OECD 207: Earthworm, Acute Toxicity Tests

OECD 222: Earthworm Reproduction Test (Eisenia fetida)

Registration of Plant Protection Products (1107/2009/EC), Chemicals (REACH Regulation
1907/2006/EC), Biocides (528/2012/EC), Veterinary Medicinal Products (2001/82/EC) and
Medicinal Products for Human Use (2001/83/EC)

Indication
Earthworms play an important role in the soil
biocenosis since they enhance soil characteristics
such as the soil function of litter breakdown, soil
fertility and soil microstructure. Therefore,
earthworms are used as a standard test species to
investigate the impact of a substance on the soil
compartment. Testing is always required when
substances are applied on soil or when a
contamination of soil is possible.
Usually, is in a first step the acute toxicity tested.
Persistent substances or substances with several
applications per season cause a longer exposure
for the earthworm community. In these cases the
performance of a study on sublethal effects is
required. Furthermore the results of the acute test
can trigger a sublethal study.
IBACON performs laboratory studies with
E. fetida under full GLP conditions. Since 1994
IBACON has performed several hundreds of each
study type and has gained vast experienced with
earthworm testing.

Test Organisms
Eisenia fetida (Savigny 1826) belongs to the
Annelida, Oligochaeta. The test organisms
originate from an internal synchronous laboratory
breeding culture. Adult hermaphrodites are used
for testing when they are between two and twelve
months old.

Fig. 1: Clitellated adult earthworm (Eisenia fetida)

Studies Common Features

Earthworm tests are performed with an artificial
soil (according to OECD requirements) consisting
of quartz sand, sphagnum peat, kaolin clay and
calcium carbonate. Testing is performed in a

climatic chamber with defined temperature, light

regime and intensity.

Fig. 2: Test start assignment of the earthworms to

the treatment groups and replicates

OECD 207:
Earthworm, Acute Toxicity Tests
The purpose of this study is to determine the acute
toxicity of a substance to earthworm (Eisenia
fetida) after 7 and 14 days of exposure. On the
basis of the results of a non-GLP pre-test, the
applied concentrations for the main test are
selected. The test item is mixed into the artificial
soil.
Usually, a dose response study with five
concentrations in a geometric row with 4
replicates is performed. In total 40 earthworms are
exposed to the test item per treatment group i.e.
ten individuals per replicate. Each test comprises a
negative control. A test with a toxic reference
substance is performed once a year to proofs the
sensitivity of the test system.
After 7 and 14 days of exposure the mortality of
earthworms is assessed. As sublethal parameters
the mean body weight and behavioural
abnormalities (e.g. lack of movement) are
recorded.
Endpoint of the study is the derivation of a
concentration-response curve for mortality. The
LC50 and if possible a NOEC is calculated.

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Fig. 3: Mortality of Eisenia fetida following treatment

with the test item (mean values of four replicates per
concentration)

Available Guidelines
OECD-Guideline for the testing of chemicals No.
207 Earthworm, Acute Toxicity Test
(adopted April 4, 1984),
ISO-Guideline 11268-1:1993 Soil quality
Effects of pollutants on earthworms (Eisenia
fetida) - Part 1: Determination of acute toxicity
using artificial soil substrate.

Typical Time Frames (GLP Study)

Protocol preparation .................................. 2 weeks
Experimental phase ............................. 4 - 5 weeks
Reporting ............................................. 2 - 4 weeks
Total................................................... 8 - 11 weeks

OECD 222:
Earthworm Reproduction Test
Usually 5 test substance concentrations with four
replicates each are tested. The control comprises 8
replicates. The application can be performed by
mixing the substance into the artificial soil or by
spraying onto the soil surface.

For plant protection products the main focus of

this study is to show that the no observed effect
concentration (NOEC) of the product is less than
the 5 fold predicted environmental concentration
(PEC), which is set as the acceptability threshold.
Otherwise, an earthworm field study is the option
to prove environmental safety.
10 adult worms are exposed for 4 weeks.
Mortality and weight changes are assessed after
this time. The offspring is exposed another 4
weeks until a total exposure period of 8 weeks is
reached.
300
Mean No. of Juveniles

Significances of body weight changes are

determined statistically.

250
200
150
100
50
0
control

25

50

100

200

400

mg Test Substance / kg Artificial Soil

Fig. 5: Mean number of juveniles per treatment group at

test end

Key endpoint of the study is the reproductive

success of the worms. NOEC and/or EC50 are
determined statistically. Additionally other
sublethal effects like body weight changes and
feeding activity are recorded.

Available Guidelines
OECD, Guideline for the testing of chemicals Nr.
222 "Earthworm, Reproduction Test" (adopted
April 13, 2004).
ISO-Guideline 11268-2, Soil quality - Effects of
pollutants on earthworm (Eisenia fetida) Part 2: Determination of effects on
reproduction (1998).

Typical Time Frames (GLP Study)

Protocol preparation.................................. 2 weeks
Experimental phase ................................... 8 weeks
Reporting ............................................. 2 - 4 weeks
Total ................................................. 12 - 14 weeks

IBACON contact:
Ulf Lhrs (Study Director)
Phone: +49 6154 697 361
Email: ulf.luehrs@ibacon.com

Fig. 4: Sampling

Sabrina Westphal
Head of Business Development
Phone: +49 6154 697 238
Email: sabrina.westphal@ibacon.com

IBACON GmbH Arheilger Weg 17 D-64380 Rossdorf, Germany all rights protected

www.ibacon.com