Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was considered to be a reliability 1 as it was conducted according to OECD Test Guideline 203 using an Acute Toxicity Test method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Test Code: FRET 09-0035
Storage condition: normal temperature

Sampling and analysis

Details on sampling:
The concentrations of test solution were measured at the start and end of exposure, before and after each renewal (include sampling times when all the fish are dead). About 10 mL samples were taken from treated groups and injected after pretreatment to analyze concentrations.

Test solutions

Vehicle:
yes
Details on test solutions:
Test solution of each group was prepared by adding appropriate quantities of stock solution and acetone respectively into water, making up to 5.5 L, and mixing with stirring. Add 5.5 L water to prepare test solution of blank group. Test solutions were prepared every 24 hours, and just before use.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species Zebrafish (Brachydanio rerio)
Supplier Hangzhou Minghong Aquarium
Receipt date 2015-06-17
Generation No. FZA000-20150617-02
Sensitivity 24 h-LC50 of potassium dichromate was 202.683 mg/L

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Nominal and measured concentrations:
According to the preliminary test, treated groups set as concentrations of 12.0, 14.0, 16.4, 19.2, 22.5 mg/L (geometric series with a factor of 1.2) and blank group were designed in the definitive test.
The measured concentrations of freshed prepared test solution were not in 80-120% of the nominal concentration adn further changed after 24 hr exposure, therefore geometric mean of measured concentration was used for LC50 calculation.
Details on test conditions:
Duration 96 hours
Loading ≤ 0.401 g fish/litre
Light 14 hours photoperiod daily
Temperature 23.2~23.7 °C
pH 7.20~7.69
Oxygen concentration 71.1~103.6 %ASV
Feeding None
Test vessel Reagebt bottle

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
12.889 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 12.032-14.878
Details on results:
Water quality parameters
Photoperiod was set as 14 hours daily during the test. The total hardness and pH of test water were 100 mg CaCO3/L and 7.66, respectively, measured before the exposure.
Detailed results of dissolved oxygen, pH and temperature measured during the test are shown in attached Table 3.

Body length and body weight
The body lengths of test fish were within the range of 2.246~2.987 cm, which met the requirement of 2.0±1.0 cm of the test Guidelines. The maximum loading of fish was 0.401 g fish/L during the test. The detailed results are shown in attached Table 7 and 8.

Mortalities and abnormalities
The mortality during acclimatization period was 0%. The results of accumulative total deaths and mortalities at 24 h, 48 h, 72 h and 96 h after the exposure of test fish are shown in attached Table 4 and 5. The mortalities in the blank and solvent control groups were 0%. The highest concentration causing no mortality was 14.0 mg/L (measured concentrations was 9.60 mg/L). The lowest concentration causing 100% mortality could not be determined. No visible abnormalities were found in blank and solvent control groups. In the treated groups, test fish showed the sign of visible abnormalities of rotating or spin motion, rollover, diminished swimming capacity, and abnormal static. The detailed results are shown in attached Table 6.

Reported statistics and error estimates:
MS Excel 2013 was used to analyze statistics of collected data (such as temperature, dissolved oxygen, pH, etc.). Probit analysis of SPSS 16.0 was employed to calculate the LC50 and the 95% confidence limits at recommended observation times of 24 h, 48 h, 72 h and 96 h after the start of exposure.

Any other information on results incl. tables

The ratios of measured concentration to nominal concentration during test period were in the range of30.5%~95.6%during the test period. The deviation from the nominal concentration was greater than 20%, and the measured concentrations had a different degree reduced after 24 hours. Results were calculated based on the measured concentrations (geometric average). Moreover, the geometric averages of measured concentrations at recommended observation times of 24 h, 48 h, 72 h and 96 h after the start of exposure are shown as follow.

Nominal concentration

 (mg/L)

Geometric average (mg/L)

0~24 h

0~48 h

0~72 h

0~96 h

12.0

6.14

7.43

7.68

8.07

14.0

8.69

9.70

9.81

9.60

16.4

7.21

9.36

10.43

11.11

19.2

10.43

11.93

11.65

12.60

22.5

11.10

12.03

12.01

13.55

Table 1Water quality parameters during the acclimatization (the second test)

Day N

(acclimatization)

Water quality parameters

Temperature(°C)

pH

Dissolved Oxygen

(% ASV)

1

23.6

7.69

99.6

2

23.7

7.72

99.2

3

23.8

7.68

99.7

4

23.6

7.75

99.6

5

23.7

7.67

99.5

6

23.6

7.74

99.2

7

23.7

7.76

99.7

Minimum

23.6

7.67

99.2

Maximum

23.8

7.76

99.7

Table3Water quality parameters during the test (the second test)

Temperature(°C)

Group

Exposure Day N

0

1*

1

2*

2                 

3*

3

4

Blank

23.5

23.4

23.7

23.6

23.7

23.6

23.4

23.4

Solvent control

23.7

23.4

23.4

23.5

23.6

23.6

23.4

23.4

Treated1

23.5

23.2

23.4

23.6

23.6

23.5

23.4

23.5

Treated2

23.4

23.2

23.2

23.5

23.5

23.2

23.5

23.2

Treated3

23.4

23.4

23.4

23.6

23.5

23.2

23.4

23.5

Treated4

23.4

23.6

23.3

23.6

23.5

23.4

23.4

23.4

Treated5

23.7

23.4

23.6

23.5

23.6

23.4

23.7

23.4

Minimum

23.2

Maximum

23.7

pH

Group

Exposure Day N

0

1*

1

2*

2                 

3*

3

4

Blank

7.69

7.53

7.62

7.52

7.63

7.60

7.53

7.48

Solvent control

7.63

7.52

7.65

7.61

7.65

7.53

7.50

7.46

Treated1

7.60

7.51

7.59

7.48

7.58

7.46

7.52

7.40

Treated2

7.62

7.55

7.55

7.46

7.51

7.48

7.42

7.38

Treated3

7.61

7.50

7.52

7.45

7.42

7.38

7.38

7.36

Treated4

7.58

7.46

7.48

7.40

7.35

7.30

7.30

7.22

Treated5

7.59

7.50

7.45

7.39

7.36

7.29

7.25

7.20

Minimum

7.20

Maximum

7.69

Dissolved Oxygen (% ASV)

Group

Exposure Day N

0

1*

1

2*

2                 

3*

3

4

Blank

103.2

80.1

100.9

82.6

93.6

78.2

98.2

80.6

Solvent control

98.6

79.6

98.2

84.2

98.4

77.4

98.6

75.2

Treated1

99.2

78.4

98.6

81.2

97.8

76.3

103.0

75.3

Treated2

101.9

77.6

103.6

83.6

95.6

80.9

99.7

80.9

Treated3

95.2

78.0

101.6

83.6

99.8

75.0

95.7

74.1

Treated4

102.3

75.2

100.2

78.8

93.8

71.2

97.7

78.1

Treated5

100.6

71.1

99.3

83.6

102.1

82.1

100.6

86.9

Minimum

71.1

Maximum

103.6

Note:* means the values of water quality parameters measured before renewal. Table4Cumulative death during exposure to test substance(the second test)

Cumulative death

Group

Exposure duration

3

6

24

48

72

96

Blank

0

0

0

0

0

0

Solvent control

0

0

0

0

0

0

Treated1

0

0

0

0

0

0

Treated2

0

0

0

0

0

0

Treated3

0

0

0

0

0

1

Treated4

0

0

0

0

0

1

Treated5

0

0

0

3

4

6

 

Table5Cumulative mortality during exposure to test substance(the second test)

Cumulative mortality

Group

Exposure duration

3

6

24

48

72

96

Blank

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

Solvent control

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

Treated1

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

Treated2

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

Treated3

0.0%

0.0%

0.0%

0.0%

0.0%

14.3%

Treated4

0.0%

0.0%

0.0%

0.0%

0.0%

14.3%

Treated5

0.0%

0.0%

0.0%

42.9%

57.1%

85.7%

 

Table6 Visible abnormalitiesduring exposure to test substance(the second test)

Visible abnormalities

Group

Exposure duration

3

6

24

48

72

96

Blank

A

A

A

A

A

A

Solvent control

A

A

A

A

A

A

Treated1

A

A

A

A

F

F

Treated2

F

F I

F I

F I

F I

F I

Treated3

F

F I

F I

F I

F I D

F I D

Treated4

F I D

F I D

F I D

F I D

F I D

F I D

Treated5

F I D

 B F I D

 B F I D

F I D

F I D

D

Note: A means no abnormality; B means spiral or spin motion; D means rollover; F means diminished swimming capacity; I means abnormal static.

Table7 Body length (total length) of test fish (the second test)

Group

Body length of test fish (mm)

Blank

25.16

27.73

28.42

25.66

24.57

28.12

29.00

Solvent control

28.42

27.71

27.44

25.87

26.43

25.92

26.60

Treated1

27.81

26.43

23.32

28.40

27.72

29.81

22.46

Treated2

25.31

26.89

27.72

29.80

25.42

26.61

26.66

Treated3

24.62

29.83

25.72

27.81

27.74

28.32

29.06

Treated4

25.32

28.44

27.76

25.81

26.62

27.83

25.99

Treated5

28.44

26.51

23.92

29.14

25.86

29.87

29.02

Minimum

22.46

Maximum

29.87

Note: Minimum body length of test fish was 2.246 cm, maximum body length of test fish was 2.987 cm.

 

Table8 Body weight (wet weight) and loading of test fish (the second test)

Group

Body weight of test fish (g)

Loading

(g/L)

Blank

0.276

0.294

0.272

0.279

0.283

0.326

0.348

0.378

Solvent control

0.309

0.296

0.284

0.292

0.283

0.274

0.284

0.368

Treated1

0.334

0.286

0.259

0.283

0.296

0.342

0.249

0.373

Treated2

0.263

0.282

0.293

0.336

0.277

0.281

0.289

0.367

Treated3

0.262

0.359

0.284

0.226

0.298

0.348

0.364

0.389

Treated4

0.276

0.295

0.321

0.284

0.290

0.282

0.274

0.368

Treated5

0.382

0.290

0.255

0.320

0.284

0.356

0.317

0.401

Minimum

0.226

0.367

Maximum

0.382

0.401

Note: The volume of test solution was 5.5 liters.The maximum loading was 0.401 g fish/L during the test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 96 h-LC50 of test substance FRET 09-0035 on Zebrafish species was 12.889 mg/L (95% confidence limits of 12.032~14.878 mg/L) based on the measured concentration (geometric average) under the test conditions in this study.
Executive summary:

The LC50of 96 h of test substance FRET 09-0035 on Zebrafish species was 12.889 mg/L (95% confidence limits of 12.032 -14.878 mg/L) based on the measured concentrations (geometric average) under the test conditions in this study, according to OECD test guideline 203. Treated group of test substance with nominal concentration of 12.0, 14.0, 16.4, 19.2, 22.5 mg/L (geometric series with a factor of 1.2) and blank control group were designed in the definitive test. 10 fish were used in each group. The duration of test was 96 hours in a semi-static exposure system with renewed test solution every 24 hours. The measured concentrations were within the range of 48.2-95.6% of the nominal concentration at the fresh preparation but changed to 32.8-122.4% after 24 hr exposure. Therefore geometic mean of measured concentration was used for LC50 calculation. According to the statistical analysis, the LC50of 96 h was 12.889 mg/L and 95% confidence limits was 12.032~14.878 mg/L based on the geometric mean of measured concentration.