Registration Dossier

Ecotoxicological information

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:
25 May 2012 to 27 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU and OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Reactive Red F03-0318
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:
Concentration of Reactive Red F03-0318 in the test solutions was determined at the beginning and at the end of each water renewal period.
Each occasion four replicate samples were taken from the test solution and one sample was taken from the control solution.

Test solutions

Vehicle:
no
Details on test solutions:
Dilution and Preparation of Testing Solutions

Prior to treatment of each renewal periods, a test solution of 160 mg/L (nominal) was prepared by diluting an appropriate amount of test item in ISO medium. An amount of 80 mg test substance was diluted in 500 mL ISO medium and then distributed into test vessels.

Untreated Control

The dilution water (ISO-medium) was used without of addition of the test item.

Reference Control

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 11/357-023DA) with reference item Potassium dichromate (batch no.: 0769128) is: 18-19 January 2012.
The 24h EC50: 1.27 mg/L, (95 % confidence limits: 1.16 – 1.39 mg/L)

Dilution Water

Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.



Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna
Source: National Institute of Public Health, 1097 Budapest Gyáli u. 2-6. Hungary
Breeding: The Daphnia are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behavior or significant decrease of population is recorded.
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period

Test conditions

Hardness:
The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start and at the end of each renewal period in each test vessel. The test temperature was in the range of 21.3 – 21.9°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 20.8 and 21.9°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of each renewal period in each test vessel and was in the range of 7.34 – 7.42.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 7.0 – 7.3 mg/L.
Salinity:
Not applicable
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
Light: The test was carried out in 16-hour light and 8-hour dark cycle.

Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
There were no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Measured concentrations were 181 and 165 mg/L at the start and 161 and 166 mg/L at the end of renewal periods. As the measured concentration deviated not more than 20 per cent from the nominal value during the experiment, the biological results are based on the nominal concentration.

Number of immobilised animals and the percentage of immobility at 24 and 48 hours are listed in the table below.

Only one immobilised animal could be observed in the test item treated group. In addition, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 11/357-023DA) with reference item Potassium dichromate (batch no.: 0769128) is: 18-19 January 2012.
The 24h EC50: 1.27 mg/L, (95 % confidence limits: 1.16 – 1.39 mg/L)
Reported statistics and error estimates:
No data

Any other information on results incl. tables

Number and percentage of immobilised animals:

Test Group

Number of treated animals

Number of immobilised animals

24 hours

48 hours

number

percent

number

percent

Control

20

0

0

0

0

160 mg/L (nominal)

20

1

5

1

5

Results of the Method Validation

Method validation results (Study code: 11/008-316AN)

Selectivity

No interfering component was observed

Reinjection repeatability
(7 injections)

Coefficient of Variation <2%

Linear range

0.1 - 10 µg/ml

Limit of Quantification

0.1µg/ml

Recovery from aqueous test media

98%, 107%
(0.7, 100 mg/L in daphnia test medium)

Stability of the samples

At least 22 hours in the autosampler

Stock solution stability

At least 14 days at 5±3°C

Stability in daphnia test medium under the conditions of thetoxicitytest

79 – 92 % after 1 day

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of Reactive Red F03-0318 was assessed in the Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours.

All validity criteria were met during this study.


Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Reactive Red F03-0318 were the followings:

Parameter (48h) Test item concentration [mg/L]
EC50 > 160
NOEC 160
LOEC > 160
EC100 > 160

Executive summary:

Acute toxicity of Reactive Red F03-0318 on Daphnia magna was assessed in an Acute Immobilisation Test, over an exposure period of 48 hours in a semi-static test system.The frequency of the water renewal periods were 24 hours.

Toxic effects were not observed during the preliminary concentration range-finding test therefore a limit test was performed in the main experiment using only one test concentration (160 mg/L nominal) and one control group. Based on the analytical method validation and the fact that the test substance is not hydrolytically stable, a nominal concentration higher than 100 mg/L was tested, in order to achieve a measured test concentration of 100 mg/L in the limit test.

The test design included four replicates at each test group (test item and control).Each group comprised twenty Daphnia, five in each of the four replicate vessels, each containing approximately 40 mL test dilution.

Only one immobilised animal could be observed in the test item treated group. In addition, no abnormal behaviour or appearance of test animals was detected.

The test item concentration was analytically determined at the start and at the end of each renewal period. Measured concentrations were 181 and 165 mg/Lat the start of the renewal periods and 161 and 166 mg/L at the end of the renewal periods. As the measured concentration deviated not more than 20 per cent from the nominal value during the experiment, the biological results are based on the nominal concentration.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Reactive Red F03-0318 were the following:

 

Parameter (48h)

Test item concentration [mg/L]

EC50

> 160

NOEC

160

LOEC

> 160

EC100

> 160

 

Based on the results of this study, the test item Reactive Red F03-0318 had no toxic effect on daphnids at up to at least 160 mg/L nominal.