2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:4)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across chemicals

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE report is prepared based on toxicity to aquatic algae study:1st, 2nd and 3rd study

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:4)- Molecular formula: C26H25N5O19S6.4Na- Molecular weight: 995.85 g/mole- Smiles : [Na][Na][Na][Na]Nc1ccc2c(cc(S(O)(=O)=O)c(N=Nc3ccc(S(=O)(=O)CCOS(O)(=O)=O)cc3)c2O)c1N=Nc1ccc(S(=O)(=O)CCOS(O)(=O)=O) cc1S(O)(=O)=O- Substance type: Organic- Physical state: Solid powder

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

1. The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring/sonication for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.2. The stock solution 100 mg/l was prepared by dissolving black powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.3. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. The solution was kept 30 min in ultrasonic bath. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

other: 1. Chlorella vulgaris, 2.3. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

1. The fresh water green alga Chlorella vulgaris, was used as the test organism. Sterile, unicellular, liquid cultures of algae. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated. The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM. 2nd and 3rd study: TEST ORGANISM- Common name: - Strain: 86.81 SAG- Source (laboratory, culture collection): Institute of botany of the ASCR- Initial biomass concentration: 5x10(3) cells /ml - Method of cultivation: No data availableACCLIMATION - No data available- Acclimation period:- Culturing media and conditions (same as test or not):- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Remarks on exposure duration:

2. ±1 hour

Post exposure observation period:

1. 24, 48, 72 hrs

Test temperature:

1. 22 °C ±2°C2nd and 3rd study: 23±2°C

pH:

1. 6.7 to 7.42. Test at highest concentration: 7.7 (no change during tests)Control: 8.0 (changed to 7.5 during test)3. Test at highest concentration: 7.9 (change to 7.8 during tests)Control 1: 8.0 (changed to 8.1 during test)Control 2: 8.2 (changed to 7.4 during test)

Nominal and measured concentrations:

1. 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l. All the six concentration were in geometric series spaced by a factor of 2.2. 0, 12, 20, 35, 60, 100 mg/l nominal concentrations3. 0, 0, 20, 30, 45, 67, 100 mg/l

Details on test conditions:

1. TEST SYSTEM - Test vessel: Conical flask - Type (delete if not applicable): open / closed: No data - Material, size, headspace, fill volume: 100 ml of conical flasks filled with 60ml of test solution - Aeration: no data - Initial cells density: 10.82 x104 cells/mL) - Control end cells density: No data - No. of organisms per vessel: No data - No. of vessels per concentration (replicates): 2 - No. of vessels per control (replicates): 3 - No. of vessels per vehicle control (replicates): 3 GROWTH MEDIUM - Standard medium used: yes - Detailed composition if non-standard medium was used: No data OTHER TEST CONDITIONS - Sterile test conditions: yes - Adjustment of pH: Yes - Photoperiod: 16 Hour Light Period : 8 Hour Dark Period - Light intensity and quality: White Fluorescent Light, 1500Lux - Salinity (for marine algae): No data EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: spectrophotometer TEST CONCENTRATIONS - Spacing factor for test concentrations: No data - Justification for using less concentrations than requested by guideline: No data - Range finding study: No data - Test concentrations: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l - Results used to determine the conditions for the definitive study: Cell growth inhibition2nd and 3rd study: TEST SYSTEM- Test vessel: 50 ml Glass vessel- Type (delete if not applicable): closed (with air permeable stopper)- Sample volume: 7.5 ml- Initial cells density: 5x10(3) cells/ml- No. of vessels per concentration (replicates): 3GROWTH MEDIUM- Standard medium used: yesOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: Continuous- Light intensity and quality: 6000-8000 lxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1st study

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

76.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 61.5 to 96 mg/l

Remarks:

2nd study

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

285.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI was 173.5 - 470.8 mg/l

Remarks:

3rd study

Details on results:

1. The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0 units.

Results with reference substance (positive control):

2nd and 3rd study: Results with reference substance valid- EC50: 0.69 mg/L

Reported statistics and error estimates:

1. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.2nd and 3rd study: ErC50 was calculated using non-linear regression by the software Prism 4.0

1.Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	24800	50000	75600
Replicate 2	23600	40800	70000
Replicate 3	32800	49600	63200
Test chemical
6.25mg/l
Replicate 1	16800	18800	53600
Replicate 2	11200	16000	50400
12.5mg/l
Replicate 1	15200	17600	45600
Replicate 2	13600	18800	49600
25mg/l
Replicate 1	11600	25600	47200
Replicate 2	21600	28000	37600
50mg/l
Replicate 1	13200	16800	36800
Replicate 2	10800	13200	34000
100mg/l
Replicate 1	11200	12800	33200
Replicate 2	11600	12800	34000
200mg/l
Replicate 1	13600	17600	32400
Replicate 2	13200	19600	33200

 

 

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/l		200mg/l
Average Specific Growth rate (µ )	R1	0.645	R1	0.412	R1	0.400	R1	0.375	R1	0.366	R1	0.338	R1	0.334
	R2	0.662	R2	0.408	R2	0.384	R2	0.366	R2	0.366	R2	0.343	R2	0.308
	R3	0.691
Mean of Avg. Specific growth rate	0.645		0.549		0.519		0.479		0.421		0.403		0.395
Percentage Inhibition (%I)	_		14.932		19.516		25.774		34.795		37.451		38.695

 

 

 

 

Validity criteria fulfilled:

not specified

Conclusions:

1. After 72 hours of exposure with test chemical to various nominal test concentration on aquatic algae, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l.2. Based on the growth rate inhibition of test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the test chemical, the effect concentration ErC50 was determine at 76.8 mg/l.3. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 285.8 mg/L on the basis of effects on growth rate in a 72 hour study.Based on all three studies chemical consider to be nontoxic.

Executive summary:

Various studies available for the test chemical were reviewed to determine the toxic nature of 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no 607724 -37 -8) on the growth of algae and cyanobacteria. The studies are as mentioned below:

In the first key study the effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L. The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring/sonication for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10000cells/ml. Care was taken to have a homogeneous solution for the experiment. Test was performed in static manner at proper requirement of pH and temperature. The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0 units. After 72 hours of exposure with test chemical to various nominal test concentration on aquatic algae, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.

Similarly Short term toxicity study of test substance to aquatic algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was conducted for 72 hrs. Test was performed according to the 201 OECD guideline in a static system. The stock solution 100 mg/l was prepared by dissolving black powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the 0, 12, 20, 35, 60, 100 mg/l nominal concentrations. Effects on the growth rate of the organism were studied. Potassium dichromate (K2Cr2O7) were used as a reference positive control. Effects on growth rate were observed for 72 hours by using non linear regression by the software Prism 4.0. Based on the growth rate inhibition of test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the chemical, the effect concentration ErC50 was 76.8 mg/l. This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 3 as per the CLP criteria. But as the test chemical was readily biodegradable in water so, on that basis chemical was consider as nontoxic and can be consider to be not classified as per the CLP classification criteria.

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. The solution was kept 30 min in ultrasonic bath. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 5x10(3) cells /ml algal culture were use in the study for total exposure period of 72hrs. Test conducted in 50 ml glass vessel filled with 50 ml of sample volume and tested at the concentrations 0, 0, 20, 30, 45, 67, 100 mg/l. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 285.8 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP criteria.

Thus based on the above studies, 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]diazenyl]-, sodium salt (1:4) (CAS no 607724 -37 -8) consider to be nontoxic and not classified as per the CLP classification criteria.

Description of key information

1. After 72 hours of exposure with test chemical to various nominal test concentration on aquatic algae, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l.

2. Based on the growth rate inhibition of test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the test chemical, the effect concentration ErC50 was determine at 76.8 mg/l.

3. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 285.8 mg/L on the basis of effects on growth rate in a 72 hour study.

Based on all three studies chemical consider to be nontoxic.

Key value for chemical safety assessment

EC50 for freshwater algae:

285.8 mg/L

Additional information

Various studies available for the test chemical were reviewed to determine the toxic nature of 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no 607724 -37 -8) on the growth of algae and cyanobacteria. The studies are as mentioned below:

In the first key study the effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, 200mg/L. The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring/sonication for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10000cells/ml. Care was taken to have a homogeneous solution for the experiment. Test was performed in static manner at proper requirement of pH and temperature. The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0 units. After 72 hours of exposure with test chemical to various nominal test concentration on aquatic algae, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.

Similarly Short term toxicity study of test substance to aquatic algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was conducted for 72 hrs. Test was performed according to the 201 OECD guideline in a static system. The stock solution 100 mg/l was prepared by dissolving black powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the 0, 12, 20, 35, 60, 100 mg/l nominal concentrations. Effects on the growth rate of the organism were studied. Potassium dichromate (K2Cr2O7) were used as a reference positive control. Effects on growth rate were observed for 72 hours by using non linear regression by the software Prism 4.0. Based on the growth rate inhibition of test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the chemical, the effect concentration ErC50 was 76.8 mg/l. This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 3 as per the CLP criteria. But as the test chemical was readily biodegradable in water so, on that basis chemical was consider as nontoxic and can be consider to be not classified as per the CLP classification criteria.

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. The solution was kept 30 min in ultrasonic bath. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 5x10(3) cells /ml algal culture were use in the study for total exposure period of 72hrs. Test conducted in 50 ml glass vessel filled with 50 ml of sample volume and tested at the concentrations 0, 0, 20, 30, 45, 67, 100 mg/l. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 285.8 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP criteria.

Thus based on the above studies, 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]diazenyl]-, sodium salt (1:4) (CAS no 607724 -37 -8) consider to be nontoxic and not classified as per the CLP classification criteria.