Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]

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:

24 January 2019 to 23 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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:

Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%

Analytical monitoring:

yes

Details on sampling:

For definitive test 1, samples were taken at 25, 47 and 72 hours. For definitive test 2, samples were taken at 0, 24, 49 and 72 hours (0 hour samples were used to check inoculation levels only). For both definitive tests, after sampling, the cell densities were determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal 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 each test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Vehicle:

no

Details on test solutions:

Definitive Test 1 - Standard Conditions
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
3.4.4.1 Experimental Preparation
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution which was highly colored.
A series of dilutions was made from this saturated solution to give stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with 2.6 mL of algal suspension (with a cell density of 8.64 x 105 cells per mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution, which had no significant dilution effect on the final test concentration.
The stock solutions and each prepared concentration 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 O and 72 hours (see Annex 5).

Definitive Test 2 -Modified Test Conditions

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.
Due to the color of the test item and the resultant test preparations, a further test was conducted using the modified test conditions according to the requirements of OECD, 2019. Colored test items may absorb biologically relevant wavelengths and thereby may impair algal growth due to a reduction in the light available for photosynthetic growth. Using the standard algal test method it is not possible to differentiate between reduced algal growth by light absorption of the colored test solutions or an inherent toxic effect of the test item.
Since the test solutions were shown to have distinct absorbance at 460 nm, the modified test conditions were applied for a second definitive test. This included reducing the test volume to 25 mL in order to shorten the light path through the test medium and increasing the light intensity to approximately 8880 Lux (±15%) in order to reduce as much as possible the light absorbance effect of the colored test solutions.
All experimental preparation and exposure conditions were as stated in section 3.4.4.1 and 3.4.4.2 except for the following. The absorbance of each stock solution was checked at
460 nm and 665 nm before inoculation. An aliquot (250 mL) of each of the stock solutions was separately inoculated with 0.91 mL of algal suspension (with a cell density of 1.38 x 106 cells per mL) to give the required test concentrations. The control medium (500 mL) was inoculated with 1.8 mL of the same algal suspension. The test volume was 25 ml and the lux was set to 8880 (±15%).
Since the first definitive test showed that the test item was unstable under test conditions extra samples of inoculated test media were taken for analysis at 24 and 48 hours and samples without algae were also prepared and incubated alongside the test vessels for sampling at 72 hours.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Test Organism Observations
For definitive test 1, samples were taken at 25, 47 and 72 hours. For definitive test 2, samples were taken at 0, 24, 49 and 72 hours (0 hour samples were used to check inoculation levels only). For both definitive tests, after sampling, the cell densities were determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal 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 each test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Test temperature:

24 °C

pH:

7.8

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32, 100% v/v
Measured concentrations (geometric means): 0.019, 0.18, 0.37, 2.5, 9.0 mg/L

Details on test conditions:

Methods
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A preliminary media preparation trial indicated that a dissolved test item concentration of 0.11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of solubility of this test item in Elendt M7 media and was considered to be representative of the worst case scenario. Solubility in ASTM media was expected to be higher and was confirmed as 7.5 mg/L during test samples analysis of the first definitive test.
Following preliminary range-finding and stability tests, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and
100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess ( 100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.
Two tests were conducted; the first test was performed under standard algal test conditions, whereas the second test used methodology designed for test items that produce colored test preparations. The second test was performed in accordance with the recommendations of OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD, 2019), 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.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 7.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Increased light and reduced volume

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Remarks on result:

other:

Remarks:

Increased light and reduced volume

Details on results:

For the first test (conducted under standard conditions) analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.086 to 7.5 mg/L. At 72 hours the measured concentrations ranged between 0.022 and 7.0 mg/L (with a range of 25% to 98% of the O hour measured test concentrations), with a decline in the measured
concentrations at the two lowest levels of 1.0 and 3.2% v/v (25% and 56% of O hours values, respectively) and therefore it was considered appropriate to use geometric mean measured test concentrations to calculate the results.

For the second test (conducted with modified test conditions), analysis of the test preparations at O hours showed measured test concentrations to range from 0.066 to 9.9 mg/L. A decline in measured test concentration was observed at 72 hours for all concentrations except
100% v/v, with measured concentrations ranging from less than the limit of quantification
(LOQ = 0.011 mg/L) to 8.2 mg/L (8.4% to 83% of the O hour measured test concentrations). Consequently, it was considered appropriate to use geometric mean measured test concentrations to calculate the results.
As for the first test, the geometric mean for the second definitive test was calculated using the 0 hours and 72 hours measured concentrations only, which provided the worst case scenario as concentrations declined over time and allowed the results from both definitive tests to be compared; the 24 hour and 48 hour measured values were not included.
Media incubated under test conditions without inoculation of algal cells also showed a general decline in the test concentrations indicating that adsorption onto algal cells was not the cause of the decrease in test concentrations during the definitive tests.

Results with reference substance (positive control):

A positive control (Study Number YB 1 TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018.
Exposure conditions and data evaluation for the positive control were similar to those in definitive test 1.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
EC50 (growth rate) (0 to 72 hour): 1.5 mg/L; 95% confidence limits 1.3 to 1. 7 mg/L
EC50 (yeild) (0 to 72 hours): 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L

No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L

The results from the positive control with potassium dichlromate were within the normal ranges for this reference item*.

*In-house data from the last 5 years shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.26) and a mean EyC50 value of0.66 mg/L (standard deviation = 0.14).

Validity criteria fulfilled:

yes

Conclusions:

Both tests demonstrated that the test item was not stable over time under test conditions at the lower test concentrations. Comparing the results from both tests showed that the test item was less toxic when tested under modified test conditions (increased light and lower test volume) based on the NOEC and LOEC values and the growth rate EC50; however, similar values were obtained for the yield EC50 in both tests. The endpoint for yield was consistent in both tests. The variation in growth rate however is more likely to be due to the difference in light intensities used in the two tests which has resulted in a higher growth rate in the second test.

Executive summary:

The study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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, in accordance with GLP.

Both tests demonstrated that the test item was not stable over time under test conditions at the lower test concentrations. Comparing the results from both tests showed that the test item was less toxic when tested under modified test conditions (increased light and lower test volume) based on the NOEC and LOEC values and the growth rate EC50; however, similar values were obtained for the yield EC50 in both tests. The endpoint for yield was consistent in both tests. The variation in growth rate however is more likely to be due to the difference in light intensities used in the two tests which has resulted in a higher growth rate in the second test.

Description of key information

The study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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, in accordance with GLP.

Both tests demonstrated that the test item was not stable over time under test conditions at the lower test concentrations. Comparing the results from both tests showed that the test item was less toxic when tested under modified test conditions (increased light and lower test volume) based on the NOEC and LOEC values and the growth rate EC50; however, similar values were obtained for the yield EC50 in both tests. The endpoint for yield was consistent in both tests. The variation in growth rate however is more likely to be due to the difference in light intensities used in the two tests which has resulted in a higher growth rate in the second test.