Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 13 January 2017. Experimental completion date: 08 April 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

EC Number:

233-316-4

EC Name:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Cas Number:

10114-86-0

Molecular formula:

C27H22N6Na2O9S2

IUPAC Name:

disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Test material form:

solid: particulate/powder

Details on test material:

Identification: CIM-42(Direct Yellow 132)
CAS No: 10114-86-0
Appearance/Physical state: Dark orange colored powder
Batch: 150909K
Purity: 90.3%
Expiry date: 01 October 2017
Storage conditions: Room temperature over silica gel, in the dark

Sampling and analysis

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. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Range-Finding Tests:
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes 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 (5.3 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.

Definitive Test:
Based on the results of the second range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

Experiment Preparation:
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes 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 50, 25, 12.5 and 6.25 mg/L. An aliquot (250 mL) of each of the stock solutions was separately inoculated with algal suspension (1.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

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 illumination at 21 ± 2 °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 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 10E4 – 10E5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

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

pH:

7.7-7.9
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 7.9 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.

Salinity:

not applicable freshwater test.

Nominal and measured concentrations:

Range finding tests:
Nominal: 0.10, 1.0, 10 and 100 mg/L.

Definitive test:
Nominal: 6.25, 12.5, 25, 50 and 100 mg/L.
Measured (0 and 72 hrs): 87% to 98% of nominal.

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 with the aid of ultrasonication for approximately 5 minutes 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 (5.3 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 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 (225 mL) of each of the stock solutions was separately inoculated with algal suspension (3.6 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.
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 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 test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

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

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 7.97 x 10^5 cells per mL. Inoculation of 250 mL of test medium with 1.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 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 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.

Assessment:
Test Organism Observations:
Samples were taken at 23, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
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.

Water Quality Criteria:
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. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Range-finding Tests:
The results of the initial range-finding test showed no effect on growth rate at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. The results of the second range-finding test showed no effect on growth rate at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. However, inhibition of yield was observed at 100 mg/L.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations taken from the initial range-finding test at 0 and 72 hours showed measured concentrations to range from 82% to 93% 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 87% to 98% of nominal indicating that the test item was stable over the test duration and as such the results were based on nominal test concentrations only.

Growth 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) : >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 differences (P≥0.05), between the control, 6.25, 12.5, 25 and 50 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 50 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/L.

Inhibition of Yield:
EyC10 (0 - 72 h) : 29 mg/L
EyC20 (0 - 72 h) : 51 mg/L
EyC50 (0 - 72 h) : >100 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, 6.25, 12.5, 25 and 50 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 50 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg/L.

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/L, however cell debris was observed to be present in the test cultures at 100 mg/L.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test. The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 7.9 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.

Observations on Test Item Solubility:
At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from very pale yellow solutions at 6.25 mg/L through to dark yellow solutions at 100 mg/L. After the 72-Hour test period all control cultures were observed to be green dispersions. The test cultures were observed to range from pale green/yellow dispersions at 6.25 mg/L through to dark yellow solutions at 100 mg/L.

Results with reference substance (positive control):

A positive control 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) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

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

Any other information on results incl. tables

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.055		2.63E+05
	R2	0.072		9.19E+05
	R3	0.072		8.90E+05
	R4	0.069	-	7.33E+05	-
	R5	0.072		9.04E+05
	R6	0.073		9.39E+05
	Mean^**	0.072		8.77E+05
	SD^**	0.002		8.25E+04
6.25	R1	0.075	[4]	1.11E+06
	R2	0.073	[1]	9.82E+05
	R3	0.078	[8]	1.38E+06
	Mean	0.075	[4]	1.16E+06	[32]
	SD	0.003		2.01E+05
12.5	R1	0.072	0	8.79E+05
	R2	0.072	0	8.88E+05
	R3	0.073	[1]	9.36E+05
	Mean	0.072	0	9.01E+05	[3]
	SD	0.001		3.08E+04
25	R1	0.070	3	7.73E+05
	R2	0.070	3	7.48E+05
	R3	0.067	7	6.20E+05
	Mean	0.069	4	7.14E+05	19
	SD	0.002		8.23E+04
50	R1	0.070	3	7.63E+05
	R2	0.063	13	4.58E+05
	R3	0.070	3	7.92E+05
	Mean	0.068	6	6.71E+05	23
	SD	0.004		1.85E+05
100	R1	0.067	7	6.31E+05
	R2	0.065	10	5.25E+05
	R3	0.064	11	5.09E+05
	Mean	0.065	9	5.55E+05	37
	SD	0.002		6.67E+04

*       In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

^**      Values calculated based on replicates R₂-R₆only.

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to the control]

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 176 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.

Nominal cell density of control at 0 hours : 5.00 x 10E3 cells per mL

Mean cell density of control at 72 hours : 8.82 x 10E5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% 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%.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
Growth rate: EC50 >100 mg/L, NOEC 50 mg/L, LOEC 100 mg/L
Yield: EC50 >100 mg/L, NOEC 50 mg/L, LOEC 100 mg/L

Executive summary:

Introduction

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 Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods.

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 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 Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 98% of nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>100	>100	>100	50	100
Yield	29	51	>100	50	100