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Long-term toxicity to fish

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Reference
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 December 2018 - 04 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to recommended OECD Guideline (OECD 215) and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.025 and 0.079 mg/L
- Sampling method: Water samples were taken from the control and each test group on two occasions prior to the addition of fish and on Days 0, 7, 13, 20 and 28 of the test for quantitative analysis (gas chromatography).
- Sample storage conditions before analysis: all samples were analysed on the day on sampling. A duplicate set of samples was taken on each occasion and stored frozen for further analysis if necessary.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (625 mg) was dissolved in dimethlyformamide and the volume adjusted to 250 mL to give a 2.5 mg/mL stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.79 and 0.25 mg/mL. The solvent stock solutions were prepared on Days -4, 3, 10, 18 and 25 of the test and were stored in a refrigerator between uses.
Each of the stock solutions was inverted several times to ensure adequate mixing and homogeneity.

Dynamic continuous flow test conditions were employed in the test. Dilution water was dosed at a flow rate of 140 mL per minute using Tacmina solenoid-driven metering pumps and the solvent stock solutions at a flow rate of 0.84 mL per hour using a Kd Scientific Syringe Pump for the solvent control and a Harvard Syringe Pump for the test concentrations. The dilution water and solvent stock solutions were combined in separate mixing vessels with minimal headspace at each concentration and delivered to the test vessels.

- Controls: The control and the solvent control group were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 μL/L of dimethlyformamide.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide

- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 μL/L in solvent control and both test groups

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): not reported
- Weight at study initiation (mean and range, SD): average weight in the batch was determined to be 3.35 g
- Method of breeding: not reported

FEEDING DURING TEST
- Food type: commercial trout pellets
- Amount: 4% body weight
- Frequency: twice daily during the week, once at weekends
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Hardness:
152 mg CaCO3/L
Test temperature:
Target 13-15 °C, actual 14-15 °C
pH:
Target (OECD Guideline): 6.5 to 8.5, but during a given test pH should stay within ± 0.5 pH units. Actual: 7.4-8.2
Dissolved oxygen:
Target: at least 60% ASV (approximately equivalent to 6 mg/L), Actual: 7.2-10.3%
Salinity:
not applicable
Conductivity:
345 μS/cm at 20 °C
Nominal and measured concentrations:
nominal concentrations of 0.025 and 0.079 mg/L; geometric mean measured concentrations 0.0083 and 0.029 mg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 64L glass test vessels with minimal headspace
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): Dilution water was dosed using a solenoid-driven metering pump
- Renewal rate of test solution (frequency/flow rate): test vessels supplied with test solution a flow rate of 140 mL per min
- No. of organisms per vessel: Eight
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): Two
- No. of vessels per vehicle control (replicates): Two
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: <1 mg C/L
- Particulate matter: not measured (study plan deviation)
- Metals:




- Pesticides: 0 μg/L
- Chlorine: 0.32 mg/L
- Alkalinity: 92 mg CaCO3/L
- Ca/mg ratio:
- Salinity: not applicable
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, pH, dissolved oxygenand light intensity recorded daily. Hardness, alkalinity and TOC were measured at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
pseudo-specific
Duration:
13 d
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
pseudo-specific
Duration:
28 d
Dose descriptor:
NOEC
Remarks:
13-28 days
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
pseudo-specific
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
13 d
Dose descriptor:
NOEC
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
- Fish weights (individual and mean values) on day 0, 14 (if measured) and 28: see Table 1
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): see table 2
- Type of and number with morphological abnormalities: none detected
- Type of and number with behavioural abnormalities: none detected
- Other biological observations: none
- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: none
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
All data were compared to a pooled control group. The Day 13 weight data was subject to a Dunnett's Multiple t-test, and the Day 28 data was subject to a Williams Multiple Sequential t-test. The Day 0-13 and 0-28 pseudo-specific growth rate data were each subject to a a Williams Multiple Sequential t-test, and the Day 13-28 interval pseudo-specific growth rate data was subject to a multiple sequentially rejective Welsh t-test. After 13 days, no significant differences (P≥0.05) in terms of relative weight were observed between the pooled control and each test group. For the period 0 to 13 Days, no significant differences (P≥0.05) in terms of pseudo specific growth rate were observed between the pooled control and 0.0083 mg/L test group; however, significant differences (P<0.05) were observed between the pooled control and 0.029 mg/L test group. After 28 days, no significant differences (P≥0.05) in terms of relative weight or pseudo specific growth rate were observed between the pooled control and 0.0083 mg/L test group; however, significant differences (P<0.05) were observed between the pooled control and 0.029 mg/L test group. For the period 13 to 28 Days, no significant differences (P≥0.05) in terms of pseudo specific interval growth rate were observed between the pooled control and each test group.

Table 1                       Fish Length and Weights on Day 0, 13 and 28

 

Day

Replicate

Fish

Geometric Mean Measured Concentration (mg/L)

Control

Solvent Control

0.0083

0.029

Length (cm)

Weight (g)

Length (cm)

Weight (g)

Length (cm)

Weight (g)

Length (cm)

Weight (g)

0

1

1

7.0

3.29

7.0

3.36

7.0

3.30

6.5

2.72

2

6.9

3.63

7.0

3.41

7.1

3.44

6.8

3.59

3

6.7

2.99

6.7

2.75

7.2

3.94

7.0

3.27

4

7.0

3.30

7.5

3.85

7.2

3.74

7.0

3.76

5

6.6

2.91

6.5

2.95

6.7

2.84

7.0

2.98

6

7.0

3.47

6.7

3.38

7.1

4.02

7.1

3.42

7

6.6

2.95

6.5

2.83

6.6

2.99

7.2

4.17

8

7.4

3.50

7.0

3.51

7.4

3.75

7.0

3.36

2

9

7.0

3.33

7.0

2.98

6.5

2.65

6.7

3.06

10

7.5

3.77

6.9

3.16

7.1

4.03

7.0

3.23

11

7.0

3.25

7.0

3.31

7.5

4.17

7.1

3.66

12

6.9

3.30

6.6

2.77

7.2

3.96

7.0

3.22

13

6.6

3.17

7.2

3.87

7.0

3.65

7.0

3.28

14

7.5

4.05

7.0

3.68

6.5

2.85

6.5

2.73

15

6.8

3.15

6.9

3.14

7.0

3.36

7.5

4.08

16

6.9

3.08

7.0

3.79

6.9

3.39

7.2

3.76

Average

7.0

3.32

6.9

3.30

7.0

3.51

7.0

3.39

St Dev

0.29

0.31

0.26

0.38

0.30

0.48

0.25

0.43

13

1

1

7.9

5.29

8.5

6.61

8.6

7.85

7.5

5.55

2

8.2

6.18

8.3

6.29

8.5

6.94

7.7

5.01

3

8.1

6.39

8.4

6.55

8.0

5.23

8.0

5.98

4

8.3

6.97

8.5

6.82

8.2

6.08

7.2

4.34

5

7.9

5.35

7.5

5.19

8.6

7.44

8.5

6.97

6

8.0

5.56

9.0

7.66

8.0

5.76

8.1

6.02

7

8.4

6.62

7.9

5.75

8.5

7.07

7.9

5.38

8

8.1

5.79

7.7

4.94

8.0

5.68

8.1

6.63

2

9

8.0

6.27

8.0

6.62

7.8

5.51

8.0

6.16

10

8.3

6.35

8.8

7.87

9.0

7.61

8.0

5.60

11

8.0

5.95

8.0

5.43

8.5

6.91

7.5

4.80

12

8.4

6.58

8.2

6.15

7.8

5.24

8.0

5.49

13

8.2

6.68

8.0

5.73

8.5

6.47

8.7

7.34

14

8.0

6.02

8.1

6.17

8.4

6.97

8.0

6.96

15

8.0

6.05

8.5

7.13

8.4

6.41

8.3

6.84

16

8.8

8.09

7.9

5.19

8.0

6.06

7.9

5.50

Average

8.2

6.26

8.2

6.26

8.3

6.45

8.0

5.91

St Dev

0.24

0.68

0.40

0.87

0.34

0.84

0.37

0.86

28

1

1

10.0

11.46

9.1

8.96

9.5

9.99

10.0

12.67

2

9.5

9.42

9.1

8.81

9.5

10.30

9.9

11.0

3

9.4

9.22

10.1

11.82

9.6

10.40

9.5

10.91

4

10.9

14.09

10.6

14.69

10.1

12.59

9.5

10.99

5

10.0

10.67

10.1

12.04

10.5

13.60

8.7

7.65

6

10.1

11.01

9.6

11.71

10.5

12.90

8.6

7.34

7

9.5

9.76

10.3

12.18

9.5

10.35

9.0

9.24

8

10.0

11.71

10.0

10.84

10.2

14.40

9.0

9.56

2

9

9.7

11.00

10.1

12.03

9.5

10.22

9.9

10.90

10

9.6

10.88

10.5

12.85

9.2

9.74

9.1

8.08

11

10.2

12.39

9.4

10.13

10.4

13.14

10.0

11.78

12

9.2

9.79

9.6

10.63

10.1

12.13

10.4

12.41

13

10.8

14.70

9.5

9.79

10.0

11.35

9.5

8.40

14

10.3

12.36

10.1

11.39

10.0

12.26

9.5

9.59

15

10.0

11.29

10.6

14.97

10.5

13.10

9.4

9.31

16

9.9

10.70

9.6

9.98

10.5

12.36

10.0

11.99

Average

9.9

11.28

9.9

11.43

10.0

11.80

9.5

10.11

St Dev

0.47

1.54

0.49

1.77

0.45

1.47

0.52

1.71

Table 2                       Pseudo-Specific Growth Rates

 

Geometric Mean Measured Concentration (mg/L)

Pseudo Specific Growth Rate

Percentage Inhibition (%)

Pseudo Specific Growth Rate

Percentage Inhibition (%)

Pseudo Specific Growth Rate

Percentage Inhibition (%)

0-13 days

13-28 days

0-28 days

Control

0.048

-

0.039

-

0.043

-

Solvent Control

0.049

-

0.040

-

0.044

-

0.0083

0.046

5

0.040

-2

0.043

-1

0.029

0.042

13

0.036

10

0.039

12

A negative value indicates an increase in growth relative to the pooled control group.

Validity criteria fulfilled:
yes
Conclusions:
The test was considered to be valid as no fish died during the test, the mean weight of fish in the control increased by 3.4 during the test period, the oxygen concentration was maintained above 60% ASV in all test vessels, and the water temperature was maintained between 13 and 15°C throughout the test.
Executive summary:

A study was performed according to OECD 215 to assess the effects of 3,3,4,4,5,5,6,6,7,7,8,8,8 -tridecafluorooctyl 2 -chloropropenoic acid ester on the growth of juvenile rainbow trout. Juvenile rainbow trout were exposed to the test substance at nominal concentrations of 0.025 and 0.079 mg/L for a period of 28 days under flow through conditions. As the test substance is poorly soluble in water, dimethylformamide was used as a co-solvent to aid test solution preparation. Geometric mean measured test concentrations during the test period were found to be 0.00083 and 0.0029 mg/L, respectively. Pseudo specific growth rates were calculated for the time periods 0 -13 days, 13 -28 days and 0 -28 days for each test concentration, and these were compared to the pooled control data.

The pseudo-specific growth rate in the 0.029 mg/L treatment group was significantly difference to the pooled control group (p<0.05) for 0 -13 and 0 -28 days and for weight at 28 days, therefore the NOEC was determined to be 0.0083 mg/L.

Description of key information

28 -day NOEC = 0.0083 mg/L; OECD 215; Sacker (2019)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.008 mg/L

Additional information

A study was performed according to OECD 215 to assess the effects of 3,3,4,4,5,5,6,6,7,7,8,8,8 -tridecafluorooctyl 2 -chloropropenoic acid ester on the growth of juvenile rainbow trout. Juvenile rainbow trout were exposed to the test substance at nominal concentrations of 0.025 and 0.079 mg/L for a period of 28 days under flow through conditions. As the test substance is poorly soluble in water, dimethylformamide was used as a co-solvent to aid test solution preparation. Geometric mean measured test concentrations during the test period were found to be 0.00083 and 0.0029 mg/L, respectively.

Pseudo specific growth rates were calculated for the time periods 0 -13 days, 13 -28 days and 0 -28 days for each test concentration, and these were compared to the pooled control data. The pseudo-specific growth rate in the 0.029 mg/L treatment group was significantly difference to the pooled control group (p<0.05) for 0 -13 and 0 -28 days and for weight at 28 days, therefore the NOEC was determined to be 0.0083 mg/L.

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