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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:
18 Jul 2019 - 30 Jun 2020
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:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
A single test concentration of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test".

- Sampling method:
Water samples were taken from the control and test group from the freshly prepared bulk test preparation on Days -1, 0, 4, 11, 18, 25 and 32 and from the old or expired media on Days 1, 6, 13, 20, 27 and 33 (from a single replicate of each treatment group, changing systematically amongst replicates throughout the test) for quantitative analysis.


- Sample storage conditions before analysis:
The samples were sent for analysis on the same day as sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Pre-experiments were performed using different test substance concentrations and solvents. The results obtained showed that no significant effect was noted in terms of increasing the test item concentration, however, grinding the test item appeared to improve the consistency of the achieved concentrations. Therefore the method described below was chosen.
- Method: On Days -1, 0, 4, 6, 8, 11, 13, 15, 18, 20, 22, 25, 27, 29 and 32 a nominal amount of test item (550 mg) was dispersed in test water and the volume adjusted to 11 liters with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter used to pre-condition the filter was discarded) to give a 100% v/v saturated solution.
- Eluate: test water
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Test concentration separation factor: Limit test conducted at a concentration of 100% v/v saturated solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: not reported
- Source: Adult fathead minnows that produced the eggs for the test were bred at Covance CRS Research Limited

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): - Method of collection of fertilised eggs: Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 12 March 2020 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.
- Subsequent handling of eggs: not reported

POST-HATCH FEEDING
- Start date: Start after larvae stage
- Type/source of feed: Day 7 - Day 11: special grade brine shrimp nauplii
Day 12 - Day 32: basic grade brine shrimp nauplii
- Amount given: Not reported
- Frequency of feeding: Not reported
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Remarks on exposure duration:
28 days post-hatch period
Hardness:
144 - 170 mg CaCO3/L
9.19 °dH
Test temperature:
24 - 25 °C
pH:
7.8 - 8.6
Dissolved oxygen:
≥ 4.9 mg/L
Conductivity:
513 µS/cm at 20 °C
Nominal and measured concentrations:
Nominal concentration:Saturated solution
Geometric mean measured concentration: 0.91 µg/L (in medium)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 liter glass vessels were used from Day 0 to Day 12 and from Day 13 to the end of the test, 5 liter glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 7, 800 mL from Day 8 to Day 12 and 4000 mL from Day 13 to the end of the test.
- Type: open
- Aeration: Via narrow bore glass tubes from Day 11 onwards
- Renewal rate of test solution (frequency/flow rate): A semi-static test regime was employed in the test involving a renewal of the test preparations three times per week. All test vessels were pre conditioned with test media for 24 hours prior to use.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total 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
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals: Aluminium < 6 µg/L
Iron < 35 µg/L
Manganese = 1.5 µg/L
Sodium = 46.4 mg/L
Copper = 0.0764 mg/L
Lead < 1.5 µg/L
Nickel = 4.6 µg/L
- Pesticides: < 0.004 µg/L
- Chlorine: 26.28 mg/L
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Salinity: not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity:593 - 732 lux

EFFECT PARAMETERS MEASURED: 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: No, as no solvent was used.

RANGE-FINDING STUDY
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD 23 Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions. The range-finding test was conducted by exposing newly fertilized fathead minnow eggs to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v
saturated solution for a period of 15 days.
The results showed there was no difference between the control and all test concentrations in terms of hatching, survival and growth.
No sub-lethal effects were observed at any of the test concentrations throughout the duration of the test.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 7
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20

FERTILIZATION SUCCESS STUDY
Not performed.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.91 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Post-hatch Survival
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.91 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks:
Body length
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.91 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
Body weight
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.91 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations. Details can be found in ‘Any other information on results incl. tables’.
- Days to hatch or time to release of young: 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.
- Numbers hatched: In control and treatment group all eggs successfully produced hatchlings.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): not reported
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
Mean body length: Control = 20.86 mm; Treatment = 21.58 mm
Mean wet weight: Control = 85.5 mg; Treatment = 86.0 mg
Details can be found in ‘Any other information on results incl. tables’.
- Type of and number with morphological abnormalities: Abnormal observations were observed in three replicates in the treatment group. The observations recorded were pale fish and bent spine; however, the numbers of fish recorded with these observations were very low. Therefore, these observations are considered to be due to defects occurring in a natural population, and not necessarily attributable to exposure to the test item. There were no sub-lethal effects observed in the control group throughout the test
- Type of and number with behavioural abnormalities: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No, as a saturated solution in test medium was used.
- Analytical verification: Analysis of the fresh test preparations on Days 0, 6, 13, 19, 27 and 32 showed measured test concentrations to range from less than the Limit of Detection (ND) of the analytical method, determined to be 0.5 µg/L or less than the Limit of Quantification (LOQ) of the analytical method. Measured test concentration of the old test preparations on Days 1, 7, 14, 20, 28 and 33 was observed to be between less than the Limit of Detection of the analytical method and 1.2 µg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentration for the 100% v/v saturated solution was determined to be 0.91 µg/L.
Results with reference substance (positive control):
No reference substance used.
Reported statistics and error estimates:
Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated by taking the square root of the product of the measured concentration at the start of the media renewal period and the measured concentration at the end of the media renewal period.

Lowest Observed Effect Concentration and No Observed Effect Concentration Determinations
As the study was conducted as a Limit Test, the LOEC could not be determined. For the estimation of the NOEC the hatching and post-hatch survival data for the control and test group were compared using Fisher’s Exact Binomial Test. The length and wet weight data for the control and each test group obtained on termination of the test were compared using the Student t-test for Homogeneous Variances incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Table 1: Number of Dead Eggs

Geometric Mean
Measured
Concentration
(µg/L)

Number of Dead Eggs (Initial Population = 20)

 

 

 

 

 

 

 

 

Replicate

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6*

Total

Control

1

1

0

0

0

0

0

1

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

0

0

0

0

0

0

0

0.91

1

2

0

0

0

0

0

2

 

2

0

0

0

0

0

0

0

 

3

1

0

0

0

0

0

1

 

4

0

0

0

0

0

0

0

* All surviving eggs hatched by Day 6

 

Table 2: Number of Dead Fish (Day 1 - Day 17)

Geometric Mean
Measured
Concentration
(µg/L)

Number of Dead Fish (Non-cumulative)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Replicate

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 10

Day 11

Day 12

Day 13

Day 14

Day 15

Day 16

Day 17

Control

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0.91

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

4

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

 

Table 3: Number of Dead Larvae/Fish (Day 18 - Day 33)

Geometric Mean
Measured
Concentration
(µg/L)

Number of Dead Fish (Non-cumulative)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Replicate

Day 18

Day 19

Day 20

Day 21

Day 22

Day 23

Day 24

Day 25

Day 26

Day 27

Day 28

Day 29

Day 30

Day 31

Day 32

Day 33

Control

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0.91

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Table 4: Hatching and Post Hatch Survival

Geometric Mean
Measured
Concentration
(µg/L)

Replicate

Hatching Rate

Mean Hatching Rate

Survival Rate

Mean Survival Rate

 

 

%

%

%

%

Control

1

95

99

100

100

 

2

100

 

100

 

 

3

100

 

100

 

 

4

100

 

100

 

0.91

1

90

96

94

97

 

2

100

 

100

 

 

3

95

 

100

 

 

4

100

 

95

 

 

Table 5: Fish Length and Weight Data - Control

Fish Length and Weight Data - Control

 

 

 

 

 

 

 

 

Replicate

1

 

2

 

3

 

4

 

Fish

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

1

19.15

49.4

21.18

88.3

20.47

73.0

20.43

75.8

2

21.39

83.3

22.84

110.1

20.99

86.4

20.73

97.7

3

22.42

105.9

20.94

78.2

20.95

84.3

21.23

95.3

4

20.61

70.7

22.02

117.5

21.44

88.0

19.57

83.5

5

21.86

93.0

20.46

72.2

21.53

94.6

20.69

95.4

6

19.38

57.9

20.87

86.6

19.91

81.4

19.99

81.7

7

19.92

75.5

21.26

100.4

19.50

65.2

20.37

91.2

8

17.32

43.6

20.64

73.3

21.81

98.9

20.21

78.2

9

20.33

82.1

20.73

80.5

20.72

84.8

20.34

72.6

10

21.56

78.8

20.93

85.7

19.66

77.7

21.01

96.5

11

21.26

84.4

21.54

107.8

20.69

93.5

21.39

93.2

12

21.53

77.1

19.84

66.3

20.14

77.6

20.03

87.3

13

21.93

87.6

21.51

81.9

21.75

113.5

20.36

93.6

14

22.00

101.1

21.12

85.8

21.97

94.1

20.45

90.1

15

22.16

100.2

21.60

103.9

21.94

103.5

20.71

83.9

16

22.23

85.1

22.27

103.4

21.81

97.2

20.88

82.0

17

18.38

42.1

21.58

87.2

21.25

72.8

21.00

97.2

18

21.09

74.5

21.17

87.0

19.76

98.8

21.20

92.8

19

18.64

59.5

21.47

96.8

19.39

68.4

21.31

102.2

20

-

-

21.20

81.9

20.53

75.1

21.82

97.5

Average

20.69

76.4

21.26

89.7

20.81

86.4

20.69

89.4

 

Table 6: Fish Length and Weight Data - Treatment

Fish Length and Weight Data 0.91 µg/L (Geometric Mean Measured Concentration)

 

 

 

 

 

 

 

 

Replicate

1

 

2

 

3

 

4

 

Fish

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

Length
(mm)

Wet
Weight
(mg)

1

22.20

94.8

21.69

86.8

19.95

83.4

18.26

50.8

2

21.10

82.5

20.62

80.6

21.39

98.5

20.03

93.7

3

21.42

101.1

21.98

90.1

22.54

105.9

21.52

101.1

4

20.94

77.5

20.81

73.1

22.51

110.1

23.04

88.6

5

20.77

83.5

20.93

85.5

22.93

87.3

22.63

95.8

6

19.89

72.1

20.21

73.7

22.88

87.9

20.39

60.1

7

19.71

63.4

20.24

77.2

22.65

82.1

20.84

70.0

8

21.31

78.8

21.78

98.9

21.23

75.8

22.29

95.3

9

21.06

94.9

21.54

72.1

21.08

71.2

22.39

87.0

10

21.44

87.7

21.55

88.6

21.25

88.3

22.69

85.6

11

20.70

75.7

21.64

83.4

20.94

82.2

22.86

96.6

12

23.16

94.3

21.82

79.1

23.46

114.0

21.30

74.9

13

21.52

82.6

21.51

85.3

22.43

96.1

22.10

95.5

14

22.90

101.1

21.54

87.4

21.08

83.0

21.07

73.4

15

21.74

79.8

21.95

90.9

21.86

88.0

20.17

74.0

16

20.61

75.1

22.21

87.6

23.16

90.1

22.08

100.7

17

21.37

83.2

22.42

92.8

22.51

84.6

22.38

103.2

18

-

-

22.65

92.7

21.87

87.3

22.44

110.4

19

-

-

21.46

77.5

20.56

71.1

22.64

103.9

20

-

-

21.54

71.6

-

-

-

-

Average

21.28

84.0

21.50

83.7

21.91

88.8

21.64

87.4

 

 

 

Table 7 Validity criteria for OECD 210

Criterion from the guideline

Outcome

Validity criterion fulfilled

The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.

>59%

Yes

The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).

+-1.5 °C

Yes

The analytical measure of the test concentrations is compulsory.

A detailed and considered analytical verification of the treatment concentrations was performed

Yes

Overall survival of fertilised eggs and post-hatch success in the controls

Hatching success rate = 99 %

Post-hatch survival = 100 %

Yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
>= 0.91 µg/L

Additional information

One study is available assessing the long-term toxicity of (Z)-N-Octadec-9-enylhexadecan-1-amide (CAS No. 16260-09-6)  to fish. The chronic toxicity the substance was investigated in a semi-static limit test according to OECD Guideline No. 210. Fish embryos were exposed for 33 days to a limit concentration of 100% v/v. The test concentrations was analytically verified by LC-MS/MS. No lethal and sublethal effects were observed. A NOEC (33 d) >= 0.91 µg/L (meas. geom. mean) was determined.