N-tert-butylbenzothiazole-2-sulphenamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-01-16 - 1997-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study. also cited in OECD SIDS.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: 96.8 %, Batch no. 612040

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test materials were stored in a refrigerator in our laboratory.
The infrared absorption spectra of the available test substance were measured and confirmed to be consistent with the structure of the test substance. At the end of the test, the infrared absorption spectrum was also measured and compared with the spectrum measured before the start of the test. As a result, there was no change in the spectrum, and the test substance was judged to have been stable during storage.

Analytical monitoring:

yes

Details on sampling:

At the beginning of the test and after 24 hours, 0.75 mL of the test solution was collected from each test tank, and 0.75 mL of acetonitrile was added to the solution and mixed well, which was used as the analysis sample. For the analysis of the test solution, a standard solution was measured for each sample measurement, and quantified from the peak area ratio.

Vehicle:

yes

Remarks:

Vehicle, solvent: HCO-30 and DMF

Details on test solutions:

To 100 mg of the test substance, 200 mg of dimethylformamide and 2300 mg of dispersing agent (HCO-30) were added, mixed and dissolved, and then the volume was fixed in 1000 mL of pure water to make a stock solution of the test substance (test substance concentration: 100 mg/L). A stock solution of solvent was prepared to keep the concentration of the solvent constant in all the concentration groups. For the stock solution of the additive, 200 mg of "methylformiate" and 2300 mg of dispersing agent (HCO-30) were mixed well, and then the volume was fixed at 1000 mL with pure water. For the test solution, the following amounts of the test substance and the solvent solution were taken and made up to 5.0 volumes with diluted water.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Size: 1.59-2.30 cm
Weight: 0.059-0.217 g

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

The hardness of the diluted water was 61 mg/L (CaCO3 equivalent).

Test temperature:

24°C +/- 1°C

pH:

7.2 - 7.9

Dissolved oxygen:

5.0 - 8.3 mg/l

Nominal and measured concentrations:

Nominal concentrations: 0.25, 0.5, 1.0, 2.0, 4.0
Measured concentrations 0 hours: 0.223, 0.465, 0.909, 2.03, 3.99
Measured concentrations 24 hours: 0.139, 0.24, 0.295, 0.351, ---*
Geometric Mean during 24 hours: 0.176, 0.334, 0.518, 0.844, 3.99

all concentrations are given in mg/L
*no measurement was made because all fish were dead at this observation time

Details on test conditions:

Concentrations: Control, vehicle control, 0.25, 0.5, 1.0, 2.0, 4.0 mg/L. Total amount of test water was replaced every 24 hours. Test was performed in 5 L test vessel per concentration with 10 fish per vessel. Irratiation was room light, 16 hours light and 8 hours dark.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.518 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

3.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The measured concentrations compared to the set-up concentrations exceeded +/-20 %, hence values were based on the geometric mean of the measured concentrations.

Results with reference substance (positive control):

The result of the acute toxicity test for 96 hours using the reference substance Copper(II) sulfate was LC50 = 0.44 mg/L and 0.80 mg/L.

Sublethal observations / clinical signs:

Abnormal swimming was observed at test concentrations over 0.518 mg/L (set-up concentration 1.00 mg/L). No abnormal symptoms were seen in the control groups during the exposure period.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity to fish ( Orizias latipes) of TBBS was determined after 96 hours at a LC50 of 1.38 mg/L under semi-static conditions.

Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 203 “Fish Toxicity Test" (1992). For N-tert-butyl-benzothiazole-2-sulphenamide, an acute toxicity test against medaka (Oryzias latipes) was performed to determine the median lethal concentration (LC50) for 96 hours under semi-static exposure conditions. At the start of exposure, the weight and scale length of 10 fish acclimatized in the same tank as the test fish were measured. No feeding was done during the dew. After measuring the pH, dissolved oxygen concentration, and water temperature of the test solution in each test container, 10 fish under test were placed in the test container per concentration group. The pH, dissolved oxygen concentration and water temperature were measured at the beginning of the test and before and after water change every 24 hours. No feeding was conducted during the exposure period. Deaths were recorded 24, 48, 72 and 96 hours after the start of exposure and any signs or abnormalities of toxicity observed were recorded. When dead individuals were found, they were promptly removed to prevent deterioration of water quality. As a result of the preliminary test, the maximum 0% mortality concentration was 0.500 mg/L, and the minimum 100% mortality concentration was 5.00 mg/L or more. With reference to this result, in this study, the concentration ratio was 2.0. Five test concentrations of 0.25 mg/L, 0.5 mg/L, 1.00 mg/L, 2.00 mg/L and 4.00 mg/L were prepared. After 96 hours exposure, the median lethal concentration (LC50) based on measured concentration was 1.38 mg/L or more. The mortality rate for 7 days before the start of exposure was less than 5.0%. In addition, the result of the acute toxicity test using the reference substance (copper sulfate (II) pentahydrate, reagent special grade) of the test fish was 0.44 mg/L and 0.80 for 96 hours LC50. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute fish toxicity study.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

also cited in OECD SIDS.

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

Purity 96.8 %, Batch No. 612040

The test materials were stored in a refrigerator in our laboratory.
The infrared absorption spectra of the available test substance were measured and confirmed to be consistent with the structure of the test substance. At the end of the test, the infrared absorption spectrum was also measured and compared with the spectrum measured before the start of the test. As a result, there was no change in the spectrum, and the test substance was judged to have been stable during storage.

Analytical monitoring:

yes

Details on sampling:

0.75 mL of test solution was taken from each test tank at the beginning (day 0), 7 days, and 14 days, and 0.75 mL of acetonitrile was added to the test solution and well mixed as the analysis sample. For the analysis of the test solution, a standard solution was measured for each sample measurement and quantified from the peak-to-peak ratio.

Vehicle:

yes

Remarks:

Hardened castor oil (HCO-30) and DMF.

Details on test solutions:

To 600 mg of the test substance, 1.2 g of dimethylformamide was added and dissolved. To this solution, 42.0 g of dispersing agent (HCO-30) was added and mixed well, and then the volume was fixed at 1000 mL with pure water to make a stock solution of the test substance (test substance concentration: 600 mg/L). (Test substance concentration: 600 mg/L). 3.6 g of dimethylformamide and 126.0 g of dispersing agent (HCO-30) were added and mixed well, and then the volume was fixed at 3000 mL with pure water to make the stock solution of the solvent. The stock solution of the test substance was made by taking and mixing the following amounts of the stock solution of the solvent. The stock solution was mixed with the following amount of the stock solution of the test substance. 1000 mL of the stock solution of the solvent was used for the solvent control group.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Test species

1) Common name : Medeka
2) Scientific name : Oryzias latipes
3) Body length : 2.10 cm (1.72～2.48 cm), n = 10
4) Weight : 0.148 g (0.072～0.222 g), n = 10
5) Source of supply : Sankyo Suisan Corporation (1-1 Ichiyadacho, Shinjuku-ku, Tokyo)
6) Date of aquisition : 1996-11-01
7) Period : 1996-11-1 – 1997-03-05
8) Result of the acute toxicity test for 96 hours
using the reference substance* (LC50): 0.44 mg/L and 0.80 mg/L
*: Copper sulfate (II), Wako Pure Chemical Industries, Ltd.

Remark:
They were reared and acclimatized under the same conditions (water quality, temperature, etc.) as the test conditions. Commercial tetramine⑳ was fed. Feeding was not performed for 24 hours prior to the start of exposure. Healthy and normal individuals were used for the tests. The mortality rate during the 7 days before the start of exposure was less than 5%.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Hardness:

The hardness of the diluted water was 61 mg/L (CaCO3 equivalent)

Test temperature:

24°C +/- 2°C

pH:

7.4 - 7.9

Dissolved oxygen:

5.5 - 8.2 mg/L

Nominal and measured concentrations:

nominal concentrations: 0.049, 0.150, 0.440, and 1.30 mg/L

mean measured concentration: 0.044, 0.127, 0.427, 1.38 mg/L

Details on test conditions:

Test method
At the beginning of the exposure, the weight and scale length of 10 fish acclimated in the same tank as the test fish were measured, and the scale length was confirmed to be 2.0 ± 0.5 cm.
The water temperature, dissolved oxygen concentration, and pH of the test solution in each test tank were measured, and then 10 test fish were introduced into the test tank per concentration group.
Water temperature, dissolved oxygen concentration, and pH of the test solution in each test tank were measured three times a week after the start of exposure.
During the exposure period, the animals were fed about 2% of their weight per day. When the number of individuals decreased due to mortality, the amount of food was reduced accordingly, but not due to a decrease in food intake.
The number of deaths was recorded three times a week after the start of 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 in quality. The weight of the dead and surviving fish at the end of exposure was recorded. The weight and scale length of dead and surviving fish at the end of exposure were measured and recorded.

*-Defined as a general case.
Death : No response when stimulated.

Abnormal breathing : Fish with different gill movements compared to fish in the control group.

Abnormal swimming : Those that clearly swam differently from the fish in the control area. Slow movement, irritability, convulsion, inversion, etc.

Inability to swim : Those who are moving at the bottom or on the surface of the water but are unable to swim in the water. Including rollover and asphyxia.


Test conditions

1) Exposure method : Flow-through type (water surface was covered with teflon sheet to prevent volatilization of the test substance from water using a continuous dilution device with a metering pump)
　　　
2) Exposure period : 14 days

3) Amount of test solution : Approx. 32 L/day (22.55 mL/min)

4) Number of sequence : 1 test vessel/concentration group
　　　
5) Number of organisms : 10 fish / concentration group

6) Aeration : None

7) Test water temperature : 24 ± 2 °C
　　　
8) Lighting : 16 hours light / 8 hours dark

9) Feeding : Commercial tetramine ⑪ was given daily at 2% of fish weight.　　　


Dilution water

Dechlorinated water (Yokohama city tap water with residual chlorine etc. removed by activated charcoal and then aerated) was used. The hardness of the diluted water was 61 mg/L (CaCO3 equivalent), and the pH was 7.8.


Test equipment and instruments, etc.

Water-based test equipment: A test water tank (5.0 L glass beaker, 180 mm φ × 270 mm) with a 12 cm glass siphon for drainage was installed in a thermostatic bath at 24°C. The diluted water was supplied at a flow rate of 22.5 mL/min and the stock solution at a flow rate of 0.05 mL/min using the metering pump shown below. Diluted water was supplied at a flow rate of 22.5 mL/min and stock solution at a flow rate of 0.05 mL/min using the metering pump shown below. The total flow rate was 22.5 mL/min, and the water change rate of the test tank was about 6 times/day.

1) Dilution water pump
Motor (drive unit) : MASTERFLEX by Cole-Parmer 7543-30
Supplier : Cole-Parmer lnSt．Co．MASTERFLEX 7016-20
Motor (unit) : Cole-Parmer lnst．Co．MASTERFLEX PharMed 6485-16

2) Standard solution pump Made in Japan by Saba Denshi Kagaku Chemica Organization SP-D-2501(S)

3) Thermostatic tank : Vinyl chloride water tank (thermostatic apparatus, made by T.I.C.)(Model CL-80F in "Cu")

4) Water thermometer : Yokogawa Denki 2455 02

5) Dissolved oxygen meter : Electrochemical measuring instrument DOL-10

6) pH meter : Toa Radio HM-40V type

Test concentration design
The concentration range was determined based on the results of the acute toxicity test. Since the 96-hour-LC50 was 1.38 mg/L, four concentration groups were established: 0.049, 0.150, 0.440, and 1.30 mg/L
(common ratio: 3.0).

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

LC0

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

1.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

14 d

Dose descriptor:

LC100

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The ratios of the concentrations measured to the set-up concentrations were within +/- 20 % in all the test doses. All test concentrations were therefore based on nominal test substance concentrations.

Validity criteria fulfilled:

not applicable

Conclusions:

The prolonged toxicity to fish (Oryzias Latipes) was determined at a LC50 of 1.02 mg/L after 14 days. A LOEL after 14 days of 0.44 mg/L was calculated.

Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 204 “Fish Toxicity Test" (1992). For N-tert-butylbenzothiazole-2-sulphenamide, a prolonged toxicity test on medaka (Oryzias latipes) is conducted for 14 days to determine the threshold value and maximum no action concentration (NOEC) of lethality and other observed effects on medaka. At the start of exposure, the weight and scale length of each of the 10 fish acclimatized 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 pH, dissolved oxygen concentration, and water temperature of the test solution in each test tank, 10 fish to be tested were put into the test tank per concentration group. After the start of exposure, the water temperature, pH, and dissolved oxygen concentration of the test solution in each test tank were measured three times a week. During the exposure period. Food was given 2% of the individual weight per fish. If the population decreased due to death, the amount of feed was reduced accordingly. Daily deaths were recorded and observed signs of toxicity or abnormalities were recorded after the start of exposure. When dead individuals were found, they were promptly removed to prevent deterioration of water quality. At the end of exposure, the weight and scale length of all surviving fish were measured and recorded. After 14 days of exposure, the median lethal concentration (LC50) was 1.02 mg/L and the NOEC was 0.15 mg/L. The above concentrations are all based on nominal concentrations. This toxicity study is classified as acceptable and satisfies the guideline requirements for the prolonged fish toxicity study.

Description of key information

Following the OECD Guideline 203 a 96h-LC50 of 1.38 mg/L was observed on Oryzias latipes (National Institute of Technology and Evaluation, 1996).
According to the OECD Guideline 204 a 14d-LC50 of 1.02 mg/L was observed on Oryzias latipes (National Institute of Technology and Evaluation, 1996). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.38 mg/L

Additional information

A hydrolysis half-life time of TBBS of 7.76 – 9.53 h was observed in deionised and environmental water buffered at pH 7.0. Benzothiazole-2-thiole (MBT) was determined as hydrolysis product. However, the effect concentrations of TBBS to Oryzias latipes are chosen as key studies, since the observed effects are most probably a combined effect from both TBBS and MBT. The effect concentration on fish reported in the study for MBT (0.73 mg/L) is less sensitive than the reported value for TBBS on algae (0.071 mg/L), and hence the value for MBT is not used in further risk assessment for the PNEC estimation.