Bi2003 Ecotoxicology
Ecotoxicological bioassays
Jakub Hofman
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Content
§Introduction – what, why, how, concept
§Types of bioassays
§Ecotoxicological bioassays’ design and results
§Aquatic bioassays - examples
§Soil bioassays – examples
§Use of bioassays in praxis
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Bioassays’ general design
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1) Prepare the organism
Culture media, standardized numbers, age, etc.
2) Prepare the sample
Dilution series
water/culture media – direct organism exposure
Include BLANK (medium only)
solvent for organic compounds – minimum to be added
Include SOLVENT CONTROL
specifics for the SOLID MATRICES
3) Expose of organisms
… for appropriate time, number of repetitions, under specified conditions
§
General scheme of bioassay
Schema testu Ps_putida
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0) culture
of organisms
4) Evaluate and report results
measure the endpoint / count organisms
validity criteria
statistical evaluation (means, ANOVA, dose-response …)
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Organisms
Ideally
§good availability (laboratory cultures, commercial availability ...)
§easy storage and breeding in laboratory conditions in sufficient quantities for experiments
§the biology of the species and the genetics of the respective culture are characterized
§the relative sensitivities of the species / culture to different classes of toxic substances are
studied
§the susceptibility of the species should be a good representative of the relevant group of
organisms (Daphnia - crustaceans, Danio rerio - freshwater fish)
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Organisms
Cultures !!!
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Obsah obrázku stůl, interiér, láhev, brýle Popis byl vytvořen automaticky Obsah obrázku místnost,
postel Popis byl vytvořen automaticky IMG_0054 IMG_0061 IMG_3421_1 005 11

Organisms
§the result of toxicity determination and interpretation is influenced by a number of other
biological parameters
ogenetically determined sensitivity of the respective culture / clone / variety ...
osize and age of individuals
osex
odevelopmental stage (eggs, embryos, larvae, adults ...)
ophysiological conditions - optimum (diseases, food - antioxidants ...)
§
§in general, the organisms must be in optimal status before the test è this is checked by the test
validity criteria and testing reference substances
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Exposure in aquatic bioassays
§Usually exposure of whole organisms (intake by body surface area, respiratory system, food) less
often: single injections (fish, input and dose are not affected by the environment)
Distribution according to the arrangement of the exposure
ostatic (without exchange of solutions - possible changes of concentrations, oxygen)
ostatic with medium change/renewal (change at defined times, ~24 h)
orecirculation (medium recirculation, more technically demanding ...)
oflow-through (continuous maintenance of concentrations, technically demanding ...)
§
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static
static – large volume
static – medium change
flow-through

Exposure of vertebrate animals in bioassays
§vertebrates - laboratory rodents, birds
§
§like in "classical" toxicology:
oinjection intramuscular (IM), intraperitoneal (IP), intravenous (IV), subcutaneous (SC)
ooral - dosing in food, application of gauze (tube directly into the stomach) ...
orespiration - air contamination - closed containers / cells, inhalation ...
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Exposure in soil bioassays
§soil, sediments - bacteria, invertebrates - contact with the whole surface (direct contact tests -
solid phase tests)
oreal soil / sediment
oartificial soil / sediment
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§plants - roots - contact with solid or liquid medium, exposure to gaseous pollutants from the air
§
§often several exposure routes can be realistically assumed at the same time
§
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Exposure in soil bioassays
it is specific:
§the fate of the contaminant in the soil environment, the influence on the real bioavailability for
soil organisms comes into play significantly
§
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Pb: 2 g/kg in soil
mortality of earthworms
Lanno et al. (2004) Ecotoxicology and Environmental Safety 57, 39-47

Exposure in soil bioassays
Bioavailability
§soil is heterogenous and there is lot of places available for sorption or sequestration of the
pollutants è fate, beahviour, distribution affected è exposure, toxicity, risks affected
§
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bioavailability

Ingestion and oral
•food and soil particles - organisms consume mineral and organic matter - an important route of
exposure for sorbed chemicals
•contaminants can be biomagnified - for example in fungi that are consumed by sprintails
•important path for arthropods
Dermal
•from the soil or soil solution - especially organisms drilling in the soil (earthworms and
enchytraeids), which have a thin cuticle and are in contact with the soil and pore water
•it is also possible to model the results of tests in an aquatic environment by supplementing the
model of the distribution of the substance between the soil solution and the sorption on particles
= the so-called Equilibrium partitioning theory (EqP)
Breathing
almost no data
Exposure in soil bioassays
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Potworms (Enchytraeids doerjesi)

Exposure in soil bioassays
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ARTIFICIAL SOIL
§10% dry fine peat
§20% caoline clay, min 30% calolinite
§70% quartz sand fine min. 50% of size 0.05 – 0.2 mm
§0.3-1% calcium carbonate à pH of 6 ± 0.5
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Exposure in soil bioassays
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IMG_4025
§artificial soil is NOT real soil

Exposure in soil bioassays
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§artificial soil is NOT real soil
http://lufa-speyer.de

Exposure in soil bioassays
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§the goal is HOMOGENITY of exposure to the test substance
water soluble chemicals
§in water which is also used to adjust soil moisture
insoluble in water
§using carrier - non-toxic, water soluble/miscible (acetone, ethanol)
§using carrier - non-toxic volatile organic solvent and evaporated rapidly
§in both cases, solution can be added to:
osmall amount (1-10%) of fine quartz sand; after evaporation of solvent, this is added to soil and
mixed
odirectly into soil (dry or wet) followed by evaporation and mixing
§in all carrier cases, it is necessary to include a control for the carrier/solvent
insoluble in water or solvent
§mixed directly with quartz sand or whole soil

Factors / conditions of the assay
§CRUCIAL !!! they affect both the organisms and the tested chemical, they significantly affect the
exposure and the final results
§must be standardized !!!
otemperature
olight / photoperiod
ooxygen (aquatic consumers)
opH
owater hardness
oclay and organic matter content in soil
ofood added/non-added
oetc.
§
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Results of the bioassays
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Parameters of evaluation - endpoints
§effects ~ response = result of the exposure to toxic chemical (stressor)
ohigher/lower with increasing stressor intenzity (except hormesis)
§endpoint = measured (measurable) response / effect
ooriginal units (numbers, weight, enzyme activity etc.) or relative (% of control)
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§acute effects
oanimals – letality/mortality, imobilization in case of Daphnia
oplants – algae: growth, chlorofyl (fluorescnence); vacular: emergence, growth
odestruents – bacteria: growth, activity ...
§chronic/sublethal effects
oanimals –growth, malformations, reproduction, behavior
oplants – growth, reproduction...
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Measures of exposure
§DOSE versus CONCENTRATION
otoxicology – dose - mg/kg b.w. - body weight, mg/kg b.w./day
oecotoxicology – usually the concentration in the medium - mg/L, mg/kgsoil etc.
o
§pure chemicals and defined mixtures
oconc. in media - mg/L, mmol/L (= mM), mg/kg etc.
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§environmental samples
oextracts of the samples and their % dilutions
o% of the sample in the reference material
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Data from ecotoxicity bioassays
§
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Error bars in the graphs indicate that regardless of the type of response, it is measured in
several replicates and the resulting data have some variability

Data from ecotoxicity bioassays
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Tested factor - qualitative, nominal
Contamination (or other stressor tested) in variants (samples) is not quantified, is not
quantifiable, or is quantified, but the aim is not to study the influence of its intensity on the
effect. Variants (samples) cannot be (or it is not the goal) arranged in any way. It is a
comparative test of several variants (samples) each other and / or against the control (e.g. soils
from monitoring from different localities, samples of different soil materials - sludge, sediments,
waste). The extreme is „the limit test“ - one tested variant (sample) is compared with the control.
Tested factor - kvalitative, ordinal
Contamination (or other stressor tested) in variants (samples) is not fully quantified, but
variants (samples) can be ranked based on some criteria. However, it is not possible to determine
how many times the variant is larger or smaller than the previous or next one - the intensity of
contamination (stress) cannot be plotted on the axis and no relationship between it and the effect
can be modeled. It is a comparative test of several variants (samples) each other and / or against
the control (e.g. soils little, medium and very far from the source; soils from little, medium and
very damaged ecosystem, etc.).
Tested factor – quantitative
Contamination (stressor) is quantified to the extent that it is possible to say how many times or
by how much its intensity is greater or less than in the previous or next variant. The contaminant
concentration (stressor intensity) can be plotted on an axis and the experiment arranged and
evaluated as a test of the relationship between concentration and effect, this relationship can
then be graphically expressed, modeled and ecotoxicity parameters calculated. Whether the tested
factor is quantified by discrete (integer) or continuous (even decimal) data is not very relevant.

Data from ecotoxicity bioassays
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Observed response – qualitative, binary
The answer is not quantifiable, it is only possible to determine whether it has occurred or not - a
typical example is mortality / survival of organisms, occurrence of some signs (lesions, swelling,
mutations,), immobilization, escape reaction, etc. Finding on a number of organisms (biological
systems), the binary result can be converted to the frequency or affected fraction and expressed as
a percentage of the effect (e.g. mortality, lethality, survival, leakage) or as an affected
fraction with values from 0 to 1. In this form, this type of data can be evaluated similarly to
quantitative continuous data (test, model, regressions, etc.), but statistical methods designed for
binomial data should be used correctly, including, for example, another formula for calculating
variance.
Observed response – qualitative, ordinal
The response is quantified to the extent that the results can be ranked, eg small, medium and large
damage. However, it is not possible to determine between the samples (variants) how many times or
by how much the result is larger or smaller, and therefore it is not possible to model the
relationship between the effect and the concentration. As with binary data, these results can be
converted to fraction or frequency and expressed as a percentage in each category.
Observed response – quantitative, continuous
The response is quantified to the extent that it is possible to say how many times or how much is
greater or less in one variant (concentration) than in another (eg weight, size, enzyme activity,
production, number of juveniles, biomarker concentration…). The results can be plotted and the
relationship between concentration and effect evaluated.

Data from ecotoxicity bioassays
§
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Draft Guidance Document for on the Statistical Analysis of Ecotoxicity Data. OECD Environmental
Health and Safety Publications, Series on Testing and Assessment, Environment Directorate, OECD,
Paris 2003.

Dose(concentration) - response relationship
AcuteToxScheme_RM
No Observed Effect Concentration (NOEC)
Lowest Observed Effect Concentration (LOEC)
ECx ( x % effects concentration)
LCx ( x % lethal concentration)
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each conc./dose is tested in several replicates !!!

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Dose(concentration) - response relationship
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