N-cyclohexylbenzothiazole-2-sulfenamide

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

Study period:

2020-09-22 - 2021-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

The test item was dissolved in a volume of acetone sufficient to prepare a stock solution with a concentration of 17.5 mg/mL. This stock solution was used to produce the various dosage solutions of the test item. An appropriate volume of the stock and the dosage solutions were used to soak 10 % quartz sand of the artificial soil, respectively. After evaporation of the solvent by placing the application vessels under a fume hood, the quartz sand was coated by the test item.

The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in for 5 minutes using a power drill.
Finally, the contaminated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g soil (dw) was used.

The earthworms 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, respiratory and alimentary uptake.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Source: ECT Oekotoxikologie GmbH since February 1994, originally delivered by Co. Landenberger
- Age at test initiation (mean and range, SD): adult worms were at least two months but no more than one year old, however the age of individuals did not differ by more than four weeks
- Weight at test initiation (mean and range, SD): Only adult earthworms (with clitellum) with a fresh weight (FW) between 300 and 600 mg were used

ACCLIMATION
- Acclimation period:The worms selected for the test were acclimatised in artificial soil under test conditions 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:

The test item was applied once at the beginning of the test. The duration of the test (exposure of earthworms to the artificial soil containing the test item) was 56 days. The adult earthworms were removed from the substrate after 28 days.

Test temperature:

Temperature: 18.7 – 22.7 °C (recommended 20 ± 2 °C)

The temperature rose above 22°C (recommended as upper limit by the guideline) up to 22.7 °C at three measurement points. Thus, the temperature was above the upper limit for around two hours within 8 weeks. This short term deviation is considered to have had no impact of the outcome and validity of this study.

pH:

Between 5.9 and 6.2 at the beginning of the test. Between 6.9 and 7.0 at the end of the test. (recommended 6 ± 0.5), see table below

Moisture:

soil moisture (day 0): 47.5 – 51.5 % of the WHCmax, (recommended 40 – 60 % of the WHCmax),
soil moisture (day 56): 59.7 – 67.0 % of the WHCmax, see table below

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Test vessels were Bellaplast containers (Company Kastelplast, D-55120 Mainz) consisting of inert (non-toxic) plastic (Polystyrol). The test vessels have a base area of 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm. The test vessels were covered with a transparent and perforated lid.
- Amount of soil or substrate: 500 g dw per container
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control:8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): The composition of this artificial soil was based on OECD Guideline No. 222 (2016):
- 5 % Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20 % Kaolin-Clay (kaolinite content > 30 %)
- 64.75 % Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50 %)
- 0.25 % Calcium carbonate (CaCO3).

The missing 10 % Quartz sand to reach the required ~ 75 % was separated before for the application of the test item. 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.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark cycle
- Light intensity: a constant light intensity at the substrate surface of 400 – 800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality: Number of dead adult worms for each test vessel and mean number of dead adult earthworms as a percentage of the initial number at the start of the test for each concentration and for the solvent control.
- Biomass: Loss/increase in the mean biomass (i.e. the fresh mass of organisms) 28 days after application expressed as a percentage change of the initial fresh mass of organisms at the start of the test for each concentration and for the solvent control.
- Reproduction: Mean number of juveniles per test vessel 56 days after application for each concentration and for the solvent control.
- Morphology and behaviour: Obvious or pathological symptoms or distinct changes in behaviour (e.g. reduced feeding activity) of both adult and juvenile earthworms were reported.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

The test item was tested in five concentrations with a spacing factor of 2. All tested concentrations were based on the test item. The following concentrations of the test item were used:
500
mg test item/kg soil (dw)
250
mg test item/kg soil (dw)
125
mg test item/kg soil (dw)
62.5
mg test item/kg soil (dw)
31.25
mg test item/kg soil (dw)

Reference substance (positive control):

yes

Remarks:

In a separate GLP study (ECT study number IRR2002), the EC50 value for reproduction was calculated by probit analysis as 268.0 mg boric acid/kg soil (dw) (95% confidence limits = 251.1 – 282.6 mg boric acid/kg soil (dw)).

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

not determinable

Remarks:

The maximum inhibition of reproduction was 3.1% compared to the solvent control. Thus, a reliable curve fit could not be applied to the data and the EC50 could not be calculated. The EC50 is considered to be > 500 mg test item/kg soil (dw).

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 500 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass change

Remarks:

The change in biomass of the earthworms was observed

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass change

Remarks:

The change in biomass of the earthworms was observed

Key result

Duration:

56 d

Dose descriptor:

LC50

Effect conc.:

> 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

not determinable

Remarks:

Since mortality of the adult test animals was < 50 % in all concentrations of the test item tested no LC50 value could be calculated and is regarded to be > 500 mg test item/kg soil (dw).

Key result

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: 0 % and 1.3 % mortality of the adult test animals were observed in the water control and the solvent control, respectively. 0 – 7.5 % mortality of the adult test animals occurred in all concentrations of the test item tested.
Fisher’s exact binomial test with Bonferroni Correction (α = 0.05, one-sided) showed no significant difference concerning mortality of the adult test animals between the solvent control and all concentrations of the test item tested.
- Total mass of adults at beginning of test: mean total mass of each group (control, solvent control, test concentrations) between 398.4 and 411.3 mg fresh weight
- Changes in body weight of live adults (% of initial weight) at end of exposure period: see table under 'any other information'
- No. of offspring produced: see table under 'any other information'
- Morphological and behavioural abnormalities: In all test vessels the administered food was almost fully consumed by the worms in the first four weeks except of the highest treatment with 500 mg test item/kg soil (dw): After 21 days, less than one half of food was consumed by the worms in the replicate D of this concentration.
In the same replicate three worms were observed at the surface after 14 days and a dead worm was removed from the soil surface after 21 days.
No further unusual effects on behaviour or morphology of the adult earthworms were observed throughout all treatments.

Results with reference substance (positive control):

In a separate GLP study (ECT study number IRR2002), the EC50 value for reproduction was calculated by probit analysis as 268.0 mg boric acid/kg soil (dw) (95% confidence limits = 251.1 – 282.6 mg boric acid/kg soil (dw)). Annex E.4 of the guideline states that boric acid should reduce reproduction by 50% at about 400 to 600 mg boric acid/kg soil (dw). The estimated EC50 value was slightly lower than recommended by the guideline. However, since the calculated EC50 is lower by factor of 1.6 than the recommended value of about 400 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Reported statistics and error estimates:

Statistical evaluation:
- Mortality: Fisher’s exact binomial test with Bonferroni Correction (one-sided, α = 0.05).
- Biomass change: Data were checked for normality by Shapiro-Wilk’s test procedure and for homogeneity by Levene’s test. Treatment means were compared by Dunnett’s-test (α = 0.05, two-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
- Reproduction: Data were checked for normality by Shapiro-Wilk’s test procedure and for homogeneity by Levene’s test. Treatment means were compared by Dunnett’s test (α = 0.05, one-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
The statistical software package ToxRat Professional 3.3.0 was used for these calculations.

Adult mortality:

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

Concentration [mg test item/kg soil dw]	Number of dead adult worms Day 28	Mortality [%] Day 28
Control	0	0.0
Solvent Control	1	1.3
31.25	0	0.0
62.5	0	0.0
125	1	2.5
250	0	0.0
500	3	7.5

 

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 the concentrations). Data are given as mean values per replicate and mean change in percent of the initial weight change per replicate.

Concentration [mg test item/kg soil dw]	Biomass [mg fresh weight] ± SD		Mean change in % of the initial weight per replicate
	Day 0	Day 28	Day 28
Control	411.3 ± 38.3	605.8 ± 29.9	48.1
Solvent Control	403.1 ± 31.2	602.9 ± 19.6	50.5
31.25	407.9 ± 25.3	625.4 ± 45.9	53.5
62.5	404.0 ± 24.6	673.6 ±42.9	67.6
125	398.4 ± 34.6	640.4 ± 51.7	61.4
250	402.4 ± 29.0	671.6 ± 32.7	67.6
500	408.1 ± 21.0	663.4 ± 46.1	63.1

Statistical analysis showed no significant difference (Dunnett’s t-test; α = 0.05, two-sided) concerning biomass development of individual adults over 28 days between the solvent control and all concentrations of the test item tested.

 

Reproduction:

Reproduction after 56 days. Data are given as mean number of juveniles ± standard deviation and as % of the control (n = 8 for solvent control; n = 4 for treatment and deionised water control).

Concentration [mg test item/kg soil dw]	Number of Juveniles per Treatement (mean ± SD)	Number of Juveniles [% of Solvent Control]
Control	367.0 ± 23.8	108.0
Solvent Control	339.8 ± 82.3	-
31.25	337.5 ± 57.0	99.3
62.5	410.5 ± 40.6	120.8
125	376.8 ± 52,5	110.9
250	356.3 ± 66.0	104.9
500	329.3 ± 135.8	96.9

The mean numbers of juveniles were 367.0 and 339.8 in the control and the solvent control, respectively. The mean number of juveniles in the test item treatment groups ranged from 329.3 to 410.5.
Statistical analysis (Dunnett’s t-test; α = 0.05, one-sided) showed no significant difference concerning the number of juveniles between the solvent control and all concentrations of the test item tested.

Validity criteria fulfilled:

yes

Conclusions:

In a GLP compliant Reproduction toxicity study to the earthworm Eisenia fetida in artificial soil including 5% peat the substance N-Cyclohexyl-2-benzothiazolesulfenamide (CAS 95-33-0) was tested according OECD Guideline No. 222 for the Testing of Chemicals (adopted July 29, 2016). The studied test parameters include the mortality and biomass of adult specimen after 28 days and the reproduction after 56 days. Additionally, the morphology and behaviour of the adult and juvenile worms was observed. For the test parameters mortality, reproduction and biomass no effect up until the highest test item concentration (500 mg test item /kg soil dw) was determined. Hence, the NOEC and LOEC for all three parameters are ≥500 mg test item/kg soil dw and >500 mg test item/kg soil dw, respectively. The LC50 (mortality) and EC50 (reproduction) are also determined as >500 mg test item/kg soil dw.

Executive summary:

To investigate the effect of N-cyclohexylbenzothiazole-2-sulphenamide on terrestrial macroorganisms except arthropods, a GLP comliant study on earthworms (eisenia fetida) according to OECD technical Guideline No. 222 (adopted July 29, 2016) was conducted.

The purpose of this study was to determine a NOEC/LOEC and if possible, EC50 for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) using a standardised artificial soil. Additionally, a LC50 was estimated. The test item was mixed with the substrate.

 

As the test item was poorly soluble in water, the volatile organic solvent acetone was used as a vehicle.
The test item was dissolved in a volume of acetone sufficient to prepare a stock solution with a defined concentration of 17.5 mg/mL. This stock solution was used to produce the various dosage solutions of the test item. An appropriate volume of the stock and the dosage solutions were used to soak a 10 % quartz sand portion of the artificial soil, respectively. Through evaporation of the solvent by placing the application vessels under a fume hood, the quartz sand was coated by the test item.

 

The earthworms 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, respiratory and alimentary uptake.

The test item was applied once at the beginning of the test. The duration of the test (exposure of earthworms to the artificial soil containing the test item) was 56 days. The adult earthworms were removed from the substrate after 28 days.
The test item was tested in five concentrations with a spacing factor of 2. All tested concentrations were based on the test item. The following concentrations of the test item were used:

 

500 mg test item/kg soil (dw)
250 mg test item/kg soil (dw)
125 mg test item/kg soil (dw)
62.5 mg test item/kg soil (dw)
31.25 mg test item/kg soil (dw)

Four replicates per concentration, eight solvent control replicates and additionally four deionised water control replicates (ten worms per replicate) were tested.

 

For the test parameters mortality, reproduction and biomass no effect up until the highest test item concentration (500 mg test item /kg soil dw) was determined. The summarized results are listed below:

 

Mortality:

NOEC_mortality ≥ 500 mg test item/kg soil dw

LOEC_mortality > 500 mg test item/kg soil dw

LC₅₀ > 500 mg test item/kg soil dw

 

Reproduction:

NOEC_reproduction ≥ 500 mg test item/kg soil dw

LOEC_reproduction > 500 mg test item/kg soil dw

EC₅₀ > 500 mg test item/kg soil dw

 

Biomass:

NOEC_biomass ≥ 500 mg test item/kg soil dw

LOEC_biomass > 500 mg test item/kg soil dw

Description of key information

In a GLP compliant reproduction toxicity study to the earthworm Eisenia fetida in artificial soil including 5% peat the substance N-Cyclohexyl-2-benzothiazolesulfenamide (CAS 95-33-0) was tested according OECD Guideline No. 222 for the Testing of Chemicals (adopted July 29, 2016). The studied test parameters include the mortality and biomass of adult specimen after 28 days and the reproduction after 56 days. Additionally, the morphology and behaviour of the adult and juvenile worms were observed. For the test parameters mortality, reproduction and biomass no effect up to the highest test item concentration (500 mg test item /kg soil dw) was determined. Hence, the NOEC and LOEC for all three parameters are ≥500 mg test item/kg soil dw and >500 mg test item/kg soil dw, respectively. The LC50 (mortality) and EC50 (reproduction) are also determined as >500 mg test item/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

500 mg/kg soil dw

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

500 mg/kg soil dw