Dibenzyl sulphoxide

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:

05.09. – 06.10.2016

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:

Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the analytical determination of the test substance concentrations was performed at the beginning and at the end of the test.

- Sampling method:
The samples for analysis were taken from the stock solution (100 mg·L-1) with and without algae.
The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory.
The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing.

- Sample storage conditions before analysis: The samples were analysed on the day of delivery. All samples were stored at laboratory temperature.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

For the preliminary experiment the solutions were prepared as follows: 100 mg of the test substance in 1000 mL of test medium was ultrasonicated for 30 minutes and stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter and the concentrations of the test substance in resulted solutions were determined.

The stock solutions of the test substance were prepared in the test medium. 100 mg of the test substance was weighed into 1 000 mL of the test medium for the preliminary and limit test. The stock solutions were ultrasonicated for 30 minutes then stirred for period 72 hours and subsequently filtered through 0.45 μm filter.
The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with dilution water.


- Differential loading:
Preliminary test:
The stock solution of the test substance in the test medium was prepared for the preliminary test. The test was performed in a range nominal concentrations from 1 mg·L-1 to 100 mg·L-1. Testing mixtures were prepared by dosing appropriate volumes of stock solution of the test substance and inoculum into volumetric flasks. Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained three parallel series of the test substance concentrations and three controls without the test substance.

Limit test:
The limit test was performed with nominal concentration 100 mg·L-1. Testing solutions were prepared by dosing of stock saturated solution of the test substance and inoculum into volumetric flasks. Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained six parallel series of the test substance and six controls without the test substance.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Test medium - the water with conductivity smaller than 5 μS·cm-1 was used for the preparation of solutions. All chemicals used were of analytical-grade.

- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
The volume of the test solution was 50 mL in each testing flask.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Desmodesmus subspicatus Brinkmann 1953/SAG 86.81
- Source: from the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň (on date 27.5.2016)
- Age of inoculum (at test initiation): The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed.
After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.


- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.0 – 21.5 °C

pH:

7.8

Conductivity:

1.73 µS·cm-1

Nominal and measured concentrations:

100 mg·L-1
100 w.a. 99.52 (0 h) 100.51(72 h) mg·L-1
100 98.97 100.27

Details on test conditions:

Preliminary test
Stock solution of the test substance: 100.03 mg/L
Test concentrations: 100, 50, 10, 5 and 1 mg/L
Conductivity of deionized water: 1.64 μS/cm
pH of the test medium: 7.7
Volume of inoculated algae culture: 2.35 mL in 200 mL of mixture
Lighting during the test: 8 325 – 8 345 lux
Temperature during the test: 21.0 – 21.5 °C
Inicial density: 5000 cells per 1mL

Limit test
The test was performed with nominal concentrations 100 mg·L-1.
Stock solution of test substance: 100.03 mg·L-1
Test concentration: 100 mg·L-1
Conductivity of deionized water: 1.73 µS·cm-1
pH of test medium: 7.8
Volume of inoculum algae culture: 1.85 mL in 500 mL mixture
Lighting during the test: 8 340 – 8 345 lux
Temperature during the test: 21.0 – 21.5 °C
Inicial density: 5000 cells per 1ml

Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained six parallel series of the test substance concentration and six controls without the test substance.The volume of the test solution was 50 mL in each testing flask.
The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber. The growth rates (u) and the yield (Y) and subsequently percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Reference test:
The sensitivity of the test species and correctness of test performance in our laboratory is periodically verified on a six-month period by testing the reference substance, potassium dichromate.
72 hour – ErC50 = 0.87 mg/L (95% confidence limit: 0.64 – 1.17 mg/L)
Interlaboratory test: 72 hour – ErC50 = 0.54 – 1.26 mg/L

Method validity evaluation:
The value EC50 for inhibition of growth rate (72h – ErC50) obtained from our last reference test meets the calculated range from the interlaboratory test.
The sensitivity of test species and proficiency of our laboratory in test performance was proved.

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 33.0 % in the limit test. Therefore exact value of ErC50 could not be calculated and the value of EC are given in the form of a range.
The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
There is stated in the guideline that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, the results can be based on nominal or measured initial values.
The nominal concentrations were used for all evaluations and results.
Test results:
72 hour – ErC50 > 100 mg·L-1                       (nominal concentration)
72 hour – NOECr < 100 mg·L-1                      (nominal concentration)

Executive summary:

The test substance, Dibenzylsulfoxide, was tested for growth inhibition on algae Desmodesmus subspicatus.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No. 2016/266.

The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg·L-1.

The analytical results showed that the test substance Dibenzylsulfoxide was stable in the test medium at conditions of the test.

The highest inhibition of growth rate was 35.5 % and the highest inhibition of yield was 79.0 % in the preliminary test.

Based on toxicity of the test substance lower than 50 % found in preliminary test (measured by growth rate), the limit test was performed subsequently. Only the value of 72 hour - E_rC₅₀is needed for the classification and risk assessment of chemicals, so the value of 72 hour - E_yC₅₀was not calculated.

The concentration of 100 mg·L-1 was tested in the limit test. The inhibition of growth rate was 33.0 %.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study.

The nominal concentrations were used for all evaluations and results.

Test results:

72 hour – E_rC₅₀> 100 mg·L-1                         (nominal concentration)

72 hour – NOEC_r< 100 mg·L-1                      (nominal concentration)

Description of key information

The inhibition of growth rate was 33.0 % in the limit test. Therefore exact value of ErC50 could not be calculated and the value of EC are given in the form of a range.

The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).

There is stated in the guideline that if evidence is available to demonstrate that the concentration of the test substance in limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, the results can be based on nominal or measured initial values.

The nominal concentrations were used for all evaluations and results.

Test results:

72 hour – ErC50 > 100 mg·L-1                       (nominal concentration)

72 hour – NOECr < 100 mg·L-1                      (nominal concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L