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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Guideline C(L1)4, Toxicity for Earthworms, Artificial Soil Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
The test substance applications were prepared in acetone. The weighed amount of test soil was spread out evenly over a sheet of "Benchkote" in a fume cupboard. The test solution was applied evenly over the soil surface from a 50 mL fan-jet propelled gas tight syringe. The acetone was allowed to evaporate for approximately 60 minutes before the soil was placed in a mixing bowl. The weighed artificial soil was then placed into a food mixer bowl. The soil was mixed for two minutes by a heavy duty food mixer to evenly incorporate the test substance into the soil. Deionized water was then added to bring the moisture content of the soil up to 50% and mixing continued for a further two minutes.

From the prepared treated soil, 750 g (fresh weight) aliquots were weighed into 1 L glass beakers. For the initial test, E. fetida were exposed in two aliquots (replicates) of treated soil and for the definitive tests four aliquots of soil per treatment.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Eisenia fetida used were clitellate adults that had been cultured in bulb fibre compost at Jealott's Hill. The earthworms were supplied by Monkfield, The Aquatic Centre, Cambridge, U.K. The cultures arrived at Jealott's Hill in spring 1990 and have since been cultured in a controlled environment room.

The mean weight ± standard deviation (SD) for E. fetida used were 0.47 ± 0.03 g in the initial and 0.55 ± 0.03 in the definitive test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
E. fetida were assessed for mortality and any abnormal behavioural effects after 14 days in the initial test and after 7 and 14 days in the definitive tests
Test temperature:
20 ± 2°C
pH:
6.2 ± 0.2
Moisture:
46-49%
Details on test conditions:
TEST SYSTEM
- Test container: 1 L glass beakers
- Amount of soil or substrate: 750 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE
The test soil was a mixture of the following ingredients in oven dry (approximately 105°C) weight proportions:
70 % fine silica sand, 20% kaolinite clay (GTY powder), 10% peat.
Calcium carbonate was incorporated into the artificial soil at 5 g/kg, Ingredients, in their natural fresh weight form, were mixed in a rotary cement mixer.

OTHER TEST CONDITIONS
- Photoperiod: 24 hour light
- Light intensity: 600 ± 200 lux

EFFECT PARAMETERS MEASURED: Mortality and abnormal behavioural effects

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0, 1, 10, 100, and 1000 mg a.i./kg
- Results used to determine the conditions for the definitive study: The results indicated that the LC50 was between 1 and 10 mg a.i./kg soil
Nominal and measured concentrations:
Nominal: 0, 1, 1.8, 3.2, 5.6, and 10 mg a.i./kg soil
Reference substance (positive control):
yes
Remarks:
2-chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
6.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 6.2-7.6 mg a.i./kg
Key result
Duration:
14 d
Dose descriptor:
other: NOEL
Effect conc.:
3.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, behaviour, body weight changes
Results with reference substance (positive control):
The LC50 value for 2-chloroacetamide was 52 mg/kg (95% CL: 40-80 mg/kg)
Validity criteria fulfilled:
yes
Conclusions:
14 d LC50: 6.7 mg a.i./kg
14 d NOEL: 3.2 mg a.i./kg
Executive summary:

Eisenia fetida (clitellate adults) were exposed to concentrations of the test substance in an artificial soil for 14 days at 20 ± 2°C. An initial and a definitive test were conducted. The test was conducted according to OECD guideline 207.

The value for the 14 day LC50 was 6.7 mg a.i./kg soil. At the 3.2 mg a.i./kg soil level, there were no deaths, behavioural effects, significant effects on external conditions or significant body weight changes (p = 5%) and thus, the No Observed Effect Level (NOEL) was at least 3.2 mg a.i./kg soil.

The LC50 value for 2-chloroacetamide, tested as a toxic standard, was 52 mg/kg.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
Controls: Untreated (using the same amount of acetone and fine quartz sand as in the test item treated groups) and moistened with deionized water.

Test substance was administered to earthworms via mixing the test item into the soil in which they are housed. They were in continuous contact with the compound.

A stock solution containing 2.100 mg/mL test substance was prepared by dissolving 105.0 mg of test substance in 50 mL acetone.

A sequential dilution series was prepared by adding 10 ml of acetone to 20 mL of the stock solution or the subsequent dilution and 5 mL of the corresponding solutions were added to 20 g fine quartz sand. The treated sand was left for at least one hour until the solvent had evaporated. The test item blended sand was added to the artificial soil to result in a final net weight of 2100 g dry weight. The compositions were moistened with deionized water and mixed for approximately 5 min in a laboratory mixer (Kenwood brand) to ensure a homogenous distribution. Each group was treated in one batch (two in the control) which was then split into the replicates.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms
- Source: ibacon
- Life Stage: Adults
- Age at test initiation: 6 to 7 months (but all within 4 weeks of the same age), with well-developed clitella
- Weight at test initiation: 300 to 600 mg, including gut contents
- Origin: Earthworms used in these tests were bred under standardized conditions (in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature) by ibacon.

ACCLIMATION
- Acclimation period: 1 day
- Acclimation conditions: Test environmental conditions
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18 to 22°C
pH:
At start 5.7 to 5.8; at experimental end 6.0 to 6.5
Details on test conditions:
TEST SYSTEM
- Test container: Plastic boxes with perforated transparent lids (18.3 cm x 13.6 cm x 6 cm) corresponding to 16.5 cm x 11.5 cm (approximately 190 cm²) at the level of the soil surface. The height of the soil layer in the containers was approximately 4-5 cm.
- Amount of soil: Test units were filled with 500 g artificial soil (dry weight) plus 129.2 g water. The wet weight of soil in each replicate was 629.2 g at the beginning of the test.
- No. of organisms per container: 10
- No. of replicates per treatment group: 8
- No. of replicates per control: 8

PREPARATION AND PROPERTIES OF SOIL
- Ingredients: 10% Sphagnum-peat, air-dried and finely ground (≤2 mm); 20% Kaolin clay, Kaolinite content >30%; 0.4% calcium carbonate (CaCO3) extra pure to adjust pH to 6.0 ± 0.5; 69.6% fine quartz-sand, F34 grain size with more than 50% by mass of particle size 0.05-0.2 mm
The artificial soil was moistened to approximately half of the final water content one day before the application. The additional water required to achieve the final water content was added when applying the test substance.
- Water Holding Capacity: 53% of the dry weight
- Water Content: At start, water content was 28.9% to 29.4% (equivalent to 54.5% to 55.4% of the maximum water holding capacity) and at experimental end water content was 28.8% to 31.6% (equivalent to 54.4% to 59.5% of the maximum water holding capacity.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours darkness per day
- Light intensity: Within the range of 400 to 800 lux

TEST CONDUCT
- Introduction of Individuals: Healthy earthworms were selected. All earthworms were washed with tap water, dried with a dry paper towel, weighed individually and randomly assigned to batches of 10 worms. The different batches were sorted into four categories on the basis of the total weight and one batch of each weight category was assigned to each treatment group (two batches for the control) to ensure weights were homogeneous. The earthworms were placed on the surface of the artificial soil after the soil was treated.
- Food: 5 g/container of finely ground cattle manure was scattered uniformly on the soil surface. The food was moistened with 5-6 g deionized water. 5 g food per container was added the same way each week (but moistened with 2-3 g deionized water)
- Soil Moisture Maintenance: Once a week the soil water content of each container was checked by weighing and water was added, if necessary with a sprayer. In this way, soil water was kept within 10% of the initial soil water content.
- Exposure Time: Adults were exposed for 28 days. After 4 weeks, adults were sorted from the soil and weighed. They were not placed back into the boxes. Juveniles which had been produced in the boxes remained in the soil for another 4 weeks without further feeding.
After a total of 8 weeks (56 days), the experiment was terminated. The number of juveniles of each container was determined. Young worms were extracted from the soil by placing the containers in a waterbath at 50-60°C. All emerging worms were removed and counted. Soil was then sorted through manually to recover any remaining living young worms.

EFFECT PARAMETERS MEASURED: Mortality of adults after 4 weeks, number of offspring surviving after 8 weeks, mean body weight change of adults, food given, behaviour

VEHICLE CONTROL PERFORMED: No
Nominal and measured concentrations:
0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33 and 5.00 mg test substance/kg dry artificial soil
Reference substance (positive control):
yes
Remarks:
Carbendazim
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
3.33 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
2.22 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight changes
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1.48 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
4.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
1.97 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits of 1.53 to 2.26 mg/kg soil
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
2.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits of 1.92 to 2.55 mg/kg soil
Key result
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
3.09 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits of 2.82 to 3.37 mg/kg soil
Details on results:
Mortality and Body Weight Changes in Adult Worms over 4 Weeks: An adult earthworm mortality of 72.5% was observed at the concentration of 5.00 mg test substance/kg dry artificial soil, which was statistically significantly different compared to the control (Fisher’s Exact Test, α = 0.05). No additional mortality of adult earthworms was observed in any other treatment group.
The mean body weight changes of the worms were not statistically significantly different compared to the control up to and including the concentration of 2.22 mg test substance/kg dry artificial soil. At 3.33 mg test substance/kg dry artificial soil and above the body weights were statistically significantly different compared to the control (Williams t-test, α = 0.05, two-sided). For the evaluation of the weight changes the highest concentration was not included due to the low number of surviving worms.

Earthworm Reproduction: The reproduction rates of the test item treated groups were not statistically significantly different compared to the control up to and including the concentration of 1.48 mg test substance/kg dry artificial soil. At 2.22 mg test substance/kg dry artificial soil the reproduction was statistically significantly reduced compared to the control (Williams t-test, α = 0.05, one-sided smaller).

Earthworm Food Consumption: The mean amount of food added to the test containers over the entire experimental time was 24.5 to 25.0 g/container in the control and in the groups treated with test substance up to and including the concentration of 2.22 mg test substance/kg dry artificial soil. At 3.33 mg test substance/kg dry artificial soil and above the food consumption appeared to be reduced compared to the control. This reduction is considered to be due to the reduced number of worms in this treatment group.
The method used here only approximately assesses food consumption of Eisenia fetida, because any food remaining cannot be weighed again. However, it reflects roughly the feeding activity of the earthworms.
Results with reference substance (positive control):
There were statistically significant effects on reproduction at a concentration of 1.95 mg carbendazim/kg dry artificial soil and higher; the EC50 for reproduction was calculated as 1.87 mg carbendazim/kg dry artificial soil
Validity criteria fulfilled:
yes
Conclusions:
28 d LC50: 4.80 mg/kg (mortality)
28 d NOEC: 3.33 mg/kg (mortality)
56 d EC50: 3.09 mg/kg (reproduction)
56 d NOEC: 1.48 mg/kg (reproduction)
Executive summary:

The sublethal toxicity of test substance to earthworms, Eisenia fetida Savigny, was estimated in a 56-day soil exposure GLP-compliant laboratory study based on OECD 222 (2004) and ISO 11268, Part 2 (2012). Eight replicates for the control and four replicates for the test item groups of ten clitellated adult earthworms each were exposed to artificial soil (prepared according to OECD 222) treated with the test item to obtain the nominal concentrations of 0.293, 0.439, 0.658, 0.988, 1.48, 2.22, 3.33 and 5.00 mg picoxystrobin/kg dry artificial soil and to an untreated control (using the same amount of acetone and quartz sand as in the test item treated groups moistened with deionized water).

The reference item (active substance carbendazim) is tested at least once a year at five concentrations.

Adult earthworms were sorted from soil and assessed for mortality, weight loss, and sublethal effects after 28 days. Soil was replaced in the test container and juveniles were allowed to grow for another 28 days, at which time they were removed from the soil and counted. Food consumption per test container was observed and tallied throughout the experiment.

All study validity criteria were met.

The mortality of 72.5% at the concentration of 5.00 mg test substance/kg dry artificial soil was statistically significantly different compared to the control (Fisher’s Exact Test, α = 0.05). No additional mortality was observed. The LC50 after 28 days was determined to be 4.80 mg test substance/kg dry artificial soil.

The body weight changes were not statistically significantly different compared to the control up to and including the concentration of 2.22 mg test substance/kg dry artificial soil. At 3.33 mg test substance/kg dry artificial soil and above the weight increase was statistically significantly lower compared to the control (Williams t-test, α = 0.05, two-sided). For the evaluation of the weight changes the highest concentration was not included due to the low number of surviving worms.

No statistically significant effects on reproduction were observed up to and including the concentration of 1.48 mg test substance/kg dry artificial soil. At 2.22 mg test substance/kg dry artificial soil and above reproduction was statistically significantly reduced compared to the control (Williams t-test, α = 0.05, one-sided smaller).

No behavioral abnormalities were observed in any treatment group in this study.

The NOEC (No-Observed-Effect Concentration) for mortality was determined to be 3.33 mg test substance/kg dry artificial soil. The NOEC for weight changes was determined to be 2.22 mg tes substance/kg dry artificial soil. The NOEC for reproduction was determined to be 1.48 mg test substance/kg dry artificial soil; therefore the overall NOEC of test substance to the earthworm Eisenia fetida was determined to be 1.48 mg test substance/kg dry artificial soil.

The LC50 after 28 days was determined to be 4.80 mg test substance/kg dry artificial soil. The EC10 for reproduction was determined to be 1.97 mg test substance/kg dry artificial soil; the EC20 was 2.30 mg substance/kg dry artificial soil and the EC50 was 3.09 mg substance/kg dry artificial soil.

Description of key information

56-day NOEC (Eisenia fetida): 1.48 mg/kg soil dw (reproduction); OECD 222; Reliability = 1

14-day LC50 (Eisenia fetida): 6.7 mg a.i./kg; OECD 207; Reliability = 1

14-day NOEL (Eisenia fetida): 3.2 mg a.i./kg; OECD 207; Reliability = 1

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
6.7 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
1.48 mg/kg soil dw

Additional information