2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test chemical was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): Laboratory for Biological Research in Aquatic Pollution (LABRAP) at the university of Ghent in Belgium
- Method of cultivation: Bold’s Basal Medium(BBM)

ACCLIMATION
- Culturing media and conditions (same as test or not): 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
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 °C±2°C

Nominal and measured concentrations:

Nominal test chemical conc. used for the study were 0, 6.25, 12.5, 25, 50, 100 and 200 mg/l.

Details on test conditions:

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 22°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.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

185.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from equation through probit analysis

Details on results:

The microscopic observations were also noted in each of the control and experimental flasks. All the cells appeared healthy, sickled shape and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 200 mg/l.

Reported statistics and error estimates:

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.

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

 

	CONTROL		0.3 mg/l		0.39 mg/l			0.507 mg/l		0.659 mg/l			0.856 mg/l			1.112 mg/l
Average Specific Growth rate (μ )	R1	1.00	R1	1.07	R1		0.84	R1	0.66	R1		0.74	R1		0.66	R1		0.47
	R2	1.06	R2	1.02	R2		0.84	R2	0.85	R2		0.71	R2		0.75	R2		0.52
	R3	1.02
Mean of Avg. Specific growth rate	1.026		1.05			0.84		0.76			0.73			0.71			0.50
Percentage Inhibition (%I)	-		18.44			26.65		29.44			32.72			31.42			51.56

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the median effect concentration (ErC50) was determined to be 185.4 mg/l.

Executive summary:

A freshwater algal growth inhibition test 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. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. Green algae were exposed to nominal concentration 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 22±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). 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 7%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) value was determined to be 185.4 mg/l. On the basis of the EC50 value, chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Experimental study report, 2019). 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. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. Green algae were exposed to nominal concentration 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 22±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). 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 7%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) value was determined to be 185.4 mg/l. On the basis of the EC50 value, chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

185.4 mg/L

Additional information

Experimental study of the test chemical and various supporting weight of evidence studies for its structurally and functionally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2019), a freshwater algal growth inhibition test 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. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. Green algae were exposed to nominal concentration 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 22±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). 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 7%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 200 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) value was determined to be 185.4 mg/l.

 

In a supporting weight of evidence study, another freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2019). 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. The study was performed under the same test conditions as mentioned in the above study. 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 17 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).

 

For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Study report, 2016). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 200 mg/l was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept 5 min in ultrasonic bath. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Nominal test chemical conc. used for the study were 0, 0, 12.5, 25.0, 50, 100 and 200 mg/l, respectively. Study was performed in a static system for 72 hrs. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessels containing OECD medium without the test chemical were also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 276.1 mg/l (95 % C. I. 149.8 to 508.9 mg/l).

 

On the basis of the above results, it can be concluded that the test chemicalwas considered as non-toxic to aquatic algae and hence, considered to be not classified as per the CLP classification criteria