05 principles tox testing factors
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PrinciplePrinciple ofof Toxicity Toxicity TestingTesting
Vladimr KoVladimr Ko
Institute Institute ofof ChemicalChemical Technology, Technology, PraguePrague
CzechCzech RepublicRepublic
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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.
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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
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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
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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. .
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Structuring of bioassaysStructuring of bioassaysDuration of expositionDuration of exposition
AcuteAcute
SemiSemi--acuteacute
ChronicChronic
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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
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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
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Structuring of bioassaysStructuring of bioassaysTested matrixTested matrix
WaterWater
SoilSoil
AirAir
SedimentSediment
WasteWaste
Chemical compoundChemical compound
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Structuring of bioassaysStructuring of bioassaysSpectrum of organismsSpectrum of organisms
Single SpeciesSingle Species
Multi Species Multi Species -- wildlife populations / laboratory wildlife populations / laboratory mixturemixture
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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)
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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)
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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
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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
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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.
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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.
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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)
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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
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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)
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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
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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)
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SamplingSampling Way of sampling importantly influence results of Way of sampling importantly influence results of bioassaybioassay
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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
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Pretreatment Pretreatment adjustment of adjustment of sample with a goalsample with a goal
Distinguish toxicantsDistinguish toxicants
Elimination of interferencesElimination of interferences
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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
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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.
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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
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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.
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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.
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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
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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
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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
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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
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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.
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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
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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
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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
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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.
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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
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Limit testLimit test To recognize if the samle is toxic or not.To recognize if the samle is toxic or not.
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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.
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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
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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
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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)
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EndpointEndpoint DeathDeath
Inhibition of growthInhibition of growth
ImmobilisationImmobilisation
Inhibition of physiological function Inhibition of physiological function (bioluminiscence)(bioluminiscence)
Etolgical observationEtolgical observation
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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).
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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.
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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
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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%%
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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
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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
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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
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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
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