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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
Study period:
11 August - 6 October 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
One validity criterion was not met however this was determined not to have affected the study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Principles of method if other than guideline:
Soil, manure and water screening analyses were conducted using US EPA procedures instead of GLP standards. The reference toxicant was not characterised by GLPs. Characteristics of the test substance (e.g. identity, stability) were not determined under GLP standards however, a certificate of analysis was provided and so these exceptions were not expected to impact the study results.
GLP compliance:
yes
Remarks:
See 'Principles of method if other than guideline'
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: Soil samples were removed on days 0, 2, 28 (before and after adults were removed) and 56 from the control and 1000 mg/kg treatment using a soil core. Extra test vessels were prepared at initiation for every analytical sample interval. After collecting three soil core samples, the extra test vessel was sacrificed at each interval and the soil samples analyzed for phenyl silsesquioxanes concentration.
- Sample storage conditions before analysis: Directly after sampling, the cores were placed in glass bottles with Teflon-lined caps prior to analytical processing.
Vehicle:
no
Details on preparation and application of test substrate:
- Method of application to soil surface (if used): The test substance was directly applied by injection below the surface of the natural sandy loam. To aid in test substance dispersal, half the total soil (approximately 1.4 kg wet weight per jar) was injected with the test substance. Afterwards, the remaining half (approximately 1.4 kg wet weight per jar) was added to the jars and the jars were then capped. The soil was allowed to mix thoroughly at a speed of 15 rpm for approximately 2 hours on a rolling table. After rolling table mixing, the contents of the rolling jars were combined where applicable and each treatment level was mixed using a Hobart mixer for approximately 10 minutes to ensure homogeneity.
- Controls: The control soil was prepared in the same manner as described for each treatment medium except that the control medium contained no test substance.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Aquatic Research Organisms, Hampton, New Hampshire
- Age at test initiation (mean and range, SD): 3-5 months old, mature with clitellum.
- Weight at test initiation (mean and range, SD): mean individual earthworm weight across all controls and treatment replicates was 0.4061g.

ACCLIMATION
- Acclimation period: Not reported
- Acclimation conditions (same as test or not): Prior to exposure initiation, earthworms were transferred to natural sandy loam for acclimation to conditions consistent with those used during the definitive test. Earthworms used for testing were healthy and uniform in size.
- Health during acclimation (any mortality observed): None reported
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
56 d
Test temperature:
20 ± 2 °C
pH:
6.5 - 7.1
Moisture:
16 - 21 %
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1000 mL glass beakers
- Amount of soil or substrate: 850g dwt soil (depth of approximately 13cm)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 8 for 1000mg/kg treatment. 1 for the 10 and 100 mg/kg additional concentrations
- No. of replicates per control: 8
- No. of replicates per vehicle control: Not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Not reported
- Pesticide use history at the collection site: Not reported
- Collection procedures: Not reported
- Sampling depth (cm): Not reported
- Soil texture (if natural soil)
- % sand: 87%
- % silt: 8%
- % clay: 5%
- Soil taxonomic classification: Not reported
- Soil classification system: Not reported
- Composition (if artificial substrate): Not reported
- Organic carbon (%): 3.6%
- Maximum water holding capacity (in % dry weight): 9.8% at 1/3 bar
- CEC: Not reported
- Pretreatment of soil: The percent moisture of the natural soil was 11% and was adjusted to be in the range of 16 to 24% for testing.
- Storage (condition, duration):
- Stability and homogeneity of test material in the medium: This was assessed separately. Please refer to stability assessment in IUCLID Section 6.3.1 for results.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of food consumption were recorded weekly, beginning on day 7, until test day 28. Pooled earthworm weight was determined for F0 earthworms on day 0 and day 28. Earthworm mortality and health assessments were performed on day 28. Juveniles were counted on days 56 and approximately 24 hours thereafter.

VEHICLE CONTROL PERFORMED: not necessary as no vehicle was used.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable as study design was a limit test.
Nominal and measured concentrations:
Nominal: control, 1000 mg/kg treatment (additional 10 and 100 mg/kg single replicates)
Measured: control, 800 mg/kg
Reference substance (positive control):
yes
Remarks:
Reference toxicant benomyl, was tested in a 56 day test performed from 5 December 2019 to 30 January 2020
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
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
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:
LC50
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
Remarks:
as earthworm weight
Details on results:
The coefficient of variation of reproduction (31%) did not meet the OECD TG 222 validity criteria (≤30%). However, it falls outside by only a marginal amount (1%). In addition, the mean number of juveniles in the 1000 mg/kg treatment group exceeeded the control, with a comparable CV (63 juveniles per replicate, CV 32%). Based on these results, it was determined that this study provided a suitable data set for risk assessment, which establishes no risk to earthworm survival and reproduction at a nominal concentration of 1000 mg/kg.
- Mortality at end of exposure period: 96% survival was observed in the control group and 94% survival in the 1000 mg/kg treatment group
- Total mass of adults at beginning of test: Not reported
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Mean percent weight change in the control group was -5.4% and -9.1% in the 1000 mg/kg treatment group
- No. of offspring produced: Mean number of surviving offspring per replicate was 62 and 63 in the controls and 1000 mg/kg treatment groups, respectively.
- No. of unhatched cocoons: Not reported
- Morphological abnormalities: not reported
- Behavioural abnormalities: Not reported
- Other biological observations:Not reported
Reported statistics and error estimates:
Fisher’s Exact Test was used to determine significant effects for survival. Wilcoxon Rank Sum Test was used to determine significant effects for adult weight and reproduction. All statistical conclusions were made at the 95% level of certainty except in the case of the basic assumption tests, e.g., Shapiro-Wilks’ Test and Bartlett’s Test, in which the 99% level of certainty was applied.

Table 1. Recovery rates of Phenyl silsesquioxanes throughout a 56-day exposure to Eisenia fetida

Nominal test concentration (mg/kg)

Measured concentration (mg/kg)

Mean measured (mg/kg)

SD

Percentage of nominal

Day 0

Day 2

Day 28a

Day 28b

Day 56

Control

<MDL

<MDL

<MDL

<MDL

<MDL

N/a

N/a

N/a

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

<MDL

1000

770

890

780

640

890

800

88

80

760

870

790

670

850

860

900

780

650

860

MDL = 150 mg/kg

Table 2. Effect of Phenyl silsesquioxanes on Eisenia fetida survival and weight after 28 days' exposure.

Nominal Concentration

(mg/kg)

Replicate

Day 28 Survival

(%)

Mean Individual Earthworm Weight

(g)

Mean Percent Weight Change (SD)

Day 0

Day 28

Control

1

100

0.4054

0.3988

-1.6

2

80

0.4120

0.4228

2.6

3

100

0.4012

0.3471

-13

4

100

0.4136

0.3697

-11

5

100

0.4648

0.3966

-15

6

100

0.4142

0.4216

1.8

7

100

0.4072

0.4245

4.3

8

90

0.4078

0.3609

-12

Mean (SD)

96 (7)

0.4158 (0.0203)

0.3928 (0.0303)

-5.4 (7.9)

Control - Analytical

1

NA

NA

NA

NA

2

NA

0.4268

NA

NA

3

NA

0.4246

NA

NA

4

100

0.3887

0.3650

-6.1

5

100

0.3594

0.4486

25

Mean (SD)

100 (0)

0.3999 (0.0321)

0.4068 (0.0591)

9.4 (22)

Pooled Control

Mean (SD)

97 (7)

0.4105 (0.0246)

0.3956 (0.0337)

-2.4 (12)

10

1

90

0.3783

0.3483

-7.9

100

1

100

0.4150

0.4147

-0.065

1000

1

100

0.4072

0.4181

2.7

2

100

0.4217

0.3391

-20

3

90

0.4440

0.3732

-16

4

100

0.4527

0.2722

-40

5

80

0.4482

0.5021

12

6

100

0.3870

0.3898

0.72

7

100

0.4088

0.3442

-16

8

80

0.4021

0.4136

2.8

Mean (SD)

94 (9)

0.4215 (0.0243)

0.3815 (0.0678)

-9.1 (17)

Table 3. Chronic exposure of Eisenia fetia to Phenyl silsesquioxanes - number of offspring

Nominal

Concentration

(mg/kg)

Replicate

Total Number of Surviving Offspring per Replicate

Day 56 - Total

Control

1

49

2

43

3

89

4

84

5

68

6

39

7

49

8

72

Mean (SD)

62 (19)

10

1

138

100

1

93

1000

1

57

2

65

3

40

4

70

5

70

6

72

7

96

8

33

Mean (SD)

63 (20)

Validity criteria fulfilled:
no
Remarks:
In the control group, mean mortality was 4% at day 28 and ≥62 juveniles per replicate were produced which meet the validity criteria (≤10% and ≥30 respectively). However, the coefficient of variation was 31% but it was determined not to affect the study.
Conclusions:
56-day NOEC, EC10 and EC50 values of ≥1000, >1000 and >1000 mg/kg dwt respectively, have been determined for the effects of the test substance on the reproduction of Eisenia fetida, based on nominal concentrations. 28-day NOEC and LC50 values of ≥1000 and >1000 mg/kg dwt have been determined for the survival and growth (as weight) of Eisenia fetia, based on nominal concentrations. These results are equivalent to ≥556, >556 and >556 mg/kg dwt respectively, when normalised to 2% organic carbon content.

Description of key information

NOEC, EC10 and EC50 (56-d): ≥1000, >1000 and >1000 mg/kg dwt (nominal), reproduction, Eisenia fetida

NOEC, LC50 (28-d): ≥1000 and >1000 mg/kg dwt (nominal), survival and growth (as weight), Eisenia fetita

These results are equivalent to  ≥556, >556 and >556 mg/kg dwt respectively, when normalised to 2% organic carbon content.

Key value for chemical safety assessment

Additional information

An OECD TG 222 earthworm reproduction test is available for the registration substance.

56-day NOEC, EC10 and EC50 values of ≥1000, >1000 and >1000 mg/kg dwt respectively, have been determined for the effects on the reproduction of Eisenia fetida, based on nominal concentrations. 28-day NOEC and LC50 values of ≥1000 and >1000 mg/kg dwt have been determined for the survival and growth (as weight) of Eisenia fetida, based on nominal concentrations. These results are equivalent to  ≥556, >556 and >556 mg/kg dwt respectively, when normalised to 2% organic carbon content.

Effect concentration values have been reported in terms of nominal concentrations because the measured concentrations were within 80% of nominal.

Due to the potential volatility of the registration substance, a stability trial under OECD 222 conditions was carried out prior to the above study.

The stability assessment was executed over a 56 day period using [14C] radiolabelled phenyl silsesquioxane test substance. The trial was carried out under OECD 222 test conditions, in natural soil, but without test organisms.

The test substance was mixed into natural soil and chemical analysis was performed by taking triplicate soil cores from one of the treated replicates at Days 0, 3, 7, 14, 28, and 56 and analysed by liquid scintillation counting (LSC) and high-performance liquid chromatography with radiometric detection (HPLC-RAM).

The measured dosing stock concentration was 414 mg/kg dry weight soil.

The measured applied test concentration was 381 mg/kg dry weight soil.

The test substance was determined to be stable in natural soil. This was based on the material balance remaining at or above 100 % AR (103 -107% of applied radioactivity) and the HPLC distribution of radioactivity showing similar chromatographic profiles (83.6 to 88.2%) over the 56 day study.

Further details are reported in the stability assessment EPSR (Smithers, 2020).