Reaction products of diazotised sodium 2-amino-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate coupled with sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate, subsequently coupled with diazotised sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate

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:

2 (reliable with restrictions)

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

Details on sampling:

- Concentrations: 0, 5, 10.5, 22.05, 46.31 and 97.25 mg/L
- Sample storage conditions before analysis: Test samples were analysed immediately after sampling

Vehicle:

no

Details on test solutions:

The test solution was prepared by dissolving 100 mg of test chemical in 1000 mL of OECD medium to get the
final concentration of 100 mg/L, which was then analytically determined. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from Denmark Technical University, Denmark and are 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.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.3°C

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 5, 10.5, 22.05, 46.31 and 97.25 mg/L (spacing factor of 2.1), respectively

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): Three replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, 6480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- 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 2.1.
- Test concentrations: test concentration were: 0, 5, 10.5, 22.05, 46.31 and 97.25 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 (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):

yes

Remarks:

Potassium dichromate (K2Cr2O7) was used as a reference substance.

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

6.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Other details not known

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 97.25 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

Results with reference substance (positive control):

- Results with reference substance valid?
- 72 hr EC50: 2.94 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: Assessment of Dose Range concentrations

Sr. No	Concentrations mg/ L	Wavelength ( nm)	Absorbance	Te mperature ( °C)
1	blank	418	0 . 0001	25 °C
2	3 . 5175	418	0 . 0927	25 °C
3	10 . 5525	418	0 . 2640	25 °C
4	21 . 1050	418	0 . 5327	25 °C
5	31 . 6575	418	0 . 8196	25 °C
6	42 . 2100	418	1 . 0806	25 °C
7	52 . 7625	418	1 . 3566	25 °C

 

The absorbance and concentrations were recorded at 418 nm.

 

Table: Concentration After Analytical Determination

 

		0 Hours	0 Hours	72 Hours	72 Hours
SR. No	concentrations ( mg/ L)	Absorbance ( mean)	Analytical concentrations	Absorbance ( mean)	Analytical concentrations
1	control	0 . 00	0 . 00	0 . 00	0 . 00
2	5	0 . 13	4 . 92	0 . 14	5 . 30
3	10 . 5	0 . 26	9 . 99	0 . 29	11 . 53
4	22 . 05	0 . 56	21 . 80	0 . 60	23 . 5
5	46 . 31	1 . 13	43 . 84	1 . 19	46 . 66
6	97 . 25	0 . 51 *	99 . 45	0 . 49 *	95 . 35
Note: * = Dilutions made, concentrations were calculated accordingly.

The test concentrations were measured and  found  to remain within 80 – 120  % of nominal. Hence, the  Er C 10 was expressed based on nominal concentrations.

 

Table: CELL COUNT AND PERCENT INHIBITION

 

Experimental Flasks and Test Concentration( mg/l)	0 Hr Cell Count	24 Hr Cell Count	48 Hr Cell Count	72 Hr Cell Count	Avg Specific Growth Rate ( µ)	Mean Avg Specific Growth Rate ( µ)	Percent Inhibition(%)
control	10000	45000	100000	320000	1 . 16	1 . 15	-
control	10000	40000	95000	310000	1 . 14
control	10000	50000	100000	325000	1 . 16
5	10000	40000	100000	230000	1 . 05	1 . 06	8 . 40
5	10000	35000	80000	255000	1 . 08
5	10000	45000	80000	230000	1 . 05
10 . 5	10000	50000	65000	200000	1 . 00	0 . 99	14 . 20
10 . 5	10000	30000	70000	195000	0 . 99
10 . 5	10000	35000	70000	190000	0 . 98
22 . 05	10000	35000	55000	160000	0 . 92	0 . 92	19 . 90
22 . 05	10000	30000	60000	160000	0 . 92
22 . 05	10000	30000	55000	160000	0 . 92
46 . 31	10000	30000	50000	90000	0 . 73	0 . 74	36 . 00
46 . 31	10000	25000	50000	90000	0 . 73
46 . 31	10000	20000	45000	95000	0 . 75
97 . 25	10000	20000	40000	75000	0 . 67	0 . 66	43 . 10
97 . 25	10000	20000	40000	70000	0 . 65
97 . 25	10000	20000	45000	70000	0 . 65

 

Table: p H AND TEMPERATURE

 

Test Concentration( mg/ L)	Experimental Flasks	p H		Temperature °C
		0 Hours	72 Hours	0 Hours	72 Hours
control	R 1	7 . 85	8 . 64	21 . 3	21 . 3
control	R 2	7 . 86	8 . 59	21 . 3	21 . 3
control	R 3	7 . 99	8 . 61	21 . 3	21 . 3
5	R 1	7 . 63	8 . 60	21 . 3	21 . 3
5	R 2	7 . 68	8 . 51	21 . 3	21 . 3
5	R 3	7 . 80	8 . 51	21 . 3	21 . 3
10 . 5	R 1	7 . 64	8 . 40	21 . 3	21 . 3
10 . 5	R 2	7 . 65	8 . 23	21 . 3	21 . 3
10 . 5	R 3	7 . 70	8 . 68	21 . 3	21 . 3
22 . 05	R 1	7 . 81	8 . 45	21 . 3	21 . 3
22 . 05	R 2	7 . 82	8 . 46	21 . 3	21 . 3
22 . 05	R 3	7 . 83	8 . 48	21 . 3	21 . 3
46 . 31	R 1	7 . 81	8 . 49	21 . 3	21 . 3
46 . 31	R 2	7 . 82	8 . 50	21 . 3	21 . 3
46 . 31	R 3	7 . 75	8 . 68	21 . 3	21 . 3
97 . 25	R 1	7 . 86	8 . 11	21 . 3	21 . 3
97 . 25	R 2	7 . 86	8 . 24	21 . 3	21 . 3
97 . 25	R 3	7 . 91	8 . 28	21 . 3	21 . 3

The p H was measured at the beginning of the test and after 72 hr of exposure. The p H of the control medium did not increase by more than 1 . 5 units during the test.

 

Validity criteria fulfilled:

yes

Remarks:

* The average increase in cell count in control group is >16 fold. * The coefficent of variation between replicates was <7 % (i.e., reported to be 0.696%) * coefficent of variation in the control % CV of Average specific <35% (i.e., reported to be 6.44%).

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr ErC10 and median effect concentration (ErC50) value was determined to be 6.69 and >97.25 mg/l (nominal conc.), respectively.

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 solution was prepared by dissolving 100 mg of test chemical in 1000 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 100 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 0, 5, 10.5, 22.05, 46.31 and 97.25 mg/l (factor of 2.1), respectively was from the stock test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21.3°C and with a continuous uniform illumination of 6480 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 2.94 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.696%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 6.443%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr ErC10 and median effect concentration (ErC50) value was determined to be 6.69 and >97.25 mg/l (nominal conc.), respectively. Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be ‘not classified' as per the CLP classification criteria.

Description of key information

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr ErC10 and median effect concentration (ErC50) value was determined to be 6.69 and >97.25 mg/l (nominal conc.), respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

6.69 mg/L

Additional information

Various experimental studies of test chemical and its read across chemical were reviewed for toxicity to aquatic algae endpoint which are summarised as below:

 

In an experimental study from study report (2022) for the target chemical, 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 solution was prepared by dissolving 100 mg of test chemical in 1000 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final stock solution obtained was 100 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 0, 5, 10.5, 22.05, 46.31 and 97.25 mg/l (factor of 2.1), respectively was from the stock test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21.3°C and with a continuous uniform illumination of 6480 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 2.94 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.696%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 6.443%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr ErC10 and median effect concentration (ErC50) value was determined to be 6.69 and >97.25 mg/l (nominal conc.), respectively. Thus, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be ‘not classified' as per the CLP classification criteria.

 

Another 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.

 

For the test chemical, toxicity to aquatic algae study was carried out for 72 hrsfor assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test organism were exposed to the test chemical (2 ml) in sterile 24-well plates for 72 hrs under static conditions. Algal stock solution containing 1010000 cells/mL was added (20 μL) to each well to obtain a starting algal density of 10000 cells/mL. Untreated ISO formulation freshwater and Potassium dichromate (0.10 to 1.8 mg/l) were used as a negative and positive control. All experiments was performed in triplicates. Plates were incubated at 25±1 °C for 72 hr on an orbital shaker (90 rpm) in a thermostatic chamber. Light was provided by a 2 W LED Unit for each plate. Algal cell density was measured at the beginning and end of treatment with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc), selecting a gate of 4–14 μm or by visual scoring with a Burker's chamber. Results were expressed as effect on biomass (EbC50) and on growth rate (ErC50). All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 152.76 mg/l (95% C. I. – 134.406 to 179.274 mg/l) & on the basis of biomass, the 72 hr NOEC and EbC50 value was determined to be  < 100 mg/l and  25.904 mg/l (95% C. I. – 22.233 to 32.632 mg/l), respectively. Thus, based on the ErC50 value (152.76 mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

Overall, on the basis of above information, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be ‘not classified' as per the CLP classification criteria.