N-tert-butylbenzothiazole-2-sulphenamide

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

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)

Version / remarks:

adopted January 21, 2000

Deviations:

yes

Remarks:

Microbial biomass carbon of the soil was slightly below the 1% minimum required by the test guideline. However, the N-transformation rates were in the normal range as observed in earlier studies and confirmed a metabolically active microbial biomass.

Principles of method if other than guideline:

Test Set-up
Freshly collected standard field soil type LUFA 2.3 will be provided by the Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer (LUFA), Germany. The soil will be stored at 20 ± 2°C in the dark for a period of up to 28 d. Microbial biomass carbon of the soil will be determined according to DIN/ISO 14240-2.
On the day of test start, the bulk soil for the nitrogen transformation test will be thoroughly mixed. The bulk soil will be amended with powdered lucerne-grass-green meal at 5 g/kg dry soil and will then be divided into six portions for the six different treatments (i.e. five test item treatments and an untreated control).
Defined quantities of the test item separately for each treatment will be weighed out and ground with fine quartz sand. The resulting mixtures of sand and test item, one for each treatment, will be mixed thoroughly into the soil for the respective treatment by using a hand mixer (or equivalent) in order to homogeneously distribute the test item in the soil.
The soil for treatment “control” will receive the same amount of sand without test item.
Thereafter, demineralised water will be mixed into the soil to adjust soil moisture to 45% of the maximum water holding capacity (WHCmax). The soil of each treatment will then be distributed to the individual test containers (replicates) in equal portions. The definitive soil moisture will be the same for all treatments and replicates and will be reported in the final report.
Four replicates, each, will be used for each of the five test item concentrations and for the control. Each test vessel will be labelled with the study number, the test item concentration and a specific letter for each replicate. Each test container will be covered with a lid to keep the moisture within a range of 45% ± 5% of the maximum water holding capacity, and to allow some gas exchange. The test vessels will be incubated at 20 ± 2 °C in the dark. The temperature will be recorded continuously or at intervals not longer than 1 h.
At least once per week the test vessels will be weighed and weight loss will be compensated with deionised water to keep the soil moisture within a range of ± 5%.

GLP compliance:

yes

Remarks:

There were no circumstances which may have affected the quality or integrity of the data. The range-finding test was not performed in compliance with Good Laboratory Practice Principles.

Analytical monitoring:

yes

Remarks:

Measurement of nitrate via photometer

Vehicle:

no

Details on preparation and application of test substrate:

Pre-treatment of the Test Item
The test item was almost insoluble in water and organic solvents such as acetone or ethanol; therefore, the test item was ground with fine quartz sand using a pestle and mortar prior to being mixed together with the sand into the test soil.

Dose Levels and Test Set-Up
Six treatments with four replicates, each, were prepared from a bulk soil sample: five test item concentrations (T1 to T5), and an untreated control (C). The test-item had been defined by the study director according to the results of a previously performed non-GLP rangefinding 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.
The test item spiked sand (or non-treated sand for the control) was mixed thoroughly into portions of 0.6 kg soil dry weight equivalent. Thereafter deionised water was added to the soil to adjusted 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 at in the dark at a temperature ranging from 18.3 to 20.2 °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.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

After 28 d the test is terminated.

Test temperature:

20 ± 2 °C

Moisture:

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

Details on test conditions:

Physical and chemical properties of the test system (mean value ± standard deviation)
Note: All data were provided by the supplier except (*) and refer to dry matter.
Batch number: F2.32017
pH (0.01 M CaCl2): 5.84 ± 0.6
Cation exchange capacity [meq/100 g]: 7.5 ± 0.8
Particle size according to USDA [mm]: Relative distribution [%]
< 0.002 (clay) 7.5 ± 0.4
0.002 - 0.05 (silt) 33.6 ± 0.5
0.05 - 2.0 (sand) 58.9 ± 0.4
Soil type (USDA): sandy loam
Max. water holding capacity (WHCmax) [g/100 g]: 35.4 ± 1.0
Initial soil moisture [% dry mass]: 6.1 (*)
Soil moisture adjusted for test [% dry mass]: 15.9 (*), equivalent to 45% of WHCmax
Microbial Biomass Carbon (Cmic) [μg Cmic g-1 soil]: 57.38 (*)
Cmic [% of Corg]: 0.84 (*)

Lucerne-Grass-Green Meal
Lucerne-green-meal pellets had been purchased from Semhof, 86753 Möttingen, Germany and were ground to a fine powder with an electric mill.
The finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) at a dosing of 5.0 g/kg dry weight soil immediately before test item application.
Identification: Semhof’s Bio Luzernepellets Turbo
Batch number: 151030
C/N Analysis: LUFA Speyer, Germany (Report BM 04/15)
Nitrogen content (%): 2.81
Carbon content (%): 35.5
C/N ratio: 12.6

Nominal and measured concentrations:

Test item concentration in soil: 80 mg/kg soil dw, 128 mg/kg soil dw, 205 mg/kg soil dw, 328 mg/kg soil dw, 524 mg/kg soil dw.

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:

EC10

Effect conc.:

248 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 220/269 mg/kg soil dry weight (lower/upper 95% confidence limit)

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

286 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 263/305 mg/kg soil dry weight (lower/upper 95% confidence limit)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

377 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 360/396 mg/kg soil dry weight (lower/upper 95% confidence limit)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

205 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

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:

Statistical Evaluation
Normal distribution and homogeneity of variances was tested by the Kolmogorov Smirnov test on normal distribution and the Cochran’s test on variance homogeneity, respectively.
The NOEC was determined by applying the Williams multiple t-test (p≤0.05, one-sided smaller).
Effective concentrations (EC10, EC20, EC50) were determined by applying the probit analysis.
All statistical tests were performed using the ToxRat® statistical software Version 2.10.

On day 0, nitrate concentration of test-item treated soils did not differ significantly from nitrate concentration of the control.

On day 28, nitrate concentration was significantly increased compared to the control in the soils treated with the test item at concentrations up to 205 mg/kg soil dw, while it was significantly reduced at 328 and 524 mg/kg soil dw.

Increased nitrate concentrations at test item concentrations of 10 and 100 mg/kg soil dw had already been observed in the previously performed non GLP range-finding test. The reason for the observed positive effect of the test item on formation of nitrate is not clear. However, the test item could have served as N-source for the soil microrganisms as it contains approximately 12% N.

Nitrate concentration in soils [mg/kg soil dw] on day 0 and day 28 after test item treatment and relative increase [%] compared to the control in soils treated with test item and incubated for 28 days.

Test item concentration	Day 0	Day 28	Day 28 Increase compared to control
[mg/kg soil dw]	mean ± sd	mean ± sd	[%]
0.0 (control)	73.8 ± 3.8	133 ± 3	n.a.
80	75.0 ± 0.6	178 ± 4	33.3
128	76.7 ± 0.6	194 ± 4	45.6
205	77.4 ± 1.6	203 ± 3	52.0
328	77.6 ± 0.6	87.1 ± 2.1*	-34.7
524	75.7 ± 1.9	22.4 ± 0.8*	-83.2

Mean ± standard deviation (sd) n = 4; dw = dry weight; * significantly smaller than the control (Williams multiple t-test, p<0.05, one sided smaller); n.a. = not applicable; negative increase = decrease.

Validity criteria fulfilled:

yes

Remarks:

The validity criterion given by the guideline was fulfilled (variability of less than 15% within the control). The coefficient of variation (CV) for nitrate of control samples was 5.2 (day 0) and 2.3 (day 28).

Conclusions:

Vulkacit NZ/TBBS is considered to have no long-term adverse effect on nitrogen transformation in soil at concentrations up to and including 205 mg/kg soil dry weight.

Executive summary:

The study was conducted in order to determine possible effects of Vulkacit NZ/TBBS on soil microorganisms through measuring microbial nitrate formation in treated versus untreated soils after 28 days of incubation following OECD guideline No. 216.

The nitrogen transformation activity will be assessed by measuring the nitrate concentration in aqueous extracts from the treated soil and the control soil. These measurements will take place after test item application at the start of the test (day 0) and following 28 days of incubation at 20 ± 2° C in the dark.

Nitrate concentration in soil increased with increasing Vulkacit NZ/TBBS concentration up to and including 205 mg/kg soil dry weight probably due to the introduction of the test item containing nitrogen.

Nitrate concentration in soil was statistically significantly reduced at test item concentrations of 328 mg/kg soil dry weight and above.

The EC10, EC20 and EC50 were 248 (220/269), 286 (263/305) and 377 (360/396) mg/kg soil dry weight, respectively (in brackets: lower/upper 95% confidence limit).

The NOEC was 205 mg/kg soil dry weight.

Therefore, Vulkacit NZ/TBBS is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 205 mg/kg soil dry weight.

Description of key information

The study was conducted in order to determine possible effects of Vulkacit NZ/TBBS on soil microorganisms through measuring microbial nitrate formation in treated versus untreated soils after 28 days of incubation following OECD guideline No. 216.

The nitrogen transformation activity will be assessed by measuring the nitrate concentration in aqueous extracts from the treated soil and the control soil. These measurements will take place after test item application at the start of the test (day 0) and following 28 days of incubation at 20 ± 2° C in the dark.

Nitrate concentration in soil increased with increasing Vulkacit NZ/TBBS concentration up to and including 205 mg/kg soil dry weight probably due to the introduction of the test item containing nitrogen.

Nitrate concentration in soil was statistically significantly reduced at test item concentrations of 328 mg/kg soil dry weight and above.

The EC10, EC20 and EC50 were 248 (220/269), 286 (263/305) and 377 (360/396) mg/kg soil dry weight, respectively (in brackets: lower/upper 95% confidence limit).

The NOEC was 205 mg/kg soil dry weight.

Therefore, Vulkacit NZ/TBBS is considered to have no long term adverse effect on nitrogen transformation in soil at concentrations up to and including 205 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

205 mg/kg soil dw