N-cyclohexylbenzothiazole-2-sulfenamide

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:

24 Aug 2020 - 01 Oct 2020 (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:

no

Vehicle:

no

Details on preparation and application of test substrate:

The solubility of the test item in either water or acetone was not sufficient to adjust the required test item concentration in soil. Therefore the test item was mixed with fine quartz and mortared. Defined quantities of the test-item sand mixture were thoroughly mixed into the soil by using an electric mixer with two counter-rotating blades in order to homogeneously distribute the test item in the soil of the respective test item treatment.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Following test item application all test vessels were incubated for 28 days in the dark at 18.9 to 22.7 °C.

Due to technical reasons, temperature in the test room was not logged from 09:57 on day 8 to 14:24 on day 12 of the test. On day 4 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 175±3 g soil fresh weight.

Test Set-Up
Quartz sand either spiked with the test item or without test item (control) 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 18.9 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 treated soils (concentrations: 0.5, 2.0, 8.0, 32.0, 128, 512 mg a.s./ kg soil dw), and a control soil which were mixed with the same amount of quartz sand but 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.

Key result

Duration:

28 h

Dose descriptor:

other: LOEC

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.:

128 mg/kg soil dw

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Nitrate concentration in test-item treated soil measured on day 0 was not significantly reduced compared to the control.
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 nitrate concentration in soil was statistically significantly reduced by 89.5% compared to the control.

Due to the adverse effects on nitrate concentration at 512 mg/kg soil dw the LOEC and NOEC were considered to be 512 mg/kg soil dw and 128 mg/kg soil dw, respectively.
Effective concentrations (ECX) could not be calculated due to the structure of the data.

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.

Reported statistics and error estimates:

Normal distribution and homogeneity of variances was tested by the Shapiro-Wilk’s test on normal distribution and the Levene’s test on variance homogeneity, respectively. The NOEC was determined by applying the Williams multiple t-test (p≤0.05, one-sided smaller). To justify the use of the Williams test a trend analysis by contrasts was performed.
Reliable effective concentrations (EC10, EC50) could not be determined due to the structure of the data (positive effects at all test concentrations except the highest tested concentration).
All statistical tests were performed using the ToxRat® statistical software Version 3.3.

Measured nitrate concentration in soil [mg/kg soil dw] on day 0 and day 28 and calculated nitrate increase (from day 0 to day 28).

Code	Test item concentration [mg/kg soil dw]	Nitrate day 0 (mean ± SD) [mg/kg soil dw]	Nitrate day 28 (mean ± SD) [mg/kg soil dw]	Mean nitrate inhibition (from day 0 to day 28) compared to the control [mg/kg soil dw]
C	0.0	48.6 ± 1.3	113.2 ± 3.5	n.a.
T1	0.5	49.6 ± 0.6	117.9 ± 2.0	-4.1
T2	2.0	49.6 ± 0.3	116.2 ± 3.8	-2.6
T3	8.0	50.9 ± 0.8	126.0 ± 4.6	-11.2
T4	32.0	51.3 ± 1.3	148.2 ± 4.6	-30.9
T5	128	49.3 ± 1.2	135.3 ± 4.0	-19.5
T6	512	50.8 ± 1.5	11.9 ± 0.2*	89.5*
C = control, T1 to T6 = test item treated; dw = dry weight; n.a. = not applicable; * = significantly reduced compared to the control (Williams multiple sequential T-test procedure, α = 0.1, one-sided smaller) negative inhibition represents increase.

 

Validity

The study was valid. The coefficient of variation (CV) of control samples regarding the nitrate concentration was 2.6% on day 0 and 3.1% on day 28 of the test. Since the maximum deviation was below 15% the validity criterion was fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

Adverse effects on N-transformation were observed at a nominal test item concentration of 512 mg/kg soil dry weight with nitrate concentration statistically significantly reduced compared to the control by 89.5%.
Effective concentrations (ECX) could not be determined due to the structure of the data.
The LOEC and NOEC were 512 mg/kg soil dry weight and 128 mg/kg soil dry weight, respectively.
In conclusion, the test item is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 128 mg/kg soil dry weight.

Executive summary:

The test item N-cyclohexyl-2-benzothiazolesulfenamide 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 31.7 ± 3.6 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 control soil was 48.6 ± 1.3 mg/kg soil dw and increased to 113.2 ± 3.5 mg/kg soil dw on day 28. Nitrate concentration in test-item treated soil measured on day 0 was not significantly reduced compared to the control. 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 nitrate concentration in soil was statistically significantly reduced by 89.5% compared to the control.
Due to the adverse effects on nitrate concentration at 512 mg/kg soil dw the LOEC and NOEC were considered to be 512 mg/kg soil dw and 128 mg/kg soil dw, respectively. Reliable effective concentrations (ECX) could not be determined due to the fact that except for the highest test concentration of 512 mg/kg soil dw no adverse effects of the test item on the nitrate concentration in soil was seen.

Description of key information

The test item N-cyclohexyl-2-benzothiazolesulfenamide was tested in accordance with OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test". Adverse effects on N-transformation were observed at a nominal test item concentration of 512 mg/kg soil dry weight with nitrate concentration statistically significantly reduced compared to the control by 89.5%.
Effective concentrations (ECX) could not be determined due to the structure of the data.
The LOEC and NOEC were 512 mg/kg soil dry weight and 128 mg/kg soil dry weight, respectively.
In conclusion, the test item is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 128 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

128 mg/kg soil dw