N-cyclohexylbenzothiazole-2-sulfenamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 28, 1997 to June 18, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

based on the age of the study minor deviations from the actual guideline might be present.

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

4 times during the exposure period (8 times in total before and after water change), that is, at the start of the test (0th), 7, 14 and 20 days. At the time of breeding and after each 24 days (before water change)

Vehicle:

yes

Remarks:

Hardened castor oil (HCO-40)

Details on test solutions:

Preparation of the solution
1) Due to the water solubility of the test substance, a test solution was prepared using hardened castor oil fish (HC0-40), which has a low separation from the test Daphnia.
2) A 28 mg/L solution of the test substance was added in diluted water to prepare the required amount of test solution at the nominal concentration.
3) A control group containing only diluted water was provided.
4) A dispersant control group having the maximum concentration (50 mg/L) of the dispersant used was provided.
5) Four test vessels were prepared for each concentration group, and 400 mL of the test solution was placed in each test vessel.
6) The state (appearance, etc.) of the test solution was observed, and it was confirmed that it was colorless and transparent and that there was no precipitation.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test used juvenile Daphnia magna (Daphnia magna) within the age of 24 hours. This species is obtained from the National Institute for Environmental Studies and is subcultured in our research department. In addition, as a result of an acute swimming inhibition test using a reference substance (potassium dichromate, special grade reagent), EiC50 for 48 hours. It was 0.42 mg/L

Daphnia breeding conditions:
- Breeding water: Dilution water
- Breeding density: 20-50 females/L breeding water (however, in the case of mature individuals, 25 females or less)
- Temperature: 20+1 °C
- Illumination: Room light, 16 hours light / 8 hours dark
- Feeding: Chlorella vulgaris
- Feeding amount: 0.1-0.2 mgC (organic carbon content/day/daphnia)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The total hardness was not measured by the tolidine method or the like when using diluted water.

Test temperature:

20.0 to 20.9 °C

pH:

7.2-8.0

Dissolved oxygen:

- 7.26 - 9.28 mg/L
- At 20.0 ° C saturated dissolved oxygen carbon: 8.84 mg/L

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

Test concentration (n): 0.18 mg/L, 0.35 mg/L, 0.70 mg/L and 1.40 mg/L (common ratio 2.0)

Details on test conditions:

I. Test conditions
- Test solution volume: 1 container 80 mL
- Number of stations: 4 vessels per 1 concentration group
- Number of organisms: 40 neonates/1 concentration group (10 per 1 vessel, 40 for 1 concentration group)
- Test water volume: 400 mL
- Test temperature: 20 ± 1 °C
- Lighting: 16 hours light (1200 lux or less9 /8 hours dark
- Feeding. 0.1-0.2 mgC (organic carbon content/day/daphnia)

II. Dilution water
The M4 prepared water described in the OECD Test Guidelines 202 Part II Draft (Shiratsuki 1995) was used. Therefore, the presence or absence of residual chlorine was not confirmed, and the total hardness was not measured by the tolidine method or the like when using diluted water. The pH of the diluted water was 7.9.

III. Test container, constant temperature bath, etc.
- Test vessel : 500 mLglass beaker
- Constant temperature bath : Constant temperature water tank and water circulation device (Yamato Scientific Coo Engraving Inn CL100)
- Coolant temperature gauge : pH meter built-in thermometer: pH meter (HORIBA, Ltd. Casta 21 LAB pH meter F-22)
- Dissolved oxygen meter : DO METE R (HORIBA, Ltd. OM-14)

IV Test concentration setting
As a result of conducting a preliminary test prior to this test, the 48-hour and half-swimming inhibition concentration (48hr-Eic50) was 0.79 mg/L. With reference to this result, a concentration ratio of 2.0 was nominal in 6 levels. Nominal concentration: 0.044 mg/L, 0.088 mg/L, 0.18 mg/L, 0.35 mg/L, 0.70 mg/L and 1.40 mg/L.

V. Observation of Daphnia:
- (Parent Daphnia) Daily observation and recording of life-and-death swimming status and the presence or absence of abnormalities in appearance were recorded. Dead individuals after counting were removed.
- (First brood) The number of surviving larvae, the presence or absence of dead larvae, the occurrence of aborted eggs, and dormant eggs were observed for each water change. The juveniles after counting were removed.

VI. Water quality measurement:
- Water temperature: All concentration groups (however, 1 test vessel each) were measured once a week before and after water change.
- Dissolved oxygen concentration (D.O.): Weekly for all concentration groups (however, 1 test vessel for each). It was measured before and after the water change.
- pH: The measurement was performed once a week before and after the water change in each section (however, each test vessel).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (special grade reagent)

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% confidence limit: 0.16 mg/L～0.24 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limit: 0.11 mg/L～0.14 mg/L

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

48-hours EiC50: 0.42 mg/L.

Reported statistics and error estimates:

- The median lethal concentration (LC50) on days 14 and 21 and its 95 by the Probit method (Yukms Statistical Library H Biological Test) using the number of deaths of parent and the number of test individuals (40) in the concentration group. % The confidence limit was calculated.
- The 50% reproductive inhibition concentration (ErC50) was calculated by Probit method (Yukms statistical library II bioassay)

Validity criteria fulfilled:

yes

Conclusions:

The reproductive inhibition of N-Cyclohexyl-2-benzothiazolylsulphenamide (95-33-0) to daphnia was investigated in a semi static GLP study following OECD 211. After 21 days of exposure, the median lethal concentration (LC50) of parent Daphnia per day was 0.20 mg/L and the NOEC was 0.058 mg/L. The above concentration is a value based on the time-weighted average value of the measured value.

Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 211 “Daphnia Magna Reproduction Test (proposed in April 1997) (1984)”. Immobilization and reproduction test for Daphnia magna in N-Cyclohexyl-2-benzothiazolylsulphenamide was performed under semi-static exposure conditions. After measuring the water temperature, dissolved oxygen concentration (DO), pH and total hardness of the test solution, the test Daphnia was added using a thick-tipped Hi-Petter, and the time point was defined as the start of exposure. At that time, the total amount of breeding water in the pipetta was set to be within 1% of the test solution volume. After that, Daphnia was transferred to a new test solution in a cocoon and bred until 21 days later. A healthy amount of food was fed daily during the exposure period. Daily observation and recording of life-and-death swimming status and the presence or absence of abnormalities in appearance were recorded. Dead individuals after counting were removed. The number of surviving larvae was counted every day from the first offspring, and the larvae after the number of words + numbers were removed. The date of birth of the first larva was recorded as the first birth. Warm-dissolved oxygen (DO), pH and total transport were measured for all concentration groups. After 21 days of exposure, the median lethal concentration (LC50) of parent Daphnia per day was 0.20 mg/L and the NOEC was 0.058 mg/L. The above concentration is a value based on the time-weighted average value of the measured value. In addition, as a result of an acute swimming inhibition test using a reference substance (potassium dichromate, special grade reagent), EiC50 for 48 hours. It was 0.42 mg/L. This toxicity study is classified as acceptable and satisfies the guideline requirements for the chronic daphnia toxicity study.

Description of key information

The chronic toxicity of CBS to aquatic invertebrates (Dapnia magna) was tested according to OECD TG 211 "Daphnia magna Reproduction test". After 21 days of exposure an EC50 of 0.12 mg/L and a NOEC of 0.058 mg/L were obtained (MOE Japan, 1997). The effect concentrations in the key study (MOE Japan, 1997) are based on the time weighted average, as CBS degrades in the test system. It was determined that CBS is still present between 39% and 45.5% of the nominal after 24 hours. The study was conducted in a semi static design with test solution renewals after 24h.
No data are available regarding the hydrolysis products from CBS, such as BT and BTon, but some are available from other relevant metabolites of CBS such as MBT. The chronic toxicity of MBT to Daphnia magna was investigated in a flow-through system (CMA, 1989). The NOEC-21d and EC50 -21d was reported as 0.24 mg/l (measured) and >0.47 mg/l (measured), respectively.
Regarding the data summarized here, the long-term toxicities of the described metabolites of CBS to aquatic invertebrate are lower than the one of CBS itself.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.058 mg/L

Additional information

A hydrolysis half-life time of CBS of 13.4 h was observed in deionized water at pH 7.0 and at 20°C (Currenta 2022). The effect concentrations in the key study (MOE Japan, 1997) are based on the time weighted average, as CBS degrades in the test system. It was determined that CBS is still present between 39% and 45.5% of the nominal after 24 hours. The study was conducted in a semi static design with test solution renewals after 24h. The reported effective concentrations are lower than water solubility of CBS (i.e. 0.32 mg/l).