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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
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:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Akzo Nobel Functional Chemicals B.V, Batch: 10000005282
- Expiration date of the lot/batch: 31 December 2021
- Purity test date: 99.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at room temperature (15 - 25°C)
Analytical monitoring:
yes
Details on sampling:
On preparation 3 x 1 mL samples of each ethylene dibenzoate test solution and the water and solvent controls were taken and placed into vials. One vial was sent for analysis of test substance concentration.

After soil treatment 3 x 30 g sample of each ethylene dibenzoate test rate and the water and solvent controls were taken and placed into amber glass jars, 1 x 30 g was sent for analysis of test substance concentration.

On Day 7 3 x 30 g soil samples were taken from replicate 5 of each ethylene dibenzoate test rate and replicate 9 of the water and solvent controls and placed into glass jars; 1 x 30 g of each was sent for analysis of test substance concentration.
Vehicle:
yes
Details on preparation and application of test substrate:
The treatments were prepared as follows:

Treatment Rate
(mg a.i/kg dry soil) Amount of test item used (mg) Final volume of
acetone (mL) mg a.i./mL (nominal)
1000 4426.54 20 220
555.6 2459.2 20 122.22
308.6 1366.2 20 67.90
171.5 759.0 20 37.72
95.3 421.7 20 20.96
52.9 234.3 20 11.64
29.4 130.1 20 6.47
16.3 72.3 20 3.59

Prior to treatment the initial moisture content of the soil was determined as 22.09% and the maximum water holding capacity (MWHC) as 66.64%. A moisture content equivalent to 55% of the MWHC was selected as providing suitable conditions for earthworm development. The soil was not pre-moistened as the moisture content was greater than 27.5% of the MWHC. The volume required to achieve 55% of the maximum water holding capacity was calculated as 482.05 mL water/3200 g dry ISO earthworm soil.
The test item (14.545 mL from each prepared solution) was placed over 72.75 g of dry sand, the treated sand was then left to allow the solvent to evaporate. After evaporation of the solvent the treated sand was placed into 3818.06 g of moist soil (equivalent to 3127.25 g dry soil) and mixed, 482.05 mL of RO water was then added to bring the soil moisture content to 55% MWHC. The treated soil was then mixed using an electric mixer.
For the water and solvent controls 783.33 mL RO water was mixed into a 5081.79 g soil dry weight equivalent of moist soil with 118.21 g of untreated dry sand for the water control and acetone (14.545 mL) treated sand for the solvent control to bring the soil to 55% of the MWHC.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Earthworms (Eisenia fetida) and dried rabbit manure were purchased from a reputable supplier of laboratory livestock. Prior to study start the worms were maintained in animal manure and fed regularly. Details of the suppliers are recorded only in the raw data to maintain supplier confidentiality.
Selection of adult worms for both the acute range finder and the reproduction study was based primarily on individual weights falling within the specified weight range of 300 – 600 mg at the start of the treatment period. Prior to treatment for the definitive phase, the batch of worms was inspected for reproductive maturity, i.e. production of egg cocoons or presence of a clitellum, the presence of which confirmed suitability for use.
Approximately 24 hours prior to study starts adult worms were transferred to untreated artificial soil.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
Minimum and maximum ambient temperatures were recorded daily throughout the study, with a range of 18.1 – 20.7°C
pH:
6.8 - 7.3
Moisture:
Moisture content was recorded at Day 1 and Day 56. A container of soil was maintained under identical conditions to the test containers and was used as a guide for maintaining the moisture content of the test containers throughout the study. At termination, group replicate mean moisture content values were found to be approximately 34.55 to 38.02%, equivalent to 51.88 – 57.09% MWHC.
Details on test conditions:
The definitve study was conducted in plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a fitted lid.
Prior to application selected worms were rinsed in reverse osmosis water, blotted dry and individually weighed before being reweighed in replicates of ten.
After application, ca 683.26 g of moist soil (equivalent to 500 g dry soil at 55% of the maximum water holding capacity) was transferred into each of ten replicate containers for the water control, solvent control, six replicate containers for each ethylene dibenzoate treatment rate and eight replicate containers for the toxic reference.
Dried rabbit manure was used as food. One day after application of the test item, 5 g of dried manure was uniformly distributed onto the surface of the soil in each test container and wetted with 5 mL of reverse osmosis water. Worms at each treatment rate were fed weekly in a similar way if two or more replicates per rate were assessed as having consumed 50% or more of the food. The quantity of water given was based on the amount of water loss from the moisture control box each week.
After four weeks (after removal of adult worms), the juvenile worms were fed by carefully mixing 5 g of food (dried manure) into the substrate of each container. Juvenile worms were not fed further during the four-week rearing period.
Nominal and measured concentrations:
Analysis of the concentrations of ethylene dibenzoate in the application treatment solutions confirmed nominal levels of the test substance were within applied limits ± 20% for all treatment groups, confirming accurate formulation. The nominal concentrations of treatment solutions were 220, 122.22, 67.90,37.72, 20.96, 11.64, 6.47 and 3.59.
Reference substance (positive control):
yes
Remarks:
Ringer (active ingredient carbendazim) at 5 mg a.i./kg dry soil.
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
555.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 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:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
56 d
Dose descriptor:
EC50
Remarks:
Not sufficient dose response relationship.
Basis for effect:
reproduction
Remarks on result:
not determinable
Remarks:
Not sufficient dose response relationship.
Details on results:
The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%.
Results with reference substance (positive control):
The application of the toxic reference Ringer (carbendazim) at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.
Validity criteria fulfilled:
yes
Conclusions:
The NOEC for the number of juvenile worms on Day 56 was 555.6 mg a.i./kg dry soil.  The EC50for the number of juveniles at Day 56 could not be established as there was not a sufficient dose response relationship.

The NOEC for mortality and mean body weight on Day 28 was 1000 mg a.i./kg dry soil. The EC50s could not be established as there was not a sufficient dose response relationship.

The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Ringer (carbendazim) at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.
Executive summary:

In the reproduction studyadult mortality of 0, 0, 2.5, 0, 2.5, 0, 0 and 2.5% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i/kg dry soil respectively. No mortality was recorded in the water control with 2.5% in the solvent control and 8.8% in the toxic reference. 

The LC50for the mortality on Day 28 could not be established as there was not a sufficient dose response relationship.

The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil.

There were no statistically significant adverse effects on mean bodyweights between the solvent control group and ethylene dibenzoate treated groups. The 5 mg a.i./kg dry soil Ringer (carbendazim) treated group had significantly lower body weight than the water control group (p<0.001).

The EC50for the mean body weight of the adult earthworms at Day 28 could not be established as there was not a sufficient dose response relationship.

The NOEC for mean bodyweight on Day 28 was 1000 mg a.i./kg dry soil.

The coefficient of variation of the number of juveniles in the solvent control group was 8.5%.

The 1000 mg a.i./kg dry soil ethylene dibenzoate treated group had significantly fewer juveniles than the solvent control group. The 5 mg a.i.kg/dry soil Ringer (carbendazim) treated group had significantly fewer juveniles than the water control group.

The EC50for the number of juveniles at Day 56 could not be established as there was not a sufficient dose response relationship.

The NOEC for the number of juvenile worms on Day 56 was 555.6 mg a.i./kg dry soil. 

The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Ringer (carbendazim) at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.

Description of key information

An earthworm reproduction study according to OECD 222 under GLP was performed.

Eisenia fetida was exposed to a series of concentrations: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i/kg dry soil. Water and solvent control groups of 80 worms were maintained in untreated soil to act as negative controls. A positive control group, 80 worms, was treated with Ringer (active ingredient carbendazim) at 5 mg a.i./kg dry soil. Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

The LC50 for the mortality on Day 28 could not be established as there was not a sufficient dose response relationship. The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil.

There were no statistically significant adverse effects on mean body weights between the solvent control group and ethylene dibenzoate treated groups. The EC50 for the mean body weight of the adult earthworms at Day 28 could not be established as there was not a sufficient dose response relationship. The NOEC for mean bodyweight on Day 28 was 1000 mg a.i./kg dry soil.

The 1000 mg a.i./kg dry soil ethylene dibenzoate treated group had significantly fewer juveniles than the solvent control group. The EC50 for the number of juveniles at Day 56 could not be established as there was not a sufficient dose response relationship. The NOEC for the number of juvenile worms on Day 56 was 555.6 mg a.i./kg dry soil. 

The study was considered valid as there was 10% adult mortality at four weeks and ≥ 30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Ringer (carbendazim) at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
555.6 mg/kg soil dw

Additional information