4,4'-Isopropylidenediphenol, ethoxylated

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 6 OCT 2020 to 29 JAN 2021

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))

Version / remarks:

Adopted version: 29 July 2016

Deviations:

yes

Remarks:

Water content measurements at the end of the test were not in the range 40% - 60% of the maximum water holding capacity. The water content variation was higher than 10% for the for the control and toxic control test soils.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: GE9061672
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: well-ventilated place, ambient temperature (<30°C) and obscurity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
As the test item is insoluble in water, test soils were prepared by addition of accurate amounts of test item previously dissolved in a volatile organic solvent (acetone) to a sub-sample of the sand: the test item was weighed separately for each rate of application and dispersed in 25 mL of acetone. This was poured over the sand and the solvent was allowed to evaporate in a fume hood until the sand was visibly dry (one night).
The sand subsamples were then added to the remainder of the soil constituents and thoroughly mixed before addition of a sufficient volume of deionised water to achieve 55% of the maximum water holding capacity and mixed for a further period.

OTHER SPECIFICS
Care was taken to ensure homogeneous distribution in the treated soil samples. After treating the soil and mixing thoroughly, equally-sized soil subsamples (approx. 650 g) from each treatment group was transferred into each of eight replicate containers for both control and solvent controls, four replicate containers for each treatment rate and the toxic control.

Analytical monitoring:

no

Remarks:

According to OECD 222, analytical measurements of the exposure concentrations are only required where there is an uncertainty in maintaining the nominal soil concentrations.

Vehicle:

yes

Remarks:

Acetone; purity: 100%; a copy of certificate of analysis is present as appendix 4 in the attached full study report.

Details on preparation and application of test substrate:

Test soils were prepared by addition of accurate amounts of test item previously dissolved in a volatile organic solvent (acetone) to a sub-sample of the sand: the test item was weighed separately for each rate of application and dispersed in 25 mL of acetone. This was poured over the sand and the solvent was allowed to evaporate in a fume hood until the sand was visibly dry (one night). The sand subsamples were then added to the remainder of the soil constituents and thoroughly mixed before addition of a sufficient volume of deionised water to achieve 55% of the maximum water holding capacity and mixed for a further period.
One untreated control group was prepared without solvent nor test item, an additional solvent control group was thus added and treated as described above but did not contain test item.
For the toxic control group, 2.5002 g of boric acid was added to a final volume of 500 mL deionised water (previously heated to help dissolution) to give a 5 g/L stock solution. 252 mL was mixed into a 2100 g dry weight soil. A further 356.7 mL of deionised water was then added to bring the soil to 55% of the maximum water holding capacity.
Care was taken to ensure homogeneous distribution in the treated soil samples. After treating the soil and mixing thoroughly, equally-sized soil subsamples (approx. 650 g) from each treatment group was transferred into each of eight replicate containers for both control and solvent controls, four replicate containers for each treatment rate and the toxic control. Acclimatised earthworms were then introduced and randomly allocated to treatment (ten per container). The test containers were weighed for the determination of the starting weight then incubated.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworms (with a clitellum)
- Source: Bias Labs Ltd (Kirkcaldy, United Kingdom).
- Age at test initiation: synchronised culture, between 2 months and 1 year old
- Weight at test initiation: wet mass of individual worms was between 300 and 600 mg

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions:
Worms were acclimatised to experimental conditions in untreated test soil for a period of 24 hours during which worms were fed with the same nature and conditions of feeding as used in the test (i.e. air-dried, finely blended and pasteurised rabbit manure).

- Health during acclimation (any mortality observed): no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

An artificial soil substrate with the following composition was used: Sphagnum peat: 10% w/w; Kaolin clay (kaolinite content preferably above 30 %): 20% w/w; Industrial sand (fine): 70% w/w (50% particles between 50 and 200 microns)

Limit test:

no

Total exposure duration:

56 d

Remarks:

The adult worms were removed after 28 days. The soil removed from the containers and containing any cocoons was then returned, then incubated for four additional weeks under the same test conditions except feeding only once at the start of this phase.

Test temperature:

20.0 – 20.8°C

pH:

5.6 - 6.1

Moisture:

23.7% to 52.4%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass, 1 litre capacity, allowing 5-6 cm depth
- Amount of soil or substrate: ~ 650 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: negative control 8; positive control 4
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: artificial soil
- Composition (if artificial substrate): sphagnum peat (10% w/w); kaolin clay (20% w/w); industrial sand fine (70% w/w)
- Organic carbon (%): not measured; sphagnum peat at 10% w/w
- Maximum water holding capacity (in % dry weigth): 52.7%
- Pretreatment of soil: prepared a few days before the test, moistened, and kept 24 h in test conditions. pH was adjusted with CaCo3 (0.4%)
- Storage (condition, duration): during the test, all test containers were covered with perforated plastic film to minimise evaporation whilst maintaining ventilation

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
adult mortality (LC50); reproduction (NOEC, EC50); adult bodyweight (NOEC)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Test concentrations: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/kg dry soil.
- Results used to determine the conditions for the definitive study:
a) each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test;
b) the coefficient of variation of reproduction to be ≤ 30 %;
c) adult mortality over the initial 4 weeks of the test to be ≤10 %.

Nominal and measured concentrations:

Nominal: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

boric acid; 100% purity; a copy of certificate of analysis is present as Appendix 3 in the attached full report.

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

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: There was not a dose-response relationship. Hence, EC50 > 1000

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

171.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: Not significant up to the highest concentration
- Changes in body weight of live adults at end of exposure period: Not significant up to the highest concentration
- No. of offspring produced in controls (mean ± SD):
control - 263 ± 41.3; solvent control - 204.1 ± 34.7; positive control - 0.3 ± 0.5

- No. of offspring produced in treatment groups (mean ± SD)
179.3 ± 10.8; 182.3 ± 39.9; 201.5 ± 32.1; 154 ± 18.4; 186.5 ± 32.8; 160.8 ± 46.9; 129.5 ± 15.3; 126.3 ± 24.8, respectively

- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: No statistically significant effect on mortality and bodyweight

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 100% inhibition in reproduction at 56 days at 600 mg/kg soil dw
- Other: 12.5% mortality with 600 mg/kg soil dw, in adults measured at 28 days

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for the test substance was 171.5 mg/kg dry soil for reproduction in the OECD compliant 56 days Earthworm Reproduction (Eisenia fetida) Test. 

Executive summary:

The OECD 222 guideline compliant study was designed to determine the effects of the test item on the reproduction and growth of the earthworm, Eisenia fetida, in an artificial soil under GLP conditions. The test was conducted at treatment rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/kg dry soil. One positive toxic control and a solvent control were included.

The adult worms were removed after 28 days after the test initiation, counted for dead worms, weighed and observed for unusual behaviour and morphology. The soil removed from the containers and containing any cocoons was then returned and incubated for four additional weeks after which, the number of juveniles hatched from the cocoons and all signs of harm or damage to the worm was recorded for each test container.

The pH and the moisture content of the soil in each test container was measured at the beginning and the end of the exposure period. The temperature was continuously recorded throughout the test.

The test met the validity criteria of the test guideline.

There were statistically significant differences between the solvent control and the treatment groups of 308.6, 555.6 and 1000.0 mg/kg dry soil; hence, the NOEC for reproduction was statistically determined to be 171.5 mg/kg dry soil. The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship and was therefore concluded to be >1000 mg/kg dry soil.

The LC50 for adult mortality could not be estimated due to a lack of significant mortality. LC50 and NOEC for adult mortality on Day 28 was therefore concluded to be >1000 and ≥1000.0 mg/kg dry soil, respectively.

There was no statistically significant adverse effect on adult bodyweight at any test concentrations in comparison to the control. A NOEC ≥ 1000 mg/kg dry soil is therefore proposed.

About 100% reproduction inhibition was observed after 56 days of exposure with the positive toxic control. The validity criteria for the reference control was therefore fulfilled (the ISO standard specifies that significant effects on reproduction should be observed between 400 and 600 mg/kg/sdw).

Description of key information

The OECD 222 guideline compliant study was designed to determine the effects of the test item on the reproduction and growth of the earthworm, Eisenia fetida, in an artificial soil under GLP conditions. The test was conducted at treatment rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/kg dry soil. One positive toxic control and a solvent control were included.

The adult worms were removed after 28 days after the test initiation, counted for dead worms, weighed and observed for unusual behaviour and morphology. The soil removed from the containers and containing any cocoons was then returned and incubated for four additional weeks after which, the number of juveniles hatched from the cocoons and all signs of harm or damage to the worm was recorded for each test container.

The pH and the moisture content of the soil in each test container was measured at the beginning and the end of the exposure period. The temperature was continuously recorded throughout the test.

The test met the validity criteria of the test guideline.

There were statistically significant differences between the solvent control and the treatment groups of 308.6, 555.6 and 1000.0 mg/kg dry soil; hence, the NOEC for reproduction was statistically determined to be 171.5 mg/kg dry soil. The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship and was therefore concluded to be >1000 mg/kg dry soil.

The LC50 for adult mortality could not be estimated due to a lack of significant mortality. LC50 and NOEC for adult mortality on Day 28 was therefore concluded to be >1000 and ≥1000.0 mg/kg dry soil, respectively.

There was no statistically significant adverse effect on adult bodyweight at any test concentrations in comparison to the control. A NOEC ≥ 1000 mg/kg dry soil is therefore proposed.

About 100% reproduction inhibition was observed after 56 days of exposure with the positive toxic control. The validity criteria for the reference control was therefore fulfilled (the ISO standard specifies that significant effects on reproduction should be observed between 400 and 600 mg/kg/sdw).

Key value for chemical safety assessment

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

171.5 mg/kg soil dw