4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

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:

2017-04-19 to 2017-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L and control
- Sampling method: Analytical samples taken at 0 hours (initial value) and 24 hours from fresh and after 24 and 48 hours from aged test solutions were analysed from control and the highest test concentration 100 mg/L.
- Sample storage conditions before analysis: After sampling, the test medium samples (10 mL with 0.5 mL formic acid) were stored deep-frozen (below -18 °C).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution S1: weight necessary amount of test item in volumetric flask and added test medium up to the bench mark, then homogenised by shaking. Lower test solutions: dilution appropriate solution with test medium
- Test concentration: 100, 50.0, 25.0, 12.5 and 6.25 mg/L and control

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone V
- Source: originally purchased from the Federal Environment Agency in Berlin/Germany
- Age at Test Start: Less than 24 hours
- Breeding Conditions: feed with single cell green algae (Desmodesmus subspicatus) at least three times a week.
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

232 mg CaCO3/L

Test temperature:

19.1 to 21.2 °C

pH:

7.61-8.01

Dissolved oxygen:

8.8-9.2 mg/L

Nominal and measured concentrations:

Nominal: 100, 50.0, 25.0, 12.5 and 6.25 mg/L and control

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel (100 mL)
- Type: closed
- Material, size, headspace, fill volume: 100 mL, filled up with ~ 50 mL test solution
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: ~ 10 mL of test solution for each animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- pH: 7.61
- Dissolved oxygen concentration: 9.2 mg/L
- Total hardness:13°dH (232 mg/L as CaCO3)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod /8 hours darkness daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Biological Assessment: after 24 h and 48 h: count the immobilised daphnids and record abnormal behaviour

RANGE-FINDING STUDY
- Test concentrations: 100, 10.0 and 1.00 mg/L and control
- Results used to determine the conditions for the definitive study: After 48 hours of exposure no immobilisation was observed in the control and at test item concentration of 1.00 and 10.0 mg/L. 35 % of the daphnids were immobile at at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

79 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Biological Assessment: After 24 hours of exposure no immobilisation was observed in the control and up to and including 100 mg/L. In the test item concentrations of 50.0 and 100 mg/L some of the daphnids were pent at the surface of the test solutions, but were still considered mobile.
After 48 hours of exposure no immobilisation was observed in the control and up to and including 100 mg/L. In the test item concentrations of 12.5 and 25.0 mg/L some of the daphnids did pend at the surface of the test solutions. Since the substance is borderline surface active (57.5 mN/m), more than the half of the daphnids pent at the surface of the test item solutions in the concentrations of 50.0 and 100 mg/L. Although the daphnids pent at the surface, they were still considered to be mobile.

- Analytical Results: The measured content of test item was between 93 % and 114 % of nominal in the fresh samples with a mean initial concentration of 104 % of nominal. In the aged samples the measured content was between 55 % and 67 % of nominal with a mean measured concentration of 61 % of nominal.

Results with reference substance (positive control):

EC50 (24 h) = 1.00 - 2.00 mg/L, in accordance with the requirements of the OECD guideline 202 and fall within the historical data, the daphnids were suitable for the determination of the toxicological effects of the test item.

Non-GLP range finder (48h):

	Nominal test concentration (mg/L)
	Control	1	10	100
Group 1	0	0	0	2
Group 2	0	0	0	2
Group 3	0	0	0	2
Group 4	0	0	0	1
Sum	0	0	0	7
%	0	0	0	35

Biological results main study:

Results of the main test, 48 h values

	nominal test item concentration [mg/L]
	Control	6.25	12.5	25.0	50.0	100
	immobilised daphnids after 48 h
Group 1	0	0	0	0	0	0
Group 2	0	0	0	0	0	0
Group 3	0	0	0	0	0	0
Group 4	0	0	0	0	0	0
Σ	0	0	0	0	0	0
%	0	0	0	0	0	0

Analytical verification of test concentrations:

Since no effects were observed up to and including 100 mg/L, the concentrations were verified only at this test concentration.

Determined concentration of the test item

Test item	Test item	Sampling	Test item
nominal [mg/L]	nominal [mg/L]	[h]	found [mg/L]	% of nominal	geometric mean [%]	measured concentration [mg/L]
Control	0	0 fresh	n.d.	-	-	-
		24 aged	n.d.	-
		24 fresh	n.d
		48 aged	n.d.	-
100	100	0 fresh	92.8	93	79	79.0
		24 aged	67.1	67
		24 fresh	114	114
		48 aged	55.4	55

Effect levels:

EC₅₀and NOEC-values of daphnids exposed to the test item evaluated using nominal and measured concentrations

	Test item
	[mg/L] (nominal)		[mg/L] (measured)
	24 h	48h	24 h	48h
NOEC	100	100	79.0	79.0
EC50	>100	>100	>79.0	>79.0
95 % confidence limit of EC50	-	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the EC50 (48 h) for immobilisation was supposed to be > 100 mg/L (nominal) and > 79.0 mg/L (measured, geometric mean). The corresponding NOEC (48 h) was 100 mg/L (nominal) and 79.0 mg/L (measured, geometric mean).

Executive summary:

The purpose of this study was to determine the immobilisation effect of the test item on the water flea Daphnia magna under worst-case exposure conditions according to OECD 202 (2004). A semi - static dose-response test with twenty organisms per test concentration (4 replicates of 5 animals each) was performed. The duration of the test was 48 hours. Nominal concentrations of 100, 50.0, 25.0, 12.5 and 6.25 mg/L and control were assessed.

Analytical samples taken at 0 hours (initial value) and 24 hours from fresh and after 24 and 48 hours from aged test solutions were analysed from control and the highest test concentration 100 mg/L. The geometric mean of the measured concentrations was used to assess the effect levels.

After 48 hours of exposure no immobilisation was observed in the control and up to and including 100 mg/L. According to the results of the test, the EC50 (48 h) for immobilisation was supposed to be > 100 mg/L (nominal) and > 79.0 mg/L (measured, geometric mean). The corresponding NOEC (48 h) was 100 mg/L (nominal) and 79.0 mg/L (measured, geometric mean).

Description of key information

One study is available in which the immobilisation effect of the test item on the water flea Daphnia magna was determined according to OECD 202 (2004). A semi - static dose-response test with 20 organisms per test concentration was used. The duration of the test was 48 hours. The tested nominal concentrations were 100, 50.0, 25.0, 12.5 and 6.25 mg/L and control.

According to the results of the test, the EC50 (48 h) for immobilisation was supposed to be > 100 mg/L (nominal) and > 79.0 mg/L (measured, geometric mean). The corresponding NOEC (48 h) was 100 mg/L (nominal) and 79.0 mg/L (measured, geometric mean).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

79 mg/L

Additional information

The purpose of this study was to determine the immobilisation effect of the test item on the water flea Daphnia magna according to OECD 202 (2004). A semi - static dose-response test with 20 organisms per test concentration (4 replicates of 5 animals each) was used. The duration of the test was 48 hours. Nominal concentrations of 100, 50.0, 25.0, 12.5 and 6.25 mg/L and control were used.

Analytical samples taken at 0 hours (initial value) and 24 hours from fresh and after 24 and 48 hours from aged test solutions were analysed from control and the highest test concentration 100 mg/L. Since the test concentrations decreased over 24h, the measured concentrations, i.e. geometric mean, were also used to evaluate the effect levels.

After 48 hours of exposure no immobilisation was observed in the control and up to and including 100 mg/L. According to the results of the test, the EC50 (48 h) for immobilisation was supposed to be > 100 mg/L (nominal) and > 79.0 mg/L (measured, geometric mean). The corresponding NOEC (48 h) was 100 mg/L (nominal) and 79.0 mg/L (measured, geometric mean).