Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

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:

2013-06-04 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:

148559-52-8 (as free acid)

IUPAC Name:

148559-52-8 (as free acid)

Details on test material:

- Name of test material (as cited in study report): Reactive Golden Yellow HF-RN 1331
- Molecular formula: C17H11F2Na2O7S2 (as sodium salt) C17H13F2N7O7S2 (as free acid)
- Physical state: Solid, red to orange powder
- Analytical purity: 100 %
- Purity test date: 2012-10-08
- Lot/batch No.: Kilo 10 & 12
- Expiration date of the lot/batch: 2014-01-17
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container; since 2013-03-01 additionally under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Limit test concentration of 100 mg/L
- Sampling method:
The limit concentration and the control were analytically verified by HPLC-DAD at the start (0 h) and at the end of the exposure (48 h) as specified below.
At the start of the exposure (0 h), samples were taken immediately after preparation of the limit concentration and analysed.
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: A stock solution was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: None, limit test item concentration
- Controls: DIlution water without test item exposed 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:

yes

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 of the limit concentration and the control)
Nominal
test item concentration
[mg/L] pH-value
100 7.73
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
100 7.64 7.72 7.74 7.74
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 of the limit concentration and the control)
Nominal
test item concentration
[mg/L] Dissolved
O2-concentration
[mg/L]
100 8.80
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
100 8.24 8.21 8.13 8.21
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 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 0 0 0
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 effects of the limit concentration of 100 mg/L were deduced directly from the observed immobilisation rates.

Any other information on results incl. tables

Immobilisation Rates of the definitive Test after 24 and 48 hours of Exposure

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

Nominal test item concentration [mg/L]	IMMOBILISATION [%]
	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
100	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:

At the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331, no effects were observed on Daphnia magna.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331(batch number: Kilo 10&12) were determined according to OECD 202 (2004). The limit test was conducted under static conditionsover a period of 48 hours from 2013-06-04 to 2013-06-06 at Dr. U. Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany .

The test item is a reddish to orange powder and has a water solubility of ≥ 7.5 g/L (at 23 °C). Twenty daphnids were exposed to the limit concentration and the control.

The test item Reactive Golden Yellow HF-RN 1331was analytically verified by HPLC-DAD at the start of the exposure (0 h) and at the end of the exposure (48 h) for the limit concentration of 100 mg/L and the control.

The measured concentration of the test item of the limit concentration of 100 mg/L at the start of the exposure (0 h) was 102 mg/L. The measured concentration at the end of the exposure (48 h) was 103 mg/L.

The effect values given are based on the nominal concentrations of the test item Reactive Golden Yellow HF-RN 1331 (Table 1).

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 guidelines were fulfilled.

EC-values, NOEC and LOEC

(based on the nominal concentration of the test item)

Reactive Golden Yellow HF-RN 1331
	Test duration [h]	Nominal test item concentration [mg/L]	Confidence interval p = 95% [mg/L]
EC10 / EC50 / EC100	24	> 100	Not applicable
	48	> 100	Not applicable
NOEC	48	100	---
LOEC	48	> 100	---

At the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331,

no effects were observed on Daphnia magna.