Ditolyl 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:

2020-09-29 - 2020-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Version / remarks:

2012

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

adopted July 29, 2016

Deviations:

yes

Remarks:

The spiked substrates and the substrate of the control showed higher pH-values of 7.0 – 7.2 at test start (recommended 6 ± 0.5). Since no conspicuities occurred, it is assumed that this deviation has no impact on the reliability of the study results.

Principles of method if other than guideline:

The spiked substrates and the substrate of the control showed higher pH-values of 7.0 – 7.2 at test start (recommended 6 ± 0.5). Since no conspicuities occurred, it is assumed that this deviation has no impact on the reliability of the study results.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

The test item was poorly soluble in water; therefore, the volatile organic solvent acetone was used. A stock solution was prepared in acetone.
The test item was dissolved in a volume of acetone sufficient to prepare a stock solution. This stock solution was used to produce the various dosage solutions of the test item. An appropriate volume of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil. After evaporation of the solvent by placing the application vessels under a fume hood, the quartz sand was coated by the test item.

The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in for 5 minutes using a power drill.
Finally, the contaminated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g soil (dw) was used.

The test item was applied once at the beginning of the test. The duration of the test (exposure of earthworms to the artificial soil containing the test item) was 56 days. The adult earthworms were removed from the substrate after 28 days.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) have been kept at ECT Oekotoxikologie GmbH since February 1994. They were originally delivered by Co. Landenberger (D-72355 Schömberg).

Only adult earthworms (with clitellum) with a fresh weight (FW) between 300 and 600 mg were used. The worms were at least two months, but not more than one year old, however the age of individuals did not differ by more than four weeks. Animals which fulfilled these requirements, were taken from a synchronised culture. The worms selected for the test were acclimatised in artificial soil under test conditions at least 24 hours before starting the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18.7 – 20.9 °C, recommended 20 ± 2 °C

pH:

soil pH-value (day 0): 7.0 – 7.2 (recommended 6 ± 0.5),
soil pH-value (day 56): 7.2 – 7.3

Moisture:

soil moisture (day 0): 50.5 – 54.0 % of the WHCmax, (recommended 40 – 60 % of the WHCmax),
soil moisture (day 56): 58.1 – 93.7 % of the WHCmax

Nominal and measured concentrations:

3.125, 6.25, 12.5, 25, 50 and 100 mg test item/kg soil dry weight (dw)

Reference substance (positive control):

yes

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

65.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits = 48.7 – 95.4 mg test item/kg soil (dw)

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

87.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence limits = 78.1 – 97.0 mg test item/kg soil (dw)

Details on results:

Adult mortality
0 % and 1.3 % mortality of the adult test animals were observed in the water control and the solvent control, respectively. 0 – 67.5 % mortality of the adult test animals occurred in all concentrations of the test item tested.

Adult biomass
The mean number of juveniles was 320.0 and 251.8 in the water control and solvent control, respectively. The mean number of juveniles in the test item treatment groups ranged from 0 to 344.0.


Food consumption and test observations

In all test vessels the administered food was almost fully consumed by the worms in the first four weeks except in the following replicates:

After 14 days, in two replicates (A and B) of the concentration with 50 mg test item/kg soil (dw) only half of the food was consumed.
After 28 days, only half of the food was consumed in two replicates of the deionised water control (A and D), in one replicate of the solvent control (G) and in the concentrations with
6.25 (B) and 12.5 mg test item/kg soil (dw) (A), respectively.
In all replicates of the highest treatment with 100 mg test item/kg soil (dw) less than half of the food was consumed by the worms after 7 days. No food was consumed after 14 and 28 days. On day 21, the food was eaten half in the replicate B, less than half in the replicates C and D, and in replicate A no food was consumed.

In the replicate A and B of the highest treatment three worms were observed at the surface after 7 days, respectively. In each of those replicates one dead worm was removed from the soil surface and three dead worms in the replicate D after 14 days. Another dead worm was removed in the replicate A on day 21.

No further unusual effects on behaviour or morphology of the adult earthworms were observed throughout all treatments.

Results with reference substance (positive control):

In a separate GLP study (ECT study number IRR2002), the EC50 value was calculated by probit analysis as 268.0 mg boric acid/kg soil (dw) (95% confidence limits = 251.1 – 282.6 mg boric acid/kg soil (dw)). Annex E.4 of the guideline states that boric acid should reduce reproduction by 50% at about 400 to 600 mg boric acid/kg soil (dw). The estimated EC50 value was slightly lower than recommended by the guideline. However, since the calculated EC50 is lower by factor of 1.6 than the recommended value of about 400 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Reported statistics and error estimates:

Step-down Rao-Scott-Cochran-Armitage test (α = 0.05, one-sided) and trimmed Spearman-Karber for mortality; Williams t-test and Weibull analysis for reproduction (α = 0.05, one-sided); and Dunnett’s t-test for biomass change (α = 0.05, two-sided);

The statistical software package ToxRat Professional 3.3.0 was used for these calculations.

Validity criteria:

Mortality of the adult test animals in the solvent control:  1.3 %
(required: equal or less than 10 %)
Mean number of juveniles per replicate in the solvent control (standard deviation): 251.8 ± 31.9
(required: ≥ 30)
The coefficient of variation for the number of juveniles in the solvent control: 12.7 %
(required: ≤ 30 %)

 The data provide evidence that the quality criteria have been fulfilled.

​

Validity criteria fulfilled:

yes

Remarks:

see "overall remarks"

Conclusions:

The test substance Ditolylether was tested according to OECD Guideline No. 222 for the Testing of Chemicals "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" adopted July 29, 2016. The following results were obtained after statistical evaluation of the data:
NOECMortality = 50 mg test item/kg soil (dw) LOECMortality = 100 mg test item/kg soil (dw) LC50 = 87.1 mg test item/kg soil (dw)
(95 % confidence limits = 78.1 – 97.0 mg test item/kg soil (dw))

NOECReproduction = 50 mg test item/kg soil (dw) LOECReproduction = 100 mg test item/kg soil (dw) EC50 = 65.1 mg test item/kg soil (dw)
(95 % confidence limits = 48.7 – 95.4 mg test item/kg soil (dw))

NOECBiomass = 50 mg test item/kg soil (dw) LOECBiomass = 100 mg test item/kg soil (dw).

Executive summary:

The test substance Ditolylether was tested according to OECD Guideline No. 222 for the Testing of Chemicals  "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" adopted July 29, 2016 in accordance with the International Standard ISO 11268-2 Part 2 (2012) "Soil Quality – Effects of Pollutants on Earthworms – Part 2: Determination of Effects on Reproduction of Eisenia fetida/Eisenia andrei".
The purpose of this study was to determine a NOEC/LOEC and if possible, EC50 for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standardised artificial soil. Additionally, a LC50 was estimated. The test item was mixed with the substrate.
The test item was tested in six concentrations with a spacing factor of 2. All tested concentrations were based on the test item. The following concentrations of the test item were used: 100 mg test item/kg soil (dw), 50 mg test item/kg soil (dw), 25 mg test item/kg soil (dw), 12.5 mg test item/kg soil (dw), 6.25 mg test item/kg soil (dw), and 3.125 mg test item/kg soil (dw). Four replicates per concentration, eight solvent control replicates and additionally four deionised water control replicates (ten worms per replicate) were tested. The data provide evidence that the quality criteria have been fulfilled.
Test parameters investigated were:
- Mortality: Number of dead adult worms for each test vessel and mean number of dead adult earthworms as a percentage of the initial number at the start of the test for each concentration and for the solvent control.
- Biomass: Loss/increase in the mean biomass (i.e. the fresh mass of organisms) 28 days after application expressed as a percentage change of the initial fresh mass of organisms at the start of the test for each concentration and for the solvent control.
- Reproduction: Mean number of juveniles per test vessel 56 days after application for each concentration and for the solvent control.
- Morphology and behaviour: Obvious or pathological symptoms or distinct changes in behaviour (e.g. reduced feeding activity) of both adult and juvenile earthworms were reported.

Mortality
0 % and 1.3 % mortality of the adult test animals were observed in the water control and the solvent control, respectively. 0 – 67.5 % mortality of the adult test animals were recorded across all concentrations of the test item tested. Step-down Rao-Scott-Cochran-Armitage test (α = 0.05, one-sided) showed statistically significant differences concerning mortality between the solvent control and the highest concentration of the test item tested, i.e. 100 mg test item/kg soil (dw). Therefore, LOECMortality was determined to be 100 mg test item/kg soil (dw). Accordingly, the NOECMortality was determined to be 50 mg test item/kg soil (dw).
The LC50 was calculated using the trimmed Spearman-Karber procedure:
LC50 = 87.1 mg test item/kg soil (dw)
(95 % confidence limits = 78.1 – 97.0 mg test item/kg soil (dw))

Biomass change
Statistical analysis showed a statistically significant difference (Dunnett’s t-test; α = 0.05, two- sided) concerning biomass development of individual adults over 28 days between the solvent control and the highest concentration of the test item tested, i.e. 100 mg test item/kg soil (dw). Therefore, the LOECBiomass  was  determined to be  100 mg  test item/kg soil (dw)  and  the NOECBiomass 50 mg test item/kg soil (dw).

Reproduction
The mean number of juveniles was 320.0 and 251.8 in the water control and solvent control, respectively. The mean number of juveniles in the test item treatment groups ranged from 0 to 344.0. Statistical analysis (Williams t-test; α = 0.05, one-sided) showed a statistically significant difference concerning the number of juveniles between the solvent control and the highest concentration of the test item tested, i.e. 100 mg test item/kg soil (dw). Therefore, the LOECReproduction was determined to be 100 mg test item/kg soil (dw). The NOECReproduction was determined to be 50 mg test item/kg soil (dw).

 

Description of key information

The test substance Ditolylether was tested according to OECD Guideline No. 222 for the Testing of Chemicals "Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)" adopted July 29, 2016. The following results were obtained after statistical evaluation of the data:
NOECMortality = 50 mg test item/kg soil (dw) LOECMortality = 100 mg test item/kg soil (dw) LC50 = 87.1 mg test item/kg soil (dw)
(95 % confidence limits = 78.1 – 97.0 mg test item/kg soil (dw))

NOECReproduction = 50 mg test item/kg soil (dw) LOECReproduction = 100 mg test item/kg soil (dw) EC50 = 65.1 mg test item/kg soil (dw)
(95 % confidence limits = 48.7 – 95.4 mg test item/kg soil (dw))

NOECBiomass = 50 mg test item/kg soil (dw) LOECBiomass = 100 mg test item/kg soil (dw).

As the values for NOECReproduction and NOECMortality were the same, the value of 50 mg/kg soil was chosen for the assessment.

Key value for chemical safety assessment

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

50 mg/kg soil dw