Disodium 4-[4-[[5-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]-2,5-dichlorobenzenesulphonate

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:

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MC-20/2014 (China)
- Expiration date of the lot/batch: 28 Oct 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Frozen in the dark

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 0-hour 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:

Definitive test
Experimental Preparation
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 (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.3 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

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 1E+03 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 1E+04 - 1E+05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

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.

Nominal and measured concentrations:

Range-Finding Tests: Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L

Definitive Test: Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

Range-Finding Tests:
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours. 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. Two replicate flasks were used for each control and test concentration. 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 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group was maintained under identical conditions but not exposed to the test item. Given that the test item formed colored test solutions, spectrophotometer readings were taken at both 460 and 665 nm in order to determine whether significant light absorption occurred. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. 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. All samples were stored frozen prior to analysis. The results of the initial range-finding test showed inhibition of growth occurred, particularly at 100 mg/L where significant light absorption was observed and so a second range-finding test was conducted in accordance with the recommendations of the OECD Guidance Document No. 23 on Aquatic Testing of Difficult Substances and Mixtures for the testing of colored substances. The test was conducted in 250 mL glass conical flasks each containing 25 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. 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 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.7 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group was maintained under identical conditions but not exposed to the test item. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test:
Based on the results of the second range-finding tests the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Exposure Conditions
As in the second range-finding test 250 mL glass conical flasks were used. Six flasks each containing 25 mL of test preparation were used for the control and three flasks each containing 25 mL were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.23 x 10e5 cells per mL. Inoculation of 450 mL of test medium with 5.3 mL of 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 plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Evaluations
Test Organism Observations
Samples were taken at 0, 24, 48 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:
m= average specific growth rate from time t1to tn
N1= cell concentration at t1
Nn= cell concentration at tn
t1= time of first measurement
tn= time of nthmeasurement
 
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
mc= mean average specific growth rate for the control cultures
mt= 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 ECxValues
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). ECxvalues were then determined from the equation for the fitted line.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Tests
The results of the range-finding tests showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth rate was observed to be reduced at 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test. Chemical analysis of the 1.0, 10 and 100 mg/L test preparations taken from the initial range-finding test at 0 and 72 hours showed measured concentrations to range from 87 % to 95 % of nominal indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 88 % to 106 % of nominal and so the results are based on nominal test concentrations only.


Growth Data
From the data given in Table 4 and Table 6 of the report, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not significantly 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): >100 mg/L
ErC20 (0 - 72 h): >100 mg/L
ErC50 (0 - 72 h): >100 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 decreases in growth rate (P0.05), between the control, 1.0, 3.2, 10 and 32 mg/L test concentrations however the 100 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 28 mg/L
EyC20 (0 - 72 h): 46 mg/L
EyC50 (0 - 72 h): >100 mg/L

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

Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield (P0.05), between the control, 1.0, 3.2, 10 and 32 mg/L test concentrations however the 100 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg/L.

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:

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).

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (growth rate) was > 100 mg/L.
The NOEC was 32 mg/L and the LOEC was 100 mg/L.
The EC50 (yield) was > 100 mg/L.

Executive summary:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period according OECD Guideline 201 and EU MEthod C.3. Following preliminary range-finding tests, 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. Given the colored nature of the test item, testing was conducted in accordance with the OECD Guidance Document No. 23 on Aquatic Testing of Difficult Substances and Mixtures whereby the light path was shortened and the light intensity increased to overcome any possible shading effects. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulterv Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 88 % to 106 % of nominal and so the results are based on nominal test concentrations only.

The EC50 (growth rate) was > 100 mg/L.

The NOEC was 32 mg/L and the LOEC was 100 mg/L.

The EC50 (yield) was > 100 mg/L.

Description of key information

The EC50 was (growth rate) wa >100 mg/L, while NOEC was 32 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period according to the OECD Guideline 201 and gave the following results:

The EC50 (growth rate) was > 100 mg/L.

The NOEC was 32 mg/L and the LOEC was 100 mg/L.

The EC50 (yield) was > 100 mg/L.