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Running Title
The “biological” component of
bioavailability: the usage of
ecotoxicological tests
Jörg Römbke
ECT Oekotoxikologie GmbH, D-65439 Flörsheim, Germany
Brussels, 2014
Running Title
Outline of this Talk
1. Introduction
Soil ecology and ecotoxicology
2. Selection of standard tests
General criteria and exposure
3. Description of individual tests
Already required and potential methods
4. Inclusion of bioavailability in ERA
Existing approaches
Focal compartment: Soil
Focal organisms: Soil invertebrates
Brussels, 2014
Running Title
Background: Soil Ecology
Brussels, 2014
Soils differ enormously in terms of
their biological diversity, but also
regarding their abiotic properties.
Macrofauna
Mesofauna + microbes
Running Title
Background: Soil Ecotoxicology
Brussels, 2014
Soil
Physical, chemical & microbiological properties
Chemicals Invertebrates Ecotoxicological
effects depend on the
class, fate and
availability of the
chemical, on the type
and behavior of the
species and on the
surrounding abiotic
factors (e.g. soil
properties, climate)
Surely: clear protection goals are needed for assessing any risks.
Running Title
Conceptual Basis: ISO 17402 (2008)
Conclusion: The only direct way of measuring bioavailability for
an organism to be protected is the use of that organism and to
measure the effect and/or the accumulation of a given chemical.
Brussels, 2014
Chemical measurements
(available fractions)
Biological
measurements
Toxicity tests Bioaccumulation
membrane
soil/water
organism
Methods to assess
bioavailability –
Relation between
chemical, biologi-
cal tests and
bioaccumulation
Running Title
Selection of Standard Tests: Overview
Brussels, 2014
So, soil biological tests are needed – but which ones?
0
5
10
15
20
25
1980 1985 1990 1995 2000
Blue = Laboratory
Red = Semi-field
Yellow = Field
Running Title
Selection of Standard Tests: Criteria
Brussels, 2014
Such tests are provided by two international organizations:
► OECD (Chemicals Group): single-chemical testing before
market authorization prospective protection of soil
► ISO (Technical Committee 190): soils contaminated by a
mixture of chemicals retrospective protection of soil
Regulatory needs <==> Problem: What was first?
Exposure pathways ==> Minimum: pore water, soil ingestion
Species ==> Different taxonomic / ecological groups
(often size: micro/meso/macrofauna)
Sensitivity ==> Chronic or behavioural endpoints
Practicability of tests ==> Simple, quick and cheap performance
Running Title
Selection of Standard Tests: Exposure I
Brussels, 2014
OECD and ISO methods are often similar. But: soils are different.
Organic matter: 10% Organic matter: 2-4%
OECD artificial soil:
Prospective tests Natural field soils:
Retrospective tests
Availability of chemicals in OECD and field soil (may) differ strongly.
Natural standard
LUFA soil
Running Title
Selection of Standard Tests: Exposure II
Brussels, 2014
Exposure of soil organisms is determined by three factors:
1. Exposure pathways:
► Pore-water: main route for plants and (many) invertebrates
► Ingestion of soil particles: relevant for saprophagous invertebrates
► Inhalation of air: possible for all organisms but rarely (?) relevant
2. Biology and ecology of species
► Morphology: e.g. soft- versus hard-bodied organisms
► Physiology: e.g. food uptake, i.e. with or without soil particles
► Behavior: e.g. vertical distribution, including avoidance patterns
3. Chemical properties (Peijnenburg et al. 2012)
Running Title
Selection of Standard Tests: Exposure II
Brussels, 2014
Examples: Exposure of soft- und hard-bodied organisms
Soft: Uptake of oxygen, water and contaminants via the body surface (skin)
Hard: Specific organs for the uptake of oxygen, water and contaminants
Both: Uptake of contaminants via food (with or without soil particles)
Running Title
Selection of Standard Tests: Battery
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A minimum battery of laboratory tests with soil invertebrates is
required in prospective soil protection in the European Union:
Soft-bodied macrofauna:
Chronic Earthworm Test (Eisenia andrei/fetida)
OECD 222 (2004); ISO 11268-2 (1998)
Hard-bodied mesofauna:
Chronic Collembola Test (Folsomia candida)
OECD 232 (2009); ISO 11267 (1999)
Chronic Predatory Mite (Hypoaspis aculeifer)
OECD 226 (2008)
In addition, at least one plant and one microbial test are required.
Running Title
Description of Individual Tests I
Brussels, 2014
Name: Earthworm reproduction test (OECD, ISO)
Substrate: Artificial Soil or many field soils, e.g. LUFA
Duration: 56 days
Parameter: Biomass, number of juveniles
Experience: High for pesticides, veterinary drugs, REACH,
contaminated soils, often very sensitive
Running Title
Description of Individual Tests II
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Name: Collembola reproduction test (OECD, ISO)
Substrate: Artificial Soil or many field soils, e.g. LUFA
Duration: 28 days
Parameter: Mortality of adults, number of juveniles
Experience: High for pesticides and contaminated soils, less for
other chemicals, often very sensitive
Running Title
Description of Individual Tests III
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Name: Predatory mite reproduction test (OECD)
Substrate: Artificial Soil or field soils, e.g. LUFA
Duration: 28 days
Parameter: Mortality of adults, number of juveniles
Experience: High for pesticides, almost none for other chemicals,
or contaminated soils; rarely very sensitive
Running Title
Which other Tests could be useful?
Brussels, 2014
Using the same criteria, behavioral tests are missing, especially
for retrospective soil protection:
Soft-bodied macrofauna:
Earthworm Avoidance Test
(Eisenia andrei/fetida)
ISO 17512-1 (2008)
Hard-bodied mesofauna:
Collembola Avoidance Test
(Folsomia candida)
ISO 17512-2 (2011)
Running Title
Further Standard Invertebrate Tests I
Brussels, 2014
These standard tests are available but are rarely used in practice,
mainly in retrospective soil protection:
Snail Growth Toxicity Test (Helix aspersa)
ISO 15952 (2003)
Nematode Reproduction Test
(Caenorhabditis elegans) ISO 10872 (2011
Chronic Enchytraeid Test
(Enchytraeus albidus/crypticus)
OECD 220 (2004); ISO 16387 (2004)
Running Title
Further Standard Invertebrate Tests II
Brussels, 2014
Originally "invented" for retrospective soil protection, but also
useful in prospective soil protection: semi-field tests.
Terrestrial Model Ecosystems (TME): ASTM (1993), PERAS (2010)
► Interaction of soil properties and the natural community of
microbes, invertebrates and plants
► Undisturbed soils from crop sites or meadows
► Usually about 16 weeks
► Wide variety of fate and effect endpoints
Running Title
Overview on Existing Tests: Plants I
Brussels, 2014
Prospective soil protection:
► Seedling emergence test. 6 – 10 crop species (or
"wild species") OECD 208 (2000). Exposure: mixing
chemicals into control soil; (e.g. LUFA "standard"
field soil). Endpoint: biomass, shoot length.
► Vegetative vigor test. OECD 227 (2000): Like 208
But: Exposure: spraying the pesticide on
the emerging plants and soil surface.
Note: Currently, the inclusion of "lower" plants
(e.g. mosses, ferns) is proposed (EFSA 2011).
Running Title
Overview on Existing Tests: Plants II
Brussels, 2014
Retrospective soil protection:
► Plant root growth test, usually grain species. ISO 11269-1 (1993)
Nowadays rarely used, considered to be not very robust
► Emergence and plant growth test. Avena sativa, Brassica rapa.
ISO 11269-2 (2008): Mixing of contaminated soil (or wastes) with a
control soil (usually LUFA).
Plant test most often used
for contaminated soils.
► Chronic plant test. ISO
22030 (2012): Like 11268-2.
But: Reproduction endpoints.
Running Title
Overview on Existing Tests: Microbes
Brussels, 2014
In contrast to other tests, in microbial studies usually the function (NOT
the structure) of the natural microbial community is tested, i.e. the
tests are performed in natural field soils.
Prospective soil protection:
Nitrogen transformation test: OECD 217 (2000)
Retrospective soil protection (only few examples are given):
Respiration test (SIR): ISO 14240-1 (1997)
Ammonium oxidation rapid test: ISO 15685 (2001)
Arthrobacter globiformis test: ISO 18187 (2012)
Only single species microbial test in soil
Running Title
Inclusion of Bioavailability in ERA I
Brussels, 2014
Prospective soil protection:
General impression: Bioavailability should be taken into account.
However, it is not yet clear how.
► The existing biological tests (earthworms, springtails, mites, plants,
microbes) and the soil(s) used are under review (e.g. pesticides).
► One possible addition: isopods (wood-lice)
(e.g. hard-bodied macrofauna feeding on litter)
► Main focus: Modified exposure calculations
- Exchange of total concentrations
in favor of pore-water concentrations.
- Omisssion of bound residues
Running Title
Inclusion of Bioavailability in ERA II
Brussels, 2014
Retrospective soil protection:
Proposal based on draft
ISO Standard 19204 (2014):
TRIAD approach
Testing is performed for each
of these three areas in three
refinement levels, starting with
simple screening tests.
Availability is covered both in
Chemistry and Ecotoxicology.
Ecotoxicology (laboratory
tests)
Chemistry
(exposure, bioaccu-
mulation)
Ecology
(monitoring,
field surveys)
Integra-tive
Evalu-ation
Running Title
Inclusion of Bioavailiability in ERA III
Brussels, 2014
Tier I (screening):
Chemistry: Refinement of soil screening values
Ecotoxicology: Arthrobacter test (ISO 18187); plant screening test (ISO
17126); earthworm or Collembola avoidance test (ISO 17512-1/2);
Tier II (refined screening):
Chemistry: Bioavailability methods, e.g. solid phase micro extraction.
Ecotoxicology: Chronic earthworm tests (ISO 11268-2), enchytraeids
(ISO 16387), springtails (ISO 11267); Plant growth test (ISO 11269-2).
Tier III (detailed assessment):
Chemistry: Modelling or simulation systems
Ecotoxicology: Multi-species test; Chronic plant test (ISO 22030).
Running Title
Acknowledgements
Thanks a lot to all colleagues
who have been contributed to this talk!
In particular the Working Group 3 of ISO TC 190 SC 7!
Brussels, 2014