Reactive Blue F08-0170

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:

08 March 2011 to 10 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU, OECD & US EPA test standards 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Details on test material:

Name: Reactive Blue F08-0170

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:

Analytical measurements were performed at the control and at the applied test concentration level at the beginning and end of the test. Four samples were taken from the Reactive Blue F08-0170 test solutions and one sample was taken from the control medium. The samples were diluted with Acetonitrile : Water = 1 : 9 mixture to fit the calibration range. The samples were then analysed by HPLC.

Test solutions

Vehicle:

no

Details on test solutions:

Prior to treatment, 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 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:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not 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 of the tests and 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.9 – 21.5°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.6 and 21.3°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 the tests in each test vessel and was in the range of 7.50 – 7.66.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test and was in the range of 6.7 – 6.8 mg/L.

Salinity:

Not applicable.

Nominal and measured concentrations:

The measured concentration in the applied test concentration level (based on mean value of four test item components) was 102 mg/L at the start and 84 mg/L at the end of the test. 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.

Details on test conditions:

Temperature: The water temperature was measured at the start of the tests and 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.9 – 21.5°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.6 and 21.3°C.
Oxygen concentration: The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test 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 the tests in each test vessel and was in the range of 7.50 – 7.66.
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 the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC100

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
Analytical measurements were performed at the control and at the applied test concentration level at the beginning and end of the test.
The test substance was not detected in the control samples at any analytical occasions.
The measured concentration in the applied test concentration level (based on mean value of four test item components) was 102 mg/L at the start and 84 mg/L at the end of the test. 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.

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

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
100 mg/L	20	0	0	0	0

 

Immobilisation of the test animals

Test group	Number of animals	Number of immobilised animals
		24 h	48 h
Control	5	0	0
	5	0	0
	5	0	0
	5	0	0
100 mg/L	5	0	0
	5	0	0
	5	0	0
	5	0	0

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the test item Reactive Blue F08-0170 had no toxic effect on daphnids up to at least the tested concentration level of 100 mg/L.

Executive summary:

This study has been performed in accordance with the study plan the OECD Guidelines for Testing of Chemicals (No. 202, 13 April 2004) and Directive 92/69/EEC, Annex Part C, C.2 and the EPA Health Effects Test Guidelines (OPPTS 850.1010) and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Acute toxicity of Reactive Blue F08-0170 was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a static test.

 

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 (100 mg/L nominal) and one control group.

 

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.

 

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.

 

Test concentration was analytically determined at the start and at the end of the experiment. The measured concentration (based on mean value of four test item components) was 102 mg/L at the start and 84 mg/L at the end of the test. 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 Blue F08-0170 were the followings:

 

Parameter (48h)	Test item concentration [mg/L]
EC50	> 100
NOEC	100
LOEC	> 100
EC100	> 100

 

Based on the results of this study, the test item Reactive Blue F08-0170 had no toxic effect on daphnids up to at least the tested concentration level of 100 mg/L. The substance is not classified as harmful to Daphnia.