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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-27 September 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. However, identity/composition data not for 100% clear and a deviation compared to guideline wrt moisture content. Conclusion valid.
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
moisture content lower than guideline
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
For the purpose of the definitive study the test material was prepared by direct addition to the
basic soil substrate.
An amount of test material (3500 mg) was incorporated into 3.5 kg (dry weight) of artificial soil
with 910 ml of deionised reverse osmosis water and mixed using a Hobart QE 200 mixer to give
the 1000 mg/kg test concentration with a nominal moisture content of 30% of dry weight. The
control was prepared in an identical manner using 2.5 kg (dry weight) of artificial soil and 650 ml
of deionised reverse osmosis water without the addition oftest material.
An amount of soil (approximately 100 g) was taken from the control and the 1000 mg/kg test
group. The remaining control soil was dispersed to four replicates R1 - R4 and the test soil was
dispersed to six replicates R1 -R6.
Analysis of the concentration, homogeneity and stability of the test material In the test
preparations were not appropriate to the Test Guideline.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
The test was carried out using earthworms of the species Eisenia foetida. Earthworms were
obtained from Blades Biological, Cowden, Edenbridge, Kent, UK and maintained in a 100 litre
plastic container at a temperature ·of 24°C on a substrate consisting of 50% by volume peat and
50% by volume animal manure. Once per week a mixture of animal manure and household
vegetable waste was added as an additional supplement..
The test organisms were transferred into artificial soil (without test material) approximately
24 hours prior to the start of the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
21 C
pH:
5.7-6.0 at initiation, 5.7-5.9 at termination
Moisture:
29% at initiation, 26-27% at termination
Details on test conditions:
A defined artificial soil was used as the basic test substrate with the following composition:
Industrial quartz sand; 69% w/w
Kaolinite clay; 20% w/w
Sphagnum moss peat; 10% w/w
Calcium carbonate 1% w/w (pH adjustment)

Experience has shown that a moisture content of 35% of dry weight, as stated in the Test Guideline, may be too high for the
preparation of the artificial soil, causing sludge formation.
Therefore, a moisture content of 30% of dry weight was considered appropriate for use during the
study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive study was 29% of dry weight of basic substrate on day 0 and 26 to 270/0 on Day 14.

As in the range-finding study 1 litre glass beakers were used. At the start of the study 10
earthworms were placed in each test and control vessel on the artificial soil, at random. The test
vessels were covered with plastic film with ventilation holes and maintained in a temperature
controlled room at 21°C under continuous illumination at 611 to 720 lux. The earthworms were
not individually identified and received no food during exposure.
Nominal and measured concentrations:
1000 mg/kg nominal
Reference substance (positive control):
yes
Remarks:
chloro acetamide
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
No mortality was observed in either control or test group. No significant effect was observed wrt the weight of the worms.
Results with reference substance (positive control):
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et a11977) on
Day 7 and the probit method (Finney 1971) on Day 14 gave the following results based on
nominal test concentrations:
7 days; LC50 31 mg/kg; 95% confidence (28-33 mg/kg)
14 days; LC50 23 mg/kg; 95 % confidence (20-25 mg/kg)
The results from the positive control study with chloroacetamide are within the normal range for
this material.
Reported statistics and error estimates:
For the positive control study, the LC50 values and associated confidence limits on day 7 were calculated by the trimmed Spearman-Karber method (Hamilton et a1 1977) using the ToxCalc computer software package (ToxCalc 1999) and on Day 14 by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). Probit analysis is used where two or more partial responses to exposure are shown. When only one partial response is shown the trimmed Spearman-Karber method is appropriate. An estimate of the LC50 values was given by inspection of the mortality data for the definitive study. Statistical analysis of the earthworm weight data was performed using Bartlett's test for
homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1996).

Table 1: Cumulative mortality in the range finding study

Nominal concentration (mg/kg)

Cumulative mortality (n = 10)

% Mortality

Day 7

Day 14

Day 7

Day 14

Control

0

0

0

0

10

0

0

0

0

100

0

0

0

0

1000

0

0

0

0

 

Table 2: Cumulative mortality in the definitive study

Nominal concentration (mg/kg)

Cumulative mortality

(n(control) = 40; n(test) = 60)

% Mortality

Day 7

Day 14

Day 7

Day 14

Control

0

0

0

0

1000

0

0

0

0

 

Validity criteria fulfilled:
yes
Conclusions:
Valid study, well reported. The moisture content difference compared to the guidance is not expected to have influenced the outcome of the study. The acute toxicity of the test material, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was >= 1000 mg/kg.
Executive summary:

A study was performed to assess the acute toxicity of the test material to the

earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the

OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests"

and EEC Commission Directive 87/302/EEC. Following a preliminary range-finding study, 60 earthworms (six replicates of 10 worms) were exposed to a single concentration of 1000 mg/kg of soil for a period of 14 days. The

number of mortalities were determined after 7 and 14 days exposure. A positive control study

using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes.

The 14-Day LC50 for the test material to earthworms (Eisenia foetida) based on nominal

test concentrations was greater than 1000 mg/kg: The No Observed Effect Concentration was >= 1000 mg/kg.

The result of the positive control study gave a 14-Day LC50 for chloroacetamide of 23 mg/kg

with 95% confidence limits of 20 - 25 mg/kg.

Description of key information

Key study is study of Safepharm, 2002. Short term toxicity was studied in 
imit test. Concentration expressed as nominal. The short-term EC50 or LC50
for soil macroorganisms is 1000 mg/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw

Additional information