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The “biological” component of

bioavailability: the usage of

ecotoxicological tests

Jörg Römbke

ECT Oekotoxikologie GmbH, D-65439 Flörsheim, Germany

Brussels, 2014

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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

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Background: Soil Ecology

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Soils differ enormously in terms of

their biological diversity, but also

regarding their abiotic properties.

Macrofauna

Mesofauna + microbes

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Background: Soil Ecotoxicology

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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.

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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.

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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

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Selection of Standard Tests: Overview

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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

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Selection of Standard Tests: Criteria

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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

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Selection of Standard Tests: Exposure I

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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

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Selection of Standard Tests: Exposure II

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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)

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Selection of Standard Tests: Exposure II

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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)

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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.

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Description of Individual Tests I

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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

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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

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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

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Which other Tests could be useful?

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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)

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Further Standard Invertebrate Tests I

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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)

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Further Standard Invertebrate Tests II

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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

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Overview on Existing Tests: Plants I

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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).

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Overview on Existing Tests: Plants II

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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.

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Overview on Existing Tests: Microbes

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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

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Inclusion of Bioavailability in ERA I

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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

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Inclusion of Bioavailability in ERA II

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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

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Inclusion of Bioavailiability in ERA III

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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).

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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!

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