Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

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:

From February 10, 2020 to April 01, 2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on sampling:

Application solutions for the different test substance concentrations were prepared by dispersing the test substance in water. Afterwards, each application solution of the respective treatment was thoroughly mixed with soil substrate (amended by lucerne meal). The study consisted of seven treatments: one control, five test substance concentrations and one reference substance concentration. One test vessel per treatment and per sampling date was used, three soil samples per test vessel were collected at DAT 0 and DAT 28 (DAT = Day(s) After Treatment).

Vehicle:

yes

Remarks:

Ultrapure water

Details on preparation and application of test substrate:

- Dosage and application:
The test substance concentrations were selected in consultation with the Sponsor as follows: 10, 32, 100, 320 and 1000 mg/kg dry soil.

- Application:
According to information of the Sponsor and pre-experiments regarding solubility of the test substance (non-GLP), the test substance is sufficiently soluble in purified water for the preparation of the application solutions of the required test concentrations. At DAT 0, the application solution of the highest test concentration was prepared by dispersing 2249.73 mg of the test substance in 50 mL of ultrapure water. Application solution 1 was stirred for 15 minutes before the application solutions of the lower test concentrations were prepared under continuous stirring of 15 minutes each. Due to a calculation error, the amount of weighed test substance was based on 449.8 g dry soil instead of 454.4 g dry soil. Therefore, the needed weight amount would have been 2272 mg which is 1.0% more than the actual weight used. This error is not considered to affect the outcome of the study negatively, all concentrations used in this study were reported as nominal. Except for the addition of the test substance, the controls were treated identically. For the reference substance treatment, 30.1 mg nitrapyrin was diluted in 100 mL ultrapure water. The volume of 10 mL of this application solution was mixed with the soil batch of 449.8 g dry soil corresponding to 6.7 mg nitrapyrin per kg dry soil. During application of the reference substance, the soil was adjusted to the moisture of 48.6% MWHC.

- Experimental conditions:
After treatment, the soil aliquots were incubated at 20 ± 2°C in the dark. The soil water content was adjusted to 48.6% MWHC and controlled weekly by weighing. Losses of water were compensated by addition of ultrapure water followed by mixing of the samples. The temperature was monitored continuously in the climate cabinet. The initial soil pH (in water) was measured in duplicate samples of the untreated and unamended soil prior to test start.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Temperature was 18.1 – 21.0°C with a mean of 20.2°C.

Moisture:

Water content of soil at test initiation: 45% of MWC

Organic carbon content (% dry weight):

0.65

Nitrogen content (% dry weight):

0.07

Details on test conditions:

Natural Soil Speyer 2.3
• pH 6.1 ± 0.4
• cation exchange capacity 6.8 ± 01.4 meq per 100 g
• particle size distribution 7.3 ± 0.9 % (< 0.002 mm), 33.3 ± 0.6% (0.002 – 0.05 mm), 59.4 ± 0.7% (0.05 – 2.0 mm).
• Maximum Water Holding Capacity (MWC) 35.2 ± 1.8 g per 100 g dry soil.

Nominal and measured concentrations:

Nominal concentrations: 10, 32, 100, 320 and 1000 mg/kg dry soil.

Reference substance (positive control):

yes

Remarks:

Nitrapyrin

Key result

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

- Microbial Biomass:
The microbial biomass of the test soil was measured by the fumigation method. The mean values of soluble total organic carbon of the fumigated soil and not fumigated soil samples were 141.3 and 79.1 mg/kg dry soil, respectively, resulting in the calculation of a microbial biomass of 163.5 mg/kg dry soil. This microbial biomass was 2.4% of the organic C content of the used test soil and was therefore above the trigger value of 1% as required by the guideline.

- Nitrate Concentration at DAT 0:
The soil nitrate concentration was measured at DAT 0 directly after application. In the control, the mean nitrate concentration was 7.2 mg/kg dry soil. In the test substance treatments, mean nitrate concentrations at DAT 0 was in the range of 7.5 and 8.0 mg/kg dry soil and did not differ from the control (Dunnett t-test, two-sided, α = 0.05) with the exception of the treatment with 320 mg/kg dry soil. This satistically significant increase at DAT 0 is considered to be caused by small-scale differences in metabolic activiation of the soil microflora directly after application and has no impact on the outcome of the study. The mean nitrate concentration at DAT 0 in the reference substance treatment was 8.0 mg/kg dry soil and differed statistically significantly from the control (Welch t-test, two-sided, α = 0.05). This statistical significance is not considered to impact the outcome of the study as already mentioned above for the test item treatment with 320 mg/kg dry soil.

Results with reference substance (positive control):

This study fulfilled the validity criterion since the coefficient of variation of the controls regarding nitrate concentration at DAT 28 (Table 3) was < 15%.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the NOEC and EC25 was determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively.

Executive summary:

A study was conducted to determine the effect on soil microflora activity of the test substance C8-18 and C18-unsatd. DEA, according to OECD Guideline 216, in compliance with GLP. Soil microorganisms in natural soil (Speyer 2.3) was used as test system. Application solutions for the different test substance concentrations were prepared by dispersing the test substance in water. Afterwards, each application solution of the respective treatment was thoroughly mixed with soil substrate (amended by lucerne meal). The study consisted of seven treatments: one control, five test substance concentrations which were: 10, 32, 100, 320 and 1000 mg/kg dry soil and one reference substance concentration. One test vessel per treatment and per sampling date was used, three soil samples per test vessel were collected at DAT 0 and DAT 28.  Soil nitrate concentration was measured at DAT 0 (after mixing the application solutions with soil substrate and lucerne meal) and DAT 28. The test substance has no statistically significant inhibiting effects on the soil microflora regarding the endpoints soil nitrate concentration and soil nitrate formation rate up to and including the highest tested concentration of 1000 mg/kg dry soil. Under the study conditions, the NOEC and EC25 was determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively (Schmidt, 2020).

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw

Additional information

A study was conducted to determine the effect on soil microflora activity of the test substance C8-18 and C18-unsatd. DEA, according to OECD Guideline 216, in compliance with GLP. Soil microorganisms in natural soil (Speyer 2.3) was used as test system. Application solutions for the different test substance concentrations were prepared by dispersing the test substance in water. Afterwards, each application solution of the respective treatment was thoroughly mixed with soil substrate (amended by lucerne meal). The study consisted of seven treatments: one control, five test substance concentrations which were: 10, 32, 100, 320 and 1000 mg/kg dry soil and one reference substance concentration. One test vessel per treatment and per sampling date was used, three soil samples per test vessel were collected at DAT 0 and DAT 28.  Soil nitrate concentration was measured at DAT 0 (after mixing the application solutions with soil substrate and lucerne meal) and DAT 28. The test substance has no statistically significant inhibiting effects on the soil microflora regarding the endpoints soil nitrate concentration and soil nitrate formation rate up to and including the highest tested concentration of 1000 mg/kg dry soil. Under the study conditions, the NOEC and EC25 was determined to be ≥ 1000 and > 1000 mg/kg dry soil, respectively (Schmidt, 2020).