Ecotoxicological bioassays
Klara Hilscherova, Ludek Blaha, Jakub
Hofman & co.
CHEMICAL
ENTERS THE
ENVIRONMENT
Bioavailable
fraction
EXPOSURE
acute
chronic
Toxikokinetics
biotransformation
bioactivation
excretion / sequestration
Target site
EFFECT
LEVELS, FATE,
PROCESSES
CHEMICAL
ENTERS THE
ORGANISM
biomonitoring
System of organs
Organ
Tissue
Cell
Organell
Biomolecule
Individual
Population
Community
Ecosystem
Biosphere
Flexibility
Ability to
determine cause
Specifity
Sensitivity
Ecological
relevance
Response duration
Longer-term
effects
HIGHHIGH
HIGHHIGH
LOWLOW
LOWLOW
Effect of Chemicals
Biotest
Bioassay is a process where a test system
(tissue,organism, population) is exposed
under defined conditions to different
known concentrations of tested compound
or sample.
In vivo effects?
Ecotoxicity Tests
Provide a direct measure of biological
uptake of the toxicants
Establish link between site
contamination and adverse ecological
effects
May provide info on synergistic or
antagonistic interactions among
chemicals
Direct extrapolation of lab to field
should be carefully evaluated
May do an in situ toxicity test under
field conditions
Bioassays
- single / multiple species
- acute / chronic effects
- standardized (practical)
vs. experimental (research)
Simulation of the ecosystem
- major trophic levels included
- producers
- consumers
- destruents
Microcosm & Mesocosm Studies
Ecotoxicity Tests
Toxicity tests can be used for both aquatic and terrestrial systems
Aquatic tests are more developed
Endpoints are mortality, growth and/or reproduction
Vertebrates
Rodents
Fish
Birds
Invertebrates
Insects
Amphipods (crustacea related to shrimp and krill)
Plankton
Microbes
Luminescent bacteria (Microtox)
Plants
Aquatic or terrestrial
Vascular or non-vascular
1
Standardized methods
Organizations associated with biotests and standardized
guidelines
OECD = Organization for Economic Cooperation Development
ISO = International Standardization Organization
US EPA = US Environmental Protection Agency
SETAC = Society for Environmental Toxicology and Chemistry
IOBC = International Organisation for Biological and Integrated Control of Noxious
Animals and Plants
EPPO = European and Mediterranean Plant Protection Organization
ASTM = American Society of Testing and Materials
ANSI = American National Standards Institute
CEN = European Commitee for Standardization
AFNOR = Association Francaise de Normalisation
EEC = European Economic Community
WHO = World Health Organisation
BBA = Biologische Bundesanstalt für Land- und Forstwirtschaft
OPPTS = The Office of Prevention, Pesticides and Toxic Substances (EPA)
DIN = German Deutsches Institut für Normung
OECD guidelines for the Testing of Chemicals
Section 2: Effects on Biotic Systems
http://www.oecd.org/document/40/0,3746,en_2649_34377_37051368_1_1_1_1,00.html
1
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
Tests with sediment
OECD guidelines
1
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
ISO guidelines
Aquatic microorganisms
ISO 10712:1995 Water quality -- Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test)
ISO 11348-1:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri
(Luminescent bacteria test) -- Part 1: Method using freshly prepared bacteria
ISO 11348-2:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri
(Luminescent bacteria test) -- Part 2: Method using liquid-dried bacteria
ISO 11348-3:2007 Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri
(Luminescent bacteria test) -- Part 3: Method using freeze-dried bacteria
ISO 13641-1:2003 Water quality -- Determination of inhibition of gas production of anaerobic bacteria -- Part 1: General test
ISO 13641-2:2003 Water quality -- Determination of inhibition of gas production of anaerobic bacteria -- Part 2: Test for low
biomass concentrations
ISO 13829:2000 Water quality -- Determination of the genotoxicity of water and waste water using the umu-test
ISO 16240:2005 Water quality -- Determination of the genotoxicity of water and waste water -- Salmonella/microsome test
(Ames test)
ISO/DIS 11350 Water quality -- Determination of the genotoxicity of water and waste water -- Salmonella/microsome
fluctuation test (Ames fluctuation test)
ISO 15522:1999 Water quality -- Determination of the inhibitory effect of water constituents on the growth of activated sludge
microorganisms
ISO 21338:2010 Water quality -- Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples
on the light emission of Vibrio fischeri (kinetic luminescent bacteria test)
ISO 8192:2007 Water quality -- Test for inhibition of oxygen consumption by activated sludge for carbonaceous and
ammonium oxidation
ISO 9509:2006 Water quality -- Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms
ISO guidelines
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
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
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
Application of bioassays in ecotoxicology
Testing chemicals
Traditional approach - bioassays developed to assess chemicals
Standardized and validated 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
Other standard guidelines
Limited ecological relevance
often acute tests only, too standardized
does not assess bioavailability, no consideration of mixtures
no consideration of specific modes of action
Testing toxicity of environmental matrices
Relatively new in ecotoxicology many open challenges
More complex and more complicated
cause-effects often not clear (many confounding factors )
Testing strategy
Battery of assays
Fast screening tests (inhibition of Vibrio fisheri bioluminiscence,
MICROTOX 30 min toxicity)
Standardized acute toxicity tests
Further studies with chronic assays
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 up for aquatic environment
algae / D. magna / fish
Basic principles of bioassays
Reproducibility
Standard design
Possibility of data extrapolation on field conditions
Cost and time feasibility
Tested matrix
Water
Soil
Air
Sediment
Waste
Chemical compound
Sample type
Single compounds (hydrophobic,
hydrophilic, volatile)
Mixture of compounds (known and/or
unknown)
Environmental samples (usually unknown,
mixtures of different compounds with
different properties complicated
interpretation)
Used to develop Water Quality Criteria (WQC) for different uses
Endpoints
Lethal effects (mortality)
sub lethal effects (immobilisation)
Physiological activity (photosynthetic activity, enzymatic
activity, biomass increase, resistance to diseases, pests
and/or parasites)
Reproductive activity, malformations
Mutagenicity/genotoxicity (microbial, vascular plants,
wildlife animals)
Teratogenity (amphibian- Xenopus laevis)
Embryotoxicity
Reproduction bioassays
Growth
Factors influencing results of bioassays
For reproducibility of results these main factors
have to be standardized:
Exposure duration
Temperature
Light:dark period
Volume
Oxygen content
Composition of cultivation media
Age of organism
Parameters of the biological system
Complexity / in vitro, in vivo, population, microcosm
Population characteristics sex, age
Aquatic vs. Terrestrial (soil)
Exposure duration & effects
Acute (often mortality), sub-acute, chronic (other endpoints)
(4 days - algae / 4 generations, fish / acute toxicity)
Exposure setup
Static / with exchange of media / flow-through
Depends on the compound stability (should be measured!)
Bioassay endpoints
Lethality, immobilization (Daphnia), growth, reproduction
Abiotic factors in the experiment
Validity criteria (pH, oxygen, temperature, humidity, water
hardness )
Biotests to be considered
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 (1% vol)
§ 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
§ statistical evaluation (means, ANOVA, dose-response )
Steps to conduct the biotest
1) Prepare organism
2) Prepare sample
3) Expose
4) Evaluate
50
100
LC50 [concentration]
in mg/L or % effluent
Parameters:
No Observed Effect Concentration (NOEC)
Dose-response curve
Lowest Observed Effect Concentration (LOEC)
ECx ( x % effects concentration)
LCx ( x % lethal concentration)
Laboratory ecotoxicology data and results
Acute Aquatic Toxicity Tests
Most frequently used (short = less expensive)
Relates dose (Cw x time of exposure) to time of
death for a particular test organism
Produce concentration/response curve
Ranges from 1 to 4 days for aquatic tests and up
to 10 days for assessment of sediment toxicity
Done in laboratory under controlled conditions
Acute Aquatic Toxicity Tests
n Growth inhibition assays with algae and
macrophyta (72 h) (Scenedesmus quadricauda,
Raphidocelis subcapitata, Selenastrum capricornutum,
Lemna minor)
n ISO 8692/2004 Water quality -- Freshwater algal growth
inhibition test with unicellular green algae
n OECD 201 Alga, Growth Inhibition Test
n microplate miniaturization
n Germination tests and root elongation
with higher plants testing toxicity in the aquatic
media (Lepidium sativum, Sinapis alba, Lactuca sativa)
n OECD 208 Terrestrial Plant Test: Seedling Emergence and
Seedling Growth Test
AQUATIC BIOTESTS with PRODUCERS
AQUATIC ASSAYS
§ Daphnia magna immobilisation (24 48h)
ISO 6341/1996 Water quality - Determination of the inhibition of the mobility
of Daphnia magna Straus (Cladocera, Crustacea) - Acute toxicity test
OECD 202 Daphnia sp. Acute Immobilisation Test
§ crustacea Ceriodaphnia dubia
§ rotifer Brachionus calyciflorus
§ToxKit assays
§Thamnotoxkit (Thamnocephalus platyurus)
§Artoxkit Artemia salina
BIOTESTS with CONSUMERS - invertebrates
BIOTESTS with CONSUMERS fish (acute 96h)
Guppy, Poecilia
reticulata
Zebrafih, Danio rerio
(syn. Brachydanio rerio)
Carassius (Goldfish)
Fathead minnow,
Pimephales promelas (USA)
(Rainbow) trout
(Onchorhynchus sp.)
Medaka, Oryzias latipes
Nile tilapia, Oreochromis
niloticus
OECD 203 Fish, Acute Toxicity Test
ISO 7346 Water quality - Determination of the acute lethal toxicity of
substances to a freshwater fish
Static/semi-static/flow through method
prolonged tests embryolarval tests
chronic tests reproduction, growth
Specific endpoints genotoxicity, endocrine disruption
BIOTESTS with CONSUMERS - amphibians
FETAX Frog Embryo Teratogenicity
Assay Xenopus (ASTM E1439-98 )
African clawed frog (Xenopus laevis)
96 h / egg and embryo exposure
§Toxicity to luminescent bacteria Vibrio
fisheri (MICROTOX®)
§ISO 11348 Water quality - Determination of the
inhibitory effect of water samples on the light
emission of Vibrio fischeri
§ Growth inhibitions (Pseudomonas
putida, Toxi-Chromotest, Toxi-ChromoPad)
§ Toxicity assays
with SOIL BACTERIA
BIOTESTS with DESTRUENTS - microorganisms
Microtox
EN ISO 11348-(1)
- based on inhibition of bioluminiscence of marine bacteria Vibrio fischeri
Chronic Aquatic Toxicity Tests
Longer tests: 7 - 30 days
Objective is to expose for at least 1/10th of
lifetime
Effect of different Cw on growth, reproduction,
behavioral, physiological or other biological
function
Sub-chronic: only exposed during part of lifecycle
(usually early stages)
Life-cycle tests have been done for only a few
contaminants
Chronic Aquatic Toxicity Tests
Chronic Aquatic Toxicity Tests
Aquatic Toxicity Tests
Sediment ecotoxicity tests
n Toxicity of pore water/eluates (several ISO / OECD
standards)
: 100 g d.w./L water, 24h slow shake, filter, test
V. fisheri (30 min), Algae, Invertebrates - D. magna (2 days)
? Aquatic eluate vs. Sediment
n Recommended at least 2 different species (e.g., Hyalella, Chironomus, Daphnia, etc) and
two different endpoints (e.g., growth, survival, reproduction, etc.)
n Direct (contact) toxicity (only few standards )
: sediment+organisms & evaluate effects - worms, snails days-weeks
Toxicity Test Species
Freshwater Sediments
Amphipods
Hyalella azteca
Midges
Chironomus tentans
Chironomus riparius
Oligochaetes
Tubifex tubifex
Mayfly
Hexagenia limbata
Sediment toxicity Acute tests
Pore water
Sediment eluates
48-96 h exposure
Preparation of eluates: 24h shaking, 100 g sediment/1L water
Species: Daphnia magna, Daphnia pulex, Ceriodaphnia dubia, fathead
minnow (Pimephales Promelas), rainbow trout (Oncorhynchus mykiss)
Endpoints: survival, immobilization
Contact tests - whole sediment
96h 10 day exposure
Species: amphipod (Hyalella azteca), mayfly (Hexagenia limbata), chironomids
(C.tentants/riparius)
10-day test with Hyalella azteca and Chironomus tentans
Endpoint: survival
Sediment toxicity Chronic tests
Pore water
Eluate from sediment
7-35 day exposure
Species: Ceriodaphnia dubia, fathead minnow (Pimephales
Promelas), rainbow trout (Oncorhynchus mykiss)
Endpoints: survival, immobilization, growth, reproduction, time to the
first reproduction, time of death, offspring survival
Contact tests - whole sediment
about 28 days exposure
Species: Hyalella azteca, Chironomids (C.tentants/riparius)
Endpoints: survival, immobilization, growth, reproduction, time to the
first reproduction, time of death, offspring survival
28- and 42-day tests with H. azteca
Sub-chronic and lifecycle tests with Chironomus tentans
10-day short term chronic test with amphibian larvae
Tests for sediment toxicity - EPA
www.epa.gov
Tests for sediment toxicity - EPA
www.epa.gov
Tests for sediment toxicity - ASTM
Sediment Toxicity - ASTM
Sediment Toxicity Test confounding factors
Potential Non-Contaminant Factors
Sediment grain size
Content and type of clay
Organic carbon content and character
Humic substances/organic matter structure and properties
pH
Oxygen content
Ammonia / Sulfide toxicity
Nutrition
Changing sediment toxicity due to sampling and experimental
procedures
Mixing of more contaminated sediments with the thin layer at the
sediment-water interface
Oxidation and precipitation of redox metals from the reaeration
required for the sediment toxicity testing
Microbioassay
Saving of
ØTime
ØSpace
ØWork
ØChemicals
1. Batching
2. Inoculation
3. Exposure
http://www.microbiotests.be/
Solid samples Water samples
ToxichromoToxichromo--test ®test ®
http://www.ebpi-kits.com/
ToxichromoToxichromo--Pad ®Pad ®
MICROBIOTESTS
Tests for specific in vivo effects
embryotoxicity
teratogenesis
developmental disorders
endocrine disruption
reproductive disorders
Aquatic tests or contact tests with sedimentdwelling
invertebrates, amphipods, molluscs,
fish, amphibian and mollusc eggs
- Specific sublethal endpoints, histology
In situ tests
Caging bivalves, fish, molluscs
Health status and specific biomarkers
assessment in species collected on site
Sublethal biomarkers, histology
Test subject: water
Test subject: sediment
Determination of
macrozoobenthos
saprobic index
functional feeding groups
life form index
evenness & diversity
Bioassays
toxicity, cytotoxicity
endocrine disrupting potential
genotoxicity, mutagenicity
Chemical analyses
organical analyses
limnochemical parameters
heavy metals
Ecosystem
native & concentrated surface water
whole sediment, sediment pore water, elutriate
and organic - extracts
... in vitro and in vivo
bioassays
Integrated assessment
of aquatic ecosystems
Terrestrial Toxicity Tests
Direct exposure of test biota to media samples from a
site
Indirect exposure to filtered water exposed to soil or
sediments samples
Exposure to leachates from a site
Controlled exposure to a specific contaminant using soil
from the site
Test biota
soil microbes and fungi - critical role in C, N, S, P cycling, plus
production of SOM and other organics
invertebrates (earthworms and insects): provide essential
ecosystem functions
These tests are fast, simple and relatively inexpensive, with relevant
results to evaluate effects on ecosystem biogeochemical functions
Terrestrial Toxicity Tests
Vertebrates:
amphibian: survival, growth and reproductive
success
avian and small mammal: reproductive success
and body burden
Feeding studies (small mammal and avian toxicity tests) are
useful to determine potential uptake and transfer within the
food web - potential human exposure route
Standard protocols have been derived from veterinary studies
and FDA methods, but many are still under development
Longer than invertebrate tests
Earthworms: Eisenia fetida/andrei (OECD 222, 2000)
Enchytraeids: Enchytraeus albidus, E.crypticus (OECD 220, 2000)
Collembolans: Folsomia candida, F.fimetaria (ISO 11267, 1999)
ØTest substrates: OECD artifical soil, real soils
Ø10 adults (synchronized) in test vessel
ØTest duration: 28 days 56 days
ØEndpoints: survival, reproduction number of juveniles, weight
changes
ØPreliminary test => Final test
Eisenia fetida
Folsomia candida
Enchytraeus crypticus
Terrestrial bioassays - exposure in soil
Terrestrial Toxicity Tests
Vegetation
mostly crops
primary endpoints are:
survival: seed germination test
growth: seedling growth rate and root elongation test
reproduction success: vascular plant toxicity
photosynthesis rates: chlorophyll fluorescence assay
can be applied in lab or in the field
nutrient, water and light limitations can complicate
analysis of results
longer term studies
Battery of bioassays
Different
Trophic level
Sensitivity
Target effect/organ
Specific toxic effect (mutagenity, neurotoxicity, etc )
The negative response in test with one species does not
mean that substance is not toxic.
Toxicity can be observed after longer exposure and/or in
different species.
Simple battery: algae, zooplankton, fish, bacteria.
Battery of bioassays
Standard acute toxicity tests representing different ecology groups
different levels of food chain microorganism, algae, invertebrates, fish
Different guidelines: Vibrio fisheri, Thamnocephalus, Daphnia,
Scenedesmus
USEPA crustacean Ceriodaphnia dubia, algae Selenastrum
capricornutum, fish Pimephales promelas
Chronic toxicity crustaceans Daphnia magna, Ceriodaphnia dubia
Specific endpoints: survival, reproduction, growth, activity, heartbeat,
respiration, biochemical markers
Micro and Mesocosm
Controlled experiments in lab or field to study changes at any level:
population
community
ecosystem
Microcosm are small studies, usually in lab
Mesocosm are large, containing many species, usually outdoors
Advantages of microcosm studies:
Better than single-species studies
More space efficient
Easier to maintain controlled conditions
Replication and standardization easier
Low chance of contaminating the environment
Issues with Microcosm:
Can t simulate certain processes (e.g. acid deposition from environment)
small population sizes => extinctions?
Extrapolation of results
May leave out a critical and/or sensitive component of ecosystem
endocrine disruption (compounds interfere
with hormonal regulation in organism),
(anti)estrogenicity ...
reproductive failure, teratogenicity
neurotoxicity
immunosuppressions
carcinogenicity (mutagenicity / tumor
promotion)
Chronic effects
How to study chronic toxicity ?
Chronic toxicity is difficult to study and predict
time and cost consuming experiments
limited number of species (laboratory vs. natural species)
effect = combination of chemical exposure and life style, habits ...
metabolites or derivatives (not parent compounds) are often the active
substances Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OH
Cl
Ecotoxicological bioassays
for chronic endpoints
In vitro studies (biochemical mechanisms)
+ easy to perform, short-term - ecotoxicological relevancy
+ highly controlled conditions - mostly with vertebrate cells
+ lower amounts of chemicals needed
(new compounds screening)
In vivo biotest testing
+ unique whole organisms - only few (ecologically
+ controlled conditions nonrelevant) organisms used
+ better ecological interpretation - mostly ACUTE assays
- chronic: long exposures
Field and in situ observations, epidemiological studies
How to study (chronic) toxicity ?
MECHANISMS of toxicity
Various chronic effects have uniform biochemical basis
principal studies with mechanistically based in vitro techniques
estimation of in vitro effects of individual compounds
understanding the mechanisms, prediction of hazard
application for risk assessment or monitoring
derivation of relative potencies ("toxic equivalents")
HORMONE
Biochemical
effects
TOXIN
In vivo
effectsRECEPTOR
Estrogen receptor
activation 1) female reproduction
disorders
2) male feminisation
3) tumor promotion
4) immunomodulations
5) developmental toxicity
SINGLE mechanism -> SEVERAL effects
=> understanding to mechanisms may predict effects
Effects in vitro ?
In vitro models
Original or genetically modified prokaryotic
or eukaryotic cells
BACTERIAL, YEAST TESTSBACTERIAL, YEAST TESTS
TESTS ON TISSUE CULTURETESTS ON TISSUE CULTURE CELL LINESCELL LINES
In vitro bioassays
Principle
Mechanism of action based
Mechanism related to toxic effects
Ø Using biological system as
if it was an instrumental detector
and/or integrator
Screening tests
Toxicity:
Bacterial models
Vibrio fisheri (Microtox) 0.5 h
Escherichia coli (Toxichromotest)
2 h
Fish/mammalian cell lines
Genotoxicity :
SOS chromotest, umuC test
Comet assay
GFP test etc.
Contact test
Flash test with Vibrio Fisheri
kinetic test
Toxicity/genotoxicityToxicity/genotoxicity
Yeast models
Fish/mammalian cell lines
Tests for presence of compounds with
hormone-like effects :
Anti/estrogenicity
Anti/androgenicity
Retinoid-like activity
Dioxin-like potency
Specific mode of actionSpecific mode of action
In vitro assays for genotoxic effects
GENOTOXICITY = toxic modification or alteration of the structure or
function of genetic material
Bacterial or yeast assays with reporter genes
Eukaryotic cells
GreenScreen® test for genotoxicity
Experimental
Design
NUCLEAR RECEPTOR MEDIATED EFFECTS
important mechanisms involved in chronic
toxicity
Dioxin-like activity: Aryl hydrocarbon receptor (AhR)-mediated
effects
PCDDs/Fs, PAHs, PCBs
Xenoestrogenity / Antiestrogenity: Estrogen receptor (ER)mediated
effects
PCDDs/Fs, PAHs, PCBs, OH-PCBs, alkylphenols, natural and
synthetic hormones ...
Xenoandrogenity / Antiandrogenity: androgen receptor (AR) mediated
effects pesticides
Interactions with retinoic acid receptor (RAR)
Receptor-mediated effects
luciferase reporter assays
LightLight Luciferase
Nuclear
Factors
1
P
+ AhR
HSP90
HSP90
Src
HSP90
HSP90
DRE-Luc
AhR
ARNT
Modulation of Gene
Expression
ARNT
Src
Activated
2
P
P
Cytosolic
Proteins
Membrane
Proteins
Increased Protein
Phosphorylation
Ligand (TCDD)
LightLight Luciferase
Nuclear
Factors
1
P
+ AhR
HSP90
HSP90
SrcSrc
HSP90
HSP90
DRE-Luc
AhR
ARNT
Modulation of Gene
Expression
ARNT
Src
Activated
2
P
P
Cytosolic
Proteins
Membrane
Proteins
Increased Protein
Phosphorylation
Dioxins
Estrogens
Androgens
Retinoids
Nuclear receptors (AhR, ER, AR, RAR/RXR) play an important
role in toxic effects of many pollutants
DIOXIN-like toxicity
Anti / estro-, Anti / andro- -genicity
Common mechanism - transcription factors:
development of mechanistically based bioassays
In vitro luciferase reporter bioassays studies of
individual chemicals (toxicity identification, IEF calculation)
complex environmental samples (estimation of toxic potential)
NUCLEAR RECEPTORS IN TOXICITY
BIOMARKERs
Sublethal effects, studied in organisms from biotests or sampled in the
environment
Early warning signals of potential damage in organism and/or the
whole population, early marker of toxicity (prior to any morphological
alterations)
Changes in cellular or biochemical components, structures or
functions caused by xenobiotics
Sensitive, fast responses, can show the mechanism of effect, precede
any visible toxicity symptoms
Most studied in vertebrates
Possible to study also in plants and invertebrates from standard
biotests (algae, macrophytes, invertebrates)
Biotransformation enzymes (phase I&II)
Induction of detoxification enzymes in plants and animals
A. Enzymes of the 1st phase of biotransformation MFO
enzymes (mixed function monooxygenases) induction of
P450 cytochrome enzymes (EROD, MROD, PROD)
B. Enzymes of the 2nd phase of biotransformation
glutathione transferases (GST), uridinedifosfoglukuronosyl
transferases, sulphotransferases
Oxidative stress parameters
Production of reactive oxygen species
Activity of antioxidant enzymes glutathion peroxidase,
glutathion reductase, superoxidase, catalase
Concentration of nonenzymatic antioxidants
Oxidative damage to macromolecules lipid peroxidation,
oxidative DNA aducts, products of protein oxidation
Protective proteins
Stress proteins: heat shock proteins (HSP), glucoseregulated
proteins (GRP)
Metallothioneins (MT): metal binding
Multi Xenobiotic Resistance (MXR): excretion of
xenobiotics; induction or inhibition by chemisensitizers
Hematological parameters
Serum transaminases: Alanine transaminase (ALT),
aspartate transaminase (AST); membrane disruption or
organ damage
Blood values: haematocrit, haemoglobin, blood sugars
(glucose), plasma lipids and proteins (albumin)
Immunological parameters
White blood cell count
Lymphocyte status
Morphology of spleen, thymus and kidney
Macrophage function
Susceptibility to bacterial infections
Reproductive & endocrine parameters
Biochemical: Fish vitellogenin (VTG), Zona radiata Protein
(ZRP), Cytochrome aromatase, spiggin (stickleback)
Morphology of gonads; sperm condition
Reproductive success (eggs, larvae)
Intersex, Imposex
Genotoxic parameters
DNA adducts
Comet assay
Micronucleus assay, sister-chromatid exchange
Flow cytometric screening (DNA, RNA, protein)
Neurotoxic parameters
Acetyl cholinesterase inhibition assay (ACHE)
Neurotransmitter impairment (e.g. SERT)
Behavioral studies
BIOASSAYS are needed to test effects of
1) Individual chemicals
Understanding toxicity + prospective studies for R.A.
2) Environmental samples
Routine analytical data (PAHs, PCBs, OCPs) provide only partial
information
Biological experiments complement chemical analyses and may
suggest elevated levels of unknown toxic chemicals (e.g. EDs)
In vitro assays are screening tools that help to understand
mechanisms (e.g. feminization / anti-androgenicity)
In vivo assays ecologically relevant results
SUMMARY BIOASSAYS
Real ecotoxicology needed
1) Use non-standardized organisms
n Laboratory - aquatic snails, chironomids, soil organisms
n Natural sample natural organisms and test ecotoxicity immediately
2) Assess parameters important for populations
n Reproduction
n Life cycle effects (including early life stages)
3) Consider natural situations
n Addapt test conditions (temperature?, water hardness? )
n Simulate real exposures (e.g. peaks during pesticide spraying)
Summary
Methods for assessing effect vary from
single chemical/single species
multiple stressors/multiple species
short-term/long-term
Ability to relate cause and effect varies accordingly
(easier for simpler system)
Need studies at all scales (temporal and spatial) to have
better understanding
Be critical of a standard developed with just one
methodology!