Disodium ar,ar'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[(4-chlorophenoxy)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 May 05 to June 27, 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:

validation of analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted on 13 April 2004

Deviations:

no

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 on day 0 (0 hour) and at end of test (48 hours) from all test concentrations 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:

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 solubility 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: in house maintained Daphnia magna procured, from Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Karnataka, India.
- Age of parental stock:young daphnids less than 24 hours old, derived from healthy stock showing no signs of stress, such as high mortality rate, discolored daphnids, presence of male and ephippia, delay in the production of the first brood.
- Feeding during test: no feeding was done during the test.
- Feeding: daphnids were fed with live algal cells (Pseudokirchneriella subcapitata) of 2 ml per liter during the acclimatization.

BREEDING
- Housing: daphnids were housed in cleaned glass beakers having water holding capacity of 100 ml, with controlled environmental conditions (temperature of 20.1 to 21.4 °C; pH between 7.13 to 7.69)
- Photoperiod: 16 hours of light and 08 hours dark cycle.
- Biomass loading rate: 20 ml of test solution was provided for each daphnid (i.e., a volume of 100 ml for five Daphnids per test vessel).
- Preparation of dilution weater: reconstituted water, pH 7.28 to 7.35.
- Total hardness: 216 mg/l (as CaCO3).
- Dissolved oxygen: the dilution water was aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.

ACCLIMATION
- Acclimation period: 48 hours prior to test.
- Acclimation conditions: 25 gravid female Daphinds were acclimatized in reconstituted water (similar for range finding and main study: dissolved oxygen between 7.41 to 7.51 mg/l, temperature of 20.1 and 20.4 °C, pH of 7.28 to 7.35 and hardness of 216 mg CaCO3/l.
- Type and amount of food: daphnids were fed with live algal cells.
- Feeding frequency: daphnid were fed at the beginning and on Day 2 of acclimatization.
- Choice of organisms: 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Total hardness of the reconstituted water prior to its use for exposure was 220 mg/l of CaCO3.

Test temperature:

20.1 to 20.9 °C

pH:

7.21 to 7.69

Dissolved oxygen:

7.20 to 7.42 mg/l

Nominal and measured concentrations:

2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l (corresponding to 2, 4.3, 9.4, 21, 45 and 100 mg a.i./l)

Details on test conditions:

TEST SYSTEM
- Test vessel: thoroughly cleaned rectangular glass beakers having the water holding capacity of 100 ml were used as test vessels.
- 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 in each group.
- No. of vessels per control: 4 replicates.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution weater: reconstituted water, according to the OECD guideline.
- pH: 7.28 to 7.35.
- Total hardness: 216 mg/l (as CaCO3).
- Dissolved oxygen: 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: no adjustments of pH.

EFFECT PARAMETERS MEASURED
Each test vessel was observed for immobilized Daphnids at 24 and 48 hours after the beginning of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. In addition to immobility, any abnormal behavior or appearance was recorded.

RANGE-FINDING STUDY
- No. of organisms per vessel: 5 daphnids per replicate.
- No. of vessels per concentration: 2 replicates in each group.
- Test concentrations: 0.013, 0.13, 1.26, 12.63, 63.13, 126.3 mg/l (corresponding to 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg a.i./l).
- Results used to determine the conditions for the definitive study: the immobilization percentages of 10, 30, 60 and 100 % were recorded at the tested concentrations of 1.26, 12.63, 63.13 and 126.3 mg/l, respectively, during the 48 hour observation period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

63.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No clinical signs of toxicity or immobilisation were observed in control during the 48 hour observation period.
Daphnids localized on bottom of container and lethargy were observed at the tested concentrations of 2.5, 5.4, 11.9, 26.1 and 57.4 mg/l during the 24 and 48 hour observation period.
Daphnids localized on bottom of container and lethargy were observed at the tested concentration of 126.3 mg/l during the 24 hour observation period.
The Immobilization percentages of 5, 5, 10, 25, 40 and 100 % were recorded at the tested concentrations of 2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l, during the 48 hour observation period.

RANGE-FINDING STUDY
No clinical signs of toxicity or immobilization were observed in control and at the tested concentrations of 0.013 and 0.13 mg/l during the 48 hour observation period.
Lethargy was observed at 48 hour observation at the tested concentration of 1.26 mg/l.
Daphnids localized on bottom of container and lethargy were observed at the tested concentrations of 12.63 and 63.13 mg/l during the 24 and 48 hour observation period.
Daphnids localized on bottom of container and lethargy were observed at the tested concentration of 126.3 mg/l during the 24 observation period.
The immobilization percentages of 10, 30, 60 and 100 % were recorded at the tested concentrations of 1.26, 12.63, 63.13 and 126.3 mg/l, respectively, during the 48 hour observation period.

APPEARANCE OF TEST MEDIUM
The test media was clear in almost all the test concentration during range finding and main study on day 0; on day 7 deposition of test item in the bottom of test vessels was observed at the highest test concentration of 126.3 and 157.5 mg/L during range finding and main study, respectively.

Results with reference substance (positive control):

The 48 hour EC50 of the reference substance Potassium dichromate on Daphnia magna was determined to be 0.57 mg/l. Thus, the 48 hour EC50 of the reference substance was within the acceptance range of the guideline (0.50 to 2.12 mg/l) confirming the suitability of the test system.

SUMMARY OF IMMOBILIZATION DURING THE MAIN STUDY

Test. Conc. (mg/l)	Replicate	Initial No. of Daphnids	No. of Daphnids immobilized		Cummulative No.		% of Immobilisation
			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
2.5	R1	5	0	1	0	1	0	5
	R2	5	0	0
	R3	5	0	0
	R4	5	0	0
5.4	R1	5	0	0	1	1	5	5
	R2	5	1	0
	R3	5	0	0
	R4	5	0	0
11.9	R1	5	0	0	2	2	10	10
	R2	5	1	0
	R3	5	1	0
	R4	5	0	0
26.1	R1	5	1	0	2	5	10	25
	R2	5	1	1
	R3	5	0	1
	R4	5	0	1
57.4	R1	5	1	1	4	8	20	*40
	R2	5	1	1
	R3	5	1	1
	R4	5	1	1
126.3	R1	5	2	3	10	20	50	*100
	R2	5	3	2
	R3	5	2	3
	R4	5	3	2

*: Statistical significance of >0.05.

TEST CONCENTRATIONS DURING THE MAIN STUDY

Day 0 (0 Hour)			Day 2 (48 Hour)
Nominal Concentration (mg/l)	Measured Conc. (mg/l)	% Recovery	Nominal Concentration (mg/l)	Measured Conc. (mg/l)	% Recovery
0	-	-	0	-	-
2.5	2.32	92.8	2.5	2.23	89.20
5.4	4.77	88.24	5.4	4.64	85.93
11.9	10.54	88.57	11.9	10.62	89.20
26.1	23.06	88.33	26.1	22.40	85.83
57.4	51.44	89.62	57.4	52.07	90.71
126.3	115.57	91.51	126.3	110.18	87.23

SUMMARY OF IMMOBILIZATION DURING THE RANGE-FINDING STUDY

Test. Conc. (mg/l)	Replicate	Initial No. of Daphnids	No. of Daphnids immobilized		Cummulative No.		% of Immobilisation
			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
0.013	R1	5	0	0	0	0	0	0
	R2	5	0	0
0.13	R1	5	0	0	0	0	0	0
	R2	5	0	0
1.26	R1	5	0	1	0	1	0	10
	R2	5	0	0
12.63	R1	5	1	0	2	3	20	30
	R2	5	1	1
63.13	R1	5	2	1	3	6	30	60
	R2	5	1	2
126.3	R1	5	3	2	6	10	60	100
	R2	5	3	2

TEST CONCENTRATIONS DURING THE RANGE-FINDING STUDY

Day 0 (0 Hour)			Day 2 (48 Hour)
Nominal Concentration (mg/l)	Measured Conc. (mg/l)	% Recovery	Nominal Concentration (mg/l)	Measured Conc. (mg/l)	% Recovery
0	-	-	0	-	-
0.013	-	-	0.013	-	-
0.13	0.11	84.62	0.13	0.15	115.38
1.26	1.09	86.51	1.26	1.08	85.71
12.63	10.71	84.76	12.63	11.04	87.37
63.13	53.43	84.64	63.13	54.41	86.18
126.3	107.64	85.23	126.3	111.16	88.08

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 7.20 mg/l

Conclusions:

EC50 (48 h): 63.45 mg/l (nominal)

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 to the test item under static condition. Daphnids were observed at 24 and 48 hours for signs of toxicity and immobilisation.

A range-finding test was run to determine the concentrations for the main experiment: the immobilization percentages of 10, 30, 60 and 100 % were recorded at the tested concentrations of 1.26, 12.63, 63.13 and 126.3 mg/l, respectively, during the 48 hour observation period.

Therefore, based on the results of the range finding study, main study was conducted at the test concentrations of 2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l (corresponding to 2, 4.3, 9.4, 21, 45 and 100 mg a.i./l).

No clinical signs of toxicity or immobilisation were observed in control during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy was observed at the tested concentrations of 2.5, 5.4, 11.9, 26.1 and 57.4 mg/l during the 48 hour observation period. Clinical signs of localized on bottom of container and lethargy was observed at the tested concentration of 126.3 mg/l during the 24 hour observation period.

The immobilization percentages of 5, 5, 10, 25, 40 and 100 % were recorded at the tested concentrations of 2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l, during the 48 hour observation period.

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

Based on the abovementioned results of the experiment and under experimental conditions employed, it is concluded that the 48 hour acute median effective concentration (EC50) of test item on Daphnia magna is 63.45 mg/l; the 95 % confidence interval with lower limit of 53.81 mg/l and upper limit of 73.07 mg/l.

Conclusion

EC50 (48 h): 63.45 mg/l (nominal)

Description of key information

EC50 (48 h): 63.45 mg/l (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

63.45 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 to the test item under static condition. Daphnids were observed at 24 and 48 hours for signs of toxicity and immobilisation.

A range-finding test was run to determine the concentrations for the main experiment: the immobilization percentages of 10, 30, 60 and 100 % were recorded at the tested concentrations of 1.26, 12.63, 63.13 and 126.3 mg/l, respectively, during the 48 hour observation period.

Therefore, based on the results of the range finding study, main study was conducted at the test concentrations of 2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l (corresponding to 2, 4.3, 9.4, 21, 45 and 100 mg a.i./l).

No clinical signs of toxicity or immobilisation were observed in control during the 48 hour observation period.

Clinical signs of localized on bottom of container and lethargy was observed at the tested concentrations of 2.5, 5.4, 11.9, 26.1 and 57.4 mg/l during the 48 hour observation period. Clinical signs of localized on bottom of container and lethargy was observed at the tested concentration of 126.3 mg/l during the 24 hour observation period.

The immobilization percentages of 5, 5, 10, 25, 40 and 100 % were recorded at the tested concentrations of 2.5, 5.4, 11.9, 26.1, 57.4 and 126.3 mg/l, during the 48 hour observation period.

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

Based on the abovementioned results of the experiment and under experimental conditions employed, it is concluded that the 48 hour acute median effective concentration (EC50) of test item on Daphnia magna is 63.45 mg/l; the 95 % confidence interval with lower limit of 53.81 mg/l and upper limit of 73.07 mg/l.