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?
Toxicity Tests
• Toxicity Tests/Bioassays
• Microcosm & Mesocosm Studies
• 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
Toxicity 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
Bioassays
- single / multiple species
- acute / chronic effects
- standardized (practical)
vs. experimental (research)
Simulation of the ecosystem
- major trophic levels included
- producers
- consumers
- destruents
Ecotoxicology: Laboratory studies Standardized methods
• ISO: International Organization for Standardization
(www.iso.ch)
• OECD: Organization for Economic Cooperation and
Development (www.oecd.org)
• EPA (US EPA) – Environmental Protection Agency USA
• US Army Corps of Engineers
• ASTM American Society of Testing and Materials
• CEN - European Commitee for Standardization (Comité Européen
de Normalisation) (www.cenorm.be)
• EEC: European Economic Community
• WHO – World Health Organisation
OECD Guidelines for the Testing of Chemicals
Section 2: Effects on Biotic Systems
Test No. 223: Avian Acute Oral Toxicity Test - describes procedures designed to estimate the acute oral toxicity of substances to birds, and it
provides three testing options: (1) limit dose test, (2) LD50-slope test, and (3) LD50-only test. The LD50-slope and LD50-only...
Test No. 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment - designed to assess the effects of
prolonged exposure of chemicals to the life-cycle of the sediment-dwelling freshwater dipteran Chironomus sp. First instar chironomid
larvae are exposed to five concentrations of the test chemical...
Test No. 229: Fish Short Term Reproduction Assay - describes an in vivo screening assay for fish reproduction where sexually mature male
and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle.
Test No. 230: 21-day Fish Assay - describes an in vivo screening assay for certain endocrine active substances where sexually mature male
and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle.
Test No. 231: Amphibian Metamorphosis Assay - describes an amphibian metamorphosis assay intended to screen substances which may
interfere with the normal functioning of the hypothalamo-pituitary-thyroid axis. The assay was validated with the species Xenopus laevis,
which is...
Test No. 232: Collembolan Reproduction Test in Soil
Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil- describes a method to assess the effects of chemical
substances in soil on the reproductive output of the soil mite species Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae).
Test No. 211: Daphnia magna Reproduction Test - described in this Test Guideline assesses the effect of chemicals on the reproductive output
of Daphnia magna Straus.
Test No. 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment - designed to assess the effects of prolonged exposure to
sediment-associated chemicals on the reproduction and the biomass of the endobenthic oligochaete Lumbriculus variegatus (Müller).
Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test - designed to assess effects on seedling emergence and
early growth of higher plants following exposure to the test substance applied to the soil surface or into the soil.
Test No. 221: Lemna sp. Growth Inhabition Test - designed to assess the toxicity of substances to freshwater aquatic plants of the genus
Lemna (duckweed).
Test No. 201: Alga, Growth Inhibition Test - purpose of this test is to determine the effects of a substance on the growth of freshwater
microalgae and/or cyanobacteria.
Test No. 202: Daphnia sp. Acute Immobilisation Test - describes an acute toxicity test to assess effects of chemicals towards daphnids (usually
Daphnia magna Staus).
Notes on practical testing
• 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
• 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 natural matrices
– Rather new in ecotoxicology – many open challenges
– More complex and more complicated
• „cause-effects“ often not clear (natural variability …)
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
• Plant protection products + biocides
• Veterinary and human pharmaceuticals
• Waste materials …
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, reproductive
activity
• Mutagenicity/genotoxicity (microbial, vascular plants,
wildlife animals)
• Teratogenity (amphibian- Xenopus laevis)
• Embryotoxicity
• Reproduction bioassays
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
Cu addition
Effect concentrations
expressed in total/dissolved Cu
Extrapolation =
PNECs or EQCs expressed in
total / dissolved Cu
Ecotoxicology – laboratory studies – experimental design
50
100
LC50 [concentration]
in mg/L or % effluent
Threshold:
No Observed Effect
Concentration (NOEC)
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
Growth inhibition assays with algae and
macrophyta (72 h) (Scenedesmus quadricauda,
Raphidocelis subcapitata, Selenastrum capricornutum,
Lemna minor)
ISO 8692/2004 Water quality -- Freshwater algal growth
inhibition test with unicellular green algae
OECD 201 Alga, Growth Inhibition Test
microplate miniaturization
Germination tests and root elongation
with higher plants – testing toxicity in the aquatic
media (Lepidium sativum, Sinapis alba, Lactuca sativa)
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
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
Toxicity Tests
Sediment ecotoxicity tests
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
Recommended at least 2 different species (e.g., Hyalella, Chironomus, Daphnia, etc) and
two different endpoints (e.g., growth, survival, reproduction, etc.)
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
Acute toxicity 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
Chronic toxicity 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
Time
Space
Work
Chemicals
1. Batching
2. Inoculation
3. Exposure
http://www.microbiotests.be/
Solid samples Water samples
ToxichromoToxichromo--testtest ®®
http://www.ebpi-kits.com/
ToxichromoToxichromo--PadPad ®®
MICROBIOTESTS
Test 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
Assessment
Identification
... 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)
Test substrates: OECD artifical soil, real soils
10 adults (synchronized) in test vessel
Test duration: 28 days – 56 days
Endpoints: survival, reproduction – number of juveniles, weight
changes
Preliminary 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
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/Toxicity/genotoxicitygenotoxicity
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
SpecificSpecific modemode ofof actionaction
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
Laboratory - aquatic snails, chironomids, soil organisms …
Natural – sample natural organisms and test ecotoxicity immediately
2) Assess parameters important for populations
Reproduction
Life cycle effects (including early life stages)
3) Consider natural situations
Addapt test conditions (temperature?, water hardness? …)
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!