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

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

88

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

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

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

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

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

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

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).

3333

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

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

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

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

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

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

Presentation of resultsPresentation of results EC50 + confidence intervals,95%EC50 + confidence intervals,95%

NOEC, EC05NOEC, EC05

LOEC, EC20LOEC, EC20

LC, EC95LC, EC95

5757