Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

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:

from 2008-08-28 to 2008-09-01

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the analysis of the test item concentrations, samples from the test media of the test concentration and the control were taken at the start and end of the study. All test medium samples were taken from the approximate centre of the aquaria without mixing of the test media.

Test solutions

Vehicle:

no

Details on test solutions:

Media according to EU guideline C.1, directive 92/69/EEC

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Age and Size:
Juveniles; The mean body lengths of the fish were 2.8 - 3.2 cm. The body length of 10 test animals from the test fish batch were measured prior to the start of the test. The mean body weight of the fish in the test groups and control ranged between 0.390 – 0.450 g. The body weight of fish was recorded on day 0 (at the start of the main study). First the weight of a vessel filled with water was determined. Then 7 fish were placed into this vessel. The weighting of all fish were registered and the average body weight was calculated for 1 fish. On the basis of the above, the loading of the testing liquid in the test-aquaria was calculated (g fish/litre testing liquid). The maximum loading was 0.630 g /L in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 d

Post exposure observation period:

not applicable

Test conditions

Hardness:

c(CaCO3) = 238 mg/L

Test temperature:

22.8 – 23.1 °C

pH:

8.39 to 8.46

Dissolved oxygen:

at least 90 % or higher

Salinity:

not applicable

Nominal and measured concentrations:

Nominal test item concentrations: 5.535; 6.912; 8.64; 10.8; and 13.5 mg/L.
Corresponding measured mean concentrations: 3.84; 4.79; 6.53; 8.49; and 10.62 mg test item/L.
The analytically determined mean test item concentrations ranged from 87 % to 92 % at the start and from 54 % to 67 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % at all concentration at the end of the study.

Details on test conditions:

The test item solution was prepared according to the WAF method (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000). A supersaturated test item stock solution (1000 mg/L) was prepared by dispersing/dissolving the test item amount without the use of any organic solvent into the Test Medium (filtered and aerated aquarium water) two days before the start of the study (on day -2). This solution was shaken for 24 hours at app. 30 ºC, than left settling for 24 hours at app. 20 ºC and thereafter filtrated through a 0.22 μm-MILLIPORE filter. The test solutions were prepared by appropriate dilution of the stock solution on day 0. The concentration of the stock solution was 4.39 mg/L. The concentration of these solutions were measured by the appropriate analytical method.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

6.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

8.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 10.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

12.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 8.21 - 18.08

Details on results:

The 96-hour LC50 value was calculated to be 12.18 mg/L (Probit analysis), the 96-hour LC0 and the overall NOEC were determined to be 6.53 mg/L. The 96-hour LC100 could not be determined because there was no 100% mortality during the test. As the deviation of the measured concentrations from the nominal values was higher than 20 %, all reported biological results are related to measured concentrations.

Results with reference substance (positive control):

no positive control investigated as not mandatory

Reported statistics and error estimates:

The LC50 and the 95 %-confidence limits were calculated by Probit analysis statistical software program Toxstat, Western EcoSystems Technology.

Any other information on results incl. tables

Sublethal observations / clinical signs:

There was no mortality in the control and in the test item treated groups.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this 96-hour static acute toxicity test to Zebrafish (Brachydanio rerio) with F 213 Red the 96h LC50 value was 12.18 mg/L (Probit analysis) based on measured concentrations. The overall NOEC was determined to be 6.53 mg/L, based on measured concentrations.

Executive summary:

Young fish were exposed to aqueous test media containing the test item at the nominal concentrations: 5.535; 6.912; 8.64; 10.8; and 13.5 mg/L. For the analysis of the test item concentrations, samples from the treatment groups and the untreated control were taken at the start and end of the study. The analytically determined mean test item concentrations in the analysed test media ranged from 87 % to 92 % at the start and from 54 % to 67 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % at all concentration at the end of the study. Therefore, all reported biological results are related to the geometric mean measured concentrations of the test item calculated by EXCEL Software Program. The corresponding measured mean concentrations were 3.84; 4.79; 6.53; 8.49 and 10.62 mg test item/L. The 96-hour LC50 value was calculated to be 12.18 mg/L (Probit analysis), the 96-hour LC0 and the overall NOEC were determined to be 6.53 mg/L. The 96-hour LC100 could not be determined because there was no 100% mortality during the test. As the deviation of the measured concentrations from the nominal values was higher than 20 %, all reported biological results are related to measured concentrations.