality — Effects of contaminants on Enchytraeidae (Enchytraeus sp.) — Determination of
effects on reproduction

Soil bioassays – consumers - invertebrates
53
Springtails
§Folsomia candida partenogenetic collembola or Folsomia fimetaria
§28 d; 20-30 g soil; 50-60% water holding capacity
§artificial soil or LUFA 2.2 or other
Test No. 232: Collembolan Reproduction Test in Soil
2016
ISO 11267:2014
Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants
ISO Logo Organisation for Economic Co-operation and Development 005 008 016

Soil bioassays – consumers - invertebrates
54
Springtails
Test No. 232: Collembolan Reproduction Test in Soil
2016
ISO 11267:2014
Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants
ISO Logo Organisation for Economic Co-operation and Development IMG_2850 005
http://www.chaloupky.cz/fotografie/foto-150-640.jpg 012 023 024 028 031 NS4 P1010056 IMG_2850 pokus
3

Soil bioassays – consumers - invertebrates
55
Springtails
Test No. 232: Collembolan Reproduction Test in Soil
2016
ISO 11267:2014
Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants
ISO Logo Organisation for Economic Co-operation and Development

Soil bioassays – consumers - invertebrates
56
Avoidance tests
§with earthworms or springtails
§very quick (1-2 days) - screening
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 Logo DSC07739

Soil bioassays – consumers - invertebrates
57
Mites
§...
Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil
2016
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 Logo Organisation for Economic Co-operation and Development
Hypoaspis
aculeifer
olog
prey
predator
cont. soil
0471971030
BOLD Systems: Taxonomy Browser - Oppia nitens {species}

Soil bioassays – consumers - invertebrates
58
Snails
§...
ISO 15952:2018
Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of the
effects on growth by soil contamination
ISO Logo haas_schnecke 散大蜗牛

Soil bioassays – consumers - invertebrates
59
Nematodes
§the most abundant soil invertebrates
§in fact aquatic organisms – pore water
§very fast tests - short life cycle - screening
C. elegans life cycle C. elegans anatomy

Soil bioassays – consumers - invertebrates
60
Nematodes
§Caenorhabditis elegans
§grown on agar plates with E. coli
§aseptic techniques and careful handling needed !
§24-48 h exposure
ISO Logo
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)
ASTM E 2172 – 01 Standard Guide for Conducting Laboratory Soil Toxicity Tests with the Nematode
Caenorhabditis elegans
J3 stage P.redivivus body1a 082

Soil bioassays – consumers - invertebrates
61
Beneficial arthropods
§testing of pesticides
§natural enemies of pests

Soil bioassays – destruents – microorganisms
62
Whole community testing for microbial activities
§real uncontaminated agricultural soil with indigenous microflora of desired properties:
osand > 70%; TOC = 1.5%; pH(KCl) = 7–7.5
omicrobial biomass 400-700 µg C . gdw-1; basal respiration 0.5-0.7 µg CO2-C . h-1 . gdw-1
§
ISO Logo Organisation for Economic Co-operation and Development
Test No. 216: Soil Microorganisms: Nitrogen Transformation Test
2000
Test No. 217: Soil Microorganisms: Carbon Transformation Test
2000
ISO 14238:2012
Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in
soils and the influence of chemicals on these processes
Lokalita_04_05b

Soil bioassays – destruents – microorganisms
63
Whole community testing for microbial activities
ISO Logo Organisation for Economic Co-operation and Development
Test No. 216: Soil Microorganisms: Nitrogen Transformation Test
2000
Test No. 217: Soil Microorganisms: Carbon Transformation Test
2000
ISO 14238:2012
Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in
soils and the influence of chemicals on these processes
Soil sampling
Storage
Pre-incubation
(7 days)
START
Substance application
Negative
control
Positive
control
7th day

14th day
21st day
28th day
Microbial parameters
OECD, ISO: respiration and N-mineralization
10 g per replicate
aerobic conditions
60% WHC; 22°C; dark
ISO 16072:2002
Soil quality — Laboratory methods for determination of microbial soil respiration
respiration by:

Soil bioassays – destruents – microorganisms
64
Whole microbial community testing
§same design of exposure as in OECD 216, 217
§also other parameters can be measured !!!
ISO Logo
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
biomass
enzyme activity
diversity
•structural
•genetic
•functional
denitrification

Soil bioassays – destruents – microorganisms
65
Whole community testing for microbial activities – quick tests
Potential ammonium oxidation
§= ammonification, first step of nitrification
§3 h oxidation of ammonium sulfate
§sodium chlorate added – stops nitrite oxidation to nitrate
§nitrite measured by colorimetric test
Respiration curves
§they indicate the growth of microbial community
§respirometry – increase of CO2 or decrease of O2
§
ISO Logo
ISO 15685:2012
Soil quality — Determination of potential nitrification and inhibition of nitrification — Rapid
test by ammonium oxidation
ISO 17155:2012
Soil quality — Determination of abundance and activity of soil microflora using respiration curves
92

Soil bioassays – destruents – microorganisms
66
Arthrobacter globiformis test
§introduced bacteria species NOT whole community
§contaminated soil or other solid materials (waste) or soil spiked with tested chemical(s)
§A. globiformis added and dehydrogenase activity measured
§
ISO Logo
ISO 18187:2016
Soil quality — Contact test for solid samples using the dehydrogenase activity of Arthrobacter
globiformis
Arthrobacter | coryneforms-2

Terrestrial species bioassays - examples
67


Terrestrial species bioassays - polinators
§bees or bumblebees
§brood development of cells (eggs or larvae)
§acute oral or contact tests, prolonged-chronic tests – mortality, behavior
§semi-field tests [tunnel tests (~100 m2)] and field tests - mortality, flight density, development
of bee colonies (brood and feed), behavior of bee colonies
§residue analysis studies in relevant feed matrices
§
§
68
Obsah obrázku květina, tráva, žlutá, pole Popis byl vytvořen automaticky Obsah obrázku tráva,
exteriér, žlutá Popis byl vytvořen automaticky Obsah obrázku osoba, interiér Popis byl vytvořen
automaticky

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
Terrestrial species bioassays - polinators
Acute oral test
§adult worker honey bees
§doses of test substance in sugar solution (100-200 μl, 50% sugar solution)
§after consuming food (3-4 hours), the feeding device is removed
§mortality is recorded daily for at least 48 h
Acute contact test
§test substance dissolved in a suitable vehicle
§applied directly to anesthetized bees – 1 µl droplets to back area of each bee chest
§control for carrier necessary
§
69
Organisation for Economic Co-operation and Development

Terrestrial species bioassays
Birds
§dietary toxicity tests
§dietary dosage + 14 days of observation
§reproduction tests - long-term
§
70
Test No. 223: Avian Acute Oral Toxicity Test
2016
Test No. 205: Avian Dietary Toxicity Test
1984
Organisation for Economic Co-operation and Development
Japanese quail
Bobwhite quail
Malard duck
Japanese quail - Wikipedia Northern Bobwhite (Colinus virginianus) – Planet of Birds Feral pigeon -
Wikipedia
Feral pigeon

Terrestrial species bioassays
Birds
§20 weeks of feeding by contaminated feed
§reproduction + 14 days observation of juveniles
71
Test No. 206: Avian Reproduction Test
1984
Organisation for Economic Co-operation and Development 1

In vitro bioassays
72


§special cell lines
§specific compound è specific cellular or intracellular reporter è reporter gene
§dioxin-like toxicity (AhR), estrogenicity (ER), thyroid-hormone-like toxicity ...
§
In vitro tests
73
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

§in vitro special cell lines: mammalian or fish
§specific compound è specific cellular or intracellular receptor è reporter gene conected with
luciferase è light
§dioxin-like toxicity (AhR), estrogenicity (ER), androgenicity (AR), thyroid-hormone-like toxicity
(RAR) ...
§
In vitro tests
74
H295R

Microcosms / mesocosms
75


Microcosms / mesocosms
§micro < 1 m3, meso > 1 m3
§usually multi-species – big benefit
§optimum: less uncontrolled than field but more realistic than lab tests
§considers also indirect effects
§enable also environmental fate endpoints
§many possible desings
76
121 C:\Documents and Settings\Ludek Blaha\Plocha\1.gif

Microcosms / mesocosms
§Water: large aquariums, tanks, ponds, or artificial stream ecosystems
§
77
C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek
Blaha\Plocha\1.gif

Microcosms / mesocosms
§Water: large aquariums, tanks, ponds, or artificial stream ecosystems
§
78
C:\Documents and Settings\Ludek Blaha\Plocha\1.gif C:\Documents and Settings\Ludek
Blaha\Plocha\1.gif C:\Documents and Settings\Ludek Blaha\Plocha\1.gif figure4 C:\Documents and
Settings\Ludek Blaha\Plocha\1.gif

Microcosms / mesocosms
§Soil: usually core/column (made of plastic, wood, metal) of soil with vegetation, contains
standard soil or real soil; can be outside (open system) or in the climate chamber (also gas
monitoring)
§
79
118 118

Microcosms / mesocosms
§Soil core microcosm
80
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(57.399 Byte)

Microcosms / mesocosms
§Soil core microcosm
81
1