Bioassay

Principle of Toxicity
Testing

Vladimr Ko
Institute of Chemical Technology, Prague
Czech Republic

Experiments with organisms targetted to solve

question how evaluated substance and/or sample
affect life in field condition.
Both inhibitive and stimulative effect is
unaccetable.
Intensity of effect is influenced by concentration
and/ot duration of exposition.

Aim of Science on the Field of

Ecotoxicological Bioassays

Basic principles of bioassays

The development of new standard methods.

Standard reproducible conditions,
Comparison of effects of different substances
and/od different organisms
Reproducible between different laboratories

Reproducibility
Standard design
Possibility of data extapolation on field conditions
Economy feasibility
Time feasibility

Structuring of bioassays
Duration of exposition

Biotest

Bioassay is a process where is a test systm

(tissue
tissue,,organism,
organism, population)
population) exposed under
defined conditions to different known
concentrations of tested compound or sample.
Ecotoxicological bioassays are such a bioassays
what for evaluation of observed phenomenon use
such a detection systems (tissue,
tissue, organism,)
organism,)
what enable relevant interpretation for
environmental conditions.
conditions.

Acute
Semi--acute
Semi
Chronic

Structuring of bioassays
Design, generation

Microbioassay

1st. generation - classic (standard)

2nd. generation - microbioassays
3rd. generation - biosenzors, biomarkers

Economy Reasons
Saving of
Time
1. Batching
Space
Work
2. Inoculation
Chemicals
3. Exposition

Structuring of bioassays
Trophic level

Structuring of bioassays
Tested matrix

Producent
Consument
Destruent

Water
Soil
Air
Sediment
Waste
Chemical compound

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Structuring of bioassays
Spectrum of organisms

Structuring of bioassays
Type of sample

Single Species
Multi Species - wildlife populations / laboratory
mixture

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Pure compounds (hydrophobic, hydrophilic,

volatile)
Mixture of compounds (known and/or unknown)
Environmental samples (usually unknown,
mixtures of different compounds with different
properties complicated interpretation)

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Structuring of bioassays
Pretreatment

Structuring of bioassays
Complexity of detection system

Defined very concentration of substances

Eluates, extracts of environmental samples
liquid final sample, Whole Effluent
Direct Tests, Solid Phase Tests
Comparative tests of sorbtion matrix (for
example SPMD)

From the simplest to the more integral

Enzymes, bioprobes, cell and tissue cultures in
vitro
Whole living organism
Population
Micro/mezo kosmos
Field study

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Structuring of bioassays
Type of endpoint

Special tests for Risc

Assessment

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

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AGP
Mutagenicity/genotoxicity (microbial, vascular
plants, wildlife animals)
Teratogenity (amphibian(amphibian- Xenopus laevis)
Embryotoxicity
Reproduction bioassays
Useful for clearly defined cases, where lethal
effect as an endpoint is not appropriate for
interpretation.

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Battery of assays

Batery of bioassays

The negative response in test with one specie

does not means that substance is not toxic.
Toxicity can be observed after longer exposition
and/or on different species.
Simple battery: algae, zooplancton, fish,
bacteria.

Different
Trophic level
Sensitivity
Target effect/organ
Specific toxic effect (mutagenity, neurotoxicity,
etc)

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Exposition tests

Growth, reproductive tests

Exposed organism do not reproduce during time

of assay.
Quick.
Influence on photosynthesis; synthesis of ATP,
movement of organs.
Mortality, immobilisation

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Better and more sensitive.

Reproductive process is the most sensitive
moment of living organism.
If no adverse effect will be observed during 3
generations, there is very probable that testing
organism is not sensitive to tested concentration
of toxicant.
Nap.: Algal assay, daphnia reproductive assay,
root prolongation test

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Factors influencing results of

bioassays

Standard toxicity tests

Battery of bioassays:
algae, plants, zooplancton, fish, (bacterium)

For reproducibility of results these main

factors has to be standardized:

Time of exposition
Temperature
Light period
Volume
Oxygen
Composition of cultivation media
Age of organism
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Sampling

Sampling

Pretreatment of sample
(eluate, extract, pH, oxygen)
positive

Limit Test
positive, response 50%

Way of sampling importantly influence results of

bioassay

negative

Verify Test

negative

Range finding Test

positive, response < 50%

Basic Test
Data treatment
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Pretreatment adjustment of
sample with a goal

Pretreatment

Pretreatment influence the chemical properties of

substances present in sample and thus their
biological activity.
The manner of pretreatment can diverse activity
od different toxicants presented in sample.
Treatment of pH, dewatering, filtration, aeration,
salinity treatment

Distinguish toxicants
Elimination of interferences

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Temperature

Osmotic pressure

Sterilization - autoclave, environmental samples

Denaturation of proteins, pesticides
Decomposition of thermo labile compounds
Pointing out thermo stabile compounds
Better do not carry out

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Low soluble sample low osmotic pressure

Marine organisms need for higher osmotic
pressure
Needed addition of cultivation salts
The change of mineralization can change form
and amount of solved substances.

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Aeration

pH treatment

Oxygen for organisms

Decrease of volatile substances
Changes in pH

Stripping elimination of interference volatile

substances

Chemical form and thus toxic properties of

substances can be modified. Metals, ammonium
ion
pH can be mentioned as a part of adverse effects
If the toxicity of sample is high, buffering ability
of media water solve pH problem.

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Pretreatment, liquid samples

Pretreatment, solid samples

Aquatic organisms direct testing

Solubility in water is not granted
Some liquid substances form foam, emulsion
and/or jelly with water.
Oganic chemicals have to be examined for
solubility in water.

A) conversion into liquid form (leachate, extract)

=> aquatic organisms
Version - solid phase test - suspension

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B) terrestrial, contact bioassays (constant

moisture) => soil organisms

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Pretreatment of solid samples

for aquatic bioassays

Leachate of solid samples

Leachate useful for in water well soluble

substances
Acid leachate for substances well soluble in
lower pH metals
Water saturated with CO2.
Extract organic, hydrophobic compounds
(solvents: methanol, aceton, DMSO).

Choice

of leachate pH influence
which substances (or chemical
form) will be eluated and thus
resulting toxicity

Systems of leachate buffers,
offering possibility of continuous
changing of pH from pH 2 to pH 12

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O
H

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Pretreatment of sample,
leachate

Extraction of solid samples

After homogenization of sample dry content is

determined, 105
105C.
24 hours of shaking in tilttilt-over shaker
Filtration, porosity 5 m.
Shelf life up to 1 month, 4
4C

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Extract = organic solvent

(water = leachate)
Extract hydrophobic contamination
DMSO, methanol, ethanol, aceton, octanol,
hexanol, isopropanol

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Pretreatment of solid samples

for terrestrial assays

Dewatering of solid samples

Toxicological data are referred to weight of soil.

It is necessary to keep exact moisture of sample.
Dewatering is usually carried out in air stream at
room temperature. Until constant weight.

Homogenisation
Agitation
Grinding, crushing down to recommended
grading

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Pretreatment - summary

Standard toxicant

Pretreatment imortantly influence final data of

bioassays.
The manner of pretreatment has to been chosen
regarding to:
Final interpretation of data
Objective of study (what we want to know)
Detailed specification of pretreatment should be a
part of final report of bioassay

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Example for Daphnia magna, 48 Hours

Standard
Average value EC50, mg.l-

ZnSO4 . 7H2O

K2Cr2O7

p-nitrofenol

9,6

0,67

12,9

1-25

0,62,1

5-20

Range EC50, mg.l-1

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Media

Composition of water media

Composition of media (water, soil) has strong

impact on results.
Standardization is needed.

Fish, daphnids

NaNO3

Ca(NO3)2 . 4H2O

K2HPO4

MgSO4 . 7H2O

Na2CO3

Fe
Fe--EDTA

Gaffron solution

CaCl2 . 2H2O

MgSO4.7H2O

NaHCO3

KCl

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Limit test

Algae

Limit test

To recognize if the samle is toxic or not.

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Unknown sample is overviewed if it is or is not

toxic.
Two parallels with two controls are usually
carrying out.
If no response is observed, the test is interpreted
as negative. Verify test follows.
If response is observed then RangeRange-finding test
follows.

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Vertification test

Range finding test

To verify negative result from previous limit test.

More parallels more organisms

To find interval, where EC50 can be expected

Wide range of concentrations
Small number of organisms

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Basic test

Endpoint

Exact determination of EC50

Usually at least 7 concentrations, based on range
finding test.
2 - 3 paralels
Accompanying parameters should be recorded at
the beggining and on the end of exposition
(pH,O2, temperature)

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Death
Inhibition of growth
Immobilisation
Inhibition of physiological function
(bioluminiscence)
Etolgical observation

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Ecotoxicological parameters

Calculated parameters

Magnitudes focused on kvantitative description

of substance effect on organism.
Function: estimate of risk, evaluation of
environmental acceptability of substances,
comparation of toxic effect of different samples
Output of toxicity testing (TIE).

EC 50 : Effective concentration of tested toxicant

causing 50% response of testing population.
Effective Concentration,
oncentration, mg/L, ml/L
Lethal Concentration, LC50
Inhibition Concentration, IC50
EC05, EC20, EC90,

50

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Determined parameters

Design of toxicity test

NOEC: No Observed Effect Concentration. The

highest tested concentration of toxicant what do
not cause statistically significant effect in
comparing to control.
LOEC: The immediate higher concentration than
NOEC.

Control

NOEL:

No Observed Effect Level.

No Observed Adverse Effect Level. The effect
is observed, but do not considered as dangerous.

NOAEL:

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6 mg/L

12 mg/L

25 mg/L

50 mg/L

100 mg/L

Exposed 20 pcs
Response after 24 hours, pcs
0
0
1
6
13
17
Response after 96 hours, pcs
0
0
4
9
15
20
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Recording of test

Dvka-Odpov
Dose - response

Concentration

Control

6 mg/L

12
mg/L

25
mg/L

50
mg/L

100
mg/L

Effect
24 hour

0%

0%

5%

30
%

65
%

85%

0%

20
%

35
%

75
%

100
%

Effect
96 hour

0%

Mortalita
Mortality

100

24
24 hrs
hod.
96 hod.
96
hrs

75
50
25
0

25

50

75

100

Concentration
Koncentrace
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Ideal dose response curve

Ideal dose response curve

100
90
NOEC
80
70
60
50
40
30
20
10
Non
0 toxic
0,1

100
effect, %

80
60

Linear scale is often blind

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20
0
0

500

1000

Concentration

Lethal effects

LOEC
LC

Estimate
of EC50
1

10

100

1000

10000

Logaritmic scale
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Presentation of results

EC50 + confidence intervals,95%

NOEC, EC05
LOEC, EC20
LC, EC95

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