Dihexyl ether

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:

01 July 2022 - 16 December 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))

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

• The maximum light level recorded in the acute range finder test was 1219 Lux, above the guideline maximum of 800 Lux. • The water content of the substrate in the definitive reproduction test had increased in both the control and test substance treatment

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

adopted 4 April 1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of the concentration of dihexyl ether in the substrate was conducted on samples taken on Days 0 and 28.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil: The test substance was applied directly to the pre-moistened substrate with additional RO water being added to achieve the final substrate wet weight required at 50% MWHC.
- Controls: Water
- Chemical name of vehicle: None
- Concentration of vehicle in test medium: None
- Evaporation of vehicle before use: No vehicle

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: culture stocks 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 and 600 mg (wet mass)

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Health during acclimation (any mortality observed):

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

• 10% Sphagnum peat (2 mm sieved) • 20% Kaolin clay (kaolinite content above 30%) • 70% Quartz (silica) sand (predominately fine) with more than 50% of the particles between 50 and 200 microns

Limit test:

no

Total exposure duration:

56 d

Remarks:

Adults were exposed for 28 days and juvenile worms were removed on Day 56.

Test temperature:

17.9 - 20.6 degrees C

pH:

5.79 - 6.00

Moisture:

31.58 - 43.89%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 L glass test vessels
- Amount of soil or substrate: 736.0 g wet weight, equivalent to 550 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10% Sphagnum peat (2 mm sieved), 20% Kaolin clay (kaolinite content above 30%), 70% Quartz (silica) sand (predominately fine) with more than 50% of the particles between 50 and 200 microns
- Maximum water holding capacity (in % dry weight): 67.49%
- 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 closed containers prior to use in the test, and the substrate pH was measured within the three weeks prior to the start of the tests to ensure it was within the required range of pH 6.0 ± 0.5.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): survival and adverse behavior at 28 days, number of juveniles at 56 days

VEHICLE CONTROL PERFORMED: No vehicle used

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry substrate

Nominal and measured concentrations:

16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i. /kg dry substrate

Reference substance (positive control):

yes

Remarks:

Earthworms from the same source culture as those used in a reference toxicity test with Carbendazim, a known toxic substance (performed under in-house GLP Smithers ERS Study Number 3203476 from November 2021 to January 2022).

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

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

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

Details on results:

After 28 days of exposure, there was no adult mortality recorded in the water control. There was 20.0 and 60.0% adult mortality at 555.6 and 1000 mg a.i/ kg dry substrate groups respectively and 0% in all other treatment groups. There were no sub-lethal effects.

At Day 28, there were mean weight gains of 59.52, 46.04, 45.56, 46.75, 44.48, 46.09, 60.69 and 93.61% at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively in comparison to an increase of 62.13% in the water control. This was equivalent to a reduction in weight gain of 4.21, 25.90, 26.66, 24.75, 28.40, 25.81 and 2.32% at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6 and 555.6 mg a.i./kg dry substrate respectively in comparison to the water control. At 1000 mg a.i./kg dry substrate the mean weight increase was 50.66% greater than in the control, which was considered to be the result of a greater availability of food due to the reduction in worm numbers.

The mean number of juveniles per vessel was 365.5, 293.3, 339.5, 332.3, 296.8, 338.3, 234.8 and 115.8 in the 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment groups in comparison to 329.0 in the water control. This corresponded to reductions of 10.9, 9.8, 28.6 and 64.8% at 29.4, 171.5, 555.6 and 1000 mg a.i./kg dry substrate respectively in comparison to the water control. There were increases in reproduction of 11.1, 3.2, 1.0 and 2.8% in the numbers of juveniles at 16.3, 52.9, 95.3, and 308.6 mg a.i./kg dry substrate respectively in comparison to the water control. The coefficient of variation in the water control was 17.70%. The reduction in the number of juveniles at 555.6 and 1000 mg a.i./kg dry substrate were considered to be the directly related to the adult mortality at these rates.

Results with reference substance (positive control):

The EC50 value of Carbendazim was calculated to be 3.045 mg/kg dry soil. This is within the given toxicity range of 1 to 5 mg/kg dry soil, indicating that the worms were responding as expected in the test system.

Reported statistics and error estimates:

Statistical analysis of the survival, in comparison to the water control, was conducted using CETIS version 1.8.6.8, based on the nominal test concentrations as mg a.i./kg dry substrate.
• The NOEC and LOEC for effects on earthworm survival were determined using a Wilcoxon/Bonferroni Adj Test with ANOVA for detection of significant difference.
• A Grubbs Extreme Value Test detected an outlier at 16.3 mg a.i./kg dry substrate and the data was analysed both with and without the outlier being included
• Variance was evaluated using a Mod Levene Equality of Variance Test (equal variances) and a Levene Equality of Variance Test (unequal variances).
• A Shapiro-Wilk W Normality test indicated a non-normal distribution.
• The LCx values were determined using Linear Interpolation (ICPIN), (Two-Point Interpolation).
• Adult weight variation over the 28-day exposure period was determined using a Bonferroni Adj t Test and the ECx values were determined using Linear Interpolation (ICPIN), (Two-Point Interpolation).

Statistical analysis of the reproduction data was undertaken using CETIS version 1.8.6.8, based on the nominal test concentrations. The following methods were used:
• The NOEC and LOEC for effects on earthworm reproduction were determined using a Dunnett Multiple Comparison Test with ANOVA for detection of significant difference.
• A Grubbs Extreme Value Test confirmed that there were no outliers in the data set.
• Variance was evaluated using a Bartlett Equality of Variance Test (equal variances).
• Normal distribution was confirmed using the Shapiro-Wilk W Normality test.
• The ECx values were determined using Linear Interpolation (ICPIN), (Two-Point Interpolation).

Validity criteria fulfilled:

yes

Conclusions:

For dihexyl ether, the 28-day NOEC value for adult E. fetida survival was 308.6 mg a.i./kg dry substrate, while the NOEC fo weight change was 1000 mg a.i./kg dry substrate. The NOEC value based on reproduction was 308.6 mg a.i./kg dry substrate.

Executive summary:

A laboratory OECD 222 guideline test was conducted in which mature E. fetida were exposed to concentrations of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg (dry substrate) dihexyl ether in 10% peat OECD substrate for a period of 28 days. After 28 days the adult earthworms were removed from the soil, and the juveniles produced allowed to mature for a further 28 days prior to extraction and counting.

After 28 days of exposure, there was no adult mortality recorded in the water control. There was 20.0 and 60.0% adult mortality at 555.6 and 1000 mg a.i/ kg dry substrate groups respectively and 0% in all other treatment groups. There were no sub-lethal effects.

The mean number of juveniles per vessel was 365.5, 293.3, 339.5, 332.3, 296.8, 338.3, 234.8 and 115.8 in the 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment groups in comparison to 329.0 in the water control. This corresponded to reductions of 10.9, 9.8, 28.6 and 64.8% at 29.4, 171.5, 555.6 and 1000 mg a.i./kg dry substrate respectively in comparison to the water control. There were increases in reproduction of 11.1, 3.2, 1.0 and 2.8% in the numbers of juveniles at 16.3, 52.9, 95.3, and 308.6 mg a.i./kg dry substrate respectively in comparison to the water control.

Analysis of the concentration of dihexyl ether in the substrate was conducted on samples taken on Days 0 and 28. Post application the mean soil concentrations were equivalent to 83.76, 79.06, 64.79, 65.96, 65.30, 78.59, 50.92 and 70.27% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively.

On Day 28 very low levels of the test substance (<0.2% of nominal) were detected in one of the four replicate samples of the 29.4, 52.9, 95.3, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment rates, while in the other three replicates no analytical signals could be detected. The moisture content was adjusted for each individual sample. No test substance was detected in any of the replicates at 16.3 and 171.5 mg a.i./kg dry substrate. Analysis was therefore not conducted at Day 56.

The 28-day NOEC value for adult E. fetida survival was 308.6 mg a.i./kg dry substrate, while the NOEC fo weight change was 1000 mg a.i./kg dry substrate. The NOEC value based on reproduction was 308.6 mg a.i./kg dry substrate.

Description of key information

In an OECD guideline 222 study, the 28-day NOEC value for adult E. fetida survival was 308.6 mg a.i./kg dry substrate for dihexyl ether, while the NOEC fo weight change was 1000 mg a.i./kg dry substrate. The NOEC value based on reproduction was 308.6 mg a.i./kg dry substrate.

Key value for chemical safety assessment

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

308.6 mg/kg soil dw

Additional information

A laboratory OECD 222 guideline test was conducted in which mature E. fetida were exposed to concentrations of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg (dry substrate) dihexyl ether in 10% peat OECD substrate for a period of 28 days. After 28 days the adult earthworms were removed from the soil, and the juveniles produced allowed to mature for a further 28 days prior to extraction and counting.

After 28 days of exposure, there was no adult mortality recorded in the water control. There was 20.0 and 60.0% adult mortality at 555.6 and 1000 mg a.i/ kg dry substrate groups respectively and 0% in all other treatment groups. There were no sub-lethal effects.

The mean number of juveniles per vessel was 365.5, 293.3, 339.5, 332.3, 296.8, 338.3, 234.8 and 115.8 in the 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment groups in comparison to 329.0 in the water control. This corresponded to reductions of 10.9, 9.8, 28.6 and 64.8% at 29.4, 171.5, 555.6 and 1000 mg a.i./kg dry substrate respectively in comparison to the water control. There were increases in reproduction of 11.1, 3.2, 1.0 and 2.8% in the numbers of juveniles at 16.3, 52.9, 95.3, and 308.6 mg a.i./kg dry substrate respectively in comparison to the water control.

Analysis of the concentration of dihexyl ether in the substrate was conducted on samples taken on Days 0 and 28. Post application the mean soil concentrations were equivalent to 83.76, 79.06, 64.79, 65.96, 65.30, 78.59, 50.92 and 70.27% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively.

On Day 28 very low levels of the test substance (<0.2% of nominal) were detected in one of the four replicate samples of the 29.4, 52.9, 95.3, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment rates, while in the other three replicates no analytical signals could be detected. The moisture content was adjusted for each individual sample. No test substance was detected in any of the replicates at 16.3 and 171.5 mg a.i./kg dry substrate. Analysis was therefore not conducted at Day 56.

The 28-day NOEC value for adult E. fetida survival was 308.6 mg a.i./kg dry substrate, while the NOEC fo weight change was 1000 mg a.i./kg dry substrate. The NOEC value based on reproduction was 308.6 mg a.i./kg dry substrate.