2-(6-methoxybenzofuran-2-yl)-1,3-dimethyl-5-(methylsulphonyl)1H-benzimidazolium acetate

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:

From August 09 to October 02, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/l. An aliquot (500 ml) of each of the stock solutions was separatelyl inoculated with algal suspension (9.8 ml) to give the required test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.
- Source: liquid cultures 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.
- Breeding conditions: the master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
- Preparation for the test: 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.
- Suspension: pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.56 x 10^5 cells per ml.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

Nominal 1.0, 3.2, 10, 32 and 100 mg/l - Main test
Nominal 6.25, 12.5, 25, 50 and 100 mg/l - initial experiments

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.
- Initial cell density: 5.00 x 10^3 cells per ml, nominal.

GROWTH MEDIUM
- Standard medium used: yes.
- Detailed composition: NaNO3 25.5 mg/l; MgCl2 x 6H2O 12.16 mg/l; CaCl2 x 2H2O 4.41 mg/l; MgSO4 x 7H2O 14.6 mg/l; K2HPO4 1.044 mg/l; NaHCO3 15.0 mg/l; H3BO3 0.186 mg/l; MnCl2 x 4H2O 0.415 mg/l; ZnCl2 0.00327 mg/l; FeCl3 x 6H2O 0.160 mg/l; CoCl2 x 6H2O 0.00143 mg/l; Na2MoO4 x 2H2O 0.00726 mg/l; CuCl2 x 2H2O 0.000012 mg/l; Na2EDTA x 2H2O 0.30 mg/l. The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination provided by warm white lighting (380 – 730 nm).
- Light intensity and quality: intensity approximately 7000 lux
- Shaking: constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED
- Cells density: samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.
- Cells appearance: to determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- pH: determined at initiation of the test and after 72 hours exposure.
- Temperature: recorded daily.
- Appearanc eof medium: the appearance of the test media was recorded daily.

RANGE-FINDING TEST
- Test vessels: the test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation.
- Test solution: the test item was dissolved directly in culture medium. A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 100 mgl stock solution.
- Test concentrations: 1.0, 10 and 100 mg/l. Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity
- No. of replicates: 2 replicate flasks were used for each control and test concentration.
- Temperature: 24 ± 1 ºC
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm).
- Duration: 72 hours.
- Measured concentration: a sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- the cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- the mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35 %.
- the coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

84 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Details on results:

It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test item over the 72-Hour exposure period.

Inhibition of growth rate
There were no statistically significant differences (P ≥ 0.05), between the control, 1.0, 3.2 and 10 mg/l test concentrations; however all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/l.

Inhibition of Yield
There were no statistically significant differences (P ≥ 0.05), between the control, 1.0, 3.2 and 10 mg/l test concentrations; however all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg/l.

MEASURED CONCENTRATIONS and APPEARANCE OF THE MEDIUM
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92 to 102 % of nominal and so the results are based on nominal test concentrations only.
At the start of the test all control, 1.0, 3.2, 10 and 32 mg/l test cultures were observed to be clear colorless solutions whilst the 100 mg/l test cultures were observed to be extremely pale yellow solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/l test cultures were observed to be pale green dispersions. The 32 mg/l test cultures were observed to be very pale green dispersions whilst the 100 mg/l test cultures were observed to be extremely pale yellow slightly opaque solutions.

INITIAL EXPERIMENT
Two initial experiments conducted at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l showed significant inhibition of growth occurred at 6.25 mg/l.

RANGE-FINDING TEST
The results showed no effect on growth at the test concentrations of 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/l.
Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 96 to 103 % of nominal indicating that the test item was stable under test conditions.

VALIDATION CRITERIA
The cell concentration of the control cultures increased by a factor of 109 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 5.00 x 103 cells per ml
Mean cell density of control at 72 hours : 5.44 x 105 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 13 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Results with reference substance (positive control):

ErC50 (72 h): 1.5 mg/l; 95 % confidence limits 1.3 - 1.7 mg/l
EyC50 (72 h): 0.79 mg/l; 95 % confidence limits 0.70 - 0.89 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 mg/l

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h): 84 mg/l (statistically estimated, based on nominal conc.)
EyC50 (72h): 43 mg/l (statistically estimated, based on nominal conc.)

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Algae and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at 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.

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 and 72 hours showed measured test concentrations to range from 92 to 102 % of nominal and so the results are based on nominal test concentrations only.

It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test item over the 72-Hour exposure period.

The "No Observed Effect Concentration" (NOEC) based on both growth rate and yield was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth and yield rate was 32 mg/l.

Conclusion

ErC50 (72h): 84 mg/l (statistically estimated, based on nominal conc.)

EyC50 (72h): 43 mg/l (statistically estimated, based on nominal conc.)

Description of key information

ErC50 (72h): 84 mg/l (statistically estimated, based on nominal conc.)

EyC50 (72h): 43 mg/l (statistically estimated, based on nominal conc.)

Key value for chemical safety assessment

EC50 for freshwater algae:

84 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Algae and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at 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.

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 and 72 hours showed measured test concentrations to range from 92 to 102 % of nominal and so the results are based on nominal test concentrations only.

It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test item over the 72-Hour exposure period.

The "No Observed Effect Concentration" (NOEC) based on both growth rate and yield was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth and yield rate was 32 mg/l.