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Ecotoxicological information

Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 MAY 2019 to 04 DECEMBER 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
yes
Remarks:
On Day 10 of the definitive test dissolved oxygen concentrations below 4.9 mg O2/L were recorded in the old test solutions in solvent control replicates 1, 3 and 4. The lowest measured dissolved oxygen concentration was 4.4 mg O2/L
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1812101500R
- Expiration date of the lot/batch: 10 December 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Initially stored at approximately 4 ˚C in the dark under nitrogen from receipt on 17 December 2018 until 14 January 2019 when it was stored at room temperature in the dark under nitrogen
Analytical monitoring:
yes
Details on sampling:
- Concentration: 0.0104 mg/L
- Sampling method: Water samples were taken from the control, solvent control and the test group from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the old media on Days 1, 7, 14, 21, 28 and 33 (from a single replicate of each treatment group, changing systematically amongst replicates throughout the test).
- Sample storage conditions before analysis: The samples were analyzed on the day of sampling or after being stored refrigerated at approximately 4°C prior to analysis
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (definitive test)
An initial solvent stock solution was prepared by dissolving a nominal amount of test item (20.8 mg) in a final volume of 10 mL of dimethylformamide (DMF) to give a 2.08 mg/mL solvent stock solution. A further solvent stock solution was prepared by dissolving 2 mL of the 2.08 mg/mL stock concentration in a final volume of 20 mL of DMF to give a 0.208 mg/mL solvent stock solution. An aliquot (1100 μL) of the 0.208 mg/mL solvent stock solution was dispersed in 22 liters of test water with the aid of magnetic stirring for approximately 5 minutes (on one occasion this was increased to approximately 6 minutes) to produce the test concentration 0.0104 mg/L.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Strain: Pimephales promelas
- Source: bred at Covance CRS Research Limited on 14 January 2019

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Subsequent handling of eggs: Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.

POST-HATCH FEEDING
- Start date: from Day 6
- Type/source of feed: freshly hatched special or basic grade brine shrimp nauplii
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Hardness:
The water hardness values were observed to range from 150 to 156 mg/L as CaCO3 at the start of the test and from 140 to 150 mg/L as CaCO3 at termination of the test
Test temperature:
The temperature measurements recorded in a surrogate vessel by a Testo temperature logger were shown to have been maintained at between 24.1 and 25.7 °C. The temperature recorded in each test vessel before and after each media renewal ranged from approximately 24 to 26 °C.
pH:
The pH ranged from 7.6 to 8.5
Dissolved oxygen:
The dissolved oxygen concentration was maintained at least at 4.4 mg/L (equivalent to approximately 53% ASV)
Nominal and measured concentrations:
For the definitive test: nominal concentration of 0.0104 mg/L
The geometric mean measured test concentration was determined to be 0.0033 mg/L (32% of nominal concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: In the definitive test 1 liter glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test 5 liter glass vessels were used.
- Type: closed (covered to reduce evaporation)
- Material, size, headspace, fill volume: The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 5, 800 mL from Day 6 to Day 14 and 4000 mL from Day 15 until the end of the test.
- Aeration: no
- Renewal rate of test solution: The test preparations were renewed daily with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and a proportion of the incoming water softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main supply to give a water hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Pesticides < 0.003 µg/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 370 to 840 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus.
At the end of the test the length and wet weight of each surviving fish was determined.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentration: 0.0104 mg/L
- Results used to determine the conditions for the definitive study: At the test concentration of 0.0104 mg/L (considered to be the limit of solubility of the test item) hatching and survival were comparable to the solvent control group.
Chemical analysis of test preparations on Days -2 and -1 (pre-dosing) of the test showed measured concentrations to range from 0.0046 to 0.0057 mg/L (between 44 and 55% of nominal). Chemical analysis of test preparations on Days 0, 4, 7 and 13 of the test showed measured concentrations to range from 0.00072 to 0.0039 mg/L (between 7 and 38% of nominal). The concentrations achieved throughout the test became progressively smaller the longer the test progressed, indicating that a flow through method of preparation was not a suitable generation method for this particular test item.
Based on this information a test concentration of 0.0104 mg/L was selected for use in the definitive test. As it was shown that a flow through system was not a suitable option a semi static approach (using a solvent spike) was considered the most appropriate generation method with daily renewal of the test media.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
LC10
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
EC50
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
EC50
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Number of eggs hatching: The numbers of dead eggs observed during the definitive test are given in Table 1. The number of dead eggs was low throughout the test.
The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 5 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test replicates.

Post-hatch Survival: The number of dead larvae and hatched (live) larvae observed during the definitive test are given in Table 2 and Table 3 respectively.The number of dead larvae were observed to be low throughout the duration of the test.
There were no significant mortalities or sub-lethal effects of exposure observed in any of the test replicates.
The percentage of eggs hatched and post-hatch survival are given in Table 4.

Length and weight: The body length and wet weight data for all fish alive at the end of the test are given in Table 5.
Reported statistics and error estimates:
Prior to analysis of the test groups, the control and solvent control group data for hatching and post-hatch survival were compared using the Chi2 2x2 Table Test.

Prior to analysis of the test groups, the control and solvent control group data for body length were compared using the Student-t test for Homogeneous Variances.

Prior to analysis of the test groups, the control and solvent control group fish weight data were compared using the Student-t test for Homogeneous Variances.

Table 1: Number of dead eggs in the definitive test

Geometric Mean Measured Concentration (mg/L)

Number of Dead Eggs (Initial Population = 20)

Days

Total

1

2

3

4

5*

 

 

 

Control

R1

1

0

0

0

0

1

R2

1

0

0

0

0

1

R3

7

0

0

0

0

7

R4

6

0

0

0

0

6

 

 

Solvent Control

R1

0

0

0

0

0

0

R2

2

0

0

0

0

2

R3

0

0

0

0

0

0

R4

1

0

0

0

0

1

 

 

 

0.0033

R1

0

0

0

0

0

0

R2

4

0

0

0

0

4

R3

2

0

0

0

0

2

R4

6

0

0

0

0

6

* All surviving eggs hatched by Day 5

R = Replicate

 

Table 2: Number of dead larvae in the definitive test

Geometric Mean Measured Concentration (mg/L)

Number of Dead Larvae (Non-cumulative)

Day

 

1

 

2

 

3

 

4

 

5

 

6

 

7

 

8

 

9

 

10

 

11

 

 

 

Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

1

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

 

 

Solvent Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

1

 

 

 

0.0033

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

1

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

1

0

0

0

0

0

Geometric Mean Measured Concentration (mg/L)

Number of Dead Larvae (Non-cumulative)

Day

 

12

 

13

 

14

 

15

 

16

 

17

 

18

 

19

 

20

 

21

 

22

 

 

 

Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

1

0

R4

0

0

0

0

0

0

0

0

0

0

0

 

 

Solvent Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

 

 

 

0.0033

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

Geometric Mean Measured Concentration (mg/L)

Number of Dead Larvae (Non-cumulative)

Day

 

23

 

24

 

25

 

26

 

27

 

28

 

29

 

30

 

31

 

32

 

33

 

 

 

Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

 

 

Solvent Control

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

0

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

 

 

 

0.0033

R1

0

0

0

0

0

0

0

0

0

0

0

R2

0

0

0

1

0

0

0

0

0

0

0

R3

0

0

0

0

0

0

0

0

0

0

0

R4

0

0

0

0

0

0

0

0

0

0

0

R = Replicate

 

Table 3: Cumulative number of hatched (live) larvae in the definitive test

Geometric Mean Measured Concentration (mg/L)

Number of Live Larvae (Cumulative)

Day

 

1

 

2

 

3

 

4

 

5

 

6

 

7

 

8

 

9

 

10

 

11

 

 

 

Control

R1

0

0

0

2

19

19

19

19

19

19

19

R2

0

0

0

6

19

19

19

19

19

19

19

R3

0

0

0

2

13

13

12

12

12

12

12

R4

0

0

0

1

14

14

14

14

14

14

14

 

 

Solvent Control

R1

0

0

0

0

20

20

20

20

20

20

20

R2

0

0

0

0

18

18

18

18

18

18

18

R3

0

0

0

3

20

20

20

20

20

20

20

R4

0

0

0

3

19

19

19

19

19

19

18

 

 

 

0.0033

R1

0

0

0

3

20

20

20

20

20

20

20

R2

0

0

0

0

15

15

15

15

15

15

15

R3

0

0

0

1

18

18

18

18

18

18

18

R4

0

0

0

3

14

13

13

13

13

13

13

Geometric Mean Measured Concentration (mg/L)

Number of Live Larvae (Cumulative)

Day

 

12

 

13

 

14

 

15

 

16

 

17

 

18

 

19

 

20

 

21

 

22

 

 

 

Control

R1

19

19

19

19

19

19

19

19

19

19

19

R2

19

19

19

19

19

19

19

19

19

19

19

R3

12

12

12

12

12

12

12

12

12

11

11

R4

14

14

14

14

14

14

14

14

14

14

14

 

 

Solvent Control

R1

20

20

20

20

20

20

20

20

20

20

20

R2

18

18

18

18

18

18

18

18

18

18

18

R3

20

20

20

20

20

20

20

20

20

20

20

R4

18

18

18

18

18

18

18

18

18

18

18

 

 

 

0.0033

R1

20

20

20

20

20

20

20

20

20

20

20

R2

15

15

15

15

15

15

15

15

15

15

15

R3

18

18

18

18

18

18

18

18

18

18

18

R4

13

13

13

13

13

13

13

13

13

13

13

Geometric Mean Measured Concentration (mg/L)

Number of Live Larvae (Cumulative)

Day

 

23

 

24

 

25

 

26

 

27

 

28

 

29

 

30

 

31

 

32

 

33

 

 

 

Control

R1

19

19

19

19

19

19

19

19

19

19

19

R2

19

19

19

19

19

19

19

19

19

19

18*

R3

11

11

11

11

11

11

11

11

11

11

12*

R4

14

14

14

14

14

14

14

14

14

14

14

 

 

Solvent Control

R1

20

20

20

20

20

20

20

20

20

20

20

R2

18

18

18

18

18

18

18

18

18

18

18

R3

20

20

20

20

20

20

20

20

20

20

20

R4

18

18

18

18

18

18

18

18

18

18

18

 

 

 

0.0033

R1

20

20

20

20

20

20

20

20

20

20

20

R2

15

15

15

14

14

14

14

14

14

14

14

R3

18

18

18

18

18

18

18

18

18

18

18

R4

13

13

13

13

13

13

13

13

13

13

13

R = Replicate

*     One fish was considered to have been accidently transferred between tanks during water change, as twelve fish were observed in Control R3, however, one fish appeared to be missing from Control R2. For the conduct of statistical analysis the numbers of fish on Day 33 was considered to be the same as those on Day 32 (in relation to these two replicates) as no fish had died.

Table 4: Hatching and post hatch survival in the definitive test

Geometric Mean Measured Concentration (mg/L)

 

Hatching Rate (%)

 

Mean Hatching Rate

(%)

 

Survival Rate (%)

 

Mean Survival Rate

(%)

 

 

 

Control

R1

95

 

 

 

81

100

 

 

 

96

R2

95

100*

R3

65

85*

R4

70

100

 

 

 

Solvent Control

R1

100

 

 

 

96

100

 

 

 

99

R2

90

100

R3

100

100

R4

95

95

 

 

 

0.0033

R1

100

 

 

 

84

100

 

 

 

97

R2

75

93

R3

90

100

R4

70

93

R = Replicate

*     One fish was considered to have been accidently transferred between tanks during water change, as twelve fish were observed in Control R3, however, one fish appeared to be missing from Control R2. For the calculation of survival rate the numbers of fish surviving on Day 33 in each replicate was considered to be the same as those on Day 32 (in relation to these two replicates) as no fish had died.

 

Table 5: Fish length and weight data

Fish

Control

R1

R2

R3

R4

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

1

21.24

91.3

20.50

90.0

21.37

86.8

22.89

105.3

2

21.98

100.8

20.97

80.3

21.20

81.0

21.64

94.9

3

20.67

79.5

20.33

71.4

19.87

66.7

21.87

97.5

4

21.35

74.9

21.33

74.7

21.74

78.5

21.61

85.3

5

21.01

80.2

22.10

86.0

21.64

92.5

22.93

110.3

6

21.68

81.4

23.23

97.8

20.85

85.4

22.24

101.5

7

21.29

90.6

21.99

86.4

21.43

77.9

22.20

80.1

8

21.15

77.2

20.27

70.7

21.76

89.0

21.31

94.3

9

22.33

90.4

21.94

90.4

21.75

88.1

23.25

105.9

10

20.68

84.6

21.18

78.8

19.57

65.4

21.05

77.5

11

21.21

88.8

23.37

109.3

20.52

76.0

22.26

96.5

12

19.52

66.2

19.48

67.5

21.90*

95.6*

22.89

129.3

13

21.64

83.7

22.69

105.3

-

-

22.23

87.6

14

21.67

85.0

22.18

85.8

-

-

12.01

13.0

15

21.09

91.8

22.79

83.4

-

-

-

-

16

22.94

97.8

23.19

103.4

-

-

-

-

17

22.95

78.6

21.13

89.0

-

-

-

-

18

19.50

58.6

22.21*

89.6*

-

-

-

-

19

19.85

72.0

-

-

-

-

-

-

20

-

-

-

-

-

-

-

-

Average

21.25

82.8

21.72

86.7

21.13

81.9

21.46

91.4

Group Mean Length:

21.39 mm

Group Mean Wet Weight:

85.7 mg

R = Replicate

*     One fish was considered to have been accidently transferred between tanks during water change, as twelve fish were observed in Control R3, however, one fish appeared to be missing from Control R2. As it was not possible to determine which fish was transferred between replicates the fish weights and lengths statistical analysis was conducted on the data as written (ie one less for Replicate 2 and one more for Replicate 3). As this was only in relation to one fish and the replicates affected were both controls it is considered that the omission and addition between replicates would not significantly alter the group means in relation to the two parameters being assessed.

-  = Dead

Fish

Solvent Control

R1

R2

R3

R4

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

1

20.45

81.5

20.59

68.6

22.46

77.1

21.81

98.2

2

22.45

98.0

22.80

105.2

19.12

58.0

22.83

108.5

3

20.19

82.0

20.06

71.8

21.99

95.7

24.42

75.3

4

20.50

75.3

22.02

92.8

20.37

77.8

21.02

76.5

5

20.10

85.9

21.71

80.9

21.48

79.0

20.73

78.6

6

21.94

91.3

20.89

75.6

23.06

116.1

22.31

109.8

7

21.05

87.2

19.96

70.0

21.34

81.4

20.64

72.3

8

19.85

72.2

21.92

98.6

22.05

86.3

20.30

93.7

9

20.68

68.7

21.11

77.2

20.17

85.4

22.99

100.5

10

22.39

98.3

20.20

77.8

21.16

89.7

22.49

96.1

11

19.82

70.4

19.73

65.6

21.85

78.8

21.16

75.6

12

20.05

67.0

21.44

88.1

21.50

74.3

20.63

68.5

13

21.95

92.2

20.38

83.8

20.07

61.1

22.11

91.7

14

22.19

111.9

20.84

96.3

21.33

89.8

20.36

82.8

15

20.35

70.6

20.95

78.6

19.64

67.1

20.80

94.5

16

21.13

89.2

20.09

80.3

21.14

84.4

19.35

94.2

17

21.10

92.8

22.41

92.6

22.03

99.6

20.27

90.0

18

22.49

101.9

22.09

95.7

20.76

67.9

22.86

94.2

19

21.42

94.9

-

-

21.56

76.6

-

-

20

20.27

89.4

-

-

19.76

68.1

-

-

Average

21.02

86.0

21.07

83.3

21.14

80.7

21.50

88.9

Group Mean Length:

21.18 mm

Group Mean Wet Weight:

84.7 mg

R = Replicate

-  = Dead

Fish

0.0033 mg/L

R1

R2

R3

R4

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

Length (mm)

Wet weight (mg)

1

21.73

90.5

21.96

93.5

20.27

81.6

19.22

59.7

2

21.47

84.4

22.90

105.0

20.40

73.8

22.91

102.8

3

21.09

77.1

22.82

96.9

21.12

86.4

22.58

101.9

4

20.13

68.8

21.93

87.9

21.25

87.5

20.78

83.3

5

22.94

95.6

23.52

122.4

22.32

114.9

20.94

74.5

6

21.56

91.5

22.26

90.7

20.09

73.9

21.98

90.8

7

22.12

97.1

21.99

91.4

20.93

82.1

19.85

61.5

8

20.92

77.3

21.01

78.9

21.17

77.1

20.87

85.2

9

20.42

77.9

24.16

111.9

20.20

86.8

23.56

119.4

10

20.56

80.1

22.41

93.1

23.34

103.8

21.55

89.9

11

20.52

106.6

18.99

64.9

21.47

89.3

21.85

93.6

12

21.73

94.1

20.35

103.7

20.93

71.9

21.58

88.9

13

20.94

96.0

19.27

61.5

21.07

78.6

21.25

90.1

14

22.01

101.3

20.10

72.0

20.45

83.8

-

-

15

20.17

83.1

-

-

20.59

84.4

-

-

16

20.31

73.9

-

-

19.44

62.6

-

-

17

20.62

80.9

-

-

21.78

84.0

-

-

18

20.76

88.7

-

-

22.24

113.6

-

-

19

20.55

69.6

-

-

-

-

-

-

20

19.86

66.6

-

-

-

-

-

-

Average

21.02

85.1

21.69

91.0

21.06

85.3

21.46

87.8

Group Mean Length:

21.31 mm

Group Mean Wet Weight:

87.3 mg

R = Replicate

-  = Dead

Validity criteria fulfilled:
no
Remarks:
The following validity criteria was not achieved during the test: Dissolved oxygen - required: equal to or greater than 60% ASV - achieved: 53-113% ASV considered to have not affected the integrity or validity of the study
Conclusions:
Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to a test concentration of 0.0033 mg/L, as such, the EC/LC10, EC/LC20 and EC/LC50 values were considered to be >0.0033 mg/L for hatching, post-hatch survival, body length and wet weight.

The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Concentration (NOEC) was 0.0033 mg/L.
Executive summary:

Introduction
A study was performed to assess the effects of the test item on the early-life stages of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.


Methods
Information provided by the Sponsor indicated the water solubility of the test item to be approximately 0.0104 mg/L.
Preliminary solubility work conducted indicated that it was possible to obtain a testable solvent stock solution of the test item at a concentration of 0.208 mg/ml in dimethylformamide (DMF).
Based on the results of a preliminary range-finding test, newly fertilized fathead minnow eggs (four replicates of 20 eggs per group) were exposed to solutions of the test item at a nominal concentration of 0.0104 mg/L for a period of 33 days (28 days post-hatch) at a temperature of approximately 25 ºC under semi-static test conditions. The test solutions were renewed daily with the exception of Day 3 when there was no water change to avoid causing premature hatching of the eggs.
An initial solvent stock solution was prepared by dissolving a nominal amount of test item (20.8 mg) in a final volume of 10 mL of dimethylformamide (DMF) to give a 2.08 mg/mL solvent stock solution. A further solvent stock solution was prepared by dissolving 2 mL of the 2.08 mg/mL stock concentration in a final volume of 20 mL of DMF to give a 0.208 mg/mL solvent stock solution. An aliquot (1100 μL) of the 0.208 mg/mL solvent stock solution was dispersed in 22 liters of test water with the aid of magnetic stirring for approximately 5 minutes (on one occasion this was increased to approximately 6 minutes) to produce the test concentration 0.0104 mg/L.
The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and wet weight of the surviving fish were measured.


Results
Analysis of the untreated fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.0084 and 0.011 mg/L. Analysis of the untreated aged test preparations on Days 1, 7, 14, 21, 28 and 33 showed measured test concentrations to be either none detected (one occasion only) or between less than the Limit of Quantification (LOQ)* and 0.0043 mg/L. This showed that the test system was correctly dosed throughout the test and that a decline was noted over each 24 hour dosing period.
Due to the decline in the measured test concentration it was considered appropriate to calculate the results based on the geometric mean measured test concentrations of the untreated samples in order to give a “worst case” analysis of the data. The geometric mean measured concentration was determined to be 0.0033 mg/L.
Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to a test concentration of 0.0033 mg/L, as such, the EC/LC10, EC/LC20 and EC/LC50 values were considered to be greater than 0.0033 mg/L for hatching, post-hatch survival, body length and wet weight.
The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Concentration (NOEC) was 0.0033 mg/L.

Description of key information

The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Concentration (NOEC) was 0.0033 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.003 mg/L

Additional information