4-methylbenzothiazol-2-ylamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-01 - 2017-06-28 (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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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: 1.0, 3.2, 10, 32 and 100 mg/L (definitive test)
- 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 diluent (methanol:water (50:50 v/v)) 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. Samples containing algal cells were filtered through a plug of glass wool situated at the top of the cartridge. 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.

Test solutions

Vehicle:

no

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.
Based on this information a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Visual assessment of the saturated solutions after stirring showed no undissolved test item to be present. Whilst the results obtained from the filtered test samples were below the expected concentration of 100 mg/L, this was considered to be due to the measured concentrations exceeding the linear range. As such it was considered that the test item had fully dissolved in both the 24 and 48 hours preparations.
Based on this information the test item was prepared using a prolonged stir method of preparation at an initial loading rate of 100 mg/L. After stirring for a period of 24 hours, as a precautionary measure, any undissolved test item was to be removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded).
A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

range from pH 7.5 at 0 hours to pH 7.8 at 72 hours

Nominal and measured concentrations:

Nominal concentrations of 1.0, 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:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 9.8-14

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 3.7-5

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0-72h) = 1.4 mg/L; 95% confidence limits 1.2-1.5 mg/L
EyC50 (0-72h) = 0.6 mg/L; 95% confidence limits 0.52 - 0.69 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.5 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 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 (72h, growth rate) = 12 mg/L
EC50 (72h, yield) = 4.3 mg/L
NOEC (72h, growth rate & yield) = 2.3 mg/L
LOEC (72h, growth rate & yield) = 6.0 mg/L

Based on this result, the substance does not need to be classified as Aquatic Acute Cat. 1, as the ErC50 (72h) is > 1 mg/L. Further, the ErC50 (72h) is >10 and ≤100 mg/L and hence, the substance does has to be classified as Aquatic Chronic Cat. 3, as it is not rapidly degradable (criteria for substances for which adequate chronic toxicity data are not available). Due to the fact that algae were not the most sensitive species tested, the actual classification of the substance is based on the results obtained with daphnids.

Executive summary:

A study according to OECD 201 and EU method C.3 (GLP) was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata.

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, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by dissolving 1100 mg of test item in 11 liters of culture medium 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 stock solution of the test item. This stock solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.84 to 38 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on 0-Hour measured test concentrations only.

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

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth Rate	12	9.8 -14	2.3	6.0
Yield	4.3	3 .7 -5.0	2.3	6.0