[No public or meaningful name is available]

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:

9 October 2017 to 6 February 2019

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:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil : The test substance was weighed separately for each rate of application and dispersed in 150 mL of acetone by warming to 60°C in a water bath. This was poured over 750 g dry soil and the solvent was allowed to evaporate in a fume hood before addition to a 1450 g dry weight equivalent of moist soil. Reverse osmosis (RO) water (410.34 mL) was then added to bring the water content to 55% of MWHC. The dry soil and water were mixed into the moist soil using a hand held electric mixer.

- Controls:
For the water control 783.72 mL RO water was mixed into a 2766.6 g soil dry weight equivalent of moist soil to which 1433.4 g dry soil had been added. The mixing procedure employed was the same as used for the test item treatments.

For the solvent control 286.7 mL of acetone was poured over 1433.4 g of dry soil and allowed to evaporate. This was mixed into 2766.6 g soil dry weight equivalent of moist soil and 783.72 mL RO water added.

For the positive control group, 42 µL of Ringer (500 g/L carbendazim) was added to a final volume of 100 mL RO water to give a solution containing 0.21 mg a.i./mL. This was mixed into a 2766.6 g dry weight equivalent of moist soil to which 1433.4 g of untreated dry soil had been added. A further 683.72 mL of RO water was added to bring the soil to 55% of the maximum water holding capacity.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone

- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms (Eisenia fetida)
- Source: Reputable supplier of laboratory livestock.
- Age at test initiation (mean and range, SD): Adult
- Weight at test initiation (mean and range, SD): Weight range of 300-600 mg

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

28 d

Test temperature:

18.1 - 21.7°C

pH:

7.1 - 7.6

Moisture:

55% of the maximum water holding capacity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic containers measuring approximately 11 x 17 x 5 cm
- Amount of soil or substrate: 500 g dry soil at 55% of the maximum water holding capacity
- No. of organisms per container (treatment): 40
- No. of replicates per treatment group: 8 Groups
- No. of replicates per control: 80
- No. of replicates per vehicle control: 80

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Artivicial soil:
Industrial quartz sand 70% w/w
Kaolin clay 20% w/w
Peat 10% w/w

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8-hour dark
- Light intensity: 521 - 798 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On Day 28 of the study the soil was removed with tweezers from the containers and the numbers and replicate weights of live adults were recorded. The number of surviving juvenile worms, unemerged cocoons and moisture content of the soil were determined on Day 56.

Nominal and measured concentrations:

Nominal concentration range : 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 a.i./kg dry soil

Reference substance (positive control):

yes

Remarks:

Treated with Ringer.

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:

other: Body weight

Key result

Duration:

26 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of juvenile worms

Details on results:

- Mortality at end of exposure period: No adult mortality was recorded with the test material. No mortality was recorded in the water control or solve control. Mortality in the positive control group was 18.8%.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: There were no statistically significatn adverse effects in mean bodyweights between the solvent control group and the test item treatment groups. The positive control group had significantly lower body weight than the water conrol group.
- No. of Juveniles: Water control 255, Solvent control 256, Test item 233-253 (range of the 8 groups)
- No. of unhatched cocoons: 1

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 5 mg/kg dry soil

Reported statistics and error estimates:

Statistical analysis was carried out using SAS 9.1.3 (SAS Institute 2002). The IT systems listed below were used on this study:
Pristima: Test substance management
SAS 9.1.3: Statistics

Test Soil pH:

 

Days	Water	Solvent control	Test material (mg a.i./kg dry soil)								Ringer (mg a.i./kg dry soil)
			16.3	29.4	52.9	95.3	171.5	308.6	555.6	1000	5
0	7,1	7.2	7.4	7.3	7.4	7.4	7.4	7.3	7.3	7.3	7.4
56	7.2	7.3	7.2	7.5	7.4	7.6	7.4	7.2	7.3	7.4	7.4

 

Moisture Content of Soil:

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 31.36 to 35.27%, equivalent to 52.88 – 59.48% MWHC.

 

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for mortality and body weight on Day 28 was 1000 mg a.i./kg dry soil. The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

Executive summary:

A study according TG OECD 222 and GLP was performed to determine the effects of the registered substance on the reproduction and growth of the earthworm, Eisenia fetida. The test material was applied to the artificial soil to give an indication of the effects on the test organism following field application.

A study was conducted at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg active ingredient (a.i.)/kg dry soil. Eleven groups of worms were allocated to the study. Eight groups of 40 worms were introduced into soil treated with the test item at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 a.i./kg dry soil. Control (water only) and solvent control (acetone) 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.

No adult mortality was recorded at any treatment rates. No mortality was recorded in the water control or solvent control. Mortality in the toxic reference group (Ringer) was 18.8%.

As there was no mortality at Day 28 in the treatment groups, no statistical analysis could be performed and therefore the LC50 was estimated to be >1000 mg a.i./kg dry soil. The NOEC for mortality on Day 28 was 1000 mg a.i./kg dry soil.

There were no statistically significant adverse effects in mean bodyweights between the solvent control group and test material treatment groups. The positive control group (5 mg a.i./kg dry soil carbendazim) had significantly lower body weight than the water control group.

The EC50 for the mean body weight of the adult earthworms at Day 28 could not be estimated because there was not a dose-response relationship. The NOEC for the mean bodyweight at 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 5.64% and 5.44% in the water control group.

No statistically significant differences in the number of juveniles were recorded on Day 56 between the test material treatment groups and the solvent control. The 5 mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group (p<0.001).

Description of key information

A study according TG OECD 222 and GLP was performed to determine the effects of the registered substance on the reproduction and growth of the earthworm, Eisenia fetida. The NOEC for mortality and body weight on Day 28 was 1000 mg a.i./kg dry soil.  The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw