S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP. Justification for Klimisch 2: see remarks.

Remarks:

Guideline study with GLP. Justification for Klimisch 2: see remarks. The study is well performed, except for using a loading rate of 111 mg of the insoluble test substance and the resulting water accommodated fraction (WAF), and not a concentration at the solubility limit. Thereby mainly the enriched impurities of the substance were tested.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The actual concentration of the test substance was determined at the start of the incubation period in aliquots of the filtered WAF, of the blank containing test substance, but no algae, and of one replicate of each of the test cultures. One sample was taken of each of the above listed aliquots and analysed in duplicate on the same day by HPLC. As all actual test substance concentrations were below the limits of quantitation and of detection, no analysis was performed at the end of the incubation period.

Vehicle:

no

Details on test solutions:

The test substance was practically insoluble in culture media (nutrient medium), therefore an aqueous extract from a "loading rate" of 111.4 mg substance per L nutrient medium was prepared by ultrasonication of the test substance in medium and subsequent filtration (cellulose acetate filter, 0.2 µm). A water accommodated fraction, WAF, was obtained.
The nutrient medium consisted of sterile deionised water and sterile concentrated nutrient medium 9+1 v/v. The conductivity of the deionised water used was below 5 "µScm-1.
The WAF was used as the highest test substance concentration. Aliquots of the WAF were further diluted with nutrient medium to obtain lower concentrations. The preparations were made freshly before the start of the exposure.
One negative control culture (reference substance: nutrient medium) was set up
concurrently.

Test organisms (species):

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

Details on test organisms:

Selenastrum capricornutum ATCC (American Type Culture Collection) 22662, obtained directly from ATCC, Rockville, Maryland 20852, USA.
Deep frozen algae were thawed and cultivated as described by the supplier in 250 mL conical flasks (Erlenmeyer) each containing 100 ml medium for stock culture at a temperature of 23 ± 2 °C under permanent light with an intensity of at least 6000 lux. In about weekly intervals 1 mL of the stock culture was diluted 100-fold with nutrient medium for precultivation and incubation was continued. Test precultures were prepared by incubating a new stock culture for three days. At the start of the experiment the cell density was determined and adjusted to about 10E5 algae/mL by diluting with nutrient medium for precultivation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None.

Hardness:

Water hardness corresponds to the standard test medium.

Test temperature:

22 +- 1 °C.

pH:

The pH of the test cultures was determined at the start of the incubation period and after 72 h.
The test was performed without adjustment of the pH of the test substance cultures.

Dissolved oxygen:

n.a.

Salinity:

N.a.

Nominal and measured concentrations:

Nominal and actual: Starting point was a WAF from loading 111 mg of the test substance to 1 L medium. This WAF was diluted 4 times by a factor of 2, to obtain 5 concentrations.

Details on test conditions:

Sterile nutrient medium was used for the negative control cultures.
The test culture and the blank were set up in 250 ml conical flasks. The total culture volume was 100 ml in each case. The complete cultures each consisted of 10 ml inoculum (10E5 algae/ml) and 90 ml of the test substance stock solution or of the nutrient medium (control).
The blank contained 10 ml of nutrient medium (instead of the algal inoculum) and 90 ml of the undiluted WAF.
There were three replicates for each test and control culture.
Incubated for three days in permanent light with an intensity of at least 6000 lux (wavelength 400-700 nm) while shaking.

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: EbC50 is between 1/4WAF and 1/2WAF

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: ErC50 is between 1/2WAF and WAF

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: NOEC is the 1/2WAF

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC is the 1/4WAF

Details on results:

Growth inhibition:
Compared to the negative controls, algal growth was inhibited by 89.8 % based on the area under the growth curves or by 57.6 % based on the average growth rates at the highest tested concentration and it was slightly enhanced by 23.2% or 4.3% at the lowest one.

Actual test substance concentration: All concentrations analysed as the pure substance T15-AE were below the limit of quantitation of the method used (0.0014 mg/L) and even below the limit of detection (0.0004 mg/L) at the start of the incubation period. Therefore no chemical analysis was performed at the end of the incubation period. However, the actual test substance concentration of the WAF, determined immediately after its preparation, was 0.123 mg/L. More than three hours passed between the end of the WAF preparation and the submission of test culture samples for analysis, as the test cultures were set up in the meantime. In a separate experiment it was shown that an actual test substance concentration of 0.088 mg/L declined to below the limits of quantitation and detection within three hours.

pH of the test cultures and of the controls: The test substance only slightly altered the pH of the test media. The pH was 7.8 or 7.9 at the start of the incubation in the test cultures and it was 7.7 in the control cultures. After 72 hours of incubation the pH was 8.1 or 8.2 in the test cultures and it was 8.2 in the control cultures.

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

n.a.

Validity of the test: The increase in cell density in the control cultures during the 72 hours incubation period was more than 16-fold, as recommended in the EC-guideline. It was about 111-fold, corresponding to about 6.8 generations and showing suitable growth conditions for the algae.

Validity criteria fulfilled:

yes

Conclusions:

NOEC(0-72h) is 1/2WAF, based on the area under the growth curves
NOEC(0-72h) is 1/4WAF, based on the average growth rate

EbC5072h is between 1/4WAF and 1/2WAF.
ErC5072h is between 1/2WAF and WAF.

Executive summary:

A Selenastrum capricornutum growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of the substance on the growth of a unicellular green algal species. As the test substance was practically insoluble in culture media (nutrient medium), an aqueous extract from a loading rate of 111 mg T15-AE per L nutrient medium was prepared. A water accommodated fraction, WAF, was obtained from the dissolved part and four dilutions of this extract were tested against one negative control (nutrient medium only).

Results:

Growth inhibition: Compared to the negative controls, algal growth was inhibited by 89.8 % based on the area under the growth curves or by 57.6 % based on the average growth rates at the highest tested concentration and it was slightly enhanced by 23.2% or 4.3% at the lowest one.

NOEC(0-72h) is 1/2WAF, based on the area under the growth curves

NOEC(0-72h) is 1/4WAF, based on the average growth rate

EbC5072h is between 1/4WAF and 1/2WAF.

ErC5072h is between 1/2WAF and WAF.

Actual test substance concentration: All concentrations analysed as the pure substance T15-AE were below the limit of quantitation of the method used (0.0014 mg/L) at the start of the incubation period.

 

Validity of the test: The increase in cell density in the control cultures during the 72 hours incubation period was more than 16-fold, as recommended in the EC-guideline. It was about 111-fold, corresponding to about 6.8 generations and showing suitable growth conditions for the algae.

Description of key information

The obtained EC50 was based on a water accommodated fraction (WAF) after loading 111 mg test substance / L, and using the dissolved part. The ErC50 is between 1/2 WAF and WAF.  ¼ WAF did not reduce the growth rate. The EC50 is higher than the solubility of the substance in the medium. The result is not directly suitable for a classification, but it demonstrates that the mixture, i.e. the pure substance and its impurities, as far as they are soluble at 111 mg mixture/L, is toxic to algae and a dilution of this solution to ¼ is not.

The impurity benzothiazole-2-thiol (typically 1.2 % in T15-AE) is classified in CLP as Aquatic Acute 1 H400 and Aquatic Chronic 1 H410. The EC50 of Benzothiazole-2-thiol = 0.50 mg/L, the water solubility is 118 mg/L at 25 °C and the substance is not readily biodegradable (results obtained from the ECHA website). Applying the additivity formula of section 4.1.3.5.2. in CLP, an EC50 of the mixture (=T15-AE) of 46 mg/L is calculated. This is in acceptable agreement with the experimentally obtained result.

 

An EC50 above the solubility limit is confirmed by a QSAR-calculation leading to an EC50 is 17 or 53 mg/L, depending on the model used.

 

Key value for chemical safety assessment

EC50 for freshwater algae:

46 mg/L