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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, OECD & US EPA test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

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

Test material

Constituent 1
Details on test material:
Name: Reactive Orange F08-0314
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:
Because toxic response was not observed during the preliminary concentration range-finding test, a limit test was carried out using only one test concentration (100 mg/L) and one control group.

Test solutions

Vehicle:
no
Details on test solutions:
Prior to treatment of each renewal periods, a test solution of 100 mg/L (nominal) was prepared by diluting an appropriate amount of test item in ISO medium. An amount of 50 mg test substance was diluted in 500 mL ISO medium and then distributed into test vessels.

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 LAB Research Ltd.
The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour 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:
yes
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 20.9 – 21.1°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.7 and 21.4 °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 7.45 – 7.60.
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 6.7 – 6.8 mg/L.
Salinity:
Not applicable.
Nominal and measured concentrations:
Measured concentrations were 102.1 and 103.5 mg/L at the start and 74.8 and 66.3 mg/L at the end of renewal periods respectively. Concentrations of test item deviated more 20 per cent from the initial values at the end of the renewal periods, however geometric mean value of measured concentrations (85 mg/L) was within ±20 per cent of the nominal concentration.
Details on test conditions:
Test Conditions
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 20.9 – 21.1°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.7 and 21.4°C.
Oxygen concentration: 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 6.7 – 6.8 mg/L.
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 7.45 – 7.60.
Light: The test was carried out in 16-hour light and 8-hour dark cycle.
Hardness: The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Description of the Test Procedure
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker) of five animals each (at least 4 mL test solution/animal) were used at the test concentration and for the control. The animals were not fed during the test.
The choice of the test concentration was done on the basis of the results of a preliminary range-finding test.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours.

Concentration Levels Investigated in the Main Test
Because toxic response was not observed during the preliminary concentration range-finding test, a limit test was carried out using only one test concentration (100 mg/L) and one control group.

Observations
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature, oxygen concentrations and pH of the controls and the test solution were measured at the beginning and at the end of each renewal periods.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Concentrations of the test item
Concentration of Reactive Orange F08-0314 was analytically determined at the start and at the end of each renewal period.
Measured concentrations were 102.1 and 103.5 mg/L at the start and 74.8 and 66.3 mg/L at the end of renewal periods respectively. Concentrations of test item deviated more 20 per cent from the initial values at the end of the renewal periods, however geometric mean value of measured concentrations (85 mg/L) was within ±20 per cent of the nominal concentration. Biological results are reported separately based on nominal as well as measured value.

IMMOBILISATION
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour .

There was no immobilisation in 20 daphnids exposed to each group. In addition to immobility, 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: 10/332-023DA) with reference item Potassium dichromate (batch no.: 0769128) is: 16-17 December 2010.
The 24h EC50: 1.61 mg/L, (95 % confidence limits: 1.34 – 1.93 mg/L)
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.
The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

Any other information on results incl. tables

In the Daphnia study, the semi-static design with change of media every 24 hours was predicted to be fully acceptable based on the stability study. The study was run as a limit test run at 100 mg/L. The end concentrations noted following analysis were unexpectedly below the 80% cutoff specified in the OECD guidance.  However, the EC50 is stated as > 100 mg/L, as no effects were seen in this study. In addition, the test was performed in a semi static system and the degradation products of the main compound are compounds which are necessary for the proper use of the dye and are very similar in structure as can be seen in the hydrolysis study (see Chapter 5.1.2 of this dossier).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In the Daphnia study, the semi-static design with change of media every 24 hours was predicted to be fully acceptable based on the stability study. The study was run as a limit test run at 100 mg/L. The end concentrations noted following analysis were unexpectedly below the 80% cutoff specified in the guidance. However, the EC50 is stated as > 100 mg/L, as no effects were seen in this study. In addition, the test was performed in a semi static system and the degradation products of the main compound are compounds which are necessary for the proper use of the dye and are very similar in structure as can be seen in the hydrolysis study (see Chapter 5.1.2 of this dossier).

The substance is therefore not considered to be harmful to Daphnia.
Executive summary:

In the Daphnia study, the semi-static design with change of media every 24 hours was predicted to be fully acceptable based on the stability study. The study was run as a limit test run at 100 mg/L. The end concentrations noted following analysis were unexpectedly below the 80% cutoff specified in the guidance. However, the EC50 is stated as > 100 mg/L, as no effects were seen in this study. In addition, the test was performed in a semi static system and the degradation products of the main compound are compounds which are necessary for the proper use of the dye and are very similar in structure as can be seen in the hydrolysis study (see Chapter 5.1.2 of this dossier).

The substance is therefore not considered to be harmful to Daphnia.