disodium 4-hydroxy-3-[2-[4-({4-[2-(1-hydroxy-4-sulfonatonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-16-04 to 2013-06-06

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1215771-98-4 (as free acid)

IUPAC Name:

1215771-98-4 (as free acid)

Details on test material:

- Name of test material (as cited in study report): Acid Red RN 2949
- Molecular formula:
C41H30N4Na2O10S2 (as sodium salt)
C41H32N4O10S2 (as free acid)
- Molecular weight:
848.82 g/mol (as sodium salt)
804.86 g/mol (as free acid)
- Physical state: Dark red powder
- Analytical purity: 100 % (composition see AZ. No. 12-2302/4928 v1., dated 2012-08-28)
- Purity test date: 2013-02-21
- Lot/batch No.: Reg (=Batch 48)
- Expiration date of the lot/batch: 2015-03-05
- Stability under test conditions: see results
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 25.0 - 50.0 - 100 - 200 - 400 mg/L
- Sampling method: All tested concentration levels and the control were analytically
the test item verified at the start of the exposure (0 h) and at the end of the exposure (48 h) in all concentration levels and in the control as specified below.
At the start of the exposure, sampling was carried out immediately after preparation of the test concentrations.
At the end of the exposure (48 h), samples were taken from additional replicates. The additional replicates were prepared with test media, but without daphnids, and incubated under test conditions until sampling.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (400 mg/L of the test item were weighed out) was freshly prepared with dilution water (0 h). Dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 25.0 - 50.0 - 100 - 200 - 400 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:
Dilution water ISO test water, according to OECD 202, Annex 3:
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123
pH-value: 7.8  0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 256 (mg CaCO3/L)

Test temperature:

19.0 °C (Water)

pH:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Nominal
test item concentration
[mg/L] pH-value


400 7.77
200 7.75
100 7.74
50.0 7.70
25.0 7.65
Control 7.82

Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Nominal
test item concentration

[mg/L] pH-values
Replicates
1 2 3 4
400 7.58 7.57 7.56 7.55
200 7.57 7.57 7.58 7.58
100 7.61 7.60 7.60 7.58
50.0 7.63 7.62 7.61 7.60
25.0 7.64 7.63 7.63 7.63
Control 7.63 7.69 7.71 7.72

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Nominal
test item concentration
[mg/L] Dissolved O2-concentration

[mg/L]
400 8.65
200 8.74
100 8.69
50.0 8.76
25.0 8.63
Control 9.07

Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Nominal
test item concentration

[mg/L] Dissolved O2 -concentration [mg/L]
Replicates
1 2 3 4
400 7.34 7.29 7.55 7.26
200 7.75 7.74 7.83 7.90
100 7.79 7.84 7.84 7.59
50.0 7.98 7.90 7.86 7.85
25.0 7.70 7.85 8.00 8.04
Control 8.16 8.27 8.27 8.28

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start:654 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was determined in all groups after 24 h and 48 h.

TEST CONCENTRATIONS
- Range finding study:
Immobilisation Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
test item concentration
[mg/L] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 0 0 0 20 30 25
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (not applicable)

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.66 (CI 1.48 - 2.01) mg/L

Reported statistics and error estimates:

The EC10-value after 24 h was calculated by sigmoidal dose-response regression. The confidence interval for the EC10 was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.
The EC50-value for the reference item was calculated by sigmoidal dose-response regression. The confidence interval for the EC50 was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

Any other information on results incl. tables

Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Acid Red RN 2949	IMMOBILISATION [%]
Nominal test item concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
400	40*	40*	20*	40*	35	40*	40*	20*	40*	35
200	0	0	0	0	0	20*	20*	0	20*	15
100	20*	0	0	0	5	20*	0	20*	0	10
50.0	0	0	0	0	0	0	0	0	0	0
25.0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Acid Red RN 2949 the 48 h-EC50 for Daphnia magna was > 400 mg/L. The NOEC after 48 h was 100 mg/L, which was also the EC10 after 48 h. The LOEC after 48 h was 200 mg/L.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item Acid Red RN 2949 (batch number: Reg(=Batch 48)) were determined according to OECD 202 (2004) from 2013-06-04 to 2013-06-06 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h with 5concentrations of the test item Acid Red RN 2949 in the range of 25.0 to 400 mg/L, prepared with dilution water in a geometric series with a separation factor of 2. Twenty daphnids were exposed to each concentration level and the control.

The test item Acid Red RN 2949 was analytically verified by HPLC-DAD in samples of all test concentrations and the control at the start of the exposure (0 h) and at the end of the exposure (48 h).
The measured concentrations of the test item at the start of the exposure (0 h) were in the range of 96 to 104 % of the nominal values. At the end of the exposure (48 h), the measured concentrations of the test item were in the range of 97 to 104 % of the nominal values.

The recoveries of the test item were within ± 20 % of the nominal values. This indicates that the test item was successfully maintained for the duration of the test. The effect values given are based on the nominal concentrations of the test item Acid Red RN 2949 (Table 1).

Typically physical effects were observed at the concentration levels of 100 to 400 mg/L. The test item precipitated after 24 h. The sedimented test item had an adverse effect on the buoyancy of the daphnids. The sedimented test item formed a densely layer of particles on the bottom of the test vessels and the affected daphnids were not able to swim into the water phase. Moreover, the test item stuck to the carapax of the daphnids.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1:    EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀-Values, NOEC and LOEC

(based on the nominal concentrations of the test item)

Endpoints	Test duration [h]	Acid Red RN 2949
		Nominal test item concentration [mg/L]	Confidence interval p = 95% [mg/L]
EC10	24	326	203 – 400
	48	100	Not applicable
EC50	24	> 400	Not applicable
	48	> 400	Not applicable
EC100	24	> 400	Not applicable
	48	> 400	Not applicable
NOEC	48	100	Not applicable
LOEC	48	200	Not applicable