1,2-Benzisothiazole, 3-methyl-, 1,1-dioxide

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

Study period:

2016-11-15 to 2016-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

7 December 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Analytical monitoring:

yes

Details on sampling:

- Concentrations: each test concentration and control
- Sampling method: from the test solutions (10 mL at the start and 3 x 2 mL at the end of the study); Three formulation samples were analysed from each test concentration and control at the start and at the end of the test.

Vehicle:

yes

Remarks:

OECD medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving the test item in OECD medium, thereafter sonicated for 30 minutes in ultrasonic bath (under cooling). The further test solutions were prepared by appropriate dilution of this stock solution.
- Method: The test solutions used in the test were prepared by mechanical dispersion without the aid of any solubilising agents. The test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.
- Controls: Untreated Control: Algal Mineral Salts Test Medium (without test item) was inoculated and examined in parallel in the study.; Toxic Reference Control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- Chemical name of vehicle: Reconstituted algal growth medium (OECD medium, according to OECD 201)
- Concentration of vehicle in test medium: not applicable as the OECD medium was used for substance dilution

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum: Cells were taken from an exponentially growing pre-culture, set up four days prior to the test start using the same conditions as in the test.
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test temperature:

21.3 – 21.5 °C measured in the flask and 21.2 – 23.1 °C measured within the climate chamber

pH:

8.03 – 8.77

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.4, 1.56, 6.25, 25, 100 mg/L

Mean of the measured concentrations (mg/L)
Start of experiment (n=3): 0.4, 1.61, 6.34, 25.6, 97.1
End of experiment (n=3): 0.35, 1.53, 6.24, 24.1, 91.3
On the basis of the analytical measurements there is evidence that the concentrations of the tested substance were maintained within ± 20 % of the nominal concentrations throughout the test. Therefore the analysis of the results was based on the nominal concentration values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: ~250 mL volume with 100 mL test medium
- Initial cells density: 10E4 cells/mL
- Control end cells density: 59.17 × 10E4 cells/mL (mean value) after 72 hours in the Untreated control
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium, according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 8016 lux, fluorescent lamps (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: counting chamber, at 24, 48 and 72 hours
- Chlorophyll measurement: No
- Other: morphological changes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 4
Range finding study: yes
- Test concentrations: 0.1, 1.0, 10.0, 50.0, 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes; The higher spacing factor of 4 in comparison to the spacing factor of 3.2 is justified because the results of the Preliminary Range-Finding Test show a flat concentration response curve.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits: 4.60 – 11.68

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

21.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits: 15.00 – 31.91

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

177.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits: 93.10 – 337.12

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits:

Remarks:

0.73 – 1.75

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits: 1.97 – 3.85

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits: 11.45 – 19.64

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: Cell density in the control cultures increased by a factor of more than 16 (by a factor of 59.17) within 72 hours.
- Observation of abnormalities: No morphological abnormalities were observed in the control and in the lowest test item concentration (0.4 mg/L) during the experiment. Swollen cells (together with cells without sign of any effect) were detected in the test group of 1.56 mg/L, while swollen, thin and small cells (together with cells without sign of any effect) were observed at 6.25 mg/L. At the test concentration of 25 mg/L swollen and small cells (together with cells without sign of any effect) were found, while less normal and more swollen and smaller cells with pinches grew in the test flasks of 100 mg test item/L.
- Unusual cell shape: yes
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: The measured concentration of the test item in the test solution was in the range of 97 – 103 % at the start and 87 – 100 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Reported statistics and error estimates:

The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.25, 25 and 100 mg/L (Bonferroni t-Test, 2 tailed, α=0.05) when compared to the control.

Validity criteria fulfilled:

yes

Conclusions:

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 177.16 (93.1 – 337.12) mg/L and 15.0 (11.45 – 19.64) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 7.33 (4.6 – 11.68) mg/L and 1.13 (0.73 – 1.75) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 1.56 mg/L.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The study was conducted under static conditions with an initial cell density of 10E4 cells/mL. Five concentrations were tested in a geometrical series with a spacing factor of 4: 0.4, 1.56, 6.25, 25 and 100 mg/L (nominal concentrations). The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-UV at the start and the end of exposure. The measured concentration of the test item in the test solution was in the range of 97 – 103 % at the start and 87 – 100 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.25, 25 and 100 mg/L (Bonferroni t-Test, 2 tailed, α=0.05) when compared to the control. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 177.16 (93.1 – 337.12) mg/L and 15.0 (11.45 – 19.64) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 7.33 (4.6 – 11.68) mg/L and 1.13 (0.73 – 1.75) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 1.56 mg/L. In conclusion the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata.

Description of key information

The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 177.16 (93.1 – 337.12) mg/L and 15.0 (11.45 – 19.64) mg/L, respectively.The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 7.33 (4.6 – 11.68) mg/L and 1.13 (0.73 – 1.75) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 1.56 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

177.16 mg/L

EC10 or NOEC for freshwater algae:

7.33 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3. The study was conducted under static conditions with an initial cell density of 10E4 cells/mL. Five concentrations were tested in a geometrical series with a spacing factor of 4: 0.4, 1.56, 6.25, 25 and 100 mg/L (nominal concentrations). The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-UV at the start and the end of exposure. The measured concentration of the test item in the test solution was in the range of 97 – 103 % at the start and 87 – 100 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.25, 25 and 100 mg/L (Bonferroni t-Test, 2 tailed, α=0.05) when compared to the control. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 177.16 (93.1 – 337.12) mg/L and 15.0 (11.45 – 19.64) mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 7.33 (4.6 – 11.68) mg/L and 1.13 (0.73 – 1.75) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 1.56 mg/L. In conclusion the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata.