Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2011 to 31 December 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:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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:
The test substance concentrations of control group, solvent control group and the theoretical nominal concentration of 159mg/L solution of the test substance were measured at 0h and 96h and 24h, 48h, 72h (before and after the renewal) after the start of the test.
The filtrate was analyzed immediately. Otherwise the samples were stored at 4℃ until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of test stock solution
0.478g test substance was weighted in the 300mL flask and dissolved completely; then transferred to a 5 L test chamber and diluted with the test water to 3000mL. The concentration of 159mg/L test substance solution was used in the test (100mg/L calculated based on a purity of 62.8% of the main component).

Controls:
Blank control containing no test substance.
A separate test with a positive control (potassium dichromate) was conducted (see under any other information on methods and materials below)

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Test fish:
(1) Species
Zebra fish (Brachydanio rerio)
(2) Reason for selection of species
The test species is recommended by "The Guidelines for the Testing of Chemicals".
(3) Supplier
Zebra fish (Brachydanio rerio) was obtained on September 28, 2011 from Young Aquarium of Guangzhou
(4) Acclimation
Before the start of the test, Zebra fish (Brachydanio rerio) were acclimated for 24 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.
(5) Size:
10 fish were randomly selected and measured on November 10, 2011. The average and standard deviation were 25.18 mm ± 1.21 mm, at total length and 0.147 g ± 0.027 g at weight in the population from which the fish were selected.
(6) 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:
126mg/L (expressed as CaCO3)
Test temperature:
Temperature of test solutions : 20.5°C-23.5°C
pH:
7.0 to 8.48
Dissolved oxygen:
79.4 to 99.7%
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.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentration: 159mg/L test substance solution (100mg/L calculated based on the purity of 62.8% for the main component)
Measured concentration at the start of the test and after the renewal: 58.73 mg/L - 88.48 mg/L test substance in test solution
Measured concentration before the renewal and at the end of the test: 54.05 mg/L - 72.13 mg/L test substance in test solution
Measured concentration (geometric mean of the actual concentration of the main component) = 64.72 mg/L
Details on test conditions:
(1) Replicate: 1 replicate/test group
(2) Number of fish: 10 fish/test group
(3) Test water: Dechlorinated tap water
(4) Type of test: Semi-static test by test solution replaced every 24 h
(5) Preparation of test stock solution: 0.478g test substance was weighted in the 300mL flask and dissolved completely; then transferred to a 5 L test chamber and diluted with the test water to 3500mL. The concentration of 159mg/L test substance solution was used in the test.
(6) Volume of test solution: 3L/test vessel
(7) Temperature of test solutions: 20.5°C-23.5°C
(8) Photoperiod: Artificial light of white fluorescent lamp, 14h light/10h darkness
(9) Feeding: No feeding
(10) Aeration: No aeration

Observation and measurements:
(1) 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, if applicable. A fish was considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction.
(2) 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, 72h (before and after the renewal) after the start of the test. Measurement of pH was carried out with a portable pH meter. Measurement of temperature and dissolved oxygen were carried out with an oxygen meter.
(3) Measurement of actual concentrations of test substance in test solution
The test substance concentrations of control group, solvent control group and the nominal concentration of 159mg/L solution of the test substance at 0h and 96h and 24h, 48h, 72h (before and after the renewal) after the start of the test were analyzed by HPLC



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

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 103 mg/L test substance solution calculated based on the geometric mean of 64.72 mg/L of the actual concentration of the main component).
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 103 mg/L test substance solution calculated based on the geometric mean of 64.72 mg/L of the actual concentration of the main component).
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 103 mg/L test substance solution calculated based on the geometric mean of 64.72 mg/L of the actual concentration of the main component).
Details on results:
Mortality
Cumulative mortality of each group at 24h, 48h, 72h and 96h after the start of the test were all 0% which shown in table 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
No abnormal response was observed in blank control and test group which shown in Table 2.

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 242 mg/L with 95% confidence limits (L95) of 215 mg/L - 274 mg/L, within the range of 200 mg/L-400 mg/L
Reported statistics and error estimates:
No data

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

159

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

159

0/10

N

0/10

N

0/10

N

0/10

N

Note: N— Normal

Table 3 Temperature of test solutions[°C]

Nominal concentration (mg/L)

0h

24h

48h

72h

96h

Old

New

Old

New

Old

New

Blank control

21.6

22.3

22.3

23.5

22.4

22.2

22.1

20.5

159

21.6

22.4

22.4

23.5

23.5

23.0

22.2

20.6

Table pH of test solutions

Nominalconcentration (mg/L)

0h

24h

48h

72h

96h

Old

New

Old

New

Old

New

Blank control

8.35

8.33

8.48

8.32

8.48

7.30

8.02

7.00

159

8.34

8.47

8.45

8.36

8.39

7.92

8.31

7.07

Table 5 Dissolved oxygen concentration of test solutions[%]

Nominal concentration (mg/L)

0h

24h

24h

48h

48h

72h

72h

96h

Old

New

Old

New

Old

New

Blank control

92.5

89.6

99.1

83.0

94.6

81.4

91.2

85.8

159

99.7

87.3

98.9

86.4

96.4

79.4

96.8

82.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the 96h-LC50 of test substance was greater than the geometric measured mean concentration of 100 mg/L (nominal concentration of 159mg/L substance solution. The geometric mean measured concentration was 64.72mg/L ) for Zebra fish (Brachydanio rerio), which calculated by the main component of 62.8% was 100mg/L). The 96h Lowest Observed Effect Concentration (LOEC) was greater than the nominal concentration of 159mg/L, which calculated by the main component of 62.8% was 100mg/L. 96h No Observed Effect Concentration (NOEC) was the nominal concentration of 159mg/L, which calculated by the main component of 62.8% was 100mg/L.
Executive summary:

The Fish, acute Toxicity Test of Reactive Red F03-0318 was conducted in accordance with OECD Guideline 203 and in compliance with the Good Laboratory Practice Guidelines.

In this test, the measured concentration of test solution was expressed by the main components of test substance. During the test, the test substance reacted with water. The main component of test substance gradually transformed into another substance, therefore the initial concentration of the test solution could not be kept steady. The measured concentration of test solution was expressed by the geometric mean concentration of the parent compound.

Under the conditions of the test, the 96h-LC50of test substance was greater than the geometric mean concentration of 100 mg/L (nominal concentration of 159 mg/L substance solution. The geometric mean measured concentration was 64.72 mg/L for Zebra fish (Brachydanio rerio),which calculated by the main component of 62.8% was 100 mg/L). The 96h Lowest Observed Effect Concentration (LOEC) was greater than the nominal concentration of 159 mg/L,which calculated by the main component of 62.8% was 100 mg/L. 96h No Observed Effect Concentration (NOEC) was the nominal concentration of 159 mg/L,which calculated by the main component of 62.8% was 100 mg/L.