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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:
Definitive study dates: 8 March 2022 - 4 May 2022.
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:
yes
Remarks:
See 'principles of method if other than guideline' for protocol deviations
Principles of method if other than guideline:
In the range finder limit test, the recorded temperatures exceeded the upper and lower limits. The upper limit was exceeded (reached maximum 23.7°C) on three occasions over three consecutive days. The lower limit was exceeded (reached a minimum of 16.6°C) on four occasions over the same 3 consecutive days and one additional occasion near the start of the test.

The water content of the substrate had increased by >10% in both the control and all test substance treatments over the course of the range finding limit and definitive tests. Similarly, the control and all test substance treatments over the course of the range finding limit and definitive tests had MWHC > 60% at Day 56. This was considered to be the result of using a moist dung feed. As additional water was standardly added, this allowed the excess moisture to move into the substrate.

As all the validity criteria were met these deviations were not considered to have had any impact on the integrity or outcome of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The test substance was applied directly to the test substrate and thoroughly mixed for a minimum of 2 minutes using a hand held electric mixer. An appropriate amount of reverse osmosis (RO) water was added to achieve the final substrate wet weight required (55% MWHC), then it was thoroughly mixed for ca. 5 minutes using a hand held electric mixer to ensure triethoxy(octyl)silane was fully incorporated into the test substrate. The treatment levels were prepared in bulk and applied in the order of the water control and then increasing concentrations of triethoxy(octyl)silane.
- Controls: untreated (water) control, treated in the same way as the test concentrations
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Laboratory stock cultures. Original worms were purchased from Bias Labs Ltd, UK
- Age at test initiation (mean and range, SD): between 2 and 12 months old with clitellum.
- Weight at test initiation (mean and range, SD): 300-600 mg (wet mass).

ACCLIMATION
- Acclimation period: worms were left for 15 minutes after addition to test vessels - no further acclimation was needed, as the worms were taken from stock cultures.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18.5-20.9 ºC
pH:
5.85 - 6.44
Moisture:
36.99 - 50.38%
Details on test conditions:
TEST SYSTEM
- Test container (material, size):1-L glass test vessels
- Amount of soil or substrate: approximately 500g dry weight soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: not applicable, no vehicle used.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% sphagnum peat (2mm sieved), 20% kaolin clay (kaolinite content above 30%) and 70% quartz (silica) sand (predominately fine).
- Organic carbon (%): not reported
- Maximum water holding capacity (in % dry weight): 71.05%
- CEC: not reported
- Pretreatment of soil: The dry ingredients were thoroughly mixed and moisture content was determined by drying a pre-weighed sample at approximately 105ºC. During the preparation process, calcium carbonate was added as necessary to adjust the substrate to pH 6.0 ± 0.5.
- Storage (condition, duration): The substrate was stored in a sealed container for three weeks prior to use in the test.
- Stability and homogeneity of test material in the medium: not reported

OTHER TEST CONDITIONS
- Photoperiod: 16:8 hour light:dark cycle
- Light intensity: 400 – 800 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality (day 28), live weight (day 0 and 28), mean number of juvenile worms and cocoons (day 56)

VEHICLE CONTROL PERFORMED: not applicable, as no vehicle used.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx 1.5
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: control, 0.1, 1, 10, 100 and 1000 mg a.i./kg dry weight.
- Results used to determine the conditions for the definitive study: 28-day NOEC adult survival: 100 mg a.i./kg soil dry weight, 28-day LC50 adult survival 100-1000 mg a.i./kg soil dry weight, 28-day EC50 adult weight change >100 mg a.i./kg soil dry weight, 56-day NOEC reproduction 100 mg a.i./kg soil dry weight and 56-day EC50 reproduction 100-1000 mg a.i./kg soil dry weight.
Nominal and measured concentrations:
Nominal: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6, 777.8 and 1000 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
Carbendazim: EC50 3.74 mg/kg soil dry weight.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
308.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
555.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LC10
Effect conc.:
351.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% C.I. 334.7-389.7 mg/kg dw
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
657.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% C.I. 420-850 mg/kg dw
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
as weight change
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
as weight change
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
308.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
555.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
44.71 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 95% C.I. N/a - 205.1 mg/kg dw
Key result
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
472.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 95% C. I. 352.5 - 563.4 mg/kg dw
Details on results:
- Mortality at end of exposure period: mortality observed in 555.6, 777.8 and 1000 mg/kg dw (see Table 1)
- Total mass of adults at beginning of test: mean live weight on day 0 ranged from 389.3 - 440.0 mg (see Table 1)
- Changes in body weight of live adults (% of initial weight) at end of exposure period: % change ranged from -12.54 to +58.43 (see Table 1)
- No. of offspring produced: mean number of juveniles ranged from 7 to 109. Mean number of cocoons ranged from 0.0 - 0.75 (see Table 2)
- No. of unhatched cocoons: not reported
- Morphological abnormalities: none reported
- Behavioural abnormalities: none reported
- Other biological observations: not reported
Reported statistics and error estimates:
NOEC/LOEC values were determined using Fisher Exact/Bonferroni-Holm Test (for mortality/sub lethal effects) and Dunnett Multiple Comparison test (for reproductive output).
Statistical analysis of the survival and reproduction data, based on the nominal test concentrations as mg/kg dry substrate, was undertaken using CETIS version 1.8.6.8.

Table 1. Mortality and weight change of earthworms after 28 days' exposure

Nominal Concentration

(mg/kg dry substrate)

Mortality

Mean live weight (mg)

Change (%)

Number of E. fetida Exposed

Day 28

Number of Mortalities

Total
(%)

Day 0

Day 28

Water control

80

0

0

427.6

374.0

-12.54

16.3

40

0

0

440.0

391.7

-10.97

29.4

40

0

0

427.5

380.7

-10.95

52.9

40

0

0

412.5

381.5

-7.52

95.3

40

0

0

389.3

357.4

-8.19

171.5

40

0

0

433.3

392.6

-9.38

308.6

40

0

0

427.0

384.9

-9.85

555.6

40

18

45.0

406.3

432.4

+6.41

777.8

40

22

55.0

403.2

423.8

+5.11

1000

40

35

87.5

433.1

686.2

+58.43

Table 2. Reproduction of earthworms after 56 days' exposure

Nominal Concentration

(mg/kg dry substrate)

Mean Number of Juveniles

Mean Number of Cocoons

% Difference when Compared to the Water Controla

Water control

106

0.38

N/A

16.3

100

0.75

-6.5

29.4

109

0.50

+2.7

52.9

92

0.50

-13.3

95.3

84

0.75

-21.5

171.5

87

0.25

-18.2

308.6

84

0.0

-21.3

555.6

42

0.0

-61.0

777.8

28

0.25

-73.7

1000

7

0.0

-93.7

aDoes not include cocoons

N/A = Not Applicable
Coefficient of variation for water control = 19.73%

Validity criteria fulfilled:
yes
Remarks:
Mortality in control group was 0%, mean number of juveniles was 106 per control container and COV of reproduction was 19.73% in the control group.
Conclusions:
56-day EC50, EC10 and NOEC values of 472.2 mg/kg soil dry weight, 44.71 mg/kg soil dry weight and 308.6 mg/kg soil dry weight, respectively, have been determined for the effects of the test substance on the reproduction of Eisenia fetida. The result is expressed as nominal concentrations of the test substance over the test period. Nominal 28-day NOEC values for adult survival and weight change were 308.6 mg/kg soil dry weight and ≥1000 mg/kg soil dry weight, respectively. 28-day LC50 and EC50 values for adult survival and weight change were 657 mg/kg soil dry weight and >1000 mg/kg soil dry weight, respectively.

Description of key information

Long-term toxicity to soil macroorganisms except arthropods:

28-day LC10 351.7 mg/kg dw, survival of Eisenia fetida;

56-day EC10 44.71 mg/kg dw, reproduction of Eisenia fetida.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
472.2 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
44.71 mg/kg soil dw

Additional information

A long-term toxicity to soil macroorganisms except arthropods study according to the OECD test guideline 222 and in compliance with GLP has been conducted with the registered substance, triethoxy(octyl)silane (CAS 2943-75-1, EC No. 220-941-2) (Smithers 2022a).

 

The study reports the following effects on earthworm survival, growth and reproduction:

            28-day NOEC value for adult Eisenia fetida survival: 308.6 mg/kg dry substrate

            28-day LOEC value for adult Eisenia fetida survival: 555.6 mg/kg dry substrate

            28-day LC10 value for adult Eisenia fetida survival: 351.7 mg/kg dry substrate (334.7 – 389.7 mg/kg dry substrate)a

            28-day LC50 value for adult Eisenia fetida survival: 657.4 mg/kg dry substrate (420 - 850 mg/kg dry substrate)a

            28-day NOEC value for adult Eisenia fetida weight change: 1000 mg/kg dry substrate

            28-day EC50 value for adult Eisenia fetida weight change: >1000 mg/kg dry substrate

            NOEC value based on reproduction: 308.6 mg/kg dry substrate

            LOEC value based on reproduction: 555.6 mg/kg dry substrate

            EC10 value for effects on reproduction: 44.71 mg./kg dry substrate (N/A – 205.1 mg/kg dry substrate)a

            EC50 value for effects on reproduction: 472.2 mg/kg dry substrate (352.5 – 563.4 mg/kg dry substrate)a

a95% Confidence limits in parentheses

 

The hydrolysis half-life of triethoxy(octyl)silane at 20-25°C is approximately 30 hours at pH 7, 0.7 hours at pH 4 and 0.4 hours at pH 9 (QSAR) to form octylsilanetriol and ethanol. However, the hydrolysis rate of the substance in soil is not known and is expected to be slower once adsorbed to organic matter.

It is therefore likely that test organisms were initially exposed predominantly to the parent substance, with hydrolysis occurring throughout the duration of the test resulting in increasing amounts of exposure to the hydrolysis products.

Chemical analysis of test substance concentrations in the soil was not conducted. Neither the substance nor its silanol hydrolysis product are volatile and the silanol is not biodegradable. Therefore, although triethoxy(octyl)silane is expected to transform to its hydrolysis products in contact with moisture in the soil, losses of test substances are not expected through other means. Consequently, the nominal concentrations of the test substance applied to the soil are expected to have remained in the soil throughout the duration of the study, either in the form of the parent substance or the silanol hydrolysis product. The non-silanol hydrolysis product, ethanol, is likely to have volatilised out of the soil. The effects on earthworms observed in the study are therefore attributed to the toxicity of the substance and its hydrolysis products, although it is not possible to assess which chemical species is causing the effects.