disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

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:

July 8, 2002 - November 12, 2002

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:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations the following samples were taken:
Just before test start:
- duplicate samples from the test medium (without daphnids)
- duplicate samples from the control (without daphnids)

After 48 hours (stability samples) :
- duplicate samples from the test medium
- duplicate samples from the control

All samples were deep-frozen (at about -20°C) immediately after sampling. Based on preexperiments for investigation of the storage stability (without GLP) the test item is sufficiently stable in the test water under these storage conditions.
The concentration of the test item Red Rwa 4565 was analyzed in the duplicate test medium samples of both sampling times (0 and 48 hours). From the control only one of the duplicate samples was analyzed from each of both sampling times. The analytical procedure and the results are described in the attached analytical report.

Vehicle:

no

Details on test solutions:

The test medium of the only test concentration of nominal 100 mg/L was prepared by dissolving 30.2 mg test item completely in 300 mL test water by intense stirring for 15 minutes at room temperature.
The test medium was prepared just before introduction of the daphnids (= start of the test).
The actual concentration of the test item in the test medium was analytically determined.
The test concentration was based on the results of a range-finding test (without GLP).
However, according to the EU Commission Directive 92l69lEEC concentrations in excess of nominal 100 mg/L were not tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Source: A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5. Since this date the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions and total hardness) and under temperature and light conditions identical to those of the tests.
- Age of parental stock: At the start of the test the used test organisms were 6-24 hours old and were not first brood progeny.
- Feeding during test: none

Test type:

static

Water media type:

other: Reconstituted test water

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

2.5 mmol/L (=250.0 mg/L) as CaC03

Test temperature:

Water temperature of 20°C during the test period.
The test was performed in a temperature-controlled room (room temperature continuously monitored).

pH:

At the beginning and the end of the test period, the pH value ranged from 7.8 to 8.2.

Dissolved oxygen:

The test water was aerated prior to the start of the study until oxygen saturation was reached.
During the test period the test water was not aerated. At the beginning and the end of the test period, the dissolved oxygen concentration in the test medium and the control was at least 8.2 mg/L.

Salinity:

CaCl2 x 2H2O: 2.0 mmol/L (= 294.0 mg/L)
MgSO4 x 7H2O: 0.5 mmol/L (= 123.0 mg/L)
NaHCO3: 0.75 mmol/L (= 65.0 mg/L)
KCI: 0.075 mmol/L (= 5.8 mg/L)

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
The analytically determined test item concentration in the test medium at the start and the end of the test was 102% of the nominal value. Under the test conditions the test item Red Rwa 4565 was stable during the test period of 48 hours. Therefore all reported results are related to the nominal concentration of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass beakers with 50 mL test medium
- Type (delete if not applicable): beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions
- Material, size, headspace, fill volume:
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period the test water was not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):

At the single test concentration and for the control 20 daphnids were used, divided into two replicates of ten daphnids each. The daphnids were randomly distributed to. the test vessels at initiation of the test. The loading rate was lower than one daphnia per 2 mL test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
CaCl2 x2H20 : 2.0 mmol/L (= 294.0 mg/L)
MgS04 x 7H20 : 0.5 mmol/L (= 123.0 mg/L)
NaHC03 : 0.75 mmol/L (= 65.0 mg/L)
KCI : 0.075 mmol/L (= 5.8 mg/L)
Water Hardness : 2.5 mmol/L (=250.0 mg/L) as CaC03
Alkalinity : 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Na : K = 1 O : 1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: A 16-hour light to 8-hour darkness photoperiod (with a 30 minute transition period).
- Light intensity: light intensity at light period between 550 and 690 Lux.

Reference substance (positive control):

yes

Remarks:

For evaluation of the quality of the Daphnia magna clone and of experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

the 48-hour EC0 was determined to be at least 100 mg/L, it might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to the EU Commission Directive.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

the 48-hour NOEC was determined to be at least 100 mg/L, it might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to the EU Commission Directive.

Details on results:

NOEC, EC0, EC50, EC100:
The 48-hour NOEC (highest concentration tested without toxic effects after the
exposure period of 48 hours), resþectively the 48-hour EC0 of Red Rwa 4565 to Daphnia magna were determined to be at least 100 mg/L. The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations in excess of 100 mg/L have not been tested, acording to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of Red Rwa 4565 at the test concentration of 100 mg/L.


IMMOBILITY/MORTALITY:
The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 secondes after gentle agitation of the test beaker are considered to be immobile).
ln the test concentration of 100 mg/L no immobilized test organisms were observed during the test period of 48 hours. tn the control, one daphnia was found to be immobile at the observation after 48 hours. However, this immobility rate (5%) is within the guideline recommended range for the test to be valid (10%). No mortality was observed.


No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear colored solution throughout the whole test duration.

Results with reference substance (positive control):

No results with the positive control.

Reported statistics and error estimates:

No statistical analysis was performed.

lnfluence of Red Rwa 4565 on the mobility of Daphnia magna

Nominal test item concentration (mg/L)	No. of daphnids tested	lmmobilized daphnids after 24 hours		lmmobilized daphnids after 48 hours
		No.	%	No.	%
Control	20	0	0	1	5
100	20	0	0	0	0

 

Appearance of the test medium during the test period

Abbreviations:

0: no remarkable observations, clear test medium

1: homogeneous dispersion in the water, turbidity observable

2: noticeable turbidity caused by the test item

3: noticeable coloration caused by the test item

4: inhomogeneous dispersion of the test item

5: precipitation of the test item

6: test item at the surface

7: test item lying at the bottom of the test beaker

Nominal test item concentration (mg/L)	Exposure time
	0 hours	24 hours	48 hours
100	3	3	3

 

Dissolved oxygen concentration, pH value and temperature in the test medium and the control

 

Nominal test item concentration (mg/L)	Start (0 hours)			End (48 hours)
	pH	Oxygen (mg/L)	Temperature (°C)	pH	Oxygen (mg/L)	Temperature (°C)
Control	7.8	9.2	20	8.0	8.2	20
100	8.2	8.8	20	8.0	8.3	20

 

Validity criteria fulfilled:

yes

Remarks:

(1) In the control: not more that 10 per cent of the daphnids should have been immobilised -> met (in absence of mortality) (2) The dissolved oxygen concentration is ≥ 3 mg/L in control and test vessels -> met (with concentration≥ 8.2 mg/L)

Conclusions:

The acute toxicity of the test item Red Rwa 4565 to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals No. 202, (1984), resulting in a 48-hour EC50 > 100 mg/L

Executive summary:

The acute toxicity of the test item Red Rwa 4565 to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202, Paft I (1984).

 

A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of nominal 100mg/L. Thus the only concentration tested was nominal 100 mg/L and a control.

 

The analytically determined test item concentration in the test medium at the start and the end of the test was 102% of the nominal value. Under the test conditions the test item Red Rwa 4565 was stable during the test period of 48 hours. Therefore all reported results are related to the nominal concentration of the test item.

 

ln the control and the test concentration of 100 mg/L no significant immobilization rate was observed at the organisms during the test period of 48 hours.

Therefore, the 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours), respectively the 48-hour EC0 of Red Rwa 4565 to Daphnia magna were determined to be at least 100 mg/L. The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations in excess of 100 mg/L have not been tested, according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of Red Rwa 4565 at the test concentration of 100 mg/L.