N-tert-butylbenzothiazole-2-sulphenamide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Principles of method if other than guideline:

Test design: 5 treatments plus control and solvent control, 4 replicates per treatment and water control, 8 for the solvent control;

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Remarks:

The test item was sparingly soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone.

Details on preparation and application of test substrate:

Pre-treatment of the Test Item
The test item was sparingly soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone.
The test item was dissolved in an amount of acetone sufficient to prepare a stock solution. This stock solution was used to produce a range of dosage solutions for each concentration. An appropriate volume of the stock or respective dosage solution was used to soak a portion of the quartz sand of the artificial soil. The solvent was allowed to evaporate at room temperature for 2 hours under a fume hood.
The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in 5 minutes.
Finally, the contaminated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw was used.
The method of pre-treatment of the test item was selected according to the characteristics of the test item and is described in the test guideline.

Method of Administration and Justification for Selection of the Method
The worms were introduced into the test vessels after the treated artificial soil was placed into the test vessels. The test animals were exposed to the test item by dermal and alimentary uptake.

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 (72355 Schömberg, Germany).
Only adult earthworms (with clitellum) with a fresh weight (FW) between 300 and 600 mg were used. The earthworms 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 earthworms selected for the test were acclimatised in artificial soil under test conditions for at least 24 hours before starting the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Biomass: 28 days after application; Reproduction:56 days after application

Test temperature:

18.2–21.4 °C (recommended 20±2 °C)

pH:

Soil pH-value (day 0): 5.9–6.2 (recommended 6±0.5),
Soil pH-value (day 56): 6.8–7.0

Moisture:

Soil moisture (day 0): 48.2–52.2% of the WHCmax (rec. 40–60%),
Soil moisture (day 56): 63.6–69.1% of the WHCmax

Details on test conditions:

The earthworms were tested in an artificial soil. The composition of this artificial soil was based on OECD Guideline No. 222 (2016) (the percentages refer to the dry weight):
- 5% Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20% Kaolin clay (kaolinite content >30%)
- 74–75% Quartz sand (fine sand content with particles between 50 and 200 μm higher than 50%)
- 0.3–1% Calcium carbonate (CaCO3).
With respect to the properties of the test item (log Pow≥2), considering the influence on bioavailability, 5% instead of 10% peat were used (EPPO 2003). The organic carbon content is considered to be 2.5% as given in the literature (Ad-hoc-AG Boden, 2005).
The air-dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. The pH value was adjusted to 6±0.5 using calcium carbonate. The moisture content was adjusted to 40–60% of WHCmax using deionised water. The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated.
The following conditions were adjusted:
Temperature: 20 +/- 2 ºC
Light/dark cycle: 16 h light:8 h dark with a constant light intensity at the substrate surface of 400–800 lx.

Nominal and measured concentrations:

Test concentrations: 75, 133.4, 237.2, 421.8 and 750 mg test item/kg artificial soil dry weight (dw);

Reference substance (positive control):

yes

Remarks:

Boric acid

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

>= 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

>= 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

133.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

237.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No mortality was observed throughout all test vessels after 28 days. Hence, no statistical analysis was performed and the NOEC(Mortality) was considered as ≥ 750 mg test item/kg soil dw (the highest concentration of the test item tested). The LOEC(Mortality) could not be determined and was regarded to be >750 mg test item/kg soil dw.

Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p≤0.05) concerning biomass development of individual adults over 28 days between the solvent control and all concentrations of the test item tested. Therefore, the NOEC(Biomass) was determined as ≥750 mg test item/kg soil dw (the highest concentration of the test item tested).
The LOECBiomass could not be determined and was regarded to be >750 mg test item/kg soil dw.

Statistical analysis (Williams t-test; 1-sided, p≤0.05) showed a significant difference concerning the number of juveniles between the control and the concentrations of 237.2, 421.8 and 750 mg test item/kg soil dw. Therefore, the NOEC(Reproduction) was determined as 133.4 mg test item/kg soil dw. Accordingly, the LOEC(Reproduction) was determined and was determined as 237.2 mg test item/kg soil dw.
Since inhibition of reproduction was <50%, no EC50 value was calculated; the EC50 was regarded to be >750 mg test item/kg soil dw.

Results with reference substance (positive control):

In a separate GLP study (ECT study number IRR1703) Eisenia fetida were exposed to Boric acid at nominal concentrations of 150, 300 and 600 mg boric acid/kg artificial soil (dw). According to the ISO guideline 11268-2 Part 2 (2011) a significant effect on reproduction should be observed between 400 and 600 mg boric acid/kg soil dry weight (dw). Concerning number of juveniles statistical analysis (William’s t-test, p = 0.05, one-sided) showed a significant difference between the control and the concentrations of 300 and 600 mg boric acid/kg soil (dw). Since this observed effect is slightly higher than mentioned in the ISO guideline the effects of boric acid confirm suitable sensitivity of the test system.

Reported statistics and error estimates:

No mortality was observed throughout all test vessels after 28 days. Hence, no statistical analysis was performed and the NOEC(Mortality) was considered as ≥750 mg test item/kg soil dw (the highest concentration of the test item tested). The LOEC(Mortality) could not be determined and was regarded to be >750 mg test item/kg soil dw.
Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p≤0.05) concerning biomass development of individual adults over 28 days between the solvent control and all concentrations of the test item tested. Therefore, the NOEC(Biomass) was determined as ≥750 mg test item/kg soil dw (the highest concentration of the test item tested). The LOECBiomass could not be determined and was regarded to be >750 mg test item/kg soil dw.
Statistical analysis (Williams t-test; 1-sided, p≤0.05) showed a significant difference concerning the number of juveniles between the control and the concentrations of 237.2, 421.8 and 750 mg test item/kg soil dw. Therefore, the NOEC(Reproduction) was determined as 133.4 mg test item/kg soil dw. Accordingly, the LOEC(Reproduction) was determined as 237.2 mg test item/kg soil dw.
Since inhibition of reproduction was <50%, no EC50 value was calculated; the EC50 was regarded to be >750 mg test item/kg soil dw.

Adult mortality

Number of dead earthworms after 28 days (n = 80 for solvent control; n = 40 for control and concentrations). Data are given as absolute values and in percent of the total number (mortality).

Concentration	Number of dead adult worms	Mortality [%]
[mg test item/kg soil (dw)]	Day 28	Day 28
Control	0	0.0
Solvent Control	0	0.0
75.0	0	0.0
133.4	0	0.0
237.2	0	0.0
421.8	0	0.0
750	0	0.0

Adult biomass

Average live weight (mg) per surviving adult earthworm at the beginning of the test and after 28 days (n = 80 for solvent control; n = 40 for control and concentrations). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Concentration	Biomass (mg fresh weight)		Mean change in % of the initial weight per replicate
[mg test item/kg soil (dw)]	Day 0	Day 28	Day 28
Control	388.4 ± 20.7	491.4 ± 20.4	26.8
Solvent Control	388.1 ± 24.3	493.1 ± 23.5	27.3
75.0	388.3 ± 19.1	509.6 ± 12.8	31.6
133.4	390.6 ± 15.0	529.5 ± 13.5	35.6
237.2	389.5 ± 14.5	527.5 ± 12.5	35.5
421.8	387.1 ± 18.2	514.0 ± 15.1	33.1
750	389.0 ± 15.3	534.4 ± 31.1	37.4

Reproduction

Reproduction (given as mean number of juveniles ± standard deviation and as % of the solvent control) in the treated test vessels.

Concentration	Number of Juveniles per Treatment	Number of Juveniles
[mg test item/kg soil (dw)]	(mean ± sd)	[% of Solvent Control]
Control	344.0 ± 50.5	109.8
Solvent Control	313.3 ± 39.3	-
75.0	295.0 ± 26.4	94.2
133.4	281.0 ± 55.4	89.7
237.2	246.5 ± 42.0	78.7*
421.8	229.8 ± 24.9	73.3*
750	167.8 ± 37.5	53.6*

* Statistically significant different to Solvent Control (Williams t-test, p = 0.05, one-sided)

Validity criteria fulfilled:

yes

Remarks:

Mortality of the adults in the solvent control = 0% (required: ≤10%); Mean number of juveniles in the solvent control = 313.3 (required: ≥30) CV for the number of juveniles in the solvent control = 12.6 (required: ≤30%)

Conclusions:

The chronic effects of VULKACIT NZ/TBBS on the earthworm Eisenia fetida were evaluated under laboratory test conditions using an artificial soil substrate containing 5% w/w peat content:
NOECMortality ≥ 750 mg test item/kg soil dw.
LOECMortality > 750 mg test item/kg soil dw.
NOECBiomass ≥ 750 mg test item/kg soil dw.
LOECBiomass > 750 mg test item/kg soil dw.
NOECReproduction = 133.4 mg test item/kg soil dw.
LOECReproduction = 237.2 mg test item/kg soil dw.
EC50 > 750 mg test item/kg soil dw.

Executive summary:

The purpose of this study was to determine a NOEC/LOEC for the effects of VULKACIT NZ/TBBS on the reproduction (56 days after application), mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standardised artificial soil with 5% peat. The test item was mixed with the substrate.

This study was designed to comply with OECD Guideline No. 222 and ISO 11268-2 Part 2. The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna.

In the present study no adverse effects on either mortality, biomass development and morphological changes were observed at concentrations up to and including 750 mg test item/kg soil dry weight. In terms of effects on earthworm reproduction the NOEC was determined as 133.4 mg test item/kg soil (dw) and LOEC as 237.2 mg test item/kg soil (dw). Since inhibition of reproduction was <50%, no EC50 value was calculated; the EC50 was regarded to be >750 mg test item/kg soil dw.

Concerning validity criteria all requirements have been fulfilled.

NOECMortality ≥ 750 mg test item/kg soil dw.

LOECMortality > 750 mg test item/kg soil dw.

NOECBiomass ≥ 750 mg test item/kg soil dw.

LOECBiomass > 750 mg test item/kg soil dw.

NOECReproduction = 133.4 mg test item/kg soil dw.

LOECReproduction = 237.2 mg test item/kg soil dw.

EC50 > 750 mg test item/kg soil dw.

Description of key information

The chronic effects of VULKACIT NZ/TBBS on the earthworm Eisenia fetida were evaluated under laboratory test conditions using an artificial soil substrate containing 5% w/w peat content.

In the present study no adverse effects on either mortality, biomass development and morphological changes were observed at concentrations up to and including 750 mg test item/kg soil dry weight. In terms of effects on earthworm reproduction the NOEC was determined as 133.4 mg test item/kg soil (dw) and LOEC as 237.2 mg test item/kg soil (dw). Since inhibition of reproduction was <50%, no EC50 value was calculated; the EC50 was regarded to be >750 mg test item/kg soil dw.

Concerning validity criteria all requirements have been fulfilled.

NOECMortality ≥ 750 mg test item/kg soil dw.

LOECMortality > 750 mg test item/kg soil dw.

NOECBiomass ≥ 750 mg test item/kg soil dw.

LOECBiomass > 750 mg test item/kg soil dw.

NOECReproduction = 133.4 mg test item/kg soil dw.

LOECReproduction = 237.2 mg test item/kg soil dw.

EC50 > 750 mg test item/kg soil dw.

Key value for chemical safety assessment

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

133.4 mg/kg soil dw