Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 11, 2020 to December 11, 2020

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not specified

Details on sampling:

Dosage: The limit test substance concentration was selected in consultation with the Sponsor based on the results of the range finding test: 1000 mg/kg dry soil. A control was tested in parallel. In addition to the eight replicates of each treatment in the test, one additional replicate was prepared for the option of chemical analysis of the test item in the soil substrate.

Details on preparation and application of test substrate:

The treated soil substrate needed for the eight replicates of the limit test treatment (+ replicate 9 for the option of chemical analysis) was prepared as one batch. The amount of 2 x 2200 g of soil with residual moisture was pre-wetted with 2 x 450 mL ultrapure water three days before application. On the application day, 7260.62 mg (target: 7261.5 mg) of the test substance was mixed thoroughly with 500 mL of purified water. The test substance was equally dispensed with the application solution, clouding was visible. The volume of 300 mL application solution was then mixed with the premoistened soil (i.e. 2 x 2178.44 mg dry soil + 2 x 21.56 mL residual moisture + 2 x 450 mL purified water for pre-moistening). The overall used water volume of 1243.12 mL corresponded to 28.53% water content (based on soil dry weight) and 52.30% MWHC (based on a 100 % MWHC = 54.55%). The moistened soil substrate was mixed by means of a laboratory mixer for five minutes to ensure homogenous distribution of the test substance. The control treatment was prepared in the same way as the test substance treatment but using only ultrapure water. The application of the soil substrates was performed first with the control and then with the limit test item treatment.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The study was performed with earthworms of the species Eisenia fetida (Annelida: Lumbricidae) which came from a stock culture maintained at IES Ltd. Synchronized E. fetida adults with an age of eight months and a clitellum were used for the study. The wet mass of individual worms was between 300 and 600 mg. Earthworms were acclimatized for two days in the artificial soil used for the test. The test method and the test species are recommended by the OECD test guideline.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Test temperature:

The test temperature of the study was set at 20 ± 2°C and recorded throughout the study using a data logger. The temperature was within the range of 20 ± 2°C.

pH:

For pH measurements of the soil substrate at test initiation and termination, approximately 5 g of soil per treatment was sampled. Soil pH was measured by mixing the soil with 25 mL of 1.0 M solution of analytical grade potassium chloride (KCl). The suspension was stirred thoroughly for five minutes and then left to settle for approximately two hours.

Moisture:

The maximum water holding capacity (MWHC) was determined to be 52.30%. Three days before application, the soil substrate was pre-moistened with purified water, i.e. the amount of 2200 g artificial substrate (for four replicates) with a residual moisture of 21.56 g water (corresponding to 2178.44 g dry artificial soil) was mixed with 450 mL purified water. During application, the soil was again moistened with 150 mL purified water for all treatments. The final water content was 621.56 mL purified water corresponding to 28.53% water content and 52.30% of MWHC.

Details on test conditions:

The test was performed under controlled light-dark cycles (16 hours light and 8 hours dark) with a set illumination of 400 to 800 lux in the area of the test units. Light was measured once at the beginning of the test. Light intensity was measured at exposure test unit level and was within the range of 545-703 lux.
Test units were weighed weekly during the exposure phase and before feeding weight loss was replenished by the addition of purified water in order to keep the initial soil humidity. After the removal of the adults only purified water was added each week up to the test unit weight of the previous week.

Reference substance (positive control):

yes

Remarks:

A reference substance is tested at regular intervals at IES Ltd to verify the performance of the test and confirm that the response of the test organisms under the test conditions has not changed significantly over time.

Key result

Duration:

56 d

Dose descriptor:

other: LC20

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

LC10

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

other: Body weight

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

other: Body weight

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

other: Body weight

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

other: Body weight

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

other: Body weight

Key result

Duration:

56 d

Dose descriptor:

other: LC20

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

LC10

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil ww

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Details on results:

- Mortality:
Adult mortality at Day 28 was 1.25% in the control. No mortality was observed in the limit test concentration of 1000 mg/kg dry soil. Therefore, a statistical analysis of differences between the control and the test item treatment was not needed.

- Body weight change and behaviour at test initiation:
All earthworms burrowed into the soil with no behavioural abnormalities. Food consumption was similar in the control and the limit test concentration (1000 mg/kg dry soil), with almost all food being consumed. At test initiation, the fresh weight of individual worms ranged from 300 to 600 mg with an overall average weight of 417 mg per earthworm and was within the target range of 300-600 mg per worm. For the replicates proposed for the control and the limit test concentration, the mean body weight per replicate (with ten worms each) ranged from 370 to 475 mg per worm. The mean body weight in the proposed test substance treatment was statistically significantly smaller compared to the proposed control (Student t-test, two-sided, α = 0.05). This statistical finding was mainly caused by the low variability of the control and is not considered to be biologically relevant since the difference is < 10%. Additionally, no statistically significant differences in body weight at Day 28 and in body weight change from Day 0 to 28 were found between the limit test concentration and the control. The mean body weight at Day 28 in the test substance treatment and the control was 553 and 559 mg and did not differ statistically significantly from each other (Student t-test, one-sided smaller, α = 0.05). The mean body weight change in the test substance treatment was +27% and was not statistically significantly smaller compared to the control (Student t-test, one-sided smaller, α = 0.05).

- Reproduction:
The average number of full cocoons on Day 56 in the control and the test substance treatment was 0.1 and 0.6, respectively. The average number of juveniles per vessel was 62.3 in the control and 60.0 in the test substance treatment and did not differ statistically significantly from each other (Student t-test, one-sided smaller, α = 0.05).

The following validity criteria from OECD 222 were met in the control:
• Each replicate produced ≥ 30 juveniles by the end of the test (actual values: 51-88 juveniles per replicate in the control).
• The coefficient of variation (reproduction) was ≤ 30% (actual value: 20.9%).
• Adult mortality over the initial four weeks of the test was ≤ 10% (actual value: 1.25%).

 

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the NOEC and LOEC values for reproduction were determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively. The EC10, EC20, and EC50 were > 1000 mg/kg dry soil.

Executive summary:

A study was conducted to determine the effects on reproduction of Eisenia fetida (Annelida: Lumbricidae) of the test substance C8-18 and C18-unsatd. DEA, according to OECD Guideline 222, in compliance with GLP. The worm species Eisenia fetida was exposed for 56 days to artificial soil treated with a limit concentration of 1000 mg/kg of test substance. Body weight and behaviour, mortality, and reproduction parameters were recorded. No mortality was observed and there were no statistically significant changes in body weight or in the average number of cocoons and juveniles compared to controls. Under the study conditions, the NOEC and LOEC values for reproduction were determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively. The EC10, EC20, and EC50 were > 1000 mg/kg dry soil (Schmidt, 2021).

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

A study was conducted to determine the effects on reproduction of Eisenia fetida (Annelida: Lumbricidae) of the test substance C8-18 and C18-unsatd. DEA, according to OECD Guideline 222, in compliance with GLP. Worms were exposed for 56 days to artificial soil treated with a limit concentration of 1000 mg/kg of test substance. Body weight and behaviour, mortality, and reproduction parameters were recorded. No mortality was observed and there were no statistically significant changes in body weight or in the average number of cocoons and juveniles compared to controls. Under the study conditions, the NOEC and LOEC values for reproduction were determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively. The EC10, EC20, and EC50 were > 1000 mg/kg dry soil (Schmidt, 2021).