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Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between September 2016 and February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Waxy solid
Details on test material:
Identification: Acetalization product between glucose and C20/22 (even numbered)-alcohol
Physical state/Appearance: Beige waxy solid (pearls)
Batch: U44635
Purity: >99.9% (dry extract)
Expiry Date: 01 November 2017
Storage Conditions: Room temperature in the dark
Specific details on test material used for the study:
Identification: Acetalization product between glucose and C20/22 (even numbered)-alcohol
Physical state/Appearance: Beige waxy solid
Batch: 160330010593
Purity: >99.9% (dry extract)
Expiry Date: 01 March 2019
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 100 mg/L Loading Rate WAF test group from the freshly prepared bulk test preparation on Days 0, 4, 8, 18, 25 and 32 and from the old or expired media on Days 2, 6, 11, 20, 27 and 33 (Replicates R1 to R4 pooled) for quantitative analysis. The samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Based on the results of a Daphnia magna reproduction test (Envigo Research Limited, Study Number: 41502364), newly laid fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a water accommodated fraction of the test item at a nominal loading rate of 100 mg/L for a period of 33 days under semi-static test conditions.

A nominal amount of test item (2200 mg) was added to the surface of 22 liters of test water to give the 100 mg/L loading rate. A glass siphoning tube was placed into the media before the addition of the test item. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Visual observations at the end of the settlement period dictated the need for a glass wool plug to be inserted into the end of the siphoning tube. After filtration through glass wool, microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were bred at Envigo Research Limited on 31 March 2016 and maintained in dechlorinated tap water with an activated carbon and biological filtration system.
The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the seven days preceding the start of the test, the water temperature was controlled at 24 °C to 26 °C with a dissolved oxygen content of greater than or equal to 7.4 mg O2/L. The breeding stock fish were fed ZM 400 flake food daily and frozen brine shrimp.
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 28 September 2016 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.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

The larvae were fed less than 24-Hour old special grade brine shrimp nauplii from Day 6 to Day 12, and less than 24-Hour old basic grade brine shrimp nauplii from Day 13 to Day 15. Basic grade brine shrimp naulplii of 24 to 48 hours old were fed to the larvae from Day 16 through to the end of the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d

Test conditions

Hardness:
144 to 156 mg/L as CaCO3
Test temperature:
25 ± 1.5 °C
pH:
7.1 to 8.5
Dissolved oxygen:
36 to 104% ASV
Nominal and measured concentrations:
Chemical analysis of the test preparations at days 0, 2, 4, 6, 8, 11, 18, 20, 15, 27, 32 and 33 showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0070mg/L, to 0.064 mg/L.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Nominal test concentration: 100 mg/L
Four replicate flasks were used for each control and test 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 14 to the end of the test 5 liter glass vessels were used.
- Type (delete if not applicable): 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 6, 800 mL from Day 6 to Day 14 and 4000 mL from Day 14 through to the end of the test.
- Aeration: The test vessels were aerated via narrow bore glass tubes from Day 5 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 from the start of the test.
- No. of fertilized eggs/embryos per vessel: Twenty eggs
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
The water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded before and after each test media renewal throughout the test. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration was measured using a Hach Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in Control Replicate R1 using a Testo temperature logger.
The water hardness was measured in the bulk test preparation at the start and in each vessel on termination of the test and was determined using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of
33 days.
- Light intensity: 514 to 720 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.


Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
33 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival and Growth
Details on results:
Number of Eggs Hatching
The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.
The start of egg hatching was observed on Day 5 of the test and completion of hatching was observed on Day 6 of the test. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.
LL50 (Hatching): >100 mg/L loading rate WAF
Statistical analysis of the hatching data was carried out for the control 100 mg/L loading rate WAF test group. There were no statistically significant differences (P≥0.05), between the control and the 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on the number of eggs hatching was 100 mg/L loading rate WAF. The "Lowest Observed Effect Loading Rate" (LOEL) was not determined as there were no effects observed at the maximum concentration required to be tested by the test guideline.

Post-hatch Survival
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 concentrations.
LL50(Post-hatch survival): >100 mg/L loading rate WAF
Statistical analysis of the post-hatch survival data was carried out for the control and the 100 mg/L loading rate WAF test group. There were no statistically significant differences (P≥0.05), between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on post-hatch survival was 100 mg/L loading rate WAF. The LOEL was not determined as there were no effects observed at the maximum concentration required to be tested by the test guideline.

Length and Weight Data
EL50 (Length): >100 mg/L loading rate WAF
Statistical analysis of the fish body length data was carried out for the control the 100 mg/L loading rate test group. There were no statistically significant differences (P≥0.05), between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on body length was 100 mg/L loading rate WAF. The LOEL was not determined as there were no effects observed at the maximum concentration required to be tested by the test guideline.
EL50 (Weight): >100 mg/L loading rate WAF
Statistical analysis of the fish wet weight data was carried out for the control the 100 mg/L loading rate test group. There were no statistically significant differences (P≥0.05), between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on wet weight was 100 mg/L loading rate WAF. The LOEL was not determined as there were no effects observed at the maximum concentration required to be tested by the test guideline.
Given this information it was considered that no effect on survival or growth attributable to the test item was observed.

Observations
There were two sub-lethal effects observed in the test, uncoordinated swimming seen on Day 9 (100 mg/L R3 1 larva) and on Day 22 to 24 one fish was observed as being small (Control R1).

Any other information on results incl. tables

Validation Criteria

The following validity criteria were achieved during the test:

 

Required

Achieved

1)     Dissolved oxygen

60% - 100% ASV

36 to 104% ASV

2)     Water temperature

 

 

Between test chambers or successive days

± 1.5oC

± 0.9oC

Range

25 ± 1.5oC

24.8 - 26.4oC

3)     Hatching success rate*

>70%

89%

4)     Post-hatch survival*

>75%

100%


*In control vessels

 Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs. At the beginning of stirring the 100 mg/L Loading Rate WAF was observed to be a clear colorless water column with beads of test item floating at the surface. At the end of the stirring period and after settling, the 100 mg/L Loading Rate WAF was observed to be a clear colorless water column with beads of test item floating at the surface and with particles of test item dispersed throughout.

After dosing all loading rates were observed to be clear, colorless solutions by visual inspection.

Hatching and Post Hatch Survival in the Definitive Test are presented in the table below.

Nominal Loading Rate
(mg/L)

Hatching Rate

(%)

Mean Hatching Rate (%)

Survival Rate

(%)

Mean Survival Rate (%)

Control

R1

95

89

100

100

R2

70

100

R3

100

100

R4

90

100

100

R1

85

86

100

99

R2

90

100

R3

80

94

R4

90

100


R1– R4= Replicates 1 to 4

Fish Length and Weight Data are displayed in the following tables.

Fish

ControlR1

Control R2

Control R3

Control R4

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

1

22.70

101.0

23.59

107.0

22.61

109.5

22.59

110.0

2

20.88

75.8

13.05

15.7

21.54

80.0

23.14

118.2

3

21.87

84.1

19.56

75.1

24.05

122.1

21.28

90.5

4

20.42

75.3

16.85

35.8

22.00

91.3

23.22

124.8

5

22.69

109.8

17.13

38.0

22.69

104.9

22.45

111.1

6

23.69

112.0

22.36

95.0

22.08

90.0

22.06

100.0

7

21.38

86.9

24.36

139.7

20.90

81.6

21.54

98.0

8

15.16

23.7

23.63

122.9

21.26

82.5

22.84

98.9

9

13.02

11.2

22.78

118.0

22.05

83.7

20.99

89.0

10

19.53

64.1

20.39

78.4

21.22

86.6

21.25

85.7

11

13.77

19.2

17.54

39.0

21.95

80.9

15.84

30.2

12

20.28

73.2

20.80

77.7

22.30

99.7

22.63

93.6

13

20.72

81.3

21.52

88.4

21.95

92.3

21.68

89.6

14

21.62

91.4

19.37

62.8

20.62

69.0

18.68

47.2

15

21.33

77.4

-

-

15.63

26.2

16.75

30.3

16

21.88

81.2

-

-

21.72

92.5

14.56

28.6

17

21.51

79.9

-

-

14.91

21.8

19.78

69.3

18

19.41

60.7

-

-

22.58

103.2

17.44

39.2

19

20.36

68.9

-

-

20.05

68.0

-

-

20

-

-

-

-

21.66

78.4

-

-

Average

20.12

72.5

20.21

78.1

21.19

83.2

20.48

80.8

Group Mean Length:               20.50 mm

Group Mean Wet Weight:      78.7 mg


R1– R4= Replicates 1 to 4

- = No measurement recorded due to fish mortality

Fish

100 mg/L LR WAFR1

100 mg/L LR WAFR2

100 mg/L LR WAFR3

100 mg/L LR WAFR4

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

Length
(mm)

Wet Weight
(mg)

1

21.82

78.7

16.68

32.5

23.54

121.7

14.21

21.5

2

16.06

31.5

13.56

32.7

22.86

107.3

16.38

34.8

3

19.60

60.5

14.04

15.5

23.67

123.5

19.60

68.4

4

22.15

100.2

22.11

108.5

21.55

96.0

24.05

132.6

5

21.90

93.2

22.09

92.9

20.88

79.8

22.53

103.0

6

17.66

42.3

21.98

94.6

23.60

105.0

24.33

127.7

7

23.60

121.0

23.37

106.8

23.24

109.6

23.98

122.9

8

23.03

101.5

23.72

106.9

14.62

23.8

23.03

90.5

9

17.50

41.7

28.41

111.0

23.37

116.8

24.49

140.8

10

21.37

87.5

24.03

120.3

21.46

103.0

21.80

96.3

11

22.24

93.5

18.60

57.0

22.25

95.2

13.12

16.6

12

19.32

65.3

23.92

124.2

20.45

85.4

19.66

64.9

13

21.75

90.2

22.67

94.4

15.41

24.1

17.72

40.6

14

19.62

60.4

21.89

86.1

14.95

23.5

17.48

39.8

15

20.12

70.9

17.58

52.7

16.95

38.2

19.88

69.0

16

19.97

65.7

17.50

48.9

-

-

20.24

73.6

17

19.54

67.4

20.94

91.7

-

-

18.59

46.1

18

-

-

20.39

66.6

-

-

16.22

35.2

19

-

-

-

-

-

-

-

-

20

-

-

-

-

-

-

-

-

Average

20.43

74.8

20.47

80.2

20.59

83.5

19.90

73.6

Group Mean Length:               20.35 mm

Group Mean Wet Weight:      78.0 mg


R1– R4= Replicates 1 to 4

- = No measurement recorded due to fish mortality

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The dissolved oxygen concentration and temperature deviated slightly outside of recommended parameters, these deviations didn't coincide with mortalities or sub-lethal effects, and was considered not to adversely affect the results
Conclusions:
The application of the test item 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 Loading Rate was 100 mg/L Loading Rate WAF.
Executive summary:

Introduction

A study was performed to assess the effects of the test item on freshly hatched larvae 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

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Based on the results of a Daphnia magna reproduction test (Envigo Research Limited, Study Number: 41502364), newly laid fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to a Water Accommodated Fraction (WAF) of the test item for a period of 33 days at a temperature of 24 ºC to 26 ºC under semi-static test conditions. The test solutions were renewed 3 times per week. The test item solution was prepared by stirring a nominal amount of test item (100 mg/L) in test water using a magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surfacefor 23 hours. After the stirring period the mixture allowed to stand for 1 hour before the aqueous phase or WAF was removed by mid-depth siphoning to give the 100 mg/L loading rate WAF.

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 fresh test preparations on days 0, 4, 8, 18, 25 and 32 showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be
0.0070 mg/L, to 0.642 mg/L. Measured test concentrations of the aged test preparations on days 2, 6, 11, 20, 27 and 33 were observed to range from less than the LOQ to 0.034 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

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 100 mg/L loading rate WAF. The application of the test item to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Loading Rate was 100 mg/L loading rate WAF. 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.