Reaction mass of sodium amino-bis{[4-(ethenylsubstituted)phenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-{[4-(ethenylsubstituted)phenyl]diazenyl}-{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-bis{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate

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:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

None

Vehicle:

yes

Details on test solutions:

None

Test organisms (species):

Daphnia magna

Details on test organisms:

Not available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Each test vessel was checked for immobilised daphnids and any abnormal behavior or appearance at 24 and 48 hours
Daphnids that were not able to swim within 15 seconds, after gentle agitation of the test vessel were considered to be immobilised (even if they can still move their antennae). On completion of test, dead daphnids found and all live daphnids (were euthanized with MS222 medium) were discarded in the drain along with the test solutions

Hardness:

213 mg/L

Test temperature:

Control : 21.7 - 21.8 °C
Treatment : 22.1 – 23.8 °C

pH:

Control : 7.5 - 7.8
Treatment : 7.4 - 7.7

Dissolved oxygen:

Control : 7.82 – 7.89 mg/L
Treatment : 7.53 – 7.85 mg/L

Salinity:

Not available

Nominal and measured concentrations:

0 hour – Mean Recovery results: 91.91 %, 93.19 % and 95.75 % for 2.5 mg/L, 26.62 mg/L and 128.84 mg/L concentrations respectively.
48 hour – Mean Recovery results: 90.43 %, 91.92 % and 94.35 % for 2.5 mg/L, 26.62 mg/L and 128.84 mg/L concentrations respectively.

Details on test conditions:

None

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

34.58 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mobility

Details on results:

None

Results with reference substance (positive control):

Acute immobilisation toxicity study in Daphnia magna, was performed with Potassium Dichromate Pure (Batch No. MG0M602109) in accordance with the guideline OECD 202. Daphnids were exposed to a range of five different test concentrations viz., 0.4, 0.72, 1.30, 2.33 and 4.20 mg/L. Four replicates (5 daphnids in each test replicate) were maintained for each test concentration and control group was maintained along with the test concentrations. The test item was formulated with M4 medium.
The daphnids with brood chambers were acclimatized one day prior to the exposure in the dilution water (M4 medium). After the exposure on day ‘0’, daphnids were observed for immobilisation approximately at ‘24’ and ‘48’ hours and the same was recorded.
No immobilisation was observed in control and daphnids exposed to 0.4 mg/L throughout the experimental period. At the end of ‘24’ hour 3/20, 5/20, 12/20 and 20/20 immobility was observed in daphnids exposed to test concentrations of 0.72, 1.30, 2.33 and 4.20 mg/L respectively. Similarly, at the end of ‘48’ hour 5/20, 8/20, 16/20 and 20/20 cumulative immobilisation was observed in daphnids exposed to test concentrations of 0.72, 1.30, 2.33 and 4.20 mg/L respectively. Thus the percent immobilisation at the end of ‘48’ hour was observed to be 0%, 25%, 40 %, 80% and 100% in daphnids exposed to 0.4, 0.72, 1.30, 2.33 and 4.20 mg/L respectively.
Dissolved oxygen, pH and temperature were measured in bulk of control and test concentrations at the beginning and end of the test in control and in all the replicates of control and test concentrations. Hardness was analyzed once at the start of the experiment in the control.
Dose verification analysis was carried out in the test concentrations of low (0.4 mg/L), intermediate (1.30 mg/L), high concentration (4.20 mg/L) and control (M4 medium) at ‘0’ and ‘48’ hours. The mean recovered active ingredient percentage in the samples were found to be 91.73%, 92.46% and 95.01% at ‘0’ hour whereas, 90.31%, 91.59% and 94.67% at ‘48’ hour for 0.4 mg/L, 1.30 mg/L and 4.20 mg/L concentrations respectively.

CONCLUSION

Based on the test results, the EC50of Potassium Dichromate Pure in Daphnia magna observed for a period of ‘24’ hour was calculated as 1.72 mg/L with upper confidence limits of 2.12 mg/L and lower confident limits of 1.40 mg/L and regression equation Y = 4.0957 + 3.8604 log (x). At ‘48’ hour, the EC50 was calculated as 1.33 mg/L with upper confidence limits of 1.63 mg/L and lower confidence limits of 1.08 mg/L and regression equation Y = 4.5239 + 3.8769 log (x).

Reported statistics and error estimates:

Hour EC50
(mg/L) LCL
(mg/L) UCL
(mg/L) Regression equation
48 34.58 21.20 60.32 Y = 1.3269 + 2.387 log (x)

 IMMOBILISATION - MAIN EXPERIMENT

Concentration (mg/L)	No. of Daphnids/Replicate	Immobility
		24 hour					48 hour (*)
		R1	R2	R3	R4	(%)	R1	R2	R3	R4	(%)
Control	10	0	0	0	0	0	0	0	0	0	0
2.5	10	0	0	0	0	0	0	0	0	0	0
5.5	10	0	0	0	0	0	0	1	2	0	7.5
12.1	10	0	0	0	0	0	1	2	2	0	12.5
26.62	10	0	0	0	0	0	3	4	3	4	35
58.56	10	0	0	0	0	0	6	7	6	5	60
128.84	10	5	7	6	7	62.5	10	10	10	10	100

Key: mg/L - milligram/liter, No. - Number, R-Replicate, % - percentage

        *- Cumulative Immobility

  PHYSICO CHEMICAL PARAMETERS – MAINEXPERIMENT 

Concentration	Control		2.5 mg/L		5.5 mg/L
	0 hour	48 hour	0 hour	48 hour	0 hour	48 hour
pH	7.5	7.8	7.5	7.7	7.4	7.6
Temperature °C	21.7	21.8	22.1	22.7	22.2	22.9
Dissolved oxygen (mg/L)	7.89	7.82	7.85	7.79	7.83	7.77
Hardness* (mg/L)	213	-	-	-	-	-
Concentration	12.1 mg/L		26.62 mg/L		58.56 mg/L
	0 hour	48 hour	0 hour	48 hour	0 hour	48 hour
pH	7.4	7.6	7.4	7.6	7.4	7.6
Temperature °C	22.3	22.9	22.3	23.1	22.7	23.5
Dissolved oxygen (mg/L)	7.76	7.70	7.76	7.68	7.62	7.53
Concentration	128.84 mg/L		-		-
	0 hour	48 hour	-	-	-	-
pH	7.4	7.5	-	-	-	-
Temperature °C	23.1	23.8	-	-	-	-
Dissolved oxygen (mg/L)	7.68	7.60	-	-	-	-

Key: °C – Degree centigrade, mg/L-milligram per liter, * - Analysed at the start of the e

 STATISTICAL TABLE 

Hour	EC50 (mg/L)	LCL (mg/L)	UCL (mg/L)	Regression equation
48	34.58	21.20	60.32	Y = 1.3269 + 2.387 log (x)

Key:   EC – Effective Concentration, mg/L – milligram/Litre, UCL – upper confidence limits,
LCL – lower confidence limits

Validity criteria fulfilled:

yes

Conclusions:

Based on the test results, the EC50 of FAT 40865/A TE in Daphnia magna observed for a period of 48 hour was calculated as 34.58 mg/L with upper confidence limits of 60.32 mg/L and lower confidence limits of 21.20 mg/L and regression equation Y = 1.3269 + 2.387 log (x).

Executive summary:

An acute immobilization study in Daphnia magna was conducted in accordance with OECD Guideline 202 with FAT 40865/A TE. A main experiment was conducted with a range of concentrations viz., 2.5, 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group was maintained. The test item was formulated with dilution water (M4 Medium). The daphnids with brood chambers were acclimatized one day prior to the exposure in M4 medium. After the exposure on day 0, daphnids were observed for immobilisation at approximately 24 hour and 48 hour and the respective data was recorded. Water Parameters viz.,pH, temperature and dissolved oxygen were recorded at the start and end of the experiment in control and all test concentrations. Hardness was recorded in control at the start of the test. Analytical verification of the test item in the dilution water (M4 medium) samples were performed in the test item concentrations of low (2.5 mg/L), intermediate (26.62 mg/L) and high (128.82 mg/L) at 0 hour and 48 hour. The mean recoveries data are given below:

0 hour – Mean Recovery results: 91.91, 93.19 and 95.75 % for 2.5, 26.62 and 128.84 mg/L concentrations respectively.

48 hours – Mean Recovery results: 90.43, 91.92 and 94.35 % for 2.5, 26.62 and 128.84 mg/L concentrations respectively.

No immobilization was observed in control and daphnia exposed to 2.5 mg/L concentration throughout the experimental period. At the end of 24 hour, no immobilization was recorded in the daphnia exposed to any of the test concentrations except 128.84 mg/L in which 25/40 immobilisation was observed. At the end of 48 hour, 3/40, 5/40, 14/40, 24/40 and 40/40 cumulative immobility were observed in daphnids exposed to 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L respectively.

Thus the percent immobilisation at the end of 48 hours was recorded to be 0, 7.5, 12.5, 35, 60 and 100% in 2.5, 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L respectively.

Based on the test results, the EC50 of FAT 40865/A TE in Daphnia magna observed for a period of 48 hour was calculated as 34.58 mg/L with upper confidence limits of 60.32 mg/L and lower confidence limits of 21.20 mg/L and regression equation Y = 1.3269 + 2.387 log (x).

Description of key information

The EC50 of  FAT 40865/A TE to Daphnia magna observed for a period of 48 hours was calculated as 34.58 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

34.58 mg/L

Additional information

A key study was performed to find out the acute immobilization toxicity in Daphnia magna in accordance with OECD guideline 202 with FAT 40865/A TE. A main experiment was conducted with a range of concentrations viz.,2.5, 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L. Forty daphnids with four replicates (10 daphnids in each replicate) for each test concentration and control group was maintained. The test item was formulated with dilution water (M4 Medium).

No immobilization was observed in control and daphnia exposed to 2.5 mg/L concentration throughout the experimental period. At the end of 24 hour, no immobilization was recorded in the daphnia exposed to any of the test concentrations except 128.84 mg/L in which 25/40 Daphnia were found to be immobilised. At the end of 48 hours, 3/40, 5/40, 14/40, 24/40 and 40/40 cumulative immobility were observed in daphnids exposed to 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L, respectively. Thus the percent immobilisation at the end of 48 hour was recorded to be 0%, 7.5%, 12.5%, 35%, 60% and 100% in 2.5, 5.5, 12.1, 26.62, 58.56 and 128.84 mg/L respectively.

Based on the test results, the EC50 of FAT 40865/A TE in Daphnia magna observed for a period of 48 hour was calculated as 34.58 mg/L with upper confidence limits of 60.32 mg/L and lower confidence limits of 21.20 mg/L and regression equation Y = 1.3269 + 2.387 log (x).