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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:
20 October 2017 to 10 July 2018.
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:
OECD Guideline for Testing of Chemicals No. 210 Fish, Early Life Stage Toxicity Test (July 2013)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Remarks:
Details of analytical information is appended below for reference.
Details on sampling:
Concentrations of LOWINOX® AH-25 were determined by diluting treated mains water samples with acetonitrile, then diluting further with acetonitrile/treated mains water, 1:4, v:v as required to bring the response within the calibration range. Samples are analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.
The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, LOWINOX® AH-25, and its oxidation product. LOWINOX® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.
Samples (10 mL) from the control, solvent control and each test concentration were taken for analysis prior to exposure and on Days 0 and 3 (pre-hatch) and Days 0, 3, 7, 10, 13, 16, 20, 23 (samples discarded as could not be analysed due to issues with the analytical instrument), 24 and 28 (post-hatch). Analysis was conducted on one replicate per concentration with replicates sampled alternatively. At each sampling occasion, duplicate samples were taken. One for chemical analysis and one as a ‘back up’ sample should further analysis be required.
Vehicle:
yes
Details on test solutions:
A solvent stock solution of 2 mg/mL was prepared by dissolving ca 100 mg of test substance in 50 mL of acetone. Serial dilutions were prepared in acetone to give further solvent stock solutions of 0.64, 0.20, 0.064 and 0.020 mg/mL. The 0.020, 0.064, 0.20, 0.64 and 2 mg/mL solvent stock solutions were each separately dosed at a rate of 0.060 mL/hour to a diluent flow rate of 20 ± 10% mL/min to give the 1.0, 3.2, 10, 32 and 100 µg/L test concentrations, respectively.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The Pimephales promelas eggs used in the early life stage test were obtained from the in-house breeding system (details maintained within the centrally held records). Viable fish eggs were obtained from breeding groups of Pimephales promelas. Prior to initiation of the test the stage of development was established. The eggs were added to the test system, typically before the first cleavage of the blastodisc or within 24 hours of fertilisation.

The breeding groups in the breeding system were held in a temperature-controlled room under artificial light (with a 16 hour light:8 hour dark photo-period with a ca 30 minute dawn/dusk period) in holding tanks appropriate to their size, under continuous water renewal (flow-through) conditions.

The breeding groups of Pimephales promelas were fed ad libitum using freshly hatched brine shrimp (Artemia salinis) nauplii and also with Tetramin® flake food, which was added to the holding tank in quantities dictated by the size of the fish. The food was not considered to contain contaminants likely to affect the outcome of the study. Uneaten food and debris was siphoned or cleaned from the tanks as required.

The water used was laboratory treated mains supply. The water was pumped to the laboratory through an activated carbon filter.

The eggs were deposited approximately 24 hours prior to testing and were collected in glass scintillation vials containing treated mains water. The embryos were acclimated to laboratory conditions for at least one hour prior to being added to the vessels. During this time the stage of embryonic development was assessed under a microscope.
Test type:
flow-through
Water media type:
freshwater
Remarks:
was treated mains water (treated via activated carbon particulate filter)
Limit test:
no
Total exposure duration:
28 d
Remarks on exposure duration:
As per test guideline.
Post exposure observation period:
Not required for the study
Hardness:
Not specified
Test temperature:
24.2 - 25.3 ºC
pH:
7.10 - 7.85
Dissolved oxygen:
90 - 105 % ASV
Salinity:
Not applicable - freshwater study
Conductivity:
200 uS/cm
Nominal and measured concentrations:
The geometric mean measured concentrations of parent test substance were calculated to be 2.1, 13, 31 and 44 µg/L for the 3.2, 10, 32 and 100 µg/L test concentrations, respectively
Details on test conditions:
Definitive Test
Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L. Control and solvent control groups were also included.
The test was conducted using a flow-through design (continual renewal of the media).
A solvent stock solution of 2 mg/mL was prepared by dissolving ca 100 mg of test substance in 50 mL of acetone. Serial dilutions were prepared in acetone to give further solvent stock solutions of 0.64, 0.20, 0.064 and 0.020 mg/mL.
The 0.020, 0.064, 0.20, 0.64 and 2 mg/mL solvent stock solutions were each separately dosed at a rate of 0.060 mL/hour to a diluent flow rate of 20 ± 10% mL/min to give the 1.0, 3.2, 10, 32 and 100 µg/L test concentrations, respectively.
A solvent control was prepared in a similar manner by dosing solvent only at a rate of 0.060 mL/hour to a diluent flow rate of 20 ± 10% mL/min.
Four replicates were prepared for the solvent control and each test concentration.
A dilution water control was prepared by continuously delivering treated mains water only into the four control vessels.
Solvent stock solution usage and diluent flow rates were checked daily during the test.

Appearance of Test Media
The appearance, colour and behaviour of the test substance in the test media were recorded daily.

Test Procedures
The test tanks were nominal 3.5 L constructed glass aquariums. Four replicate tanks were used for the control, solvent control and each test concentration each filled with ca 3 L of the appropriate media.
Egg incubation chambers used in the test were 17 cm long glass cylinders (6.5 cm internal diameter) with a 0.5 - 1.0 mm non-reactive mesh attached to one end (bottom).
The glass cylinders had opposing holes approximately 6.5 cm from the top (at the opposite end to the mesh), through which a stainless steel rod was inserted to enable each chamber to be suspended across the width of each replicate test vessel. Each replicate test tank contained one incubation chamber.
To ensure adequate movement of water within the test vessels and maintain dissolved oxygen concentrations in the egg incubation chambers, two aeration tubes were added to each replicate vessel. The first was maintained in the test medium surrounding the incubation chamber and the second aerator was maintained within the incubation chamber, positioned with the tube end approximately 1 cm from the mesh on the end of the cylinder. The agitation/turbulence caused by the aerators on the water column within the chambers ensured that a dynamic equilibrium of concentration was achieved between the environment within the chamber and surrounding test medium. The turbulence created slight movement of the embryos and maintained good levels of aeration within the chamber.

Egg Addition and Observations
At the start of the test, groups of 20 eggs were added to glass scintillation vials half filled with treated mains water and each vial was randomly assigned to a test vessel.
The eggs were transferred using a discrete pipette into the corresponding incubation chamber. The eggs were released under the surface of the test media, as close to the mesh as possible.
During the pre-hatch period, where possible, non viable or necrotic eggs were removed, avoiding disturbance of adjacent viable eggs.
The post-hatch phase started once all of the viable eggs were considered to have hatched in the control groups. Once all viable eggs had hatched across all treatments and fish were actively feeding and mobile, the chambers were removed.
On Day 28 the total numbers of surviving larvae were counted and individual total fish lengths and wet weights of all remaining fish were determined. Prior to initial weighing, the fish were blotted dry to make sure that all surface water was removed.
The percentage post-hatch survival was determined by expressing the number of surviving larvae on Day 28 post-hatch as a percentage of the hatched larvae at Day 0 (post-hatch).

Feeding
The developing larvae were fed using brine shrimp (Artemia salinis) nauplii, freshly cultured during the test. Initially the larvae were fed approximately 24-hour old shrimp. From Day 6 post-hatch onwards the larvae were fed approximately 48-hour old shrimp.
Surplus feed and faecal material were siphoned from the test vessel on at least daily intervals during the test.

Water Quality and Environmental Conditions
The test was conducted in a light-controlled facility, with a 16-hour light:8 hour dark period with an approximate 30 minute dawn:dusk transition period.
The water temperature (°C), pH and dissolved oxygen concentration, expressed as a percentage of the air saturation value (% ASV), were measured at the start of the test and at weekly intervals in each test vessel.
Water temperatures (°C) were monitored continuously in a vessel maintained in the test area using a minimum/maximum digital thermometer.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
44 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Hatching succes, grwoth (length and weight)
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
2.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: post-hatch survival
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 44 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Hatching success, growth (length & weight)
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Details on results:
Test Organism
The approximate age of the fertilised eggs on addition to the test vessels was ca 24 hours post-fertilisation. They were considered acceptable for use as their stage of embryonic development at the time of addition was at the early embryo/tail bud stage.

Definitive Test
Hatching Success
First egg hatch, in all treatment, solvent control and the control vessels occurred between Days 3 and 4 (post-egg addition). Completion of hatching occurred between Days 4 and 5 (post-egg addition) in all vessels. NOEC and LOEC values for time to first egg hatch and the egg-hatching period were therefore not statistically derived. This indicated no difference in the time to first hatch or hatching period across all treatments when compared to the control groups.
The mean hatching success of embryos in the control and solvent control vessels was 100% and 99% respectively, which was in excess of the validity criterion of ≥70%. It was therefore considered that the validation criterion had been achieved.
Mean hatching success in the treatments ranged between 98% - 100%. There were no statistically significant effects on hatching success at any of the test concentrations employed in the test compared to the combined controls. The NOEC and LOEC values for hatching success were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Survival
The mean post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was in excess of the validity criterion of ≥75%.
Mean post-hatch survival in the treatments ranged between 12% - 90%. Statistical analysis showed significant effects (p<0.05) in terms of post-hatch survival between the 10, 32 and 100 µg/L test concentrations and the combined controls. The NOEC and LOEC values for post-hatch survival were considered to be 3.2 and 10 µg/L, respectively, based on nominal concentrations (2.1 and 13 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Fish Total Lengths and Wet Weights
No significant differences were observed between the combined controls and test groups in terms of length and wet weight.
Taking both length and weight into consideration, the NOEC and LOEC values for growth were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Abnormalities
A small number of fish were observed to be small or pale or have a bent spine. However, these observations were not concentration specific and were therefore not considered to be due to any toxic effect but rather natural variation within the population.
Results with reference substance (positive control):
Not performed
Reported statistics and error estimates:
No statistically significant differences were observed in terms of hatching success or final length and wet weight at any of the test concentrations employed in the test compared to the combined controls. Statistically significant differences were observed in terms of post-hatch survival at the 10, 32 and 100 µg/L test concentrations compared to the combined controls.

Hatching Success

The results for hatching success are presented in Table 4 and summarised below.



Nominal concentration

(µg/L)

Geometric mean measured concentration (µg/L)

Hatching success

(%)

Control

Control

100

Solvent control

Solvent control

99

1.0

-

100

3.2

2.1a

100

10

13

100

32

31

98

100

44

98

- Not calculated
aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution

 

Survival

The results for survival of hatched larvae at Day 28 post-hatch are presented in Table 4 and summarised below.



Nominal concentration

(µg/L)

Geometric mean measured concentration (µg/L)

Post-hatch survival Day 28

(%)

Control

Control

88

Solvent control

Solvent control

76

1.0

-

90

3.2

2.1a

73

10

13

58*

32

31

51*

100

44

12*

- Not calculated
aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution

* Significant (p <0.05) difference compared to the solvent control

 

Measured concentrations of LOWINOX® AH-25 in test media during the definitive test

Nominal concentration (µg/L)

Measured concentration (µg/L)

Day 0 (pre-hatch)*

Day 3 (pre-hatch)

Day 0 (post-hatch)

Day 3 (post-hatch)*

Day 7 (post-hatch)*

Day 10 (post-hatch)

Control

-

-

-

-

-

-

Solvent control

-

-

-

-

-

-

1.0

0.14

0

0.35

0.23

0

0

3.2

0.79

4.28

1.72

0

0.58

2.46

10

6.4

8.83

6.8

6.81

10.9

102

32

0

97.1

25.2

22

17.6

29.1

100

26.7

26.3

34.2

43.4

50.3

67.2

– Non detected above limit of quantification (LOQ), assessed as 5 µg/L
* Analytical batch failed acceptance criteria. Results have been provided for information only and were not used in the calculation of the geometric mean measured concentrations

 

Nominal concentration (µg/L)

Measured concentration µg/L)

Geometric mean measured concentration (µg/L)

Day 13 (post-hatch)

Day 16 (post-hatch)*

Day 20 (post-hatch)

Day 24 (post-hatch)*

Day 28 (post-hatch)

Control

-

-

-

-

-

-

Solvent control

-

-

-

-

-

-

1.0

0.24

0

0

0.69

<0

NC

3.2

0

1.5

0.78

4.56

<0

2.1a

10

9.3

10.7

10.7

10.8

6.76

13

32

37.9

29.3

15.2

34.3

22.8

31

100

25.6

34.1

88.3

77

52.8

44

– Non detected above limit of quantification (LOQ), assessed as 5 µg/L
* Analytical batch failed acceptance criteria. Results have been provided for information only and were not used in the calculation of the geometric mean measured concentrations

NC = Not calculated
aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ where no test substance was detected. Result should be treated with caution

 

Values for the 1.0 and 3.2 µg/L have been provided for information purposes only as values were <LOQ.

 

Water quality determinations during the fish early life stage test

Nominal concentration (µg/L)

Day

Day 0

pre-hatch

Day 3

post-hatch

Day 10 post-hatch

Day 17 post-hatch

Day 24

post-hatch

Day 28

post-hatch

Temperature (ºC)

Control

24.5 – 24.9

24.9 – 25.0

25.1 – 25.3

24.9 – 25.1

24.7 – 24.8

24.6 – 24.8

Solvent control

24.4 – 24.7

24.8 – 25.0

24.8 – 25.1

24.7 – 25.1

24.7 – 24.9

24.4 – 24.7

1.0

24.6 – 24.8

24.7 – 24.8

24.8 – 25.1

24.9 – 25.1

24.8 – 24.9

24.4 – 24.7

3.2

24.4 – 24.7

24.5 – 24.7

24.9 – 25.0

24.9 – 25.1

24.9 – 25.2

24.6

10

24.2 – 24.8

24.4 – 24.8

24.9 – 25.1

24.9 – 25.2

24.9 – 25.0

24.5 – 24.7

32

24.3 – 24.6

24.5 – 24.7

24.7 – 24.9

24.9 – 25.2

24.9 – 25.0

24.5 – 24.9

100

24.2 – 24.3

24.2 – 24.4

24.6 – 24.8

24.9 – 25.0

24.7 – 24.8

24.5 – 24.6

pH

Control

7.67 – 7.72

7.55 – 7.63

7.33 – 7.39

7.76 – 7.85

7.20 – 7.29

7.47 – 7.58

Solvent control

7.53 – 7.63

7.50 – 7.54

7.24 – 7.38

7.66 – 7.78

7.19 – 7.32

7.44 – 7.57

1.0

7.61 – 7.67

7.52 – 7.57

7.26 – 7.28

7.61 – 7.71

7.21 – 7.25

7.44 – 7.48

3.2

7.65 – 7.67

7.57 – 7.59

7.27 – 7.34

7.65 – 7.74

7.12 – 7.32

7.40 – 7.43

10

7.65 – 7.66

7.51 – 7.53

7.26 – 7.33

7.65 – 7.70

7.20 – 7.28

7.33 – 7.38

32

7.58 – 7.68

7.51 – 7.54

7.34 – 7.35

7.60 – 7.66

7.10 – 7.21

7.42 – 7.49

100

7.63 – 7.69

7.55 – 7.57

7.40 – 7.46

7.62 – 7.71

7.25 – 7.30

7.34 – 7.35

Dissolved oxygen concentration (%ASV)

Control

101 – 104

100

99

98 – 105

94 – 98

93 – 99

Solvent control

101 – 102

98 – 99

82 – 99

99 – 102

92 – 101

92 – 98

1.0

102 – 103

99 – 100

93 – 94

88 – 103

92 – 94

93 – 97

3.2

101 – 103

100 – 101

92 – 96

98 – 108

90 – 102

94 – 96

10

101 – 103

94 – 99

87 – 95

91 – 103

90 – 97

90 – 94

32

100 – 102

97 – 99

95 – 96

92 – 102

88 – 92

94 – 98

100

100 – 101

96 – 97

96 – 101

94 – 96

92 – 98

86 – 89

ASV – Air Saturation Value
Values stated are the range across the four replicates

 

Continuously recorded temperature range during the fish early life stage test

Day number

*Recorded Temperature (°C)

 

 

0

25.2

1

25.1 – 25.6

2

25.1 – 25.6

3

25.2 – 25.6

4

25.2 – 25.8

5

24.9 – 25.3

6

25.5 – 25.9

7

25.0 – 25.5

8

24.9 – 25.6

9

25.1 – 25.4

10

25.0 – 25.6

11

25.0 – 25.8

12

25.0 – 25.4

13

25.2 – 25.8

14

25.1 – 25.7

15

25.2 – 25.7

16

25.2 – 25.7

17

25.2 – 25.7

18

25.3 – 25.7

19

25.5 – 25.7

20

25.5 – 25.7

21

25.3 – 25.8

22

25.3 – 25.6

23

25.4 – 25.6

24

25.3 – 25.7

25

25.3 – 25.8

26

25.2 – 25.9

27

25.0 – 26.0

28

25.0 – 26.0

29

24.5 – 25.6

30

25.1 – 25.6

31

25.2 – 25.6

32

25.4 – 25.8

* Recorded using a min/max digital thermometer

 

Hatching success and fish larval survival during the fish early life stage test

Nominal Concentration (µg/L)

Vessel number

Number of eggs added at start of test

Number of hatched larvae

Percentage of hatched larvae (hatching success)

Number of larvae surviving at 28 days post-hatch

Percentage of larvae surviving at 28 days post-hatch (post-hatch survival)

Individual vessels

Pooled vessels

Individual vessels

Pooled vessels

Individual vessels

Pooled vessels

Individual vessels

Pooled vessels

Individual vessels

Pooled vessels

Control

V1

20

80

20

80

100

100

16

70

80

88

V2

20

20

100

19

95

V3

20

20

100

17

85

V4

20

20

100

18

90

Solvent control

V5

20

80

19

79

95

99

11

60

58

76

V6

20

20

100

14

70

V7

20

20

100

17

85

V8

20

20

100

18

90

1.0

V9

20

80

20

80

100

100

19

72

95

90

V10

20

20

100

18

90

V11

20

20

100

17

85

V12

20

20

100

18

90

3.2

V13

20

80

20

80

100

100

17

58

85

73

V14

20

20

100

15

75

V15

20

20

100

13

65

V16

20

20

100

13

65

10

V17

20

80

20

80

100

100

8

46

40

58

V18

20

20

100

14

70

V19

20

20

100

16

80

V20

20

20

100

8

40

32

V21

20

80

20

78

100

98

14

39

70

51

V22

20

20

100

13

65

V23

20

20

100

0

0

V24

20

18

90

12

67

100

V25

20

80

*

59

*

98

*

7

*

12

V26

20

19

95

0

0

V27

20

20

100

3

15

V28

20

20

100

4

20

* Eggs were lost possibly due to cleaning error. Not included in statistical analysis

 

Total length of fish (mm) at 28 days post-hatch

Fish
Number

Nominal concentration (µg/L)

Control

Solvent control

1.0

Vessel number

V1

V2

V3

V4

V5

V6

V7

V8

V9

V10

V11

V12

1

19.8

21.3

20.6

18.4

23.4

25.2

20.1

20.7

21.1

13.8

20.7

19.9

2

21.1

14.1

21.2

23.2

27.8

25.4

21.8

19.7

21.3

17.9

20.2

22.7

3

21.6

16.5

16.1

24.3

24.2

22.1

21.0

26.0

20.5

22.2

22.4

22.2

4

22.9

14.0

24.2

14.3

24.4

23.7

19.6

21.1

20.9

22.1

14.1

21.1

5

24.8

21.3

20.9

20.6

28.0

23.5

21.0

21.8

20.9

22.3

14.2

21.4

6

22.0

22.6

19.6

20.2

18.9

22.9

20.8

16.1

21.8

19.9

23.4

23.2

7

23.4

14.6

22.0

21.7

24.9

22.7

23.9

20.1

20.7

21.8

22.6

22.2

8

23.3

19.4

22.9

16.4

16.5

20.4

22.2

20.5

21.7

21.5

22.1

20.8

9

19.3

21.4

22.2

21.0

22.5

22.4

21.3

21.1

21.6

21.7

20.5

21.7

10

22.0

21.0

23.1

22.1

25.9

23.5

20.0

22.4

16.2

21.6

25.4

20.3

11

21.8

20.5

19.8

21.4

23.9

21.5

22.4

23.7

20.9

15.3

20.9

20.0

12

23.3

22.4

23.3

19.9

-

23.9

19.6

22.3

19.4

22.3

14.7

22.0

13

23.6

20.7

13.1

22.7

-

19.5

18.5

16.7

23.8

21.7

24.0

21.3

14

21.3

20.1

23.3

20.8

-

22.1

20.8

19.5

22.2

20.6

23.7

22.5

15

20.9

21.8

22.0

22.5

-

-

21.2

19.2

22.0

24.1

17.4

20.8

16

20.0

23.7

21.3

21.2

-

-

16.0

16.7

15.6

17.5

21.4

20.0

17

-

21.9

24.5

21.0

-

-

20.5

19.0

15.7

10.1

14.0

22.0

18

-

20.3

-

22.0

-

-

-

18.6

21.3

18.6

-

22.7

19

-

20.4

-

-

-

-

-

-

14.3

-

-

-

20

-

-

-

-

-

-

-

-

-

-

-

-

Mean

21.9

19.9

21.2

20.8

23.7

22.8

20.6

20.3

20.1

19.7

20.1

21.5

SD

1.5

2.9

2.9

2.4

3.4

1.6

1.7

2.5

2.6

3.6

3.8

1.0

Overall mean

20.9

21.6

20.4

Overall SD

2.6

2.7

3.0

-

Not applicable

SD

Standard deviation

 

Total length of fish (mm) at 28 days post-hatch (continued)

Fish
Number

Nominal concentration (µg/L)

3.2

10

32

Vessel number

V13

V14

V15

V16

V17

V18

V19

V20

V21

V22

V23*

V24

1

21.5

16.6

13.1

22.4

20.9

22.3

20.2

24.6

25.7

23.4

-

23.8

2

24.5

20.8

23.3

24.0

20.1

22.0

20.5

11.5

10.9

22.3

-

26.4

3

21.4

22.9

17.3

18.2

27.2

20.5

19.4

25.8

22.2

24.4

-

25.4

4

22.4

18.5

23.7

23.0

23.4

22.0

21.2

18.0

24.4

22.0

-

24.3

5

20.5

22.2

25.8

23.2

24.3

22.7

23.9

11.2

18.9

21.3

-

21.3

6

21.5

21.2

23.7

22.9

24.5

24.9

21.1

12.8

22.3

25.6

-

24.2

7

20.2

22.4

24.7

24.3

22.0

20.3

23.2

10.7

23.0

24.3

-

24.9

8

20.5

23.2

24.9

22.6

19.6

23.8

22.4

11.4

21.8

23.3

-

24.3

9

23.9

19.8

25.0

23.5

-

24.1

23.0

-

22.0

23.6

-

22.4

10

20.6

23.0

19.3

23.7

-

22.3

22.5

-

24.2

22.8

-

22.2

11

20.6

22.1

23.2

24.9

-

22.9

22.5

-

21.9

22.7

-

21.6

12

22.5

22.2

20.2

23.3

-

21.0

23.9

-

24.6

21.0

-

21.6

13

21.7

21.9

20.6

24.3

-

22.4

23.0

-

24.2

21.4

-

-

14

19.7

22.7

-

-

-

22.4

20.0

-

22.6

-

-

-

15

22.4

22.5

-

-

-

-

24.3

-

-

-

-

-

16

23.0

-

-

-

-

-

23.9

-

-

-

-

-

17

22.0

-

-

-

-

-

-

-

-

-

-

-

18

-

-

-

-

-

-

-

-

-

-

-

-

19

-

-

-

-

-

-

-

-

-

-

-

-

20

-

-

-

-

-

-

-

-

-

-

-

-

Mean

21.7

21.5

21.9

23.1

22.8

22.4

22.2

15.8

22.1

22.9

-

23.5

SD

1.3

1.9

3.7

1.6

2.6

1.3

1.6

6.3

3.6

1.4

-

1.7

Overall mean

22.0

21.2

22.8

Overall SD

2.3

3.9

2.5

-

Not applicable

SD

Standard deviation

*

Eggs were lost possibly due to cleaning error. Not included in statistical analysis

 

 

Total length of fish (mm) at 28 days post-hatch (continued)

Fish
Number

Nominal concentration (µg/L)

100

 

Vessel number

V25

V26

V27

V28

 

1

-

-

26.5

24.9

 

2

-

-

27.5

26.0

 

3

-

-

9.9

26.1

 

4

-

-

-

27.4

 

5

-

-

-

-

 

6

-

-

-

-

 

7

-

-

-

-

 

8

-

-

-

-

 

9

-

-

-

-

 

10

-

-

-

-

 

11

-

-

-

-

 

12

-

-

-

-

 

13

-

-

-

-

 

14

-

-

-

-

 

15

-

-

-

-

 

16

-

-

-

-

 

17

-

-

-

-

 

18

-

-

-

-

 

19

-

-

-

-

 

20

-

-

-

-

 

Mean

-

-

21.3

26.1

 

SD

-

-

9.9

1.0

 

Overall mean

24.0

 

Overall SD

6.3

 

-

Not applicable

 

SD

Standard deviation

 

 

Wet weights of fish (mg) at 28 days post‑hatch

Fish
Number

Nominal concentration (µg/L)

Control

Solvent control

1.0

Vessel number

V1

V2

V3

V4

V5

V6

V7

V8

V9

V10

V11

V12

1

74.6

85.9

77.7

57.1

119.2

143.1

100.5

80.9

85.8

22.0

68.6

73.1

2

93.8

16.3

86.9

122.4

207.3

156.3

87.8

87.0

86.5

71.2

75.7

102.3

3

120.2

30.8

38.9

116.9

144.8

100.5

81.3

171.7

78.3

113.1

94.3

96.6

4

126.6

19.7

131.9

23.9

148.6

118.3

73.5

93.8

75.3

112.3

17.6

90.6

5

159.3

91.1

94.8

75.4

214.9

124.6

94.5

101.1

78.5

109.8

17.5

86.2

6

105.7

121.4

69.0

72.0

79.8

140.0

101.3

38.9

86.1

81.0

115.9

114.7

7

105.7

18.7

120.8

95.7

142.2

107.1

131.3

103.2

87.6

109.1

88.2

90.9

8

120.3

73.5

119.1

35.6

27.9

89.0

103.8

89.2

86.4

104.2

121.4

76.1

9

94.5

80.2

82.8

101.6

101.5

110.2

85.5

95.7

86.0

95.0

84.0

87.6

10

107.4

89.8

131.2

95.3

166.3

130.4

63.9

105.5

226.0

112.7

133.9

69.9

11

78.2

75.6

75.8

89.0

153.6

99.7

104.3

128.8

81.1

25.2

75.3

67.0

12

122.4

111.1

112.5

68.5

-

135.8

70.0

110.9

76.2

107.2

21.4

100.4

13

144.5

82.8

18.4

118.4

-

76.9

66.1

27.2

106.3

92.4

132.9

91.8

14

87.2

92.3

125.5

82.3

-

115.9

69.8

76.0

95.3

92.2

120.5

99.4

15

92.3

109.2

94.5

109.8

-

-

96.9

52.3

101.4

120.6

53.5

66.9

16

66.1

133.6

84.2

90.5

-

-

22.3

32.0

21.5

41.9

117.0

71.5

17

-

111.0

125.5

92.2

-

-

93.5

68.5

23.6

10.1

17.3

95.8

18

-

78.1

-

109.1

-

-

-

87.2

83.5

81.6

-

100.5

19

-

83.7

-

-

-

-

-

-

16.1

-

-

-

20

-

-

-

-

-

-

-

-

-

-

-

-

Mean

106.2

79.2

93.5

86.4

136.9

117.7

85.1

86.1

83.2

83.4

79.7

87.9

SD

25.3

34.7

32.3

27.4

54.0

22.3

23.7

35.2

43.1

35.2

41.9

14.1

Overall mean

90.7

102.5

83.6

Overall SD

31.3

39.5

34.9

-

Not applicable

SD

Standard deviation

 

Wet weights of fish (mg) at 28 days post‑hatch (continued)

Fish
Number

Nominal concentration (µg/L)

3.2

10

32

Vessel number

V13

V14

V15

V16

V17

V18

V19

V20

V21

V22

V23*

V24

1

92.2

41.3

15.4

105.7

71.8

103.4

75.5

194.2

137.7

127.0

-

125.6

2

132.4

73.2

108.8

126.9

78.2

96.2

80.5

11.3

8.4

102.9

-

161.6

3

87.2

104.6

34.7

53.2

221.2

84.1

63.1

195.6

104.2

135.4

-

150.1

4

105.7

53.6

120.0

116.9

123.2

85.1

90.1

49.0

131.8

116.7

-

141.4

5

78.0

106.5

135.3

119.2

150.2

95.9

112.7

10.5

59.0

98.3

-

85.8

6

81.5

86.9

112.7

128.5

138.5

134.3

82.3

12.6

107.6

165.7

-

147.0

7

71.5

91.9

138.2

129.0

98.7

86.5

103.4

10.7

111.1

140.8

-

139.9

8

69.4

107.3

139.8

108.6

67.1

110.5

96.3

9.5

95.7

125.6

-

137.1

9

124.9

58.7

154.1

107.3

-

134.2

95.5

-

102.3

125.8

-

115.6

10

80.8

84.4

66.0

112.2

-

95.0

81.7

-

147.4

114.1

-

107.5

11

75.9

96.7

112.1

144.0

-

214.4

111.2

-

86.5

109.7

-

93.9

12

100.3

87.1

67.0

126.4

-

81.1

107.5

-

141.0

92.2

-

102.0

13

87.7

95.1

80.4

138.0

-

102.0

124.0

-

123.8

92.6

-

-

14

77.1

100.1

-

-

-

92.6

60.3

-

111.1

-

-

-

15

92.8

99.9

-

-

-

-

145.8

-

-

-

-

-

16

106.5

-

-

-

-

-

123.9

-

-

-

-

-

17

86.7

-

-

-

-

-

-

-

-

-

-

-

18

-

-

-

-

-

-

-

-

-

-

-

-

19

-

-

-

-

-

-

-

-

-

-

-

-

20

-

-

-

-

-

-

-

-

-

-

-

-

Mean

91.2

85.8

98.8

116.6

118.6

108.2

97.1

61.7

104.8

119.0

-

125.6

SD

17.8

20.4

42.8

22.4

51.8

34.8

23.3

83.3

36.4

21.0

-

24.3

Overall mean

97.2

98.1

115.9

Overall SD

28.5

48.7

29.0

-

Not applicable

SD

Standard deviation

*

Eggs were lost possibly due to cleaning error. Not included in statistical analysis

 

 

Wet weights of fish (mg) at 28 days post‑hatch (continued)

Fish
Number

Nominal concentration (µg/L)

100

Vessel number

V25

V26

V27

V28

1

-

-

212.3

160.2

2

-

-

236.7

213.1

3

-

-

6.9

190.5

4

-

-

-

234.2

5

-

-

-

-

6

-

-

-

-

7

-

-

-

-

8

-

-

-

-

9

-

-

-

-

10

-

-

-

-

11

-

-

-

-

12

-

-

-

-

13

-

-

-

-

14

-

-

-

-

15

-

-

-

-

16

-

-

-

-

17

-

-

-

-

18

-

-

-

-

19

-

-

-

-

20

-

-

-

-

Mean

-

-

152.0

199.5

SD

-

-

126.2

31.7

Overall mean

179.1

Overall
SD

80.4

-

Not applicable

SD

Standard deviation

Validity criteria fulfilled:
yes
Conclusions:
The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species, Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.
No statistically significant differences were observed in terms of hatching success or final length and wet weight at any of the test concentrations employed in the test compared to the combined controls. Statistically significant differences were observed in terms of post-hatch survival at the 10, 32 and 100 µg/L test concentrations compared to the combined controls.
Given that the organisms were exposed to a mixture of both parent test substance and oxidation product, the results have been calculated based on nominal concentrations and geometric mean measured concentrations of the parent test substance.
The NOEC and LOEC values for hatching success were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).
The NOEC and LOEC values for post-hatch survival were considered to be 3.2 and >10 µg/L, respectively, based on nominal concentrations (2.1 and 13 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).
Taking both length and weight into consideration, the NOEC and LOEC values for growth were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).
Executive summary:

The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.

 

Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L. Control and solvent control groups were also included.

 

The test was conducted under flow-through conditions with continual renewal of the test media, using embryos produced by in-house brood fish which, on exposure, were at the early embryo/tail bud stage of development.

 

The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, Lowinox® AH-25, and its oxidation product. Lowinox® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.

 

Measured concentrations of the parent test substance showed variability throughout the duration of the test. This was due to the unstable nature of the test substance in the test media. The use of flow-through conditions and daily renewal of the solvent stock solutions assisted in maintaining relatively consistent concentrations in each test group. The peak area ratio of the parent to the oxidation product was also monitored during chemical analysis which indicated consistency throughout the duration of the test.

 

Given that the organisms were exposed to a mixture of both parent test substance and oxidation product, the results have been calculated based on nominal concentrations and geometric mean measured concentrations of the parent test substance. The geometric mean measured concentrations of parent test substance were calculated to be 2.1, 13, 31 and 44 µg/L for the 3.2, 10, 32 and 100 µg/L test concentrations, respectively. As analytical results were below the limit of quantification at all sampling occasions for the 1.0 µg/L test group, the this concentration was below the NOEC, a geometric mean measured concentration was not calculated. 

 

The mean hatching success of embryos in the control and solvent control groups was 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. The post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was greater than the validity criterion of ≥75%. The test is therefore considered to be valid.

 

No significant differences were observed between the control and solvent control in terms of hatching success, post-hatch survival, lengths and weights. As such, the test groups were compared to the combined control groups.

No statistically significant differences were observed in terms of hatching success or final wet weight and length at any of the test concentrations employed in the test compared to the combined controls. Significant differences were observed between the 10, 32 and 100 µg/L test concentrations and the combined controls in terms of post-hatch survival.

 

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint

Nominal concentration (µg/L)

Geometric mean measured concentration of parent compound (µg/L)

NOEC

LOEC

NOEC

LOEC

Hatching success

100

>100

44

>44

Post-hatch survival

3.2

10

2.1*

13

Growth (Length)

100

>100

44

>44

Growth (Weight)

100

>100

44

>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.

Description of key information

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint

Nominal concentration (µg/L)

Geometric mean measured concentration of parent compound (µg/L)

NOEC

LOEC

NOEC

LOEC

Hatching success

100

>100

44

>44

Post-hatch survival

3.2

10

2.1*

13

Growth (Length)

100

>100

44

>44

Growth (Weight)

100

>100

44

>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
3.2 µg/L

Additional information

The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.

 

Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L. Control and solvent control groups were also included.

The test was conducted under flow-through conditions with continual renewal of the test media, using embryos produced by in-house brood fish which, on exposure, were at the early embryo/tail bud stage of development.

 

The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, Lowinox® AH-25, and its oxidation product. Lowinox® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.

Measured concentrations of the parent test substance showed variability throughout the duration of the test. This was due to the unstable nature of the test substance in the test media. The use of flow-through conditions and daily renewal of the solvent stock solutions assisted in maintaining relatively consistent concentrations in each test group. The peak area ratio of the parent to the oxidation product was also monitored during chemical analysis which indicated consistency throughout the duration of the test.

 

The mean hatching success of embryos in the control and solvent control groups was 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. The post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was greater than the validity criterion of ≥75%. The test is therefore considered to be valid.

No statistically significant differences were observed in terms of hatching success or final wet weight and length at any of the test concentrations employed in the test compared to the combined controls. Significant differences were observed between the 10, 32 and 100 µg/L test concentrations and the combined controls in terms of post-hatch survival.

 

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint

Nominal concentration (µg/L)

Geometric mean measured concentration of parent compound (µg/L)

NOEC

LOEC

NOEC

LOEC

Hatching success

100

>100

44

>44

Post-hatch survival

3.2

10

2.1*

13

Growth (Length)

100

>100

44

>44

Growth (Weight)

100

>100

44

>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.

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