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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2011 to 19 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD Guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 203 Fish Acute Toxicity Test. China Environmental Science Press. 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: State Environmental Protection Administration of China. The Guidelines for the test of chemical (HJ/T 153-2004). Beijing: China Environmental Science Press.2004
Deviations:
no
GLP compliance:
yes

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:
Nominal concentration of 195mg /L test solution (129 mg/L calculated based on 66% purity of main constituent)
The filtrate was analyzed immediately. Otherwise the samples were stored at 4℃ until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
0.585g test substance was dissolved in a certain amount test water with ultrasonic treatment for 5 min. Then the solution was diluted with the test water to 3000mL, the nominal concentration of 195mg /L test solution (129 mg/L calculated based on 66% purity of main constituent) was obtained. The test solution was prepared and was used every day.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebra fish (Brachydanio rerio)
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 January 25 2011. Before the start of the test, Zebra fish (Brachydanio rerio) were acclimated for 31 days by flow-through under the same conditions of water quality (Dechlorinated tap water), temperature (23°C±2°C), 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 0.8% during 7 days before the start of the test, which met the requirement.
Size: 10 fish were randomly selected and measured on March 1, 2011. The average and standard deviation were 24.76 mm±2.10 mm at total length and the average and standard deviation were 0.208 g±0.053 g at weight in the population from which the fish were selected.
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:
Not applicable

Test conditions

Hardness:
132mg/L (expressed as CaCO3)
Test temperature:
21.2℃ to 24.7℃
pH:
7.56 to 8.11
Dissolved oxygen:
73% to 109%
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 195mg /L test solution (129 mg/L calculated based on 66% purity of main constituent).
The measured concentrations of main constituent in nominal concentration of 195 mg/L test substance solutions at 0h, 96h and 24h, 48h, 72h (after and before the renewal) after the start of the test were 106.3 mg/L (0h), 67.39 mg/L (24h old), 119.1 mg/L (24h new), 65.11 mg/L (48h old), 117.9 mg/L (48h new), 69.70 mg/L (72h old), 110.0 mg/L (72h new) and 72.80 mg/L (96h), which exceeded ±20% of the nominal concentration of main constituent (129 mg/L).
Details on test conditions:
Conditions of exposure:
Type of test: Semi-static test (renewal at every 24h)
Exposure duration: 96h
Test concentration: Nominal concentration of 195 mg/L of test substance solution (129 mg/L calculated based on 66% purity of main constituent).
Control: A blank control (test water without test substance) was also 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:
Water temperature: 23.0℃±2.0℃
Dissolved oxygen concentration: The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test was kept at least 60 % of the air saturation value.
pH: 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.

Observation and measurements:
Observation of test organisms
Mortality and visible abnormality of the test fish were recorded at 24h, 48h, 72h and 96h after the start of the test during test period. The dead fish were removed as soon as they were found. A fish was considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produced no reaction.
Measurement of temperature, pH and dissolved oxygen of test solution
The temperature, pH and dissolved oxygen of each test group were measured at 0h, 96h and 24h, 48h and 72h (before and after the renewal) after the start of the test. Measurements of pH were carried out with a portable pH meter. Measurements of temperature and dissolved oxygen were carried out with an oxygen meter.
Measurement of actual concentrations of main constituent.
The actual concentrations of main constituent of nominal concentration of 195 mg/L test solution was measured at 0h, 96h and 24h, 48h and 72h (before and after the renewal) after the start of the test by a High Performance Liquid Chromatography (HPLC).
Data process
Cumulative mortality of each group at 24h, 48h, 72h and 96h after the start of the test were calculated; The LC50 values at 24h, 48h, 72h and 96h after the start of the test were calculated by Binomial test;.
Quality assurance
(1) The mortalities of the controls were less than 10% at the end of the test.
(2) The dissolved oxygen concentration in the test solutions was kept at least 60 percent of the air saturation value throughout the test.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 133.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test.

No abnormal response was observed in blank control and test substance group.

Results with reference substance (positive control):
The test result of Zebra fish (Brachydanio rerio) sensitivity test revealed that 24h-LC50 of the reference substance K2Cr2O7 was 274 mg/L with 95% confidence limits (L95) of 241mg/L - 311mg/L, within the range of 200 mg/L-400 mg/L.
Reported statistics and error estimates:
None

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table Cumulative mortality (%)

Nominal concentration

(mg/L)

Number of test fish

Cumulative mortality%

24h

48h

72h

96h

Blank control

10

0

0

0

0

195

10

0

0

0

0

 

Table 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

195

0/10

N

0/10

N

0/10

N

0/10

N

Note: NNormal

Table Temperature of test solutions()

Nominal concentration

(mg/L)

0h

24hold

24h new

48h old

48h new

72hold

72h new

96h

Blank control

23.2

24.7

23.5

22.9

21.9

22.3

21.4

22.0

195

23.5

24.6

23.0

22.8

21.2

22.1

21.5

21.9

 

Table pH of test solutions

Nominal concentration

(mg/L)

0h

24h old

24h new

48h old

48h new

72h old

72h new

96h

Blank control

8.00

7.83

8.11

7.68

7.97

7.87

8.10

7.86

195

7.59

7.56

7.68

7.66

7.64

7.63

7.76

7.67

 

Table Dissolved oxygen concentration of test solutions (%)

Nominal concentration

(mg/L)

0h

24h old

24h new

48h old

48h new

72h old

72h new

96h

Blank control

109

89

108

73

103

91

103

95

195

107

82

107

84

102

90

103

95

 

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

Exposure duration

LC50(mg/L)1

95% confidence limits

24 h

88.18

48 h

88.18

72 h

88.18

96 h

88.18

Note: 1— Calculated based on geometric meanmeasured concentrations ofmain constituent.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 96h-LC50of test substance to Zebra fish (Brachydanio rerio) was greater than nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentration of main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance).The highest concentration causing 0% mortality at 96h (LC0) was nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentration of main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance), the lowest concentration causing 100% mortality at 96h (LC100) was greater than nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentrationof main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance).
Executive summary:

The acute toxicity to fish of the substance, Reactive Yellow F01-0555, was evaluated in accordance with OECD Guideline 203.

Under the conditions of the test, the 96h-LC50of test substance to Zebra fish (Brachydanio rerio) was greater than nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentration of main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance).The highest concentration causing 0% mortality at 96h (LC0) was nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentration of main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance), the lowest concentration causing 100% mortality at 96h (LC100) was greater than nominal concentration of 195 mg/L test substance solution (88.18 mg/L calculated based on the geometric mean of actual concentrationof main constituent and 133.6 mg/L based on the geometric mean of the actual concentration of the test substance).