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

Endpoint summary

Description of key information

Short-term toxicity to fish: Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l.

 

Short-term toxicity to aquatic invertebrates: Based on the effect on mobility of test daphnids, the 48-h EC50 of test chemical to Daphnia magna was determined to be >100 mg/L.

 

Toxicity to aquatic algae: 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.

 

Toxicity to microorganisms: On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l. 

Additional information

Short term toxicity to fish:

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

In an experimental study from study report (2022) for the target chemical, the study was conducted in accordance with OECD Guideline 203, Fish, Acute Toxicity Test (2019). A study was performed to assess the Acute toxicity of test chemical, to Danio rerio (Zebra fish) under static conditions. Source of test organisms was Anu Pet Shop Nagpur. Length at study initiation 1.9 cm (avg).  Weight at study initiation was 0.15 g (avg). Test organism was acclimatized for a period of 7 days. Feeding frequency during acclimation once in a day.  Food was provided during acclimatization. No mortality observed was observed during acclimatization. Continuous aeration was also provided during acclimatization.  The test solution was prepared by dissolving 400 mg of test chemical in 4000 ml of RO water to get final concentration of 100 mg/l. The same stock solution was used as test concentration. The main study (i.e. limit test) was conducted using 0 (control) and 100 mg/L test concentration. Test vessel was taken as Glass aquaria having 7 lit capacity. Type of test vessel was of open type. Total volume of solution was 4 L. Number of organisms per vessel was taken as  seven. Number of vessel per concentration was taken in replicate. The number of vessels per control was taken in the replicate. The biomass loading rate was of 7 fishes/ 4 L of test solution. The analytical determinations were performed by UV- VIS spectrophotometer. Analytical assessments were performed for selected test concentrations at 0 Hr and 96 Hr. Environmental parameters such as pH (7.7), temperature (21 to 25 °C), dissolved oxygen (86.83 %), hardness (100 mg of CaCO3), photoperiod (16 h light- 8 h dark) with light intensity of 821.25 lux was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria includes, no mortality was found in the control by the end of the test. The dissolved oxygen concentration remained above 60 % of the air- saturation value throughout, exposure period. And the test concentrations were measured and found to remain at 120-80 % of nominal concentration. Based on mortality effect of test chemical on test organism Danio rerio (Zebra fish) the 96 hr LC50 value was determined to be > 100 mg/l. As per value of LC50 the  test chemical was  considered to be ‘not classified' as per the CLP classification criteria Thus, considered as non-toxic to fishes. 

Another acute study to fish has been carried for assessing the toxic effect of test chemical. The study was conducted by following the OECD Guideline 203 (Fish, Acute Toxicity Test) in a static system for a period of 96 days. Brachydanio rerio (Zebra fish) was used as a test organism.Test chemical concentrations were verified analytically. On the basis of the effect of the test chemical on mortality of test organism Brachydanio rerio (Zebra fish), the 96 hr LC0 and LC50 value was determined to be 500 and >500 mg/l (nominal conc.), respectively. Thus on the basis of these values, test chemical was considered as non-toxic to aquatic fish and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

 

Another acute toxicity study was conducted for 96 hrs for assessing the effect of test chemical on fish. The test was performed following the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.066 g and average length of 1.7 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.0 mg/l, pH 7.4, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was were 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24°C, pH 7.2, hardness of water 150.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. No mortalities were observed in the control and test vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) value was determined to be > 100 mg/l. Thus, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be not classified as per the CLP classification criteria.

On the basis of the above-mentioned information, the test chemical was considered to be 'not classified' as per the CLP classification criteria. Thus, test chemical was considered as non-toxic to fishes.

 

 

Short term toxicity to aquatic invertebrates

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

 

In an experimental study from study report (2022) for the target chemical, this study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The test daphnids were acclimatized 48 hours prior to the test chemical exposure. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test chemical formulation. 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (i.e. limit test) was conducted using 0 (control) and 100 mg/L concentrations. 2 replicates/concentration having 5 daphnids/replicate was used for the main study. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.0), temperature (21.1-21.3°C), dissolve oxygen (6.5 - 7.9 mg/L), hardness (>140 mg CaCO3/L), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Thus, fulfilling the validity criteria. Feed was not provided during the test. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control and test groups. The 48-h EC50 of test chemical to daphnid, Daphnia magna was >100 mg/l. The 48-h EC50 of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.65 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified' as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical.The study was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)under static conditions using Daphnia magna (Water flea) as a test organism. On the basis of the effect of chemical on mobility of the test organism Daphnia magna,the 48 median effect concentration (EC50) value was determined to be >1000 mg/l.Thus, test chemical was considered as non-toxic to aquatic invertebratesat environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

 

 

For the test chemical, short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical.The test was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test).The study was performed under static conditions using Daphnia magna (Water flea) as a test organism. Test organism was taken from Daphtoxkit F™ magna, a commercial kit that provides daphnids in the form of dormant eggs (ephippia) that need to be incubate 3 days before the use to obtain neonates used for the test. Neonates less than 24 h old (25 for each condition) were fed with lyophilized spirulina as dietary supplement for 2 h prior to the test and then moved to the exposure chamber. Test chemical solutions were prepared by directly dissolving the test chemical in specific test medium (ISO 6341 Standard Freshwater). Test chemical concentration used for the study was 100 mg/l. Thus, a limit test was performed using 100 mg/l test chemical concentration. Selected bottled water was used only in the daphnid assays and satisfied the acceptability criteria for holding and dilution water described by the OECD 202 guideline. ISO 6341 Standard Freshwater was used as a test medium for the study. Media were aerated for at least 1 h prior to use in order to ensure that the dissolved oxygen concentration has reached saturation. Test daphnids (25 test organisms) were exposed with the test chemical at 20±1°C for a period of 48 hrs. No illumination of light was provided to the test vessel during the study. No significant changes in pH were recorded in the test solutions. Untreated ISO formulation freshwater was used as negative control. Potassium dichromate (0.32 – 3.2 mg/L) was used as a reference substance during the study. 48 h LC50 were derived by non-linear regression analysis. 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. Dissolved oxygen concentration was measured with HI9142 Portable Dissolved Oxygen Meter (Hanna Instruments srl) in all test chambers at the end of the test and was higher than 3 mg/l, according to the OECD 202 guidelines.On the basis of the toxic effect of the test chemical on mobility of the test organism Daphnia magna,the 48 hr median lethal concentration (LC50) value was determined to be >100 mg/l (nominal conc.). Thus, test chemical was consideredas non-toxic to aquatic invertebrates at environmental relevant concentrations andhence, considered to be ‘not classified’ as per the CLP classification criteria.

 

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

 

Toxicity to aquatic algae and cyanobacteria

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.

Toxicity to microorganism: 

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

The first study of toxicity to microorganisms was carried out according to OECD 209 guideline. Activated sludge (sludge of a Husman apparatus) was taken as test organism. Duration of study was 3 hr. Concentrations of the test substance were 1, 3.2, 10, 32 and 100 mg/l. Temperature was 21.1°C. Oxygen-consumption was measured with an electrode system. 3,5-Dichlorophenol was used as reference substance and EC50 was within the required range (10-25 mg O2/l)On the basis of toxicity to microorganism study the 3 hr EC0 and EC50 value of test chemical was determined to be 100 and > 100 mg/l respectively.  

 

The second includes study of  potential inhibitory effect of test chemical on ammonia-N oxidation by nitrosomonas was investigated. Source of inoculum was surface water samples (20ml) were collected in duplicate sets of 50ml sterile plastic containers from the upstream zone of Aba River located in Aba, Nigeria. The isolation procedure was a modification of the method adapted from Colwell and Zambruskii (1972). The target microorganisms were first enriched by inoculating 10ml of water samples into 100ml of  broth contained in a triplicate set of 250 ml Erlenmeyer flasks. Incubation was  at 28±2°C for 4 days in the dark. The culture (10ml) was then transferred into fresh sterile 100ml broth in a triplicate set of 250ml Erlenmeyer flasks and incubated as above. Two   additional   enrichments   in   broth   as described  above  were  carried  out  after  which, 0.1 ml of  enriched culture was inoculated onto a replicate  set  of  agar  plates by spread-plate method.  Plates were incubated at 28±2°C and observed for growth. Discrete rnucoid colonies which developed were picked, purified by repeated streaking and Gram stained. Isolates which were Gram-negative rods were picked and presumed to be Ntrosomonas. Isolates were further  identified by growth in nitrite-free broth. Cultures that were positive  for  presence of  nitrite  after  3  days  of incubation in the dark were tentatively identified as Nitrosomonas. Stock  cultures  were  prepared on Winogradsky agar slants and stored at 4°C. Cells were transferred from stock cultures into 20ml Winogradsky broth and incubated at 28±2°C with shaking for 48h for maximum biomass yield. The cells were washed in nitrite-free physiological saline using a vortex mixer and allowed to stand for  1 h.   The cell sediment was resuspended in fresh sterile physiological saline and washed.  The washing procedure was done repeatedly until nitrite-N was undetected thus ensuring no residual nitrite-N. Cell viability was determined by placing 1 ml inoculum into 20ml sterile ammonium sulphate solution (0.05mgL"1  ammonia-N) contained in a duplicate set of 150ml Erlenmeyer flasks and incubated as above for 3h. Samples (1ml) of the culture were tested periodically for the presence of nitrite-N. Cultures that were positive confirmed the viability of the cells and were used for bioassay. Controls consisted of autoclaved cultures. This was to show that the accumulation of nitrite-N in the experimental flasks was due to the metabolic activities of the cells and not abiotic factors. Initial biomass concentration was 3000,000 CFU. Duration of test was 8 hr. Temperature was of 28±2°C. Test vessel was  Erlenmeyer flasks. Fill volume was of 250 ml.  Number of vessels per concentration (replicates)was in  triplicate. Number of vessels per control (replicates) was in triplicate.  The Winogradsky medium phase II contained (gL·1): (NH4)2S04 2g; K2HP04 1.0; MgS04. 7H20 0.5g; NaCl 2g; FeS04. 7H20; 0.49; ZnCl2 trace amounts and deionised water 1,000ml. The pH of medium was 7.5. Sterilization was by membrane filtration (0.2µm pore size, Acrodisc). Solid medium was prepared by adding autoclaved agar No.1 (1.5%w/v) to the broth medium. The solution was prepared by dissolving 0.32mg ammonium sulphate in 970 ml deionised water and dispensed in 97ml  amounts into triplicate 250ml Erlenmeyer flasks. Into each triplicate set of flasks was added the appropriate toxicant concentration of 0.01, 0.1, 1.00, 10 and 100 mg/l. EC50 was estimated from the linear regression of the plot of ammonia oxidation rate against the concentration of toxicant. The median effective concentration EC50 of test chemical was determined to be 332.208 mg/l in the duration of 8 hr. 

 

On the basis of the experimental studies of the structurally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, based of toxicity to microorganism study the 3 to 8 hr EC50 value of test chemical was determined to be > 100 to < 332.208 mg/l. As per the value of EC50 test chemical was consider as non-toxic to microorganisms.