Ditolyl ether

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-14- 2020-09-22 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on preparation and application of test substrate:

The test item was dissolved in acetone and applied to fine quartz sand. After complete evaporation of the solvent the spiked sand was mixed into the test soil.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Due to technical reasons, temperature in the test room was not logged from day 17 (09:57) to day 21 (14:24) of the test. On day 13 of the test, temperature exceeded the required maximum temperature of 22°C during three hours by maximal 0.7°C
The deviation is considered negligible and has no impact on the integrity of the study.

Moisture:

Soil moisture was kept within a range of ± 5% of 45% of the maximum water-holding capacity (WHCmax)

Organic carbon content (% dry weight):

0.66

Nitrogen content (% dry weight):

0.08

Details on test conditions:

Test Vessels
Glass jars with lids (volume 370 mL, Weck, Germany) were used as test vessels. The lids allowed for some gas exchange. Each test vessel was loaded with 174±2 g soil fresh weight.

Test Set-Up
Quartz sand either spiked with the test item or with the solvent only was mixed thoroughly into portions of 0.6 kg soil dry weight equivalent. Thereafter deionised water was added to adjust soil moisture to 45% WHCmax. After further thorough mixing the soils were distributed equally to four test vessels which constituted the replicates of the respective treatment.
Following test item application all test vessels were incubated for 28 days in the dark at
19.5 to 22.7 °C. Test vessels were weighed once per week and weight loss was compensated by adding deionised water. By doing so, the soil moisture was kept within a range of ± 5 % of the water content at test start.

Nominal and measured concentrations:

Seven treatments with four replicates each, were prepared from a bulk soil sample: six test item concentrations (0.5, 2.0, 8.0, 32.0, 128, 512 mg a.s./ kg soil dw), and a solvent control which received only the solvent without test item.
The test-item concentration had been defined by the study director according to the results of a previously performed non-GLP range-finding test, and in consultation with the sponsor.
The bulk soil for all treatments was amended with lucerne meal at 5.0 g/kg soil dry weight.

Reference substance (positive control):

no

Remarks:

The test guideline does not require the use of a reference item. Therefore, the test was performed without a reference item.

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 512 mg/kg soil dw

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 512 mg/kg soil dw

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

On day 28 increasing nitrate concentrations were observed with increasing test item concentrations up to 128 mg/kg soil. The reason for the observed increase in nitrate is not clear. The test item might have had some kind of stimulating effect on the nitrification process. However, the focus of the OECD test guideline No. 216 is explicitly on “adverse effects” i.e. on negative effects on nitrogen transformation. A positive effect (increase) on nitrate formation would therefore not be seen as critical.
At 512 mg/kg soil dw the measured nitrate concentration in soil was comparable to the nitrate concentration of the solvent control. This result is not in accordance with observations made in the previously performed non-GLP range-finding test. In this pre-test test increased nitrate concentrations were observed at nominal test item concentrations of up to 1000 mg/kg soil dry weight . The result therefore may suggest that no test item had been applied. The strong test item typical smell however, clearly confirmed the presence of the test item in the soil of treatment T6. Moreover, additional (non-GLP) measurements of nitrate in soil were conducted on day 57.
The additional measurements revealed mean nitrate concentrations of 199 mg/kg soil dw in soil from SC and 324 mg/kg soil dw in soil from T6. These results indicate that the initially reduced nitrogen transformation seen in soil of treatment T6 on day 28 was made up, while N-transformation in the solvent control seemed to slow down. It can be assumed that on day 57 the lucerne bound nitrogen in the soil of the solvent control had mainly been used-up, while in soil of T6 there was still plenty of nitrogen from lucerne available due to the postulated initial delay in transformation. This portion of nitrogen remaining in soil of T6 was transformed to nitrate by soil microorganisms recovering from the initial reduction.
The reason for the observed discrepancy between the results of the range finding test and the main test at test item concentrations ≥500 mg/kg soil dw may be the use of different batches of the field soil used for the two tests which may differ in the quality and quantity of soil microbial biomass and, consequently, in metabolic activity and the sensitivity against the test item.
Due to the lack of adverse effects on the measured nitrate concentration in soil ECX values could not be determined. The EC50 is assumed to be higher than the highest tested concentration of 512 mg/kg soil dw.

Results with reference substance (positive control):

The test guideline does not require the use of a reference item. Therefore, the test was performed without a reference item.

Validity

The coefficient of variation (CV) of control samples regarding the nitrate concentration was 3.0% on day 0 and 6.4% on day 28 of the test. Since the maximum deviation was below 15% the validity criterion was fulfilled.

 

Validity criteria fulfilled:

yes

Conclusions:

Due to the lack of adverse effects on nitrate concentration the LOEC and NOEC were considered to be >512 mg/kg soil dw and ≥512 mg/kg soil dw, respectively.
The lack of adverse effects was also the reason why ECX values could not be determined. The EC50 is assumed to be higher than the highest tested concentration of 512 mg/kg soil dw.

Executive summary:

The test item Ditolyl ether, was tested in accordance with OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test".

Throughout the 28 day study, soil moisture of all treatments was within the range of ±5% of the soil moisture adjusted at the start of the test. Nitrate was measured in sub-samples of all test-item treated soil and control soils on day 0 and day 28 after test item application. The nitrate concentration in the bulk soil before adding lucerne meal was determined at day 0.
Nitrate concentration of the bulk soil which was used to set up the different test item treatments and the control was 34.8 ± 1.2 mg/kg soil dw (measured before adding lucerne meal; mean and standard deviation, n = 4). After adding lucerne meal on day 0 of the test, mean nitrate concentration of the solvent control soil was 52.0 ± 1.6 mg/kg soil dw and increased to 120.5 ± 7.7 mg/kg soil dw on day 28 (mean and standard deviation, n = 4).
Nitrate concentration in test-item treated soil measured on day 0 and on day 28 was not significantly reduced compared to the solvent control.
On day 28 increasing nitrate concentrations were observed with increasing test item concentrations up to 128 mg/kg soil. At 512 mg/kg soil dw the nitrate concentration in soil was comparable to the nitrate concentration of the solvent control. This appeared remarkable and may suggest that no test item had been applied; the strong test item typical smell of the soil however confirmed the presence of the test item.
Due to the lack of adverse effects on nitrate concentration the LOEC and NOEC were considered to be >512 mg/kg soil dw and ≥512 mg/kg soil dw, respectively.
The lack of adverse effects was also the reason why ECX values could not be determined. The EC50 is assumed to be higher than the highest tested concentration of 512 mg/kg soil dw.

Description of key information

The test item Ditolyl ether, was tested in accordance with OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test".

Nitrate concentration in test-item treated soil measured on day 0 and on day 28 was not significantly reduced compared to the solvent control. On day 28 increasing nitrate concentrations were observed with increasing test item concentrations up to 128 mg/kg soil dw. At 512 mg/kg soil dw the nitrate concentration was comparable to the solvent control. Therefore the LOEC and NOEC were considered to be >512 mg/kg soil dw and ≥512 mg/kg soil dw, respectively.

Due to the lack of adverse effects on nitrate concentration in soil ECX values could not be determined. The EC50 is assumed to be higher than the highest tested concentration of 512 mg a.s./kg soil dw

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

512 mg/kg soil dw