Registration Dossier

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20th November 2008 to 31st December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Experimental Additive 20735-35

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Not applicable

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Method of application to filter paper (if used): Not applicable
- Method of mixing into soil (if used): An amount of test material (3500 mg) was added to approximately 10 g of artificial soil and carefully mixed on a watch glass using a spatula until homogenous prior to being incorporated into 3.5 kg (dry weight) of artificial soil with 960 ml of deionised reverse osmosis water and mixed using a Hobart A200N mixer to give the test concentration of 1000 mg/kg with a nominal moisture content of 30% of dry weight.
An amount of soil (approximately 100 g) was taken for moisture content determination 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.
- Method of application to soil surface (if used): Not applicable
- Controls: The control was prepared in an identical manner using 2.5 kg (dry weight) of artificial soil and 680 mL of deionised reverse osmosis water, without the addition of test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evaporation of vehicle before use: Not applicable

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms
- Source: Blades Biological, Cowden, Edenbridge, Kent, UK
- Age at test initiation (mean and range, SD): at least 2 months old with clitellum
- Weight at test initiation (mean and range, SD): 0.37-0.44 g (SD 0.06-0.11 g)

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): The test organisms were transferred into artificial soil (without test material) approximately 24 hours prior to the start of the test.
- Health during acclimation (any mortality observed): Not specified

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
Not performed

Test conditions

Test temperature:
21°C
pH:
Day 0: pH 6.1-6.2
Day 14: pH 5.4-5.8
Moisture:
The moisture content of the soil used in the definitive test was 29% of dry weight of basic substrate on Day 0 and 25 to 26% on Day 14.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1 L glass beakers were used. The test vessels were covered with plastic film with ventilation holes. The earthworms were not individually identified and received no food during exposure.
- Amount of soil or substrate: 600g per replciate for control; approximately 560 g per replicate for test samples
- No. of organisms per container (treatment): 10 earthworms/ container
- No. of replicates per treatment group: 6 replicates
- No. of replicates per control: 4 replicates
- No. of replicates per vehicle control: Not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition of artificial substrate:
Industrial quartz sand: 70% w/w
Kaolinite clay: 20% w/w
Sphagnum moss peat: 10% w/w
- Organic carbon (%):
- Maximum water holding capacity (in % dry weight): 30%
- CEC: Not specified
- Pretreatment of soil: The pH of the artificial soil was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate.
- Storage (condition, duration): Not specified
- Stability and homogeneity of test material in the medium: Not specified

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity: 621 to 712 lux

EFFECT PARAMETERS MEASURED:
The weight of each earthworm was recorded on Day 0 before addition to the test or control vessels. Each surviving earthworm was weighed on Day 14.
Any mortalities were recorded 7 and 14 days after the start of treatment and any sublethal effects are recorded daily. The criterion of death was taken to be the absence of any reaction to a physical stimulus at either end of the body.
Room temperature and light intensity were recorded daily throughout the test. Soil moisture content and pH were recorded at the start and termination of the test. The pH was measured using a VVfW pH 330 pH meter.

VEHICLE CONTROL PERFORMED: Not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable
- Justification for using less concentrations than requested by guideline: Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/kg of soil to confirm that at the maximum concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
- Range finding study
- Test concentrations: In the range-finding test earthworms were exposed to a series of nominal concentrations of 100 and 1000 mg/kg. Amounts of test material (50 and 500 mg) were each separately added to approximately 10 g of artificial soil in a scintillation vial and mixed by shaking by hand for approximately 2 minutes prior to being incorporated into 0.5 kg (dry weight) of artificial soil with 140 mL of deionised reverse osmosis water and mixed using a Kenwood Chef mixer to give the test concentrations of 100 and 1000 mg/kg with a nominal moisture content of 30% of dry weight. The control was prepared in an identical manner using 0.5 kg (dry weight) of artificial soil and 140 mL of deionised reverse osmosis water, without the addition of test material.
For the control and each test concentration 10 earthworms were added to each 1 litre glass beaker containing approximately 0.5 kg dry weight of substrate. The vessels were covered by plastic film with ventilation holes and maintained under continuous illumination (690 to 713 lux) in a temperature controlled room at approximately 21°C for a period of 14 days. After 7 and 14 days, the number of dead earthworms at each test concentration were determined.
- Results used to determine the conditions for the definitive study: Cumulative mortality data from the exposure of Eisenia foetida to the test material during the range-finding test are given in Table 1.
The results showed no mortalities at the test concentration of 100 mg/kg and 2 mortalities at 1000 mg/kg.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
Nominal and measured concentrations:
In the range-finding test earthworms were exposed to a series of nominal concentrations of 100 and 1000 mg/kg.
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/kg of soil to confirm that at the maximum concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
Reference substance (positive control):
yes
Remarks:
Chloroacetamide

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
LC50
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
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
Details on results:
- Mortality at end of exposure period: Cumulative mortality data from the exposure of Eisenia foetida to the test material during the definitive test are given in Table 2.
There were no mortalities or sub-lethal effects of exposure in 60 earthworms exposed to a test concentration of 1000 mg/kg for a period of 14 days.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 1000 mg/kg and the lowest test concentration resulting in 100% mortality to be greater than 1000 mg/kg.
- Total mass of adults at beginning of test: Mean earthworm weight at Day 0: 0.37-0.44 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period: There were no significant differences (P>=0.05) between the control and 1000 mg/kg test groups in terms of weight on Days 0 and 14.
Mean earthworm weight at Day 14: 0.34-0.40 g
- Morphological abnormalities: There were no observed sub-lethal effects of exposure of Eisenia foetida to the test material.
- Behavioural abnormalities: There were no observed sub-lethal effects of exposure of Eisenia foetida to the test material.
- Other biological observations: Not applicable
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Relevant effect levels: Cumulative mortality data from the exposure of Eisenia foetida to chloroacetamide during the positive control are given in Table 3.
LC50 (7 days): 25 mg/kg
LC50 (14 days): 24 mg/kg
NOEC (14 days): 18 mg/kg
- Other: Not specified
Reported statistics and error estimates:
95% confidence limits could not be determined for the test material.

Results for the positive control are as follows:
LC50 (7 days): 25 mg/kg (95% cofidence limits: 23-27 mg/kg)
LC50 (14 days): 24 mg/kg (95% cofidence limits: 23-25 mg/kg)

Any other information on results incl. tables

Table 1: Cumulative Mortality Data in the Range-finding Test

Nominal concentration (mg/kg)

Cumulative mortality (initial population: 10)

% mortality

Day 7

Day 14

Day 7

Day 14

Control

0

0

0

0

100

0

0

0

0

1000

2

2

20

20

Table 2: Cumulative Mortality Data in the Definitive Test

Nominal concentration (mg/kg)

Cumulative mortality (initial population: 10)

% mortality

Day 7

Day 14

Day 7

Day 14

Control

R1-R4

0

0

0

0

1000

R1-R6

0

0

0

0

Table 3: Cumulative Mortality Data in the Positive Control with Chloroacetamide

Nominal concentration (mg/kg)

Cumulative mortality (initial population: 40)

% mortality

Day 7

Day 14

Day 7

Day 14

Control

0

0

0

0

5.6

0

0

0

0

10

0

1*

0

3

18

1*

1*

3

3

32

37

39

93

98

56

40

40

100

100

* mortality not considered to be significant compared to the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
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 NOEC 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.

The 14-Day LC50 for the test material to earthworms (Eisenia foetida) based on nominal test concentrations was greater than 1000 mg/kg. The NOEC was 1000 mg/kg.