N,N-dicyclohexylbenzothiazole-2-sulphenamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

- Dilution Water Source: Dilution water was prepared from tap water. The tap water was dechlorinated and treated by activated carbon. Before using the dilution water, aeration was fully carried out.
- Dimethylformamide was used as solvent. 100uL/L of dimethylformamide was contained in the each test soltion.
- Stock Solutions Preparations and Stability: 0.2mg of test chemical was dissolved in the 5,000mL of dilution water contained 500uL of dimethylformamide. Infrared absorption spectrum of the refrigerated test chemical was detected at the start and the end of the test, and both spectrums are not contradictory to each other. Solvent was prepared as 40mg/10mL of dimethylformamide diluted to 10 times by dimethylformamide.

Test organisms (species):

Oryzias latipes

Details on test organisms:

- Supplier: Test organisms were obtained from Tsunashima Fishing (5-18-1 Tsunashima-Nishi, Kohoku-ku, Yokohama City, Kanagawa Prefecture)
- Size (length and weight): 2.06 cm (1.80 - 2.31 cm) in length; 0.122 g (0.066 - 0.173 g) in weight
- Age: Not described
- Any pretreatment: Test organisms were acclimated for one month before testing. During acclimination, test fishes were fed with TETRAMINE equivalent to 2% of weight per day. These test organisms were not fed for 24 hours before the test started. The mortality of the test organisms for 7 days before testing was less than 5%.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total hardness (as CaCO3): = 68 mg/L

Test temperature:

23.5 - 24.0ºC

pH:

6.6 - 7.3

Dissolved oxygen:

6.3 - 8.7 mg/L

Nominal and measured concentrations:

- Nominal concentration: 0.04 mg/l
- The measured concentration of 0.04mg/L at start of test was 0.0368 mg/L and decreased to 0.0334 at the end of the test. Mean measured concentration was 0.0351 or 84% of nominal concentration.

Details on test conditions:

Exposure method : Semi-static system (replace the entire volume of test solution every Water surface covered with Teflon sheeting
Exposure period: 96 hours
Test water tank: 5.0 L/test vessel (The weight of the test fish should not exceed 1.0 g per 1.0 L of test solution)
Number of sequence: 1 test vessel/concentration group
Number of organisms : 10 fish / concentration group
Test water temperature: 24 ± 1 °C
Dissolved oxygen concentration: maintained above 60% of saturation concentration (no aeration)
pH: No pH adjustment of the test solution was made.
Lighting: Room light, 16 hours bright (below 1000 lux) / 8 hours dark
Feeding: None

Dilution water:
Dechlorinated water (tap water treated with activated carbon to remove residual chlorine, etc., and thoroughly aerated) was used as dilution water. At the time of use, it was confirmed that there was no residual chlorine. It was judged that the water quality was appropriate for fish breeding water.

Test concentration design:
Concentration in the main test: control group, solvent control group, 0.0400 mg/L (limit test of the highest concentration at which the test solution can be prepared).

Preparation of the test solution:
The dilution water for the preparation of the test solution was kept at 24 ± 1°C in a thermostatic bath prior to preparation.The stock solution of the test substance and the stock solution of the solvent were prepared according the following table:

Test substance stock solution
Amount of test substance taken 0.20 mg
Solvent (Dimethylformamide) 500 µL
Constant volume 5000mL
(Dilution water: OECD medium)
Test substance concentration 0.0400 mg/L
Solvent concentration 100 µL/L

In the control group, only diluted water was used, and in the solvent control group, the solvent (dimethylformamide) was added directly, and the one containing only the solvent (solvent concentration: 100 µL/L) was prepared. The condition (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.

Analysis of test solution:
At the start of exposure and 24 hours later (before water change), 3.5 mL of test solution was taken from each test vessel, mixed with an equal volume of acetonitrile, and analysed by HPLC. The concentration of the test substance in each test solution was determined from the ratio of the peak area of the standard solution.

Test method:
At the beginning of the exposure, the scale length and weight of 10 fish that had been standardized in the same tank as the test fish were measured, and it was confirmed that the scale length was 2.0 ± 0.5 cm.
After measuring the water temperature, dissolved oxygen concentration and pH of the test solution, 10 test fish were placed in the solution per test plot. Water temperature, dissolved oxygen concentration and pH were measured at least once daily during the exposure period (including before and after water change). No feeding was carried out during the exposure period.
The number of dead animals was recorded at 24, 48, 72 and 96 hours after exposure and any signs of toxicity or abnormalities observed were recorded.
The cases and definitions to be described in general are given below.
If dead individuals were found, they were removed as soon as possible to avoid deterioration of water quality.

*-Defined as a general case.
Death : No response to stimulation.
Abnormal breathing : Fish with different behaviour of their gills compared to the control fish.
Abnormal swimming : Swimming behaviour clearly different from that of the fish in the control group.
Slow movements, hypermobility, convulsions, inversions, etc. reversal, etc.
Inability to swim : Moving on the bottom or at the surface, but unable to swim in the water.
Includes rollover and asphyxia.

.

Reference substance (positive control):

yes

Remarks:

copper sulfate pentahydrate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.033 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

None of test organisms were killed and no abnormalities are detectedduring exposure period at control and 0.0334 mg/L.

As a result of the analysis of the test solution, the ratio of the measured value to the nominal value was 92% at the beginning of the exposure and 76% after 24 h, which is above ±20%. was used. The half-life in purified water was 30 hours (0.04 mg/L) and the decrease was mainly due to hydrolysis. The main reason for the decrease was considered to be hydrolysis.
The mortality rate in the control group was 0%, and that in the solvent control group was 0%, satisfying the test conditions. LC50 at 96 hours: >0.0334 mg/L (95% confidence limit: not calculated).
Maximum concentration of 0% mortality: >0.0334 mg/L
Minimum concentration of 100% mortality: >0.0334 mg/L
No unusual symptoms were observed during the exposure period in all test concentrations. The water temperature was 24±1°C in all test vessels, and the dissolved oxygen concentration was more than 60% of the saturated dissolved oxygen concentration (8.25 mg/L at 24.0°C) in all test plots, which met the test criteria. pH was within the appropriate range (6.0-8.5) for rearing Japanese medaka.
.

Results with reference substance (positive control):

LC50(96 hr) for a reference substance (copper sulfate pentahydrate) was 1.2 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

A limit test with a test concentration of 0.0334 mg/l was performed on Oryzas latipes and a 96-hour LC50 of >0.033 mg/L can be concluded.

Executive summary:

A limit test with a test concentration of 0.0334 mg/l (highest achievable test concentration) was performed on Oryzias latipes in a semi-static test system according to OECD 203 (MITI, 2001) and no toxic effect was observed; and hence a 96-hour LC50 of >0.033 mg/L can be concluded (MITI, 2001).

Description of key information

A limit test with a test concentration of 0.0334 mg/L was performed on Oryzias latipes in a semi-static test system according to OECD 203 (MITI, 2001) and no toxic effect was observed; and hence a 96-hour LC50 of >0.033 mg/L can be concluded (MITI, 2001).
As the hydrolysis products of DCBS, a 96-hour LC50 of 0.73 mg/L on Oncorhynchus mykiss was observed in a flow-through system for MBT (Monsanto, 1981) and 12 mg/L on Oryzias latipes was observed in a semi-static system for Dicyclohexylamine (MITI, 1998).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 0.033 mg/L