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PrinciplePrinciple ofof Toxicity Toxicity TestingTesting
Vladimr KoVladimr Ko
Institute Institute ofof ChemicalChemical Technology,
Technology, PraguePrague
CzechCzech RepublicRepublic
11
BioassayBioassay Experiments with organisms targetted to solve
Experiments with organisms targetted to solve question how
evaluated substance and/or sample question how evaluated substance
and/or sample affect life in field condition. affect life in field
condition.
Both inhibitive and stimulative effect is Both inhibitive and
stimulative effect is unaccetable.unaccetable.
Intensity of effect is influenced by concentration Intensity of
effect is influenced by concentration and/ot duration of
exposition.and/ot duration of exposition.
22
Aim of Science on the Field of Aim of Science on the Field of
Ecotoxicological BioassaysEcotoxicological Bioassays
The development of new standard methods.The development of new
standard methods.
Standard Standard reproducible conditions, reproducible
conditions,
Comparison of effects of different substances Comparison of
effects of different substances and/od different organismsand/od
different organisms
Reproducible between different laboratoriesReproducible between
different laboratories
33
Basic principles of bioassaysBasic principles of bioassays
ReproducibilityReproducibility
Standard designStandard design
Possibility of data extapolation on field conditionsPossibility
of data extapolation on field conditions
Economy feasibilityEconomy feasibility
Time feasibilityTime feasibility
44
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BiotestBiotest BioassayBioassay isis a a processprocess
wherewhere isis a test systm a test systm
((tissuetissue,,organismorganism, , populationpopulation) )
exposedexposed underunderdefineddefined conditionsconditions to to
differentdifferent knownknownconcentrationsconcentrations ofof
testedtested compoundcompound oror sample.sample.
EcotoxicologicalEcotoxicological bioassaysbioassays are such a
are such a bioassaysbioassayswhatwhat forfor evaluationevaluation
ofof observedobserved phenomenonphenomenon use use such a such a
detectiondetection systemssystems ((tissuetissue, ,
organismorganism,) ,) whatwhat enableenable relevantrelevant
interpretationinterpretation forforenvironmentalenvironmental
conditionsconditions. .
55
Structuring of bioassaysStructuring of bioassaysDuration of
expositionDuration of exposition
AcuteAcute
SemiSemi--acuteacute
ChronicChronic
66
Structuring of bioassaysStructuring of bioassaysDesign,
generationDesign, generation
1st. generation 1st. generation -- classic (standard)classic
(standard)
2nd. generation 2nd. generation --
microbioassaysmicrobioassays
3rd. generation 3rd. generation -- biosenzors,
biomarkersbiosenzors, biomarkers
77
MicrobioassayMicrobioassay Economy ReasonsEconomy Reasons
Saving ofSaving of
TimeTime
SpaceSpace
WorkWork
ChemicalsChemicals
1. Batching
2. Inoculation
3. Exposition
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Structuring of bioassaysStructuring of bioassaysTrophic
levelTrophic level
ProducentProducent
ConsumentConsument
DestruentDestruent
99
Structuring of bioassaysStructuring of bioassaysTested
matrixTested matrix
WaterWater
SoilSoil
AirAir
SedimentSediment
WasteWaste
Chemical compoundChemical compound
1010
Structuring of bioassaysStructuring of bioassaysSpectrum of
organismsSpectrum of organisms
Single SpeciesSingle Species
Multi Species Multi Species -- wildlife populations / laboratory
wildlife populations / laboratory mixturemixture
1111
Structuring of bioassaysStructuring of bioassaysType of
sampleType of sample
Pure compounds (hydrophobic, hydrophilic, Pure compounds
(hydrophobic, hydrophilic, volatile)volatile)
Mixture of compounds (known and/or unknown)Mixture of compounds
(known and/or unknown)
Environmental samples (usually unknown, Environmental samples
(usually unknown, mixtures of different compounds with different
mixtures of different compounds with different properties
properties complicated interpretation)complicated
interpretation)
1212
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Structuring of bioassaysStructuring of
bioassaysPretreatmentPretreatment
Defined very concentration of substancesDefined very
concentration of substances
Eluates, extracts of environmental samples Eluates, extracts of
environmental samples liquid final sample, Whole Effluentliquid
final sample, Whole Effluent
Direct Tests, Solid Phase TestsDirect Tests, Solid Phase
Tests
Comparative tests of sorbtion matrix (for Comparative tests of
sorbtion matrix (for example SPMD)example SPMD)
1313
Structuring of bioassaysStructuring of bioassaysComplexity of
detection systemComplexity of detection system
From the simplest to the more integralFrom the simplest to the
more integral
Enzymes, bioprobes, cell and tissue cultures in Enzymes,
bioprobes, cell and tissue cultures in vitrovitro
Whole living organismWhole living organism
PopulationPopulation
Micro/mezo kosmosMicro/mezo kosmos
Field studyField study
1414
Structuring of bioassaysStructuring of bioassaysType of
endpointType of endpoint
Lethal effects (mortality)Lethal effects (mortality)
sub lethal effects (immobilisation)sub lethal effects
(immobilisation)
Physiological activity (photosynthetic activity, Physiological
activity (photosynthetic activity, enzymatic activity, biomass
increase, resistance enzymatic activity, biomass increase,
resistance to diseases, pests and/or parasitesto diseases, pests
and/or parasites
Reproductive activity, malformations, Reproductive activity,
malformations, reproductive activityreproductive activity
1515
Special tests for Risc Special tests for Risc
AssessmentAssessment
AGP AGP
Mutagenicity/genotoxicity (microbial, vascular
Mutagenicity/genotoxicity (microbial, vascular plants, wildlife
animals)plants, wildlife animals)
Teratogenity (amphibianTeratogenity (amphibian-- Xenopus
laevis)Xenopus laevis)
EmbryotoxicityEmbryotoxicity
Reproduction bioassaysReproduction bioassays
Useful for clearly defined cases, where lethal Useful for
clearly defined cases, where lethal effect as an endpoint is not
appropriate for effect as an endpoint is not appropriate for
interpretation.interpretation.
1616
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Battery of assaysBattery of assays The negative response in test
with one specie The negative response in test with one specie does
not means that substance is not toxic.does not means that substance
is not toxic.
Toxicity can be observed after longer exposition Toxicity can be
observed after longer exposition and/or on different species.and/or
on different species.
Simple battery: algae, zooplancton, fish, Simple battery: algae,
zooplancton, fish, bacteria.bacteria.
1717
Batery of bioassaysBatery of bioassays DifferentDifferent
Trophic levelTrophic level
SensitivitySensitivity
Target effect/organTarget effect/organ
Specific toxic effect (mutagenity, neurotoxicity, Specific toxic
effect (mutagenity, neurotoxicity, etc)etc)
1818
Exposition testsExposition tests Exposed organism do not
reproduce during time Exposed organism do not reproduce during time
of assay.of assay.
Quick.Quick.
Influence on photosynthesis; synthesis of ATP, Influence on
photosynthesis; synthesis of ATP, movement of organs.movement of
organs.
Mortality, immobilisationMortality, immobilisation
1919
Growth, reproductive testsGrowth, reproductive tests Better and
more sensitive. Better and more sensitive.
Reproductive process is the most sensitive Reproductive process
is the most sensitive moment of living organism. moment of living
organism.
If no adverse effect will be observed during 3 If no adverse
effect will be observed during 3 generations, there is very
probable that testing generations, there is very probable that
testing organism is not sensitive to tested concentration organism
is not sensitive to tested concentration of toxicant.of
toxicant.
Nap.: Algal assay, daphnia reproductive assay, Nap.: Algal
assay, daphnia reproductive assay, root prolongation testroot
prolongation test
2020
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Standard toxicity Standard toxicity teststests Battery of
bioassays:Battery of bioassays:
algae, plants, zooplancton, fish, (bacterium)algae, plants,
zooplancton, fish, (bacterium)
2121
Factors influencing results of Factors influencing results of
bioassaysbioassays
For reproducibility of results these main For reproducibility of
results these main factors has to be standardized:factors has to be
standardized:
Time of expositionTemperatureLight period
VolumeOxygen
Composition of cultivation mediaAge of organism
2222
Limit Test negative
Range finding Test
Basic Test
Data treatment
Verify Test
positive
negativepositive, response 50%
positive, response < 50%
Sampling
Pretreatment of sample(eluate, extract, pH, oxygen)
2323
SamplingSampling Way of sampling importantly influence results
of Way of sampling importantly influence results of
bioassaybioassay
2424
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PretreatmentPretreatment Pretreatment influence the chemical
properties of Pretreatment influence the chemical properties of
substances present in sample and thus their substances present in
sample and thus their biological activity.biological activity.
The manner of pretreatment can diverse activity The manner of
pretreatment can diverse activity od different toxicants presented
in sample.od different toxicants presented in sample.
Treatment of pH, dewatering, filtration, aeration, Treatment of
pH, dewatering, filtration, aeration, salinity treatmentsalinity
treatment
2525
Pretreatment Pretreatment adjustment of adjustment of sample
with a goalsample with a goal
Distinguish toxicantsDistinguish toxicants
Elimination of interferencesElimination of interferences
2626
TemperatureTemperature Sterilization Sterilization -- autoclave,
environmental samplesautoclave, environmental samples
Denaturation of proteins, pesticidesDenaturation of proteins,
pesticides
Decomposition of thermo labile compoundsDecomposition of thermo
labile compounds
Pointing out thermo stabile compoundsPointing out thermo stabile
compounds
Better do not carry outBetter do not carry out
2727
Osmotic pressureOsmotic pressure Low soluble sample Low soluble
sample low osmotic pressurelow osmotic pressure
Marine organisms Marine organisms need for higher osmotic need
for higher osmotic pressurepressure
Needed addition of cultivation saltsNeeded addition of
cultivation salts
The change of mineralization can change form The change of
mineralization can change form and amount of solved substances.and
amount of solved substances.
2828
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AerationAeration Oxygen for organismsOxygen for organisms
Decrease of volatile substancesDecrease of volatile
substances
Changes in pHChanges in pH
Stripping Stripping elimination of interference volatile
elimination of interference volatile substancessubstances
2929
pH treatmentpH treatment Chemical form and thus toxic properties
of Chemical form and thus toxic properties of substances can be
modified. Metals, ammonium substances can be modified. Metals,
ammonium ionion
pH can be mentioned as a part of adverse effectspH can be
mentioned as a part of adverse effects
If the toxicity of sample is high, buffering ability If the
toxicity of sample is high, buffering ability of media water solve
pH problem. of media water solve pH problem.
3030
Pretreatment, liquid samplesPretreatment, liquid samples Aquatic
organisms Aquatic organisms direct testingdirect testing
Solubility in water is not grantedSolubility in water is not
granted
Some liquid substances form foam, emulsion Some liquid
substances form foam, emulsion and/or jelly with water. and/or
jelly with water.
Oganic chemicals have to be examined for Oganic chemicals have
to be examined for solubility in water.solubility in water.
3131
Pretreatment, solid samplesPretreatment, solid samples A)
conversion into liquid form (leachate, extract) A) conversion into
liquid form (leachate, extract) => aquatic organisms=>
aquatic organisms
Version Version -- solid phase test solid phase test --
suspensionsuspension
B) terrestrial, contact bioassays (constant B) terrestrial,
contact bioassays (constant moisture) => soil organismsmoisture)
=> soil organisms
3232
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Pretreatment of solid samples Pretreatment of solid samples for
aquatic bioassaysfor aquatic bioassays
Leachate Leachate useful for in water well soluble useful for in
water well soluble substancessubstances
Acid leachate Acid leachate for substances well soluble in for
substances well soluble in lower pH lower pH metalsmetals
Water saturated with CO2. Water saturated with CO2.
Extract Extract organic, hydrophobic compounds organic,
hydrophobic compounds (solvents: methanol, aceton, DMSO).(solvents:
methanol, aceton, DMSO).
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Leachate of solid samplesLeachate of solid samplesChoice of
Choice of leachateleachate pH influence pH influence which
substances (or chemical which substances (or chemical form) will be
form) will be eluatedeluated and thus and thus resulting
toxicityresulting toxicity
Systems of Systems of leachateleachate buffers, buffers,
offering possibility of continuous offering possibility of
continuous changing of pH from pH 2 to pH 12changing of pH from pH
2 to pH 12
HO
H
3434
Pretreatment of sample, Pretreatment of sample,
leachateleachate
After homogenization of sample dry content is After
homogenization of sample dry content is determined, 105determined,
105C. C.
24 hours of shaking in tilt24 hours of shaking in tilt--over
shakerover shaker
Filtration, porosity 5 Filtration, porosity 5 m.m. SShelf
lifehelf life up to 1 month, 4up to 1 month, 4CC
3535
Extraction of solid samplesExtraction of solid samples Extract =
organic solvent Extract = organic solvent
(water = leachate)(water = leachate)
Extract Extract hydrophobic contaminationhydrophobic
contamination
DMSO, methanol, ethanol, aceton, octanol, DMSO, methanol,
ethanol, aceton, octanol, hexanol, isopropanolhexanol,
isopropanol
3636
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Dewatering of solid samplesDewatering of solid samples
Toxicological data are referred to weight of soil.Toxicological
data are referred to weight of soil.
It is necessary to keep exact moisture of sample.It is necessary
to keep exact moisture of sample.
Dewatering is usually carried out in air stream at Dewatering is
usually carried out in air stream at room temperature. room
temperature. Until constant weight.Until constant weight.
3737
Pretreatment of solid samples Pretreatment of solid samples for
terrestrial assaysfor terrestrial assays
HomogenisationHomogenisation
AgitationAgitation
Grinding, crushing down to recommended Grinding, crushing down
to recommended gradinggrading
3838
Pretreatment Pretreatment -- summarysummary Pretreatment
imortantly influence final data of Pretreatment imortantly
influence final data of bioassays. bioassays.
The manner of pretreatment has to been chosen The manner of
pretreatment has to been chosen regarding to:regarding to:
Final interpretation of dataFinal interpretation of data
Objective of study (what we want to know)Objective of study
(what we want to know)
Detailed specification of pretreatment should be a Detailed
specification of pretreatment should be a part of final report of
bioassaypart of final report of bioassay
3939
Standard toxicantStandard toxicant
Standard ZnSO4 . 7H2O K2Cr2O7 p-nitrofenol
Average value EC50, mg.l-1
9,6 0,67 12,9
Range EC50, mg.l-1 1-25 0,62,1 5-20
Example for Daphnia magna, 48 Hours
4040
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MediaMedia Composition of media (water, soil) has strong
Composition of media (water, soil) has strong impact on
results.impact on results.
Standardization is needed.Standardization is needed.
4141
Composition of water mediaComposition of water media Fish,
daphnidsFish, daphnids
CaCl2 . 2H2OCaCl2 . 2H2O
MgSO4.7H2OMgSO4.7H2O
NaHCO3NaHCO3
KClKCl
AlgaeAlgae
NaNO3NaNO3
Ca(NO3)2 . 4H2OCa(NO3)2 . 4H2O
K2HPO4K2HPO4
MgSO4 . 7H2OMgSO4 . 7H2O
Na2CO3Na2CO3
FeFe--EDTAEDTA
Gaffron solutionGaffron solution
4242
Limit testLimit test To recognize if the samle is toxic or
not.To recognize if the samle is toxic or not.
4343
Limit testLimit test Unknown sample is overviewed if it is or is
not Unknown sample is overviewed if it is or is not
toxic.toxic.
Two parallels with two controls are usually Two parallels with
two controls are usually carrying out.carrying out.
If no response is observed, the test is interpreted If no
response is observed, the test is interpreted as negative. Verify
test follows. as negative. Verify test follows.
If response is observed then RangeIf response is observed then
Range--finding test finding test follows.follows.
4444
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Vertification testVertification test
To verify negative result from previous limit test.To verify
negative result from previous limit test.
More parallels more organismsMore parallels more organisms
4545
Range finding testRange finding test To find interval, where
EC50 can be expectedTo find interval, where EC50 can be
expected
Wide range of concentrationsWide range of concentrations
Small number of organismsSmall number of organisms
4646
Basic testBasic test Exact determination of EC50Exact
determination of EC50
Usually at least 7 concentrations, based on range Usually at
least 7 concentrations, based on range finding test.finding
test.
2 2 -- 3 paralels3 paralels
Accompanying parameters should be recorded at Accompanying
parameters should be recorded at the beggining and on the end of
exposition the beggining and on the end of exposition (pH,O2,
temperature)(pH,O2, temperature)
4747
EndpointEndpoint DeathDeath
Inhibition of growthInhibition of growth
ImmobilisationImmobilisation
Inhibition of physiological function Inhibition of physiological
function (bioluminiscence)(bioluminiscence)
Etolgical observationEtolgical observation
4848
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Ecotoxicological parametersEcotoxicological parameters
Magnitudes focused on kvantitative description Magnitudes focused
on kvantitative description of substance effect on organism.of
substance effect on organism.
Function: estimate of risk, evaluation of Function: estimate of
risk, evaluation of environmental acceptability of substances,
environmental acceptability of substances, comparation of toxic
effect of different samplescomparation of toxic effect of different
samples
Output of toxicity testing (TIE).Output of toxicity testing
(TIE).
4949
Calculated parametersCalculated parameters EC 50 : Effective
concentration of tested toxicant EC 50 : Effective concentration of
tested toxicant causing 50% response of testing population. causing
50% response of testing population.
EEffective ffective CConcentrationoncentration, mg/L, ml/L,
mg/L, ml/L
Lethal Concentration, LC50Lethal Concentration, LC50
Inhibition Concentration, IC50Inhibition Concentration, IC50
EC05, EC20, EC90, EC05, EC20, EC90,
5050
Determined parametersDetermined parameters NOEC: No Observed
Effect Concentration. The NOEC: No Observed Effect Concentration.
The highest tested concentration of toxicant what do highest tested
concentration of toxicant what do not cause statistically
significant effect in not cause statistically significant effect in
comparing to control.comparing to control.
LOEC: The immediate higher concentration than LOEC: The
immediate higher concentration than NOEC.NOEC.
NOEL: No Observed Effect Level.NOEL: No Observed Effect
Level.
NOAEL: No Observed Adverse Effect Level. The effect NOAEL: No
Observed Adverse Effect Level. The effect is observed, but do not
considered as dangerous.is observed, but do not considered as
dangerous.
5151
Design of toxicity testDesign of toxicity testControl 6 mg/L 12
mg/L 25 mg/L 50 mg/L 100 mg/L
Exposed 20 pcsExposed 20 pcs
Response after 24 hours, pcsResponse after 24 hours, pcs
00 00 11 66 1313 1717
Response after 96 hours, pcsResponse after 96 hours, pcs
00 00 44 99 1515 2020
5252
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Recording of testRecording of test
ConcentrationConcentration ControlControl 6 mg/L6 mg/L12 12
mg/Lmg/L25 25
mg/Lmg/L50 50
mg/Lmg/L100 100 mg/Lmg/L
EffectEffect
24 hour24 hour 0%0% 0%0% 5%5%3030%%
6565%%
85%85%
EffectEffect
96 hour96 hour 0%0% 0%0%2020%%
3535%%
7575%%
100100%%
5353
Dvka-Odpov
0 25 50 75 1000
25
50
75
100 24 hod.96 hod.
Koncentrace
M
o
r
t
a
l
i
t
a
Dose - response
24 hrs96 hrs
M
o
r
t
a
l
i
t
y
Concentration
5454
Ideal dose Ideal dose response curveresponse curve
0
20
40
60
80
100
0 500 1000Concentration
e
f
f
e
c
t
,
%
Linear scale is often blind
5555
Ideal dose Ideal dose response curveresponse curve
0102030405060708090
100
0,1 1 10 100 1000 10000
Logaritmic scale
Estimate of EC50
Lethal effects
LC
Non toxic
NOECLOEC
5656
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Presentation of resultsPresentation of results EC50 + confidence
intervals,95%EC50 + confidence intervals,95%
NOEC, EC05NOEC, EC05
LOEC, EC20LOEC, EC20
LC, EC95LC, EC95
5757