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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 7, 1997 to May 9, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Remarks:
HPLC
Details on sampling:
Approximately 1.5 mL of test water was collected from each test tank at the start of the test and 24 hours later and analyzed by HPLC. In the analysis of the test water, the standard solution (concentrations 0.10, 1.00,
4.00 mg/L) was measured for each sample measurement, and the calibration coefficient was obtained to measure the test substance concentration.
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 35 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 (100 mg/L) of the dispersant used was provided.
5) Four test vessels were prepared for each concentration group, and 100 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 0.87 mg/L.

Female adults of macroscopically healthy and sufficiently large size with juveniles were selected from those in the passage and transferred to a separately prepared beaker, and the juveniles produced the next day were separated into separate beakers. This juvenile was used as the parent of the test Daphnia and was acclimated for 28 days under the following conditions. After maturity and larval birth, the larvae were removed at least twice a week. The larvae were removed the day before the start of exposure, and the larvae produced the next day (within 24 hours) were used for the test. There were few dead individuals, and no dormant eggs or males were produced. Female adults of macroscopically healthy and sufficiently large size with juveniles were selected from those in the passage and transferred to a separately prepared beaker, and the juveniles produced the next day were separated into separate beakers. This juvenile was used as the parent of the test Daphnia and was acclimated for 28 days under the following conditions. After maturity and larval birth, the larvae were removed at least twice a week. The larvae were removed the day before the start of exposure, and the larvae produced the next day (within 24 hours) were used for the test. There were few dead individuals, and no dormant eggs or males were produced.

- 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: Chlorella vulgaris was given per Daphnia at a rate of 0.1~0.2 mgC (organic carbon content) / day
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
No data
Test temperature:
20.5 °C to 20.9° C
pH:
7.6 to 7.9
Dissolved oxygen:
- 7.75 mg/L to 8.58 mg/
- saturated dissolved oxygen concentration at 20.0 °C: 8.84 mg/L
Salinity:
n.a.
Conductivity:
No data
Nominal and measured concentrations:
Test concentration (n): 0.44 mg/L, 0.88 mg/L, 1.75 mg/L and 3.50 mg/L (common ratio 2.0)
Details on test conditions:
I. Test conditions
- Number of stations: 4 vessels per 1 concentration group
- Number of organisms: 20 neonates/1 concentration group (5 neonates per 1 vessel, 20 neonates for 1 concentration group)
- Test water volume: 100 mL
- Test water temperature: 20+1 °C
- Illumination: Room light, 16 hours light / 8 hours dark
- Feeding: No feeding

II. Dilution water
The M4 prepared water described in the OECD Test Guidelines 202 Part II Draft (August 1995) was used. Therefore, the presence or absence of residual chlorine and the total hardness were not measured by the tolidine method when using diluted water. The pH of the diluted water was 7.8.

III. Test container, constant temperature bath, etc.
- Test container: 100 m L glass beaker
- Constant temperature bath: Constant temperature water tank and water circulation device (Yamato Scientific Coo Engraving Inn CL1OO)
- Coolant temperature gauge: pH meter built-in thermometer (HORIBA, Ltd. Castany Takeshi B pH meter F-22)
- Dissolved oxygen meter: HORIBA-DO Meter 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 0.47 mg/L. With reference to this result, the concentration ratio is 2.0. Six levels of concentration were prepared. (Nominal concentration: 0.11 mg/L, 0.22 mg/L, 0.44 mg/L, 0.88 mg/L, 1.75 mg/L and 3.50 mg/L)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (special grade reagent)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-confidence limit: 0.64 mg/L~0.98 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.251 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
2.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
The result of the acute swimming inhibition test using the reference substance (potassium dichromate, special grade reagent) was 0.42 mg/L for EiC50 for 48 hours.
Validity criteria fulfilled:
yes
Conclusions:
The infuence of N-Cyclohexyl-2 benzothiazolylsulphenamide (95-33-0) on the immobility to daphnia was investigated in a semi-static GLP study following OECD 202. After 48 hours of exposure the EC50 value was 0.79 mg/L and the NOEC 0.251 mg/L by comparison of growth rate. The above concentrations are all based on the geometric mean of the measured values.
Executive summary:

This test is based on the OECD Guidelines for Testing of Chemicals No. It was conducted in accordance with 202 “ Daphnia, Swimming Inhibition Test and Reproductive Test” (1984)”. Acute immobilization test for Daphnia magna in N-Cyclohexyl-2 benzothiazolylsulphenamide is performed. 50% immobilization inhibitory concentration (Eic50) and maximum no-effect concentration (NOECi) at 24 and 48 hours are determined


under semi-static exposure conditions. After measuring the temperature of the test solution, the residual oxygen concentration (DO), and the pH, the test Daphnia was added, and the time point was set as the start of exposure. The test Daphnia was put in using a pipettor with a thick tip. At that time, the total amount of breeding water for Pipetta Ichihaku should be within 1% of the test water volume. It was bred for up to 48 hours. The number of Daphnia swimming inhibitors was observed 24 and 48 hours after the start of exposure. If the test vessel could not be swam for 15 seconds after being gently moved, it was considered to be inhibited swimming (however, swimming means swimming in water, and those crawling on the bottom of the water were included in the inhibition. If swimming in water even once every 15 seconds, even if it was not normal swimming, it was not included in the inhibition). Water temperature, DO, and pH were measured at the start of exposure and 48 hours after the test solution in all concentration groups (however, one test container each). As a result of conducting a preliminary test prior to this test, the 48-hour and half-swimming inhibition concentration (48hr-Eic50) was 0.47 mg/L. As a result of conducting a preliminary test prior to this test, the 48-hour and half-swimming inhibition concentration (48hr-Eic50) was 0.47 mg/L. With reference to this result, the concentration ratio is 2.0. Six levels of concentration were prepared. (Nominal concentration: 0.11 mg/L, 0.22 mg/L, 0.44 mg/L, 0.88 mg/L, 1.75 mg/L and 3.50 mg/L). After 48 hours of exposure the EC50 value was 0.79 mg/L and the NOEC 0.251 mg/L by comparison of growth rate. The above concentrations are all based on the geometric mean of the measured values. 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 acute daphnia toxicity study.

Description of key information

The acute toxicity of CBS to aquatic invertebrates (Dapnia magna) was tested according to OECD TG 202 "Daphnia sp. Acute Immobilisation test". After 48 hours of exposure an EC50 of 0.79 mg/L was obtained (MOE Japan, 1997). The effect concentrations in the key study (MOE Japan, 1997) are based on the geometric mean, as CBS degrades in the test system. It was determined that CBS is still present between 34.1% and 42.8% of the nominal after 24 hours. The study was conducted in a semi static design with test solution renewals after 24h.
CBS is rapidly degraded by hydrolysis and the main degradation products appearing during the time frame of acute tests are benzothiazole (BT) and benzothiazolone (BTon). The acute toxicity of BT to Daphnia magna was tested according to a guideline of U.S. Environmental Protection Agency (1975). The LC50 of 20 mg/l (nominal) and NOEC of 5.6 mg/l (nominal) were obtained (Analytical Bio-Chemistry Laboratories, Inc.,1978). According to OECD TG 202, the acute toxicity of BTon to Daphnia magna was determined to be a 48h-EC50 of 16.1mg/l (nominal). The nominal concentration was confirmed analytically (AKZO NOBEL, 2004).
There are some other benzothiazole derivatives as metabolites from CBS are considered as relevant for environmental exposure and risk assessment, such as MeBT, MBT, MBTS and MeSBT. The acute toxicities of MeBT and MeSBT to Daphnia magna were tested according to OECD TG 202 and the 48h- EC50 of 29.8 mg/l and 8.5mg/l (nominal) were determined, respectively, which were analytically confirmed (AKZO NOBEL, 2004). A 48h-LC50 of 4.1 mg/l of MBT to Daphnia magna was reported (Monsanto, 1979) as a nominal concentration with the application of acetone. For MBTS no acute toxic effect was observed to Daphnia magna up to its water solubility (Bayer AG, 1993).
Regarding the data summarized here, the described metabolites of CBS are less toxic to aquatic invertebrates than CBS itself.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
0.79 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 EC50 is higher than the water solubility of CBS (i.e. 0.32 mg/l). To achieve higher test concentrations a vehicle (hardened castor oil HCO-40) used in the study.


 


The LC50 reported in Monsanto 48 h acute Daphnia test is much higher than water solubility of CBS (i.e. 0.32 mg/l) with the help of co-solvent of acetone. Therefore, in 48 h of test CBS is expected to have been hydrolyzed and further dissolved in water and so on. Unfortunately this is not analytically confirmed. The observed effect (EC50 = 18 mg/l) is hence also a combined effect from both CBS and its hydrolysis products.