Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

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 substances

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: as mentioned below

Principles of method if other than guideline:

WoE report is based on three toxicity to aquatic algae studies as-
WoE 2., WoE 3 and WoE 4.

GLP compliance:

not specified

Analytical monitoring:

yes

Remarks:

WoE 2 and WoE 3: yes, WoE 4: not specified

Vehicle:

no

Remarks:

WoE 2 and WoE 3: no, WoE 4: not specified

Details on test solutions:

WoE 2: The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 894.47 mg/L. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml.

WoE 3: The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD Media This stock solution was stirred for 48 hours to obtain a homogenous solution for the experiment.

WoE 4: no data available

Test organisms (species):

other: WoE 2 and WoE 3: Pseudokirchneriella subcapitata, WoE 4: green algae

Details on test organisms:

WoE 2:
TEST ORGANISM
- Common name: green algae
- Size: 8 – 14 μm length 2 - 3 μm width
- Cell density/biomass concentration: 5000-10000 cells/ml
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the university of
Ghent in Belgium.

WoE 3:
TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained at Unique Ecotox Research Laboratory, Nagpur.
- Method of cultivation: OECD Medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium. 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 OECD Medium.
- Any deformed or abnormal cells observed: no

WoE 4: no data available

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

WoE 2: 21 to 24 ± 2°C
WoE 3: 22 °C±2°C
WoE 4: no data available

Nominal and measured concentrations:

WoE 2: Test chemical concentrations used for the study were 0, 6.25, 12.5, 25, 50, 100, 200 mg/L (nominal concentrations), respectively.
WoE 3: Nominal concentration - 100 mg/l
WoE 4: no data available

Details on test conditions:

WoE 2:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer - The absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to
observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 0, 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 24° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

WoE 3:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000 cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Three replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was OECD medium. 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 OECD Medium.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination (3000-4000Lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer - The absorbance values of each test vessel and control vessel was noted at 684nm.The OECD Media was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a automated cell counter.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms

Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (3000-4000Lux)
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.

WoE 4: no data available

Reference substance (positive control):

yes

Remarks:

WoE 2: Potassium dichromate (K2Cr2O7). WoE 3 and WoE 4: no data available

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: WoE 2: calculated from equation through probit analysis

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: AUG (biomass integral)

Remarks on result:

other: WoE 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: AUG (biomass integral)

Remarks on result:

other: WoE 4

Reported statistics and error estimates:

WoE 2: 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.
WoE 3: The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, sickle shaped and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.
WoE 4: no data available

Validity criteria fulfilled:

not specified

Conclusions:

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 96 hr LC50 value can be expected to be > 65 mg/l. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.

Executive summary:

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic fishes. The studies are as mentioned below:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 6.25, 12.5, 25, 50, 100 and 200 mg/L) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) was determined to be > 200 (nominal concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. The study was performed using the same test procedure and under test conditions as mentioned in the above study. The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD Media This stock solution was kept for stirring for 48 hours to obtain a homogenous solution for the experiment. The stock solution was analyzed analytically by using double beam VWR spectrophotometer at day 0 and day 3, which has been satisfactorily maintained within ± 20 % of the nominal initial concentration throughout the test. Green algae were exposed to nominal concentration of test chemical (100 mg/l) in 100 ml conical flasks for 72 hrs. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 7%, thus, fulfilling the validity of the criteria. In control and test vessel, all cells appeared healthy, sickle shape and green throughout the study duration. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs EC50 value was determined to be > 100 mg/l. Thus, based on the EC50 value, chemical can be considered as non-toxic to aquatic algae and thus can be considered to be not classified as per CLP classification criteria.

For the test chemical, toxicity to green algae study was carried out for assessing the effect of the test chemical. The study was conducted under static conditions for 72 hrs. On the basis of growth rate and AUG (biomass integral) obtained after exposure period of 72 hours, the median effect concentration (EC50) was determined to be > 65 and 20 mg/l & the NOEC value was evaluated to be 6.3 and 3.3 mg/l, respectively. Thus, based on the EC50 value, chemical can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 96 hr LC50 value can be expected to be > 65 mg/l. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.

Description of key information

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 96 hr LC50 value can be expected to be > 65 mg/l. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.

Additional information

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic fishes. The studies are as mentioned below:

 

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 6.25, 12.5, 25, 50, 100 and 200 mg/L) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). Apart from this, the cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) was determined to be > 200 (nominal concentration). On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. The study was performed using the same test procedure and under test conditions as mentioned in the above study. The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD Media This stock solution was kept for stirring for 48 hours to obtain a homogenous solution for the experiment. The stock solution was analyzed analytically by using double beam VWR spectrophotometer at day 0 and day 3, which has been satisfactorily maintained within ± 20 % of the nominal initial concentration throughout the test. Green algae were exposed to nominal concentration of test chemical (100 mg/l) in 100 ml conical flasks for 72 hrs. As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 7%, thus, fulfilling the validity of the criteria. In control and test vessel, all cells appeared healthy, sickle shape and green throughout the study duration. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs EC50 value was determined to be > 100 mg/l. Thus, based on the EC50 value, chemical can be considered as non-toxic to aquatic algae and thus can be considered to be not classified as per CLP classification criteria.

 

For the test chemical, toxicity to green algae study was carried out for assessing the effect of the test chemical. The study was conducted under static conditions for 72 hrs. On the basis of growth rate and AUG (biomass integral) obtained after exposure period of 72 hours, the median effect concentration (EC50) was determined to be > 65 and 20 mg/l & the NOEC value was evaluated to be 6.3 and 3.3 mg/l, respectively. Thus, based on the EC50 value, chemical can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 96 hr LC50 value can be expected to be > 65 mg/l. Since the test chemical is readily biodegradable in nature, test chemical was considered as non-toxic and hence, considered to be not classified as per CLP classification criteria.