Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

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:

The study was conducted between 12 January 2015 and 21 January 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from Experiment A only 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.

Analysis of the test preparations from Experiment B was not performed as this was not a requirement of the test guidelines.

Vehicle:

no

Details on test solutions:

Culture Medium
The culture medium used for the definitive test was the same as that used to maintain the stock culture.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 ± 1 °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 103 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 104 - 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

The temperature within the incubator was recorded daily.

pH:

The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

Procedure
Definitive Test
Based on the results obtained from the range-finding test performed for an Algal Growth Inhibition Test (Harlan Study Number 41403251), the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.


Experiment A – Experimental Preparation
In order to determine the inhibition of algal growth due to a combination of the toxic effects of the test item and the reduction in light intensity, algae were exposed to the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, with glass petri dishes containing culture medium alone being placed above the test vessels.

Nominal amounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 100 and 32 mg/L stock solutions respectively. A series of dilutions were made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of these stock solutions was separately inoculated with algal suspension (3.2 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Experiment B – Experimental Preparation
In order to determine the inhibition of algal growth due to light intensity alone, the test vessels contained algal cells in culture medium alone, whilst separate glass petri dishes containing the test item solutions at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were placed above the test vessels.

Nominal mounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give 100 and 32 mg/L stock solutions respectively. A series of dilutions were made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (80 mL) of each of the 1.0, 3.2, 10, 32 and 100 mg/L stock solutions was separately placed in a glass petri dish, the overall depth of which was approximately half the depth of the test solutions in the conical flasks below.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells.

Exposure Conditions
In the definitive test 250 mL glass conical flasks were used.

For Experiment A six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. Glass petri dishes containing 80 mL of culture medium were placed above each of the conical flasks.

For Experiment B three flasks each containing 100 mL were used for each treatment group. Glass petri dishes containing 80 mL of each test preparation were placed above each of the conical flasks. The control data obtained for Experiment A was shared with Experiment B. The control was maintained under identical conditions but not exposed to the test item.

The depth of preparation (15 mm) in the petri dishes for both Experiment A and B was half the depth of the preparation in the conical flasks (30 mm).

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.89 x 105 cells per mL. Inoculation of an appropriate volume of test medium with this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were incubated (ETAD incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) and constantly stirred at approximately 150 rpm for 72 hours.

Evaluations
Test Organism Observations
Samples were taken at 0, 23, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

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.

Data Analysis
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1n Nn – 1n N1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:
Ir = ((µc - µt) /µc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture


Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group


Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

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

Remarks on result:

other: This reflects the the true toxic effect of the test item, rather than a reduction in growth due to light absorption of the coloured test substance.

Details on results:

Experiment A - Growth Data

From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:


Inhibition of Growth Rate
ErC10 (0 - 72 h): 1.8 mg/L
ErC20 (0 - 72 h): 5.0 mg/L
ErC50 (0 - 72 h): 27 mg/L*

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P>0.05), between the control and 1.0 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.0 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 3.2 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 1.4 mg/L
EyC20 (0 - 72 h): 1.8 mg/L
EyC50 (0 - 72 h): 4.0 mg/L; 95% confidence limits 3.0 – 5.4 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 4.1.2.1. There were no statistically significant differences (P>0.05), between the control and 1.0 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.0 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.2 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 1.4 mg/L
EyC20 (0 - 72 h): 1.8 mg/L
EyC50 (0 - 72 h): 4.0 mg/L; 95% confidence limits 3.0 – 5.4 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences (P>0.05), between the control and 1.0 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.0 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.2 mg/L.


Experiment B - Growth Data
From the data, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): 3.2 mg/L
ErC20 (0 - 72 h): 7.7 mg/L
ErC50 (0 - 72 h): 35 mg/L*
where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P0.05), between the control, 1.0 and 3.2 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 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 1.6 mg/L
EyC20 (0 - 72 h): 3.0 mg/L
EyC50 (0 - 72 h): 8.6 mg/L; 95% confidence limits 7.0 – 11 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

There were no statistically significant decreases in yield (P>0.05), between the control, 1.0 and 3.2 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 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10 mg/L.

Observations on Cultures
All test and control cultures for Experiment A and B were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

True Toxic Effect
The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells.
Accordingly the following results were determined from the data:


Inhibition of Growth Rate
ErC10 (0 - 72 h): >100 mg/L
ErC20 (0 - 72 h): >100 mg/L
ErC50 (0 - 72 h): >100 mg/L

Inhibition of Yield
EyC10 (0 - 72 h): >100 mg/L
EyC20 (0 - 72 h): >100 mg/L
EyC50 (0 - 72 h): >100 mg/L

It is therefore evident from these results that the test item was not truly toxic to algae, rather a reduction in growth was observed due to light absorption of the colored test substance.

*It was not possible to calculate 95% confidence limits for these ErC50values as the data generated did not fit the models available for the calculation of confidence limits.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41403074) 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 and data evaluation 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 – 72 h): 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/L
EyC50 (0 – 72 h): 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 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.

Reported statistics and error estimates:

Statistical Analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 – 2001).

Verification of Test Concentrations

Analysis of the test preparations taken from Experiment A at 0 and 72 hours showed measured test concentrations to range from 89% to 99% of nominal and so the results are based on nominal test concentrations only.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 22 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: 5.59 x 10³ cells per mL

Mean cell density of control at 72 hours: 1.22 x 10⁵ cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 31% 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 5 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Water Quality Criteria

The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 8.2 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.

 

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

Observations on Test Item Solubility

Experiment A

At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from pale blue solutions at 1.0 mg/L through to dark blue solutions at 100 mg/L. After the 72-Hour test period the control cultures were observed to be pale green dispersions. The test cultures were observed to range from pale blue/green dispersions at 1.0 mg/L through to dark blue solutions at 100 mg/L. 

 

Experiment B

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be pale green dispersions. The 10 and 32 mg/L test cultures were observed to be very pale green dispersions whilst the 100 mg/L test cultures were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item in Experiment A on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
The 72 hour EC50 (growth rate) was 27 mg/L. It was not possible to calculate 95% confidence limits for these EC50 values as the data generated did not fit the models available for the calculation of confidence limits. The NOEC was 3.2 mg/L and the LOEC was 10 mg/L.

These results indicate the combined toxic nature of the test item and the reduction in light intensity.

In Experiment B, reduction in light intensity resulted in reduction of algal growth. The following values were calculated based on the concentration of the test item in the glass petri dishes above the test cultures:

The 72 hour EC50 (growth rate) was 35 mg/L. It was not possible to calculate 95% confidence limits for these EC50 values as the data generated did not fit the models available for the calculation of confidence limits. The NOEC was 3.2 mg/L and the LOEC was 10 mg/L.

The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells and gave the following results:

The 72 hour EC10, EC20 and EC50 (growth rate) were > 100 mg/L.
It is therefore evident from these results that the test item was not truly toxic to algae, rather a reduction in growth was observed due to light absorption of the colored test substance.

Executive summary:

A study according to OECD guideline 201 and EU Method C.3, was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item for 72 hours, under constant illumination and stirred continuously via magnetic stirrer at a temperature of 24 ± 1 ºC. The test was conducted using two experimental methods performed in parallel. In Experiment A the algae were exposed to test item concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate vessels per concentration). Glass petri dishes above the test vessels contained culture medium alone. Therefore, inhibition of algal growth in these test vessels can be attributed to a combination of both the toxic effects of the test item and reduction in light intensity. In Experiment B the glass petri dishes above the test vessels contained test item solutions at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The test vessels (three replicates per concentration) contained algal cells in culture medium alone. Therefore inhibition of algal growth can be attributed to a reduction in light intensity alone. The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells. Analysis of the test preparations taken from Experiment A at 0 and 72 hours showed measured test concentrations to range from 89% to 99% of nominal and so the results are based on nominal test concentrations only. These results indicate the combined toxic nature of the test item and the reduction in light intensity.

 

The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells and gave the following results:

 

Response Variable	EC10(mg/L)	EC20(mg/L)	EC50(mg/L)
Growth Rate	>100	>100	>100
Yield	>100	>100	>100

 

It is therefore evident from these results that the test item was not truly toxic to algae, rather a reduction in growth was observed due to light absorption of the colored test substance.

*It was not possible to calculate 95% confidence limits for these E_rC₅₀values as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

The EC50 for the test item to the algae was determined to be > 100 mg/L.    
    
    
    
    
    

The 72 hour EC10, EC20 and EC50 (growth rate) were > 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

A study was performed according OECD Guideline 201 to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata over a duration of 72 hours.

In Experiment A the algae were exposed to test item concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate vessels per concentration). Glass petri dishes above the test vessels contained culture medium alone. Therefore, inhibition of algal growth in these test vessels was due to a combination of both the toxic effects of the test item and reduction in light intensity. In Experiment B the glass petri dishes above the test vessels contained test item solutions at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The test vessels (three replicates per concentration) contained algal cells in culture medium alone. Therefore, inhibition of algal growth was due to a reduction in light intensity alone. The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test item on the algal cells and gave the following results:

The 72 hour EC10, EC20 and EC50 (growth rate) were > 100 mg/L.