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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control) and 100 mg/l
- Sampling method: The stock solution was prepared by dissolving 10 mg of test chemical in 100 ml of M7 medium to get the final concentration of 100 mg/L, followed by analytical determination. The remaining test solutions were prepared by dilution from the above stock solution.
- Sample storage conditions before analysis: sample was analysed immediately after preparation

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Method: 10 mg of test item was transferred and made up to 100 mL in a volumetric flask using M7 medium (Stock solution). From the stock solution 0 (control) and 100 mg/L was prepared using M7 medium in a glass test vessel.
- Controls: M7 Medium (Control),
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
Not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Daphnia magna were obtained from Microbio test Belgium and maintained at Unique Ecotox Research Laboratory, Nagpur.
- Age at study initiation (mean and range, SD): <24 hr
- Length at study initiation (length definition, mean, range and SD): 0.45 cm
- Feeding during test
: No
Others: A population of Daphnia magna of synchronized age structure has been maintained for more than 2 months in the test facility under constant temperature conditions (18 to 22°C) at a 16 : 8 hour light-dark photoperiod (illumination: > 1000 lux). The culture media (M7 medium') was partly renewed twice a week. During the maintenance of test organism, test daphnids were fed with unicellular green algae (Selenestrum capricornutum).

ACCLIMATION
- Acclimation period: 48 hours prior test
- Acclimation conditions (same as test or not): Yes (Test daphnids were acclimatized)
- Type and amount of food: Food was provided during acclimatization.
- Health during acclimation (any mortality observed): healthy no mortality was observed

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

>140 mg/l as CaCO3

Test temperature:

20°C

Dissolved oxygen:

6. 5 mg/l

Nominal and measured concentrations:

100 mg/l (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 mL Beakers
- Type (delete if not applicable): Loosely covered with glass lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glass, volume 20 mL
- Volume of solution: 20 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): Duplicates
- No. of vessels per control (replicates): Duplicates
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 5 daphnids/20 mL test medium


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 1337-1346 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation
VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
Based on the available data on the test chemical, range finding test was not conducted directly main study was intuited to draw the conclusion on test substance toxicity.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as a reference substance

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Other details not known

Details on results:

- Mortality of control: No mortality in control
- Other adverse effects control: None
- Immobilisation of control: No immobilisation
- Abnormal responses: None in control

Results with reference substance (positive control):

- Results with reference substance valid?
Yes valid
24 hr EC50: 0.65 mg/L

Reported statistics and error estimates:

48h EC50 value were calculated by probit analysis using STAT Plus Software, version 8.

Table: Result of Daphnia toxicity study.

 

Nominal conc. (mg/l)

Number of Daphnids/replicate

Immobility

0h

24h

48h

Cumulative Immobilization

Percent Inhibition

control

5

R1=0

R2=0

R3=0

R4=0

R1=

R2=0

R3=0

R4=0

R1=0

R2=0

R3=0

R4=0

-

-

100

5

R1=0

R2=0

R3=0

R4=0

R1=0

R2=0

R3=0

R4=0

R1=0

R2=0

R3=0

R4=0

-

-

Any abnormal behavior(if any)

All are actively moving

All are actively moving

All are actively moving

 

Table: Temperature, DO and pH values.

 

0 Hour

Nominal conc. (mg/l)

Temperature(° C)

DO(mg/l)

pH

control

R1=21.3

R2=21.3

R3=21.3

R4=21.3

R1=7.9

R2=7.9

R3=7.9

R4=7.9

R1=8.0

R2=8.0

R3=8.0

R4=8.0

100

R1=21.3

R2=21.3

R3=21.3

R4=21.3

R1=7.7

R2=7.7

R3=7.7

R4=7.7

R1=8.1

R2=8.1

R3=8.1

R4=8.1

 

24 Hours

Nominal conc. (mg/l)

Temperature(° C)

DO(mg/l)

pH

control

R1=21.3

R2=21.3

R3=21.3

R4=21.3

R1=6.9

R2=6.9

R3=6.9

R4=6.9

R1=7.9

R2=7.9

R3=7.9

R4=7.9

100

R1=21.3

R2=21.3

R3=21.3

R4=21.3

R1=6.7

R2=6.7

R3=6.7

R4=6.7

R1=8.0

R2=8.0

R3=8.0

R4=8.0

 

 

48 hours

Nominal conc. (mg/l)

Temperature(° C)

DO(mg/l)

pH

control

R1=21.1

R2=21.1

R3=21.1

R4=21.1

R1=6.6

R2=6.6

R3=6.6

R4=6.6

R1=7.0

R2=7.0

R3=7.0

R4=7.0

100

R1=21.1

R2=21.1

R3=21.1

R4=21.1

R1=6.5

R2=6.5

R3=6.5

R4=6.5

R1=7.3

R2=7.3

R3=7.3

R4=7.3

 

 

Validity criteria fulfilled:

yes

Remarks:

In the control, there was no daphnid found immobilized or other clinical signs throughout the test. The dissolved oxygen concentration at the end of the test was reported as 6.6 mg/l in control and 6.5 mg/l in test vessel.

Conclusions:

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.

Executive summary:

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.

 

Description of key information

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.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

100 mg/L

Additional information

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.