Post on 12-Feb-2017
What are bioassays and how do they work?
Cornelia Kienle
Swiss Centre for Applied Ecotoxicology Eawag-EPFL
This research has received funding from the European Union’s Seventh Framework Programme under the grant agreement no. 308339.
Environmental pollutants are affecting ecosystem and human health
by Darren Hester
by capl@washjeff.edu
by Anna Warren
How do pollutants reach surface water bodies?
Abegglen et al. 2011, Umweltwissen, BAFU
http://web.uni-frankfurt.de/fb15/didaktik/umat/See/SeiteLoesungNahrungsnetz.htm
Potential Effects on Communities and Food Webs
Molecules
Organelles
Cells
Organs
Organisms
Populations
Ecosystem
Organisms living there are exposed to various factors
etc.
chemical
stress
temperature
oxygen
pH
abiotic
reproduction
food availability
predators
biotic
competition
Specificity
Ecolocical
Relevance
and show reactions on
various levels.
Bioassays are applied to detect those reactions.
Which influences are acting on organisms in the ecosystem?
o Sensitive detection of effects specific to
groups of toxicants with similar modes of
action,
o Extrapolation on possible consequences
for organisms more difficult.
o Integrate effects of all substances in a
water sample,
o Allow conclusions on biological/
ecological effects,
o Give limited information about
responsible substance classes.
Why Using Bioassays?
For an ecotoxicological performance review
With chemical analytics:
Determination of substance concentration possible
But: only limited conclusions about ecotoxicological
consequences possible
Hence: bioassays as reasonable amendment of chemical analytics
Various types of bioassays: in vitro and in vivo
Measuring Ecotoxicological Effects in Bioassays
Bioassay definition:
A "Standardized" process of an experiment with a defined
procedure and evaluation.
YES (yeast estrogen screen)
Solid Phase Extraction
(SPE)
Assay with water flea (Daphnids)
e.g. according to OECD guideline 202
From the Ecosystem to the Lab – Hormone Active Substances
Sensitivity
Test duration
Specificity
Ecological Relevance
In Vitro
Reproducibility
Costs
Animal welfare & ethics
In Vivo &Field assays
Intersex from In Vivo to an In Vitro Bioassay
Sperms
Egg
Nucleus
Egg yolk protein
mRNA
Liver cell
Liver
Egg yolk protein
Intersex from In Vivo to an In Vitro Bioassay
11
Receptor
Ligand
DNA
mRNA
Intersex from In Vivo to an In Vitro Bioassay
1-3 weeks
1-3 years
<1-3 days
Time window
Intersex from In Vivo to an In Vitro Bioassay
In Vitro Bioassay:
Genetically Modified Yeast Cells
EREGalactosidase
ER
From the Lab to the Ecosystem – Hormone Active Substances
Sensitivity
Test duration
Specificity
Ecological Relevance
Reproducibility
Costs
Animal welfare & ethics
In Vitro In Vivo
Which parameters are assessed in bioassays?
Endpoints:
Measured parameter e.g. enzyme activity, hatching rate, mortality
www.fischerweb.ch
Main uptake through water
Examples for endpoints:
Changes in the production of
enzymes or proteins (e.g.
vitellogenin)
Changes in sexual organs
Reduced fertility
Reduced egg/sperm
production
Weight reduction
Increased mortality
respirationskin food
How are the parameters evaluated in bioassays?
EC/ED50= the effective
concentration/dose for 50% of test
organisms (calculated)
LC/LD50 = the lethal
concentration/dose for 50% of
test organisms (calculated)
LOEC/LOEL = the lowest observed
effect concentration/level
(measured)
NOEC/NOEL = the no observed
effect concentration/level
(measured)
cu
mu
lative
re
sp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
50 %
EC50/LC50
LOEC
NOEC*
*
*
*
*
** * *
Equivalent Concentrations: e.g. Yeast Estrogen Screen (YES)
log (dilution/enrichment factor)
cu
mu
lative
resp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
TEQ
Environmental SampleReference substance
log (dilution/enrichment factor)
cu
mu
lative
resp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
TEQ
log (dilution/enrichment factor)
cu
mu
lative
resp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
TEQ
cu
mu
lative
resp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
cu
mu
lative
resp
on
se
log (concentration)
100 %
40 %
20 %
60 %
80 %
TEQ
Environmental SampleReference substance
Cum
ula
tive
Response
log (concentration) log (dilution/enrichment factor)
17β-Estradiol (E2) Ambient Sample
EEQ= Estradiol-
Equivalents
(ng/L)
EEQ
17β-Estradiol
EEQ:
17β-Estradiol-Equivalents
Biological Approaches for Measuring Ecotoxicity of Chemicals
Connon et al., Sensors 2012, 12, 1
Genotoxicity: Micronucleus assay
Mutagenicity: Ames assay
Hormonal YES, ER-, AR-, GR-,
effects: PR- and PPAR-CALUX,
H295R
Molecules Organelles Cells Organs Organisms Populations Ecosystem
General Bacteria, Algae,
toxicity: Trout
Herbicidal effects: Algae
Hormonal Vitellogenin in
effects: Trout
Different Levels and Mechanisms of Effect
Ecological RelevanceSpecificity
Molecules Organelles Cells Organs Organisms Populations Ecosystem
Ecological RelevanceSpecificity
General Bacteria, Algae,
Toxicity: Trout
Herbicidal Effects: Algae
Hormonal Vitellogenin in
Effects: Trout
Different Levels and Mechanisms of Effect
Primary producers Primary consumers Secondary consumers
Detritus feeders
ShreddersDestruents
Algae
Water flea
Gammarus
SnailLumbriculus
Effects on Various Trophic Levels
Rainbow trout
Duckweed
Bacteria – Luminescence Inhibition Assay (ISO 11348-3, 2007)
■ Test organism: Aliivibrio fischeri (marine luminescence bacterium)
■ Principle:
■ Endpoints: Inhibition of luminescence (%)
■ Toxicity parameter: ECx
■ Methodology for 96 well microplates (Richter et al. 2008)
www.pnas.org
www.sergeyphoto.com
Luciferin
Luciferase
measurement in luminometer
Examples for Bioassays with Bacteria
Algae 72h Growth Inhibition Assay (OECD 201, ISO 8692, 2004)
Combined Algae Assay (Escher et al. 2008)
Examples for Bioassays with Primary Consumers - Green Algae
• Test organism: single-celled freshwater green algae (e.g. Pseudokirchneriella
subcapitata)
• Principle:
Determination of effects on
1) Photosynthesis activity and/or
2) Growth of algae
• Duration: 24 -72 h
www.nies.go.j
p
■ Endpoints Photosynthesis-, growth inhibition (%)
■ Toxicity parameter: ECx, Diuron Equivalent
Concentration (DEQ)
Diuron-Equivalents (DEQ; ng/L)
TEQs for growth inhibition (mg/L)
Diuron
Terminology: Toxicity Tests
Term Term
Exposure Time acute chronic
Endpoint lethal sublethal
Effect direct indirect
Acute: < 7 days Chronic: 7+ days
(dependent on organism)
Testorganism Exposure Time Test Type Endpoint
Green algae:Pseudokirchneriellasubcapitata
72 h chronic Growth
WaterfleaDaphnia magnaCeriodaphnia dubia
48 h / 21 d96 h / 7 d
acute/chronicacute: mortality
chronic: mortality/ immobilisation/fecundity
Fisch:Zebrafish(D. rerio)
Rainbow trout(O. mykiss)
Fathead minnow(P. promelas)
96 h / 14 d
96 h /28 d
96 h /7 d
acute/chronic
acute/chronic
acute/chronic
acute: mortalitychronic: mortality/ growth/
development
Standardised laboratory bioassays for regulatory purposes use
few model species from different trophic levels and population-
relevant endpoints
Whole Effluent Toxicity
Testing and Environmental
Monitoring (US)
Guidelines for Performing Ecotoxicological Tests
European and International Guidelines (Examples)
• OECD (Organisation for Economic Co-operation and
Development): Guidelines for the testing of chemicals
• ISO (International Organisation for Standardisation): Guidelines
for the testing of environmental samples
• AFNOR (Association française de normalisation)
• DIN (Deutsches Institut für Normung)
• ASTM (American Society for Testing and Materials)
• US EPA (US Environmental Protection Agency)
TAKE HOME MESSAGES Pollutants elicit negative effects on organisms.
These effects occur on various levels of biological
organisation from the molecular up to the ecosystem
level.
Bioassays are well suited to assess pollutant effects.
In vivo bioassays are frequently applied, regulatory
acceptance of in vitro bioassays is still lacking.
Potential effects on communitiesand food webs
Contact: cornelia.kienle@oekotoxzentrum.ch