4'-aminoazobenzene-4-sulphonic acid

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 from various test chemicals

Justification for type of information:

Experimental data of test chemical from collection of data

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 short term toxicity to algae and cyanobacteria study:
2 and 3 and 4th reports

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

2: PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution 200 mg/L was prepared by dissolving light grey powder in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

- Controls: OECD medium as specified in OECD 201, used as a control and for dilution of the stock solution.

3rd: The test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium.

4th: 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

other: 2, 3rd: Desmodesmus subspicatus (previous name: Scenedesmus subspicatus), 4th: Chlorella vulgaris

Details on test organisms:

2nd and 3rd: TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Age of inoculum (at test initiation): 5x103 cells/ml
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not): No data
- Any deformed or abnormal cells observed: No data

4th:
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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Post exposure observation period:

24, 48, 72 hrs in 4th study

Test temperature:

2nd and 3rd: 23°C ± 2°C
4th: 22 °C ±2°C

pH:

2nd: The sample at concentration 120 mg/L : pH = 7.1 changed to pH = 7.3 during the test,
Control : pH = 8.2 changed to pH = 7.4 during the test

3rd: Test at highest concentration: 8 (Change to 7.8 during tests)
Control: 8.0 (changed to 7.8 during test)

4th: 6.7 to 7.4

Nominal and measured concentrations:

2nd: 0, 2.2, 11, 25, 55 and 120 mg/L
3rd: 100 mg/l
4th: 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.

Details on test conditions:

2: TEST SYSTEM
- Test vessel: 50ml glass vessel
- Type : closed (with air-permeable stopper)
- Material, size, headspace, fill volume: 15 ml
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): No data
- Renewal rate of test solution (frequency/flow rate): No data
- Initial cells density: 5x103 cells/mL
- Control end cells density: No data
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): No data

3rd: TEST SYSTEM
- Test vessel: 50 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: Without adjustment
- Photoperiod: Continuous
- Light intensity and quality: 6000 lx - 8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell counting: Electronic particle counter.
- Chlorophyll measurement: No
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

4th: 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 x10000 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 inhibition

Reference substance (positive control):

yes

Remarks:

2nd and 3rd: Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

129.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2nd study

Duration:

72 h

Dose descriptor:

other: IC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Only 1.6 % inhibition was observed after 72 hrs.

Remarks:

3RD STUDY

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: 4th study

Details on results:

4th: 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):

2: - Results with reference substance valid
- ErC50: 0.69 mg/L K2Cr2O7
3rd: - EC50: 0.77 mg/L (24 hours)

Reported statistics and error estimates:

4th: 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.

Validity criteria fulfilled:

no

Conclusions:

2nd: The median effective concentration (ErC50) for the test substance, on a freshwater algae Desmodesmus subspicatus was determined to be 129.4 mg/L on the basis of effects on growth rate in a 72 hour study.
3rd: Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the inhibitory concentration was 100 mg/l on that only 1.6 % inhibition was observed after the 72hrs incubation period.
4th: After 72 hours of exposure of test chemical to various nominal test concentration, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l.
Based on the overall data from various sources, it can be concluded that the substance is considered to be not toxic to aquatic environment (algae and cyanobacteria) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

Executive summary:

Various short term studies and other data available for the test chemical were reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 2.2, 11, 25, 55 and 120 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 129.4 mg/L. Potassium dichromate (K2Cr2O7) were used as a reference positive control. Effects on immobilisation were observed for 48 hours by using nonlinear regression by the software Prism 4.0. Based on this ErC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance does not exhibit toxicity to aquatic algae (Desmodesmus subspicatus) and not classified as toxic as per the CLP classification criteria. 

 

Similarly in the second first freshwater study was to evaluate the nature of test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium. Effects on the growth rate of the organism were studied. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the inhibitory concentration was > 100 mg/l. At 100 mg/l only 1.6 % inhibition was observed after the 72hrs incubation period. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

 

Above studies was supported by the experimental study report from experimental data. 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 10000 cells/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 of test chemical to various nominal test concentration, 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.

 

Based on the overall data from various sources, it can be concluded that the substance is considered to be not toxic to aquatic environment (algae and cyanobacteria) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

Description of key information

2nd: The median effective concentration (ErC50) for the test substance, on a freshwater algae Desmodesmus subspicatus was determined to be 129.4 mg/L on the basis of effects on growth rate in a 72 hour study.

3rd: Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the inhibitory concentration was 100 mg/l on that only 1.6 % inhibition was observed after the 72hrs incubation period.

4th: After 72 hours of exposure of test chemical to various nominal test concentration, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l.

Based on the overall data from various sources, it can be concluded that the substance is considered to be not toxic to aquatic environment (algae and cyanobacteria) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

Key value for chemical safety assessment

EC50 for freshwater algae:

129.4 mg/L

Additional information

Various short term studies and other data available for the test chemical including structurally and functionally similar read across chemical were reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 2.2, 11, 25, 55 and 120 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 129.4 mg/L. Potassium dichromate (K2Cr2O7) were used as a reference positive control. Effects on immobilisation were observed for 48 hours by using nonlinear regression by the software Prism 4.0. Based on this ErC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance does not exhibit toxicity to aquatic algae (Desmodesmus subspicatus) and not classified as toxic as per the CLP classification criteria. 

 

Similarly in the third study freshwater study was to evaluate the nature of test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium. Effects on the growth rate of the organism were studied. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the inhibitory concentration was > 100 mg/l. At 100 mg/l only 1.6 % inhibition was observed after the 72hrs incubation period. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

 

Above studies was supported by the experimental study report from experimental data. 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 10000 cells/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 of test chemical to various nominal test concentration, 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.

 

Based on the overall data from various sources, it can be concluded that the substance is considered to be not toxic to aquatic environment (algae and cyanobacteria) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.