Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May to 19 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD test guidance in compliance with GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Reactive Red F08-0146
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:
The actual concentrations of nominal concentration of 120 mg/L test substance solution and blank control was measured at 0h, 96h and 24h, 48h, 72h (after and before the renewal) after the start of the test by a High Performance Liquid Chromatography (HPLC).

The test solution was centrifugal by 12000r/min for 3min, supernatant was collected, this procedure was repeated twice. The supernatant diluted 4 times was analyzed immediately, otherwise the sample stored at 4℃ until analyzed.

Test solutions

Vehicle:
no
Details on test solutions:
Test water
The chlorine of drinking water was removed by aeration.
Hardness: 112mg/L (expressed as CaCO3)
pH: 7.81

Preparation of test solution
0.600g test substance was dissolved in a certain amount test water with ultrasonic treatment for 3 min , then the solution was diluted with the test water to 5000mL and was stirred with a magnetic stirrer for 1h, nominal concentration of 120mg /L test solution (101 mg/L calculated based on 84.10% purity of main constituent) was obtained.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Reason for selection of species
The test species is recommended by "The Guidelines for the Testing of Chemicals".
Supplier: Kang Da Breeding Farm.

Acclimation
Zebra fish (Brachydanio rerio) was obtained on April 26, 2011.
Before the start of the test, Zebra fish (Brachydanio rerio) were acclimated for 17 days by flow-through under the same conditions of water quality (Dechlorinated tap water), temperature (23℃±2℃), light and dark period (14h light / 10h dark). The dissolved oxygen concentration during acclimation was kept at least 80 % of the air saturation value. The test fish were fed daily until 24 hours before the start of the test.
The mortality was 0% during 7 days before the start of the test, which met the requirement.

Size:
10 fish were randomly selected and measured on May 18, 2011. The average and standard deviation were 26.84 mm±1.97 mm at total length and the average and standard deviation were 0.295 g±0.117 g at weight.

Allocation to the test groups
Zebra fish (Brachydanio rerio) were allocated at random to each test group within 30 minutes.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None

Test conditions

Hardness:
112mg/L (expressed as CaCO3)
Test temperature:
24.1℃ to 24.8℃
pH:
7.55 to 8.4
Dissolved oxygen:
64% to 95%. The dissolved oxygen concentrations were kept at least 60 percent of the air saturation value, which met the criterion for the validity of the test.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentration of 120 mg/L of test substance solution (101 mg/L calculated based on 84.10% purity of main constituent).
Details on test conditions:
Test conditions:
Conditions of exposure
Type of test: Semi-static test (renewal at every 24h)
Exposure duration: 96hr
Test concentration: Nominal concentration of 120 mg/L of test substance solution (101 mg/L calculated based on 84.10% purity of main constituent).
Control: A blank control (test water without test substance) was run in parallel to the test concentration.
Replicate: No replicate
Number of fish: 10 fish / test group
Volume of test solution: 3L / test vessel

Conditions of test environment:
The test was performed without adjustment of pH.
Light: Artificial light of white fluorescent lamp, 14h light / 10h darkness
Feeding: The fish were not fed during the exposure.

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Mortality
Cumulative mortality of each group at 24h, 48h, 72h and 96h after the start of the test are shown in Table 1. below.
The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test .

Observed abnormal response
The abnormal response observed during the test are shown in Table 2 below.
No abnormal response was observed in blank control and test substance group.

Measurement of temperature, pH and dissolved oxygen of test solution
The temperature, pH and dissolved oxygen of each test group at 0h, 96h and 24h, 48h, 72h (after and before the renewal) after the start of the test are shown in Tables 3 - 5 below.
During the test, the measurement values of pH, temperature and dissolved oxygen ranged from 7.55 to 8.40, 24.1℃ to 24.8℃, and 64% to 95%, respectively. The dissolved oxygen concentrations were kept at least 60 percent of the air saturation value, which met the criterion for the validity of the test.

Concentration of main constituent in test solution
The measured concentrations of main content in blank control was all < 0.01720mg/L (LOD) at 0h, 96h and 24h, 48h, 72h (after and before the renewal) after the start of the test.
The measured concentrations of main content in nominal concentration of 120 mg/L test substance solutions at 0h, 96h and 24h, 48h, 72h (after and before the renewal) after the start of the test were 59.56 mg/L (0h), 42.24 mg/L (24h old), 71.72 mg/L (24h new), 42.04 mg/L (48h old), 82.52 mg/L (48h new), 44.44 mg/L (72h old), 77.12 mg/L (72h new) and 41.64 mg/L (96h). During renewing the test solution at 24 hour intervals, the actual concentration of main content cannot be maintained within 80%-120% of the nominal concentration of main content (86.4mg/L).

Median Lethal Concentration (LC50)
The LC50 at 24h, 48h, 72h and 96h after the start of the test are shown in Table 6 below.
At the end of the test, the cumulative mortality of nominal concentration of 120 mg/L test substance group was 0%, so the 96h-LC50 of test substance to Zebra fish (Brachydanio rerio) was greater than nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main constituent).
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
None.

Any other information on results incl. tables

Table 1 Cumulative mortality (%)

Nominal concentration

(mg/L)

Number of test fish

Cumulative mortality%

24h

48h

72h

96h

Blank control

10

0

0

0

0

120

10

0

0

0

0

 

Table 2 Observed abnormal response

Nominal concentration

(mg/L)

Result of observation

(Left column: Number of affected fish / Total survival number, Right column: Symptom detail)

24h

48h

72h

96h

Blank control

0/10

N

0/10

N

0/10

N

0/10

N

120

0/10

N

0/10

N

0/10

N

0/10

N

Note: N — Normal

Table 3 Temperature of test solutions()

Nominal concentration

(mg/L)

0h

24h

48h

72h

96h

old

new

old

new

old

new

Blank control

24.1

24.1

24.1

24.4

24.4

24.2

24.7

24.3

120

24.1

24.3

24.2

24.6

24.8

24.2

24.5

24.3

Table pH of test solutions

Nominal concentration

(mg/L)

0h

24h

48h

72h

96h

old

new

old

new

old

new

Blank control

8.40

8.04

7.97

7.55

8.36

7.96

8.36

7.80

120

8.29

7.80

7.81

7.78

8.15

7.78

8.17

7.83

 

Table 5 Dissolved oxygen concentration of test solutions (%)

Nominal concentration

(mg/L)

0h

24h

48h

72h

96h

old

new

old

new

old

new

Blank control

94

80

93

74

89

87

91

85

120

93

64

95

79

93

82

94

81

Table 6 Median lethal concentration (LC50) and the 95% confidence limits

Exposure duration

LC50(mg/L)1

95% confidence limits

24 h

> 64.76

48 h

> 64.76

72 h

>64.76

96 h

>64.76

Note:1 — Calculated based on geometric mean of actual concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 96h-LC50 of test substance to Zebra fish (Brachydanio rerio) was greater than nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main constituent). 96h Lowest Observed Effect Concentration (LOEC) was greater than nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main constituent), 96h No Observed Effect Concentration (NOEC) was nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main constituent).
Executive summary:

An acute fish toxicity test of Reactive Red F08 -0146 was conducted according to OECD Guidelines for the Testing of Chemicals, 203 Fish Acute Toxicity Test. Paris: OECD, Adopted 17 July 1992.

Under the conditions of the test, the 96h-LC50 of test substance to Zebra fish (Brachydanio rerio) was greater than nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main content). The 96h Lowest Observed Effect Concentration (LOEC) was greater than nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of main content), the 96h No Observed Effect Concentration (NOEC) was nominal concentration of 120 mg/L test substance solution (64.76 mg/L calculated based on the geometric mean of actual concentration of the main constituent).

The nominal values for the LC50 and the NOEC have been used for to determine classification for the following reasons:

Reactive dyes do not historically cause toxicity to fish and no effects on fish were observed in this study.

The lower than expected geometric mean value noted in this study is probably due to hydrolysis of Reactive Red F08-0146 under test conditions therefore it is not an accurate value. The results of the hydrolysis study included in Chapter 5.1.2 of this dossier shows that Reactive Red F08-0146 undergoes significant hydrolysis at neutral pH. The results of this study indicate that the hydrolysis products of the test substance do not appear to harm fish.

The test substance is not classified based on the 96h LC50 > 120 mg/L (nominal concentration).