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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:
17 Jan - 26 Mar 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
yes
Remarks:
Deviations of validity criteria were given for dissolved oxygen concentration (on Day 8 for some replicates) and temperature (maximum variations were 2.0 and 1.8°C). See remarks in section "See other information on results"
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and each surviving test group
- Sampling method: Water samples were taken from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the aged or expired media on Days 1, 7, 14, 21, 28 and 33 (Replicates R1 to R4 pooled) for quantitative analysis. All samples were analyzed on the day of sampling with the exception of Day 27 and 32 which were stored frozen prior to analysis.
Duplicate sets of samples were taken on each sampling occasion and stored frozen for further analysis if necessary.
Vehicle:
yes
Remarks:
Dimethylformamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: On Days 0, 1, 2 and 4 to 32, a nominal amount of test item (100 mg) was dissolved in DMF and the volume adjusted to 10 mL to give a 10 mg/mL solvent stock solution. Further dilutions were performed in DMF to give further stock solutions of 5.0, 2.5, 1.25, 0.625 mg/mL. The stock solutions were inverted several times to ensure adequate mixing and homogeneity.
On Days 0, 1, 2 and 4 to 14, an aliquot (200 µL) of each solvent stock solution was dispersed in 4 liters of test water with the aid of magnetic stirring for approximately 15 minutes to give test concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.50 mg/L.
On Days 15 to 32, an aliquot (1000 µL) of each solvent stock solution was dispersed in 20 liters of test water with the aid of magnetic stirring for approximately 15 minutes to give test concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.50 mg/L.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 µL/L of DMF.
- Controls: control, solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After dosing and throughout the test all control and test concentrations were observed to be clear, colorless solutions by visual inspection.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: The adult fathead minnows that produced the eggs for the test were bred at test facility on 14 Sep 2018.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED 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 13 Feb 2019 and used for the definitive test.
- Subsequent handling of eggs: The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.

POST-HATCH FEEDING
- Frequency of feeding: The larvae were fed freshly hatched special grade brine shrimp nauplii from Day 7 to Day 12. On Day 13 onwards, the larvae were fed freshly hatched basic grade brine shrimp nauplii.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Hardness:
120 - 146 mg/L as CaCO3 (test start)
120 - 160 mg/L as CaCO3 (test end)
Test temperature:
23.3 to 25.7 °C
pH:
7.2 to 9.0
Dissolved oxygen:
The dissolved oxygen concentration was maintained at least at 4.9 mg O2/L (equivalent to 60% ASV) during the test.
Exception of eight test vessels (two solvent controls, two at 0.024 mg/L, one at 0.050 mg/L, two at 0.21 mg/L and one at 0.39 mg/L) on Day 8 where the lowest dissolved oxygen concentration was recorded at 3.2 mg O2/L (equivalent to 39% ASV at 25 ºC).
See remarks in section "See other information on results".
Nominal and measured concentrations:
nominal: 0.03125, 0.0625, 0.125, 0.25 and 0.50 mg/L
measured: 0.024, 0.050, 0.11, 0.21 and 0.39 mg/L (geometric mean measured concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 liter glass vessels (Day 0 to Day 14), 5 liter glass vessels (Day 15 to the end of the test)
- Material, size, headspace, fill volume: volume of test preparation: 400 mL from Day 0 to Day 5, 800 mL from Day 6 to Day 14 and 4000 mL from Day 15 to Day 33
- Aeration: As a result of the low dissolved oxygen measured concentrations recorded in the old solutions on Day 8, the contents of the test vessels were aerated via narrow bore glass tubes from Day 8 onwards.
- Renewal rate of test solution (frequency/flow rate): daily, with the exception of Day 3 when 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: dechlorinated laboratory tap water
- Intervals of water quality measurement: Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The light intensity during the light period was measured daily (with the exception of Day 1 which was not measured in error) using an ATP Instrumentation Lux meter. 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).
- Conductivity: 345 μS/cm at 20 °C

OTHER TEST CONDITIONS
- Photoperiod: 16/ 8 light/dark cycle with 20 min dawn and dusk transition periods.
- Light intensity: 329 to 583 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
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).
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 concentrations: 0.50, 0.050 and 0.0050 mg/L, solvent control
- Results used to determine the conditions for the definitive study: The results showed there was no difference between the control and test concentrations of 0.0050, 0.050 and 0.50 mg/L in terms of hatching, survival and growth. No sub-lethal effects were observed at the test concentration of 0.050 mg/L. However, sub-lethal effects were observed in the control, solvent control and at test concentrations of 0.0050 and 0.50 mg/L. The sub lethal effects observed were pale coloration and bent spines. The number of fish displaying sub-lethal effects was low and did not follow a dose-response, therefore these observations are considered to be naturally occurring and not as a result of exposure to the test item.
Based on this information test concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.50 mg/L were selected for the definitive test.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.21 mg/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.:
>= 0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Mortality/survival at embryo, larval, juvenile, and adult stages:
- Days to hatch or time to release of young: start of egg hatching on Day 3, completion of hatching on Day 5
- Type of and number with morphological abnormalities: Two larvae were observed to have bent spines. This observation was recorded from Day 15 onwards in control replicate R3 (one larva) and from Day 16 onwards in solvent control replicate R1 (one larva). The overall number of larvae with bent spines was very low (two in total) and was not concentration dependent. Therefore this observation is considered to be due to naturally occurring defects occurring in a population, and not attributable to the test item. An abnormal observation was also observed in one replicate in the 0.39 mg/L test group. The observation recorded was in relation to the small size of the fish; however, the numbers of fish recorded with this observation was very low, one small fish. Therefore, this observation was considered to be due to defects occurring in a natural population, and not necessarily attributable to exposure to the test item.

Reported statistics and error estimates:
For the estimation of the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) the hatching and post hatch survival data for the pooled control and each test group were compared using the Chi2 2x2 Table Test with Bonferroni Correction. The body length and wet weight data for the pooled control and each test group obtained on termination of the test were compared using Williams Multiple Sequential t test procedure. All results were calculated using the ToxRat Professional computer software package (ToxRat).

Deviations:

The validity criteria for the test specified that the dissolved oxygen concentration should remain greater than 60% ASV (equivalent to 4.9 mg O2/L at 25 ºC), however, on Day 8 of the definitive dissolved oxygen concentrations below this were recorded in the old test solutions in the following test vessels: solvent control replicates 1 and 4, 0.024 mg/L replicates 1 and 4, 0.11 mg/L replicate 2, 0.21 mg/L replicates 2 and 3 and 0.39 mg/L replicate 3. The lowest measured dissolved oxygen concentration was 3.2 mg O2/L (equivalent to approximately 39% ASV). As such the test did not achieve the test validity criteria for this parameter; however, as the reduced dissolved oxygen level did not coincide with any mortalities or sub lethal effects, this was considered not to have adversely affect the results of the test.

The water temperature between test vessels on the same day and on successive days exceeded the limit of 1.5 °C as specified in the study plan, the maximum variations recorded were 2.0 and 1.8 °C respectively. In addition, the range of water temperatures recorded over the duration of the test was 23.3 to 25.7 °C and therefore exceeded the recommended range of 25 ± 1.5 °C given in the study plan. Given that the deviation was only slightly outside of the recommended temperature range and did not coincide with any mortalities or sub-lethal effects, this was considered not to have adversely affect the results of the test.

No details of embryonic development were recorded during the range-finding test on days 1 to 4. The number of live and dead eggs and hatching were recorded on each day, therefore this deviation was considered not to have adversely affected the results of the study.

On a number of occasions throughout the definitive test the number of fish in some test vessels was incorrectly recorded resulting in incorrect volumes of feed being provided. At the end of the test the weight and length of the surviving fish in the affected replicates were inspected and no overall impact was identified. This error was considered not to have adversely affected the results of the test.

These deviations were considered to have not affected the integrity or validity of the study.

The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed. 

The number of dead larvae was observed to be low throughout the duration of the test with no concentration dependent effects being observed. 

There were no statistically significant differences for post-hatch survival, between the pooled control and 0.024, 0.050, 0.11 and 0.21 mg/L test concentrations; however the 0.39 mg/L test concentration was significantly different. Therefore the NOEC based on posthatch survival was 0.21 mg/L. 

For the body length and the wet weight, there were no statistically significant differences, between the pooled control and all test groups.

Table 1: Hatching and Post Hatch Survival in the Definitive Test (R = Replicate)

Geometric Mean Measured Concentration
(mg/L)

Hatching Rate
(%)

Mean Hatching Rate
(%)

Survival Rate
(%)

Mean Survival Rate
(%)

Control

R1

100

96

100

100

R2

100

100

R3

95

100

R4

90

100

Solvent Control

R1

95

98

100

100

R2

100

100

R3

100

100

R4

95

100

0.024

R1

100

94

100

99

R2

90

100

R3

95

95

R4

90

100

0.050

R1

95

95

100

100

R2

95

100

R3

100

100

R4

90

100

0.11

R1

95

95

95

98

R2

90

100

R3

100

95

R4

95

100

0.21

R1

100

98

100

99

R2

95

100

R3

95

95

R4

100

100

0.39

R1

85

90

100

93

R2

95

100

R3

95

89

R4

85

82

 

Table 2: Number of eggs hatching (R = Replicate)

Geometric Mean Measured Concentration (mg/L)

Eggs Introduced

Eggs Hatched

Control

R1 - R4

80

77

Solvent Control

R1 - R4

80

78

Pooled Control

160

155

0.024

R1 - R4

81

76

0.050

R1 - R4

80

76

0.11

R1 - R4

80

76

0.21

R1 - R4

80

78

0.39

R1 - R4

80

72

 

 Table 3: Post-Hatch Survival (R = Replicate)

Geometric Mean Measured Concentration (mg/L)

Eggs Hatched

Post-Hatch Survival

Control

R1 - R4

77

77

Solvent Control

R1 - R4

78

78

Pooled Control

155

155

0.024

R1 - R4

76

75

0.050

R1 - R4

76

76

0.11

R1 - R4

76

74

0.21

R1 - R4

78

77

0.39

R1 - R4

72

67

 

Table 4: Fish Length (R = Replicate)

Geometric Mean Measured Concentration (mg/L)

Fish Length (mm)

Mean

Standard Deviation

Control

R1 - R4

20.53

0.16

Solvent Control

R1 - R4

20.32

0.83

Pooled Control

20.42

0.56

0.024

R1 - R4

20.69

0.43

0.050

R1 - R4

21.02

0.44

0.11

R1 - R4

20.50

0.45

0.21

R1 - R4

20.36

0.35

0.39

R1 - R4

20.18

0.65

 

Table 5: Fish wet weight (R = Replicate)

Geometric Mean Measured Concentration (mg/L)

Fish Weight (mg)

Mean

Standard Deviation

Control

R1 - R4

72.8

3.23

Solvent Control

R1 - R4

75.9

11.8

Pooled Control

74.3

8.15

0.024

R1 - R4

74.7

0.777

0.050

R1 - R4

77.4

3.92

0.11

R1 - R4

75.4

2.58

0.21

R1 - R4

73.3

0.638

0.39

R1 - R4

70.9

3.45

 


Description of key information

NOEC (33 d) = 0.21 mg/L (meas. geom. mean, P. promelas, OECD 210)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.21 mg/L

Additional information

Results investigating the long-term toxicity of Propane-1,2,3-triyl 2-ethylhexanoate (CAS No. 7360-38-5) to freshwater fish are available. One GLP-study was conducted under semi-static conditions according to OECD guideline 210 using Pimephales promelas as test organism.

P. promelas was exposed to five nominal test concentrations (0.03125, 0.0625, 0.125, 0.25 and 0.50 mg/L) using Dimethylformamide as solvent, which were analytically verified resulting in actual exposure concentrations of 0.024, 0.050, 0.11, 0.21 and 0.39 mg/L (geometric mean). Effects were observed for post-hatch survival resulting in a NOEC of 0.21 mg/L (geom. mean). For hatching, length and wet weight no effect was observed (NOEC ≥ 0.39 mg/L, geom. mean).

The validity criteria for the test specified that the dissolved oxygen concentration should remain greater than 60% ASV (equivalent to 4.9 mg O2/L at 25 °C). However, on Day 8 of the definitive dissolved oxygen concentrations below this were recorded in the old test solutions in the following test vessels: solvent control replicates 1 and 4, 0.024 mg/L replicates 1 and 4, 0.11 mg/L replicate 2, 0.21 mg/L replicates 2 and 3 and 0.39 mg/L replicate 3. The lowest measured dissolved oxygen concentration was 3.2 mg O2/L (equivalent to approximately 39% ASV). As such the test did not achieve the test validity criteria for this parameter. However, as the dissolved oxygen level was only slightly outside of the recommended parameter range and did not coincide with any mortalities or sub lethal effects, this was considered not to have adversely affect the results of the test and did not affect animal welfare.

The water temperature between test vessels on the same day and on successive days exceeded the limit of 1.5 °C as specified in the study plan, the maximum variations recorded were 2.0 and 1.8 °C respectively. In addition, the range of water temperatures recorded over the duration of the test was 23.3 to 25.7 °C and therefore exceeded the recommended range of 25 ± 1.5 °C given in the study plan. Given that the deviation was only slightly outside of the recommended temperature range and did not coincide with any mortalities or sub-lethal effects, this was considered not to have adversely affect the results of the test.

No details of embryonic development were recorded during the range-finding test on days 1 to 4. The number of live and dead eggs and hatching were recorded on each day, therefore this deviation was considered not to have adversely affected the results of the study.

On a number of occasions throughout the definitive test the number of fish in some test vessels was incorrectly recorded resulting in incorrect volumes of feed being provided. At the end of the test the weight and length of the surviving fish in the affected replicates were inspected and no overall impact was identified. This error was considered not to have adversely affected the results of the test.

Overall, these deviations were considered to have not affected the integrity or validity of the study.