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 fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-22 to 2013-04-26

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Not available

IUPAC Name:

Not available

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: 100 mg/L as limit test concentration
- Sampling method: Analytical evaluation of the limit concentration of Acid Red RN 2949 and the control was carried out via
HPLC-DAD from freshly prepared test media at start of the exposure and from old test media at the end of the exposure.
- 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: Direct weighing and the test media was mixed with an ultraturrax (1 min, 17000 rpm).
- Eluate: Dilution water
- Differential loading: 100 mg/L as limit test concentration
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und NAturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 1.93 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.161 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

Total Hardness at day 0: 46 mg/L

Test temperature:

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

pH:

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

Dissolved oxygen:

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

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria (3 L)
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal (after 0, 24, 48, 72 hours)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study:
Cumulative Mortality [%] in the Preliminary Test
(n = 5)
Nominal Test Item Concentration
[mg/L] Cumulative mortality [%]
after different exposure periods [hours]
24 48 72 96
100 0 0 0 0
10.0 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solutions were clearly solved throughout exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The concentrations leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. LC10,20,50-values have not to be calculated in a limit test.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Observations in the Definitive Test

Nominal test item concentration [mg/L]	Effect*	Effects on behaviour after different exposure periods [hours]
		24	48	72	96
100	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

Cumulative Mortality [%] in the Definitive Test

Nominal test item concentration [mg/L]	Cumulative mortality [%] after different exposure periods [hours]
	24	48	72	96
100	0	0	0	0
Control	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Acid Red RN 2949 was found to cause no toxic effects to Zebrafish after 96 hours when tested with the limit concentration of 100 mg/L.
The LC50 after 96 hours is > 100 mg/L. All effect levels are given based on the nominal test item concentration of Acid Red RN 2949.

Executive summary:

The acute toxicity of the test item Acid Red RN 2949 (batch no.: Reg (=Batch 48)) to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2013-04-22 to 2013-04-26, at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany .

A static limit test was performed with the nominal test item concentration of 100 mg/L. Duration of the test was 96 hours. 7 test organisms were exposed to the limit concentration and the control. Water quality parameters pH-value, temperature and oxygen-saturation, measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The test item Acid Red RN 2949 was analytically verified by HPLC-DAD at the start of the exposure (0 h) and at the end of the exposure (96 h).
The measured concentrations of the test item at the start and end of the exposure were 106 % and 105 % of the nominal value, respectively.

All effect levels are given based on the nominal test item concentration of the test item (see Table 1).

Table 1:           LC-Values with 95 % Confidence Intervals

                         based on the nominal test item concentration [mg/L]

Test duration [h]	LC50	Confidence Interval p = 95 %
24	> 100	n.a.
48	> 100	n.a.
72	> 100	n.a.
96	> 100	n.a.
LC100 = Lowest test item concentration with 100 % mortality after 96 h	> 100
LC0 = Highest test item concentration with 0 % mortality after 96 h	100