4-methylbenzothiazol-2-ylamine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-17 - 2017-06-29 (Experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

4-methylbenzothiazol-2-ylamine

EC Number:

216-028-3

EC Name:

4-methylbenzothiazol-2-ylamine

Cas Number:

1477-42-5

Molecular formula:

C8H8N2S

IUPAC Name:

4-methyl-1,3-benzothiazol-2-amine

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 3.2, 10, 32 and 100 mg/L (nominal)
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis
- Preparation of Calibration Standards:
The test item (nominal 100 mg) was dissolved in methanol (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. This stock solution was further diluted with diluent (methanol:water (50:50 v/v)) to obtain a nominal 100 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 100 mg/L. These duplicate calibration standards were used to determine the recovery and test sample concentrations.
- Preparation of Linearity Standards:
The test item (nominal 100 mg) was dissolved in methanol (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. Defined volumes of this stock solution were diluted with diluent1 to obtain standards in the range of 1 to 250 mg/L. A second standard was similarly prepared at a nominal concentration of 100 mg/L. These standards were used to evaluate the linearity of the analytical system.
- Preparation of Test Samples:
The test samples were thawed with the aid of a waterbath. The test item was extracted from the test samples using a solid phase extraction cartridge (Strata Phenyl, 500 mg/3 mL). The cartridge was pre-conditioned with 10 mL of methanol and 10 mL of water. The samples (200 mL) were drawn through the cartridge under reduced pressure. Subsequently, the cartridge was eluted with 5 mL of methanol into a 10 mL volumetric flask. The solution was made up to the mark with water.
- Preparation of Accuracy and Precision Samples:
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in methanol to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1 mg/L. Five replicates at this concentration level were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that the test item was readily soluble in deionized reverse osmosis water with the aid of prolonged stirring.
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After stirring, as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 32, 10, 3.2 and 1.0 mg/L. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Reconstituted water (ISO medium) used for both the range-finding and definitive test

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

22 °C

pH:

7.8 - 8.0

Dissolved oxygen:

8.3 - 9.1 mg O2/L

Nominal and measured concentrations:

Nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

see "Any other information on materials and methods incl. tables"

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Exposure conditions for the positive control were similar to those in the definitive test, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

EC50 (24h) = 1.3 mg/L, NOEC (24h) = 1.00 mg/L, LOEC (24h) = 1.8 mg/L
EC50 (48h) = 1.2 mg/L, NOEC (48h) = 0.56 mg/L, LOEC (48h) = 1.0 mg/L
The No Observed Effect Concentration is based upon equal to or less than 10% immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 202 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive controls gave the appropriate response. Hence, the results can be considered as reliable. Results:
EC50 (48h) = 4.2 mg/L
NOEC (48h) = 0.66 mg/L
LOEC (48h) = 2.3 mg/L

Based on this result, the substance does not need to be classified as Aquatic Acute Cat. 1, as the EC50 (48h) is > 1 mg/L. Further, the EC50 (72h) is >1 and ≤10 mg/L and hence, the substance does has to be classified as Aquatic Chronic Cat. 2, as it is not rapidly degradable (criteria for substances for which adequate chronic toxicity data are not available).

Executive summary:

A study according to OECD 202 and EU method C.2 (GLP) was performed to assess the acute toxicity of the test item to Daphnia magna.  

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.  A preliminary media preparation trial indicated that the test item was readily soluble in deionized reverse osmosis water with the aid of prolonged stirring.

Following a preliminary range-finding test and initial experiment, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 22 °C under static test conditions.  The test item solutions were prepared by dissolving 1100 mg of test item in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours.  After stirring, as a precautionary measure, any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter)  to produce a 100 mg/L solution of the test item.  This solution was then further diluted as necessary, to provide the remaining test groups.

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.66 to 34 mg/L.  There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-Hour measured test concentrations only.

Exposure of Daphnia magna to the test item gave the following results based on the 0-Hour measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	4.2	3.2	-	5.5	0.66	2.3