Reaction mass of sodium 3-[[4-[(4-ethoxy-m-tolyl)azo]-1-naphthyl]azo]benzenesulphonate and sodium 3-[[4-[(4-ethoxyphenyl)azo]-1-naphthyl]azo]benzenesulphonate

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:

From February 15 to March 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted on 13 April 2004

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of test concentration and stability of the test substance was made at the start (0 hour) on day 0 and at end of test (48 hours) from all test concentration during range finding and main study.
15 ml of samples were collected in duplicates from each group during range finding and main study.

Vehicle:

no

Details on test solutions:

Based on the in house dissolution test, the substance forms suspension in reconstituted water. Hence rno vehicle was used.
Stock solution was freshly prepared prior to exposure, by weighing required quantity of test item in a beaker, a small volume of reconstituted water was added and mixed well using a glass rod. After complete suspension of the test item, test formulation was transferred into a measuring cylinder and the beaker was rinsed with reconstituted water and transferred again to the measuring cylinder, rinsing process was repeated until the complete transfer of test contents. Stock solution prepared kept on magnetic stirrer to maintain homogeneity. Test medium of chosen concentration was prepared by dilution of stock solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Karnataka, India.
- Age of parental stock: young daphnids less than 24 hours old were selected for the treatment. Progenies produced on the first day are not used. Progenies produced on day two (less than 24 hours old) are transferred into the test containers and these Daphnids were used for range finding as well as main study respectively.
- Feeding during test: no feeding was done during the test.

ACCLIMATION
- Acclimation period: 25 gravid female daphinds were acclimatized in reconstituted water for 48 hours prior to test (similar for range finding and main study).
- Type and amount of food: daphnids were fed with live algal cells (Pseudokirchneriella subcapitata), 2 ml per liter during the acclimatization.
- Feeding frequency: daphnid was fed with live algal cells at the beginning and on Day 2 of acclimatization.
- Conditions: dissolved oxygen was between 6.53 to 6.57 mg/l, temperature of 20.1 and 20.4 °C, pH of 6.69 to 6.73 and hardness of 216 mg CaCO3/l.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Control: 20.1 - 20.4 °C
Test vessels: 20.0 - 20.9 °C

pH:

Control: 7.34 - 7.54
Test vessels: 7.34 - 7.77

Dissolved oxygen:

Control: 6.69 - 7.10 mg/l
Test vessels: 6.64 - 7.21 mg/l

Nominal and measured concentrations:

Nominal 5.3, 11.7, 25.8, 56.8 and 125.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: daphnids were housed in cleaned glass beakers having water holding capacity of 100 ml, with controlled environmental conditions.
- Aeration: test vessels was not be aerated during the test.
- No. of organisms per vessel: 5 daphnids per replicate.
- No. of vessels per concentration: 4 replicates.
- No. of vessels per control: 4 replicates.
- Biomass loading rate: 20 ml of test solution was provided for each daphnid.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: reconstituted water with the pH 7.34 to 7.77, total hardness 216 mg/l(as CaCO3). The dilution water was aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.

OTHER TEST CONDITIONS
- Adjustment of pH: none.
- Photoperiod: 16 hours of light and 08 hours dark cycle.

EFFECT PARAMETERS MEASURED
- Clinical Signs of Toxicity and Immobility: each test vessel was observed for immobilized Daphnids at 24 and 48 hours after the beginning of the test, in addition any abnormal behavior or appearance was recorded.
- Environmental Parameters: total hardness of the diluent water (reconstituted water) prior to its use for exposure was analyzed. Temperature, pH and dissolved oxygen were recorded at the beginning and at end of the test in control and in all the tested concentrations.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.
- Results used to determine the conditions for the definitive study: percentage of immobilization of 10, 40 and 80 % was recorded at the tested concentrations of 10.0, 50.0 and 100.0 mg/l.

VALIDITY CRITERIA OF THE TEST
- Immobilisation in the control group should not exceed 10 %
- Dissolved oxygen concentration at the end of the test should be ≥ 3 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Remarks:

Finney’s probit analysis

Effect conc.:

28.15 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No clinical signs of toxicity or immobility were observed in control and in the tested concentration of 5.3 mg/l during the 48 hour observation period.
Clinical signs of localized on bottom of container and lethargy was observed in the tested concentrations of 11.7, 25.8 and 56.8 mg/l during the 48 hour observation period.
Clinical sign of localized on bottom of container was observed at 24 hour observation in the tested concentration of 125 mg/l.
The immobilization percentages of 15, 45, 65 and 100 % were recorded at the tested concentrations of 11.7, 25.8, 56.8 and 125 mg/l, respectively, during the 48 hour observation period.

RANGE-FINDING
During dose range finding study, no immobilization was observed in control and in the tested concentrations of 0.01, 0.1 and 1.0 mg/l during the 48 hour observation period. Clinical signs of localized on bottom of container and lethargy was observed in the tested concentrations of 10.0, 50.0 and 100.0 mg/l; the immobilization percentages of 10, 40 and 80 % was recorded at the tested concentrations of 10.0, 50.0 and 100.0 mg/l, respectively.

MEASURED CONCENTRATIONS
Results reported that test concentrations at the 0 hour and stability at 48 hour were in the acceptable range of ± 20 % recovery to the nominal concentrations.

Results with reference substance (positive control):

The 48 hours acute median effective concentration (EC50) of Potassium dichromate on Daphnia magna is 0.57 mg/l.

SUMMARY OF CLINICAL SIGNS AND IMMOBILIZATION DURING THE MAIN STUDY

Conc. (mg/l)	Replicate	Initial no. of Daphnids	No. of Daphnids immobilized		Cummulative No.		% of Immobilization
			24 h	48 h	24 h	48 h	24 h	48 h
0	R1	5	0	0	0	0	0	0
	R2	5	0	0
	R3	5	0	0
	R4	5	0	0
5.3	R1	5	0	0	0	0	0	0
	R2	5	0	0
	R3	5	0	0
	R4	5	0	0
11.7	R1	5	0	1	2	3	10	15
	R2	5	1	0
	R3	5	1	0
	R4	5	0	0
25.8	R1	5	2	2	6	9	30	45
	R2	5	2	0
	R3	5	1	1
	R4	5	1	0
56.8	R1	5	3	1	8	13	40	65
	R2	5	2	2
	R3	5	2	1
	R4	5	1	1
125	R1	5	4	1	13	20	65	100
	R2	5	3	2
	R3	5	3	2
	R4	5	3	2

Conc. (mg/l)	Replicate	No. of Daphnids per replicate at start 0 h	Signs of toxicity and immobility of Daphnids Observed at
			24 h		48 h
			Toxic signs	Immobilization	Toxic signs	Immobilization
0	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
	R3	5	N(5)	0	N(5)	0
	R4	5	N(5)	0	N(5)	0
5.3	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
	R3	5	N(5)	0	N(5)	0
	R4	5	N(5)	0	N(5)	0
11.7	R1	5	N(4), B(1)	0	N(4)	1
	R2	5	N(3), B(1)	1	N(3), B(1)	0
	R3	5	N(3), B(1)	1	N(2), B(1), L(1)	0
	R4	5	N(2), B(1), L(2)	0	N(2), B(1), L(2)	0
25.8	R1	5	N(1), B(2)	2	N(1)	2
	R2	5	N(1), B(1), L(1)	2	N(1), B(1), L(1)	0
	R3	5	N(1), B(2), L(1)	1	N(1), B(2)	1
	R4	5	N(2), B(1), L(1)	1	N(2), B(1), L(1)	0
56.8	R1	5	N(1), B(1)	3	B(1)	1
	R2	5	N(1), B(2)	2	L(1)	2
	R3	5	N(1), B(2)	2	L(1), B(1)	1
	R4	5	N(1), B(3)	1	L(1), B(2)	1
125	R1	5	B(1)	4	-	1
	R2	5	B(2)	3	-	2
	R3	5	B(2)	3	-	2
	R4	5	B(2)	3	-	2

N: Normal; B: Localized on Bottom; L: Lethargy

Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

SUMMARY OF CLINICAL SIGNS AND IMMOBILIZATION DURING RANGE FINDING STUDY

Conc. (mg/l)	Replicate	Initial No. of Daphnids	N. daphnids immobilized		Cummulative No.		% of Immobilization
			24 h	48 h	24 h	48 h	24 h	48 h
0	R1	5	0	0	0	0	0	0
	R2	5	0	0
0.01	R1	5	0	0	0	0	0	0
	R2	5	0	0
0.1	R1	5	0	0	0	0	0	0
	R2	5	0	0
1.0	R1	5	0	0	0	0	0	0
	R2	5	0	0
10	R1	5	0	1	0	1	0	10
	R2	5	0	0
50	R1	5	1	1	2	4	20	40
	R2	5	1	1
100	R1	5	3	1	5	8	50	80
	R2	5	2	2

Conc. (mg/l)	Replicate	No. of Daphnids per Replicate at Start 0 h	Signs of Toxicity and Immobility of Daphnids Observed at
			24 h		48 h
			Toxic signs	Immobilization	Toxic signs	Immobolization
0	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
0.01	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
0.1	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
1.0	R1	5	N(5)	0	N(5)	0
	R2	5	N(5)	0	N(5)	0
10	R1	5	N(4), B(1)	0	N(4)	1
	R2	5	N(5)	0	N(4), B(1)	0
50	R1	5	N(3), L(1)	1	N(2), L(1)	1
	R2	5	N(3), B(1)	1	N(2), B(1)	1
100	R1	5	B(2)	3	B(1)	1
	R2	5	B(1), L(2)	2	L(1)	2

N: Normal; B: Localized on Bottom; L: Lethargy

Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

TEST CONCENTRATION ANALYSIS IN TEST MEDIA DURING RANGE FINDING AND MAIN STUDY (RECONSTITUTED WATER)

Main study

Nominal Concentration (mg/l)	Day 0 (0 Hour)		Day 2 (48 Hour)
	Measured conc. (mg/l)	% Recovery	Measured conc. (mg/l)	% Recovery
0	-	-	-	-
5.3	5.44	102.55	5.35	100.85
11.7	11.65	99.57	11.65	99.57
25.8	27.11	105.06	26.97	104.52
56.8	57.65	101.5	58.32	102.68
125	119.08	95.27	119.77	95.82

Range finding study

Nominal Concentration (mg/l)	Day 0 (0 Hour)		Day 2 (48 Hour)
	Measured conc. (mg/l)	% Recovery	Measured conc. (mg/l)	% Recovery
0	-	-	-	-
0.01	-	-	-	-
0.1	-	-	-	-
1.0	0.97	97.7	0.97	96.5
10	9.67	96.7	9.60	96.0
50	48.34	96.67	47.49	94.98
100	96.47	96.47	96.71	96.71

TEST CONDITIONS

Main study

Concentration (mg/l)	Replicate	Dissolved Oxygen (mg/l)		Temperature (°C)		pH
		0 h	48 h	0 h	48 h	0 h	48 h
0	R1	6.81	6.69	20.1	20.4	7.54	7.43
	R2	6.91	6.73	20.4	20.3	7.49	7.41
	R3	6.89	6.77	20.4	20.1	7.5	7.39
	R4	7.10	6.89	20.3	20.4	7.53	7.47
5.3	R1	6.84	6.73	20.2	20.3	7.5	7.44
	R2	6.87	6.74	20.0	20.2	7.52	7.46
	R3	7.21	6.81	20.9	20.8	7.54	7.5
	R4	6.94	6.73	20.7	20.4	7.51	7.41
11.7	R1	6.79	6.64	20.4	20.6	7.53	7.49
	R2	6.83	6.69	20.1	20.3	7.55	7.47
	R3	6.91	6.72	20.4	20.6	7.54	7.39
	R4	6.97	6.81	20.1	20.3	7.47	7.41
25.8	R1	6.88	6.67	20.3	20.4	7.55	7.47
	R2	6.98	6.81	20.4	20.6	7.56	7.34
	R3	6.91	6.73	20.5	20.4	7.55	7.43
	R4	7.21	6.61	20.3	20.7	7.56	7.44
56.8	R1	6.84	6.73	20.4	20.6	7.61	7.56
	R2	6.83	6.73	20.9	20.4	7.59	7.47
	R3	6.94	6.81	20.3	20.5	7.6	7.53
	R4	6.84	6.73	20.5	20.6	7.57	7.51
125	R1	6.89	6.77	20.9	20.4	7.77	7.69
	R2	6.81	6.75	20.4	20.5	7.61	7.54
	R3	6.84	6.64	20.3	20.6	7.77	7.63
	R4	6.93	6.81	20.4	20.5	7.76	7.67

Range finding study

Concentration (mg/l)	Replicate	Dissolved Oxygen (mg/l)		Temperature (°C)		pH
		0 h	48 h	0 h	48 h	0 h	48 h
0	R1	7.39	7.24	20.4	20.1	7.39	7.37
	R2	7.43	7.29	20.9	20.7	7.4	7.41
0.01	R1	7.32	7.26	20.8	20.5	7.43	7.39
	R2	7.38	7.28	20.4	20.1	7.45	7.42
0.1	R1	7.37	7.18	20.9	20.8	7.69	7.71
	R2	7.29	7.09	20.6	20.4	7.46	7.39
1.0	R1	7.43	7.27	20.4	20.3	7.64	7.57
	R2	7.46	7.31	20.5	20.4	7.66	7.61
10	R1	7.33	7.29	20.9	20.7	7.66	7.61
	R2	7.41	7.30	20.8	20.6	7.69	7.57
50	R1	7.38	7.28	20.4	20.4	7.71	7.63
	R2	7.3	7.18	20.4	20.3	7.74	7.71
100	R1	7.29	7.09	20.8	20.6	7.69	7.61
	R2	7.27	7.17	20.4	20.1	7.77	7.67

Validity criteria fulfilled:

yes

Remarks:

Immobilisation in the control group was 0 % at the termination of the test during main study and the dissolved oxygen concentration at the end of the test was 7.86 mg/l

Conclusions:

EC50 (48h): 28.15 mg/l (estimated, based on nominal conc.)

Executive summary:

The test item was evaluated for acute toxicity on Daphnia magna as per the OECD Guideline No. 202, “Daphina sp., Acute Immobilisation Test”. Daphnids were exposed for the test item under static conditions, over a period of 48 hours.

In order to set the concentrations for the main experiment, a range-finding test was conducted. At 0.01, 0.1 and 1.0 mg/l no clinical signs of toxicity or immobility were observed in both test vessels and control. Clinical signs of localized on bottom of container and lethargy was observed in the tested concentrations of 10.0, 50.0 and 100.0 mg/l.

Based on the results of the range finding study, the main study was conducted at the test concentrations of 5.3, 11.7, 25.8, 56.8 and 125.0 mg/l. No clinical signs of toxicity or immobility were observed in control and in the tested concentration of 5.3 mg/l during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy was observed in the tested concentrations of 11.7, 25.8 and 56.8 mg/l during the 48 hour observation period. Clinical sign of localized on bottom of container was observed at 24 hour observation in the tested concentration of 125 mg/l.

Immobilization percentages were 15, 45, 65 and 100 % at the tested concentrations of 11.7, 25.8, 56.8 and 125.0 mg/l during the 48 hour observation period.

During the range finding and the main studies, samples from all the test concentrations was collected at 0 hour and at 48 hour analyzed for the test concentrations and stability. The test concentrations were in the acceptable range of ± 20 % recovery to the nominal concentrations.

Conclusion

EC50 (48h): 28.15 mg/l (estimated, based on nominal conc.)

Description of key information

EC50 (48h): 28.15 mg/l (estimated, based on nominal conc.)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

28.15 mg/L

Additional information

The test item was evaluated for acute toxicity on Daphnia magna, as per the OECD Guideline No. 202. Daphnids were exposed for the test item under static conditions, over a period of 48 hours. Based on the results of the range finding study, the main study was conducted at the test concentrations of 5.3, 11.7, 25.8, 56.8 and 125.0 mg/l. No clinical signs of toxicity or immobility were observed in control and in the tested concentration of 5.3 mg/l during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy was observed in the tested concentrations of 11.7, 25.8 and 56.8 mg/l during the 48 hour observation period. Clinical sign of localized on bottom of container was observed at 24 hour observation in the tested concentration of 125 mg/l.

Immobilization percentages were 15, 45, 65 and 100 % at the tested concentrations of 11.7, 25.8, 56.8 and 125.0 mg/l during the 48 hour observation period.

During the range finding and the main studies, samples from all the test concentrations was collected at 0 hour and at 48 hour analyzed for the test concentrations and stability. The test concentrations were in the acceptable range of ± 20 % recovery to the nominal concentrations.