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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:
12 December 2018 - 02 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Testing was initiated as requested in ECHA decision TPE-D-2114359620-51-01/F.
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Two different batches were used for the dose-range finding test and for the main test. For the batch used in the DRF, a correction factor of 2.554 was used to correct for solid content. For the batch used in the main study, a correction factor of 2.525 was used to correct for solid content.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and the control
- Sampling method: 2.0 mL.
- Frequency: at the start and after 7, 14, 21, 28 and 33 days of exposure; all test concentrations on the day before the start of exposure to check the functioning of the system, and on days 14, 21, 28 and 33 of exposure from the 24 hour old stock solutions.
- Sample storage conditions before analysis: No storage, samples were transferred to the analytical laboratory of the Test Facility and analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions were prepared by preparing a 25 fold dilution of the test item in purified water. A 15 minute period of magnetic stirring was applied to assure homogenous distribution of the test item. Stocks solutions were prepared daily. The test item was completely soluble in test medium at the concentrations tested.
- Controls: Test medium without test item or other additives
- Other relevant information: A correction factor of 2.525 was used to correct for the purity/composition of the test substance (39.6% solids in water). Concentration/doses are expressed based on solids.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas, Teleostei Cyprinidae, Rafinesque.
- Source: In-house culture

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Ratio male/female fish: 1:2
- Spawning tank: The spawning tank was equipped with a substrate (pvc-tube), which enabled collection of the fertilised eggs.
- Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands).
- Time of fertilisation: Males and females were placed together in spawning tanks and spawning started the following day approximately 1 to 2 hours after lights have been switched on.

POST-HATCH FEEDING
- Embryonic phase: no feeding.
- Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48-hours old. Food was supplied ad libitum.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Hardness:
196 - 214 mg CaCO3 per litre
Test temperature:
Range in test vessels: 23.8 - 25.7 °C
Range continuously measured in control vessel: 24.1 - 25.1 °C
pH:
7.2 - 7.5
Dissolved oxygen:
7.7 - 9.7 mg O2/L
Nominal and measured concentrations:
Target concentrations: 0.050, 0.013, 0.31, 0.78 and 2.0 mg solids/L
Mean measured concentrations: 0.035, 0.090, 0.22, 0.61 and 1.6 mg solids/L. Samples at the start of the test were in agreement with the target concentrations, i.e. were at 97-119% relative to the target concentrations. The analyses of the samples taken on Days 7 to 33 showed lower measured concentrations ranging at 35-131% relative to the target concentrations. The effect parameters were based on mean measured concentrations. See Table 1 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Stainless steel vessels (~1.7 L)
- Aeration: no aeration
- Flow-through system: Exact volumes of the test item stock solutions were dosed with syringes via a computer-controlled system consisting of five dispensers (Gilson). The dosed volumes of the stock entered a mixing flask separately from the dilution water supply (adjusted ISO-medium). The dilution water was supplied applying flow-meters at a constant rate of 5 litres per hour.
In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the eggs/fish larvae/juvenile fish. The flow meters were calibrated before the start of the exposure and weekly thereafter. The whole system was checked twice daily on working days and once per day during weekends.
- Renewal rate of test solution (frequency/flow rate): continuously at a rate of 1 L per hour.
- No. of fertilized eggs/embryos per vessel: 20 fertilized eggs per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using tap-water purified by reverse osmosis, with the following composition: CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L.
- Total organic carbon: not detectable in sample taken from the adjusted ISO medium at the start of the test.
- Culture medium different from test medium: no, Adjusted ISO medium.
- Intervals of water quality measurement: Oxygen, pH and temperature: At the start of the test, at weekly intervals and at the end of the test in one replicate per group. In addition, temperature was recorded continuously in one of the control replicates. Hardness: At the start and the end of the test in all test groups.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h photoperiod daily
- Light intensity: 407 - 652 Lux
- Euthanasia: At the end of the test, the surviving larvae were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water

EFFECT PARAMETERS MEASURED
- Stage of embryonic development: Daily, from the beginning of the exposure
- Hatching and survival: Daily, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording.
- Criteria for death: Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance; Embryos: Absence of body movement and/or absence of heartbeat; Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
- Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical swimming behaviour.
- Body weight: At the end of the test, all surviving fish were weighed on a replicate basis (blotted dry weight).
- Body length: At the end of the test, individual lengths from the surviving fish were measured.

RANGE-FINDING STUDY
- Test concentrations: 0.05, 0.50 and 5.0 mg solids/L (nominal) and untreated control.
- Results used to determine the conditions for the definitive study: yes, based on the results of this preliminary test, it was decided to test a range of target concentrations between 0.050 and 2.0 mg solids/L in the final study. See Table 2 in 'Any other information on results' for results of the range-finding test.
Reference substance (positive control):
no
Key result
Duration:
8 d
Dose descriptor:
EC10
Effect conc.:
> 1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
number hatched
Remarks:
Embryonic survival
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
> 1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
adult mortality
Remarks:
Post-hatch mortality
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
morphology
Remarks:
Malformation of caudal fin
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
other: Growth based on length and weight
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
weight
Remarks on result:
other: 95% confidence interval: 0.46 - 1.0 mg solids/L
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
length
Remarks on result:
other: 95% confidence interval: 0.64 - 1.0 mg solids/L
Details on results:
- Measured concentrations: Analyses of samples taken at the start of the test showed that the measured concentrations were generally in agreement with the target concentrations, i.e. were at 97-119% relative to the target concentrations. The analyses of the samples taken on Days 7 to 33 showed lower measured concentrations ranging at 35-131% relative to the target concentrations. Consequently, samples taken from the stock solutions were taken on Days 14, 21, 28 and 33. The concentrations measured in these samples were at 94-126% of the expected concentrations and thus indicated proper preparation of stock solutions. The reason for the lower measured concentrations is not known, but the observed build-up of microbial biomass could be responsible for this. The effect parameters were thus based on mean measured concentrations, see Table 1 in 'Any other information on results'. It should be noted that small responses were measured in the samples taken from the control group. Those were, however, at the level of the observed carry-over within the analytical system and thus judged to not originate from a contamination of the control group.

- See Table 3 in 'Any other information on results' for all effect parameters.

- Embryonic survival: 99% survival in control. Hatching started on Day 3 and was completed by Day 8 of exposure. Hatchability ranged from 90 to 100% and was generally not treatment related. On Day 5, the percentage of hatching was higher in the test item treated groups than in the control treatment.

- Larval survival and development: 94% larval survival in control. Post-hatch mortality at the end of the test in test item treated groups ranged from 5 to 16% and was not concentration-related. Statistical analyses confirmed that larval survival was not significantly affected up to and including 1.6 mg solids/L (NOEC). In the highest test concentration, all larvae showed malformation of the caudal fin, first observed at day 19 (pictures of the malformations compared with the control larvae are in the attached illustration). At lower test concentrations, a number of larvae showed abnormalities of the skeleton, the eyes or the swim bladder. Other individuals showed changes of coloration, or yolk-sac or cardiac oedemas. These observations were however singular and not test item related.

- Effects on larval growth: The test item caused a dose-related reduction in both body weight and length. At the highest concentration, the reduction of weight reached 38% while length was reduced by 18%. Statistically significant reduction of body weight as well as body length was observed at 1.6 mg solids/L. Hence, the NOEC for growth was 0.61 mg solids/L based on body weight as well as based on body length.

- All environmental paramenters remained within the limits prescribed by the study plan throughout the test.
Reported statistics and error estimates:
Hatchability (embryonic survival):
- ECx values could not be calculated as ≥94% embryos hatched in all treatments.
- Differences between treatments and the control (Day 7): Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).

Larval survival:
- ECx values could not be calculated as ≤10% larvae died in any of the test concentrations compared to the control group.
- Differences between treatments and the control: Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).

Body length and body weight:
- ECx values: Probit analysis.
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- NOEC: Williams Multiple Sequential t-test Procedure (α=0.05, one-sided smaller) after trend analysis by contrasts (monotonicity of concentration/response).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Target, Measured and Mean Measured Concentrations During the ELS Test

 

Target conc.

 (mg solids/L)

Measured concentration (mg solids/L)

Mean measured conc.(mg solids/L)

Day

0

Day

7

Day

14

Day

21

Day

28

Day

33

0.050

0.058

0.034

0.039

0.029

0.020

0.031

0.035

0.013

0.14

0.098

0.10

0.069

0.070

0.061

0.090

0.31

0.34

0.25

0.28

0.15

0.18

0.15

0.22

0.78

0.76

0.60

0.50

0.48

0.27

1.0

0.61

2.0

2.4

1.7

1.6

1.6

1.4

1.2

1.6

Table 2: Survival of Fish Embryos and Larvae During the Range-Finding Test

Target concentrations

(mg solids/L)

No. of eggs

Day 0

Number of survivors (at nominal day)

Total survival (%)

1

2

3

4

5

6

7

Control

10

10

10

10

10

10

10

10

100

10

10

10

10

10

10

10

10

100

0.05

10

10

10

10

10

10

10

10

100

10

9

9

9

9

9

9

9

90

0.50

10

9

9

9

8

8

8

8

80

10

10

10

10

10

10

10

10

100

5.0

10

7

6

6

6

6

3

2

20

10

7

6

6

6

5

1

1

10

 

Table 3: Effect parameters (mg solids/L)

 

Parameter

 

Mean measured concentration (mg solids/L)

LOEC

NOEC

EC10

EC50

Embryonic survival

Value(a)

>1.6

≥1.6

>1.6

>1.6

Post-hatch

survival

Value(b)

>1.6

≥1.6

>1.6

>1.6

Malformation of caudal fin

Value(b)

1.6

0.61

n.a.

n.a.

Growth

(weight)

Value(b)

1.6

0.61

0.80

>1.6

lower 95%-cl

 

 

0.46

*

upper 95%-cl

 

 

1.0

*

Growth

(length)

Value(b)

1.6

0.61

0.79

>1.6

lower 95%-cl

 

 

0.64

*

upper 95%-cl

 

 

1.0

*

(a)for Day 8 of exposure,(b)for Day 33 of exposure, cl: confidence limit, * no confidence intervals could be determined, n.a. – not applicable

Table 4: Percentage of Embryonic Survival at the End of Hatching (Day 8)

 

Mean measured concentration

(mg solids/L)

Total Introduced

Hatched

Not Hatched

% Hatched

Control

80

79

1

99

0.035

80

80

0

100

0.090

80

75

5

94

0.22

80

80

0

100

0.61

80

76

4

95

1.6

80

76

4

95

Table 5: Post-Hatch Mortalityat the End of Exposure (Day 33)

 

Mean measured concentration

(mg solids/L)

Total*

introduced

Survived

Dead

% Post-hatch

mortality

Control

79

74

5

6

0.035

80

67

13

16

0.090

75

71

4

5

0.22

80

72

8

10

0.61

76

67

9

12

1.6

76

66

10

13

*Total number of hatched larvae.

Table 6: Mean Body Weight and Body Weight Reduction atthe End of Exposure (day 33)

 

Mean measured concentration

(mg solids/L)

Mean

Std. Dev.

n

%Reduction

Control

74.450

5.7356

4

 

0.035

73.850

4.9776

4

0.81

0.090

76.550

2.0469

4

-2.8

0.22

69.150

4.6658

4

7.1

0.61

74.475

8.9481

4

-0.034

1.6

46.025

4.9026

4

38 *

* effect statistically significant

Table 7: Mean Body Length and Body Length Reduction atthe End of Exposure (Day 33)

 

Mean measured concentration

(mg solids/L)

Mean

Std. Dev.

n

%Reduction

Control

21.21

0.395

4

 

0.035

21.29

0.335

4

-0.38

0.090

21.43

0.326

4

-1.0

0.22

20.48

0.268

4

3.5

0.61

21.26

0.826

4

-0.26

1.6

17.50

0.709

4

18 *

* effect statistically significant

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
Embryonic and larval survival was not affected at concentrations up to and including 1.6 mg solids/L; EC10 values were >1.6 mg solids/L. Consistent malformations of the caudal fin were observed in the highest test concentration of 1.6 mg solids/L, and thus the NOEC was considered to be 0.61 mg solids/L. EC10 values for growth reduction based on weight and length were 0.80 and 0.79 mg solids/L, respectively. The NOEC based on weight and length was 0.61 mg solids/L.
Executive summary:

A flow-through fish early-life stage toxicity test was performed in order to assess possible lethal and sub-lethal effects during the embryonic and early larval development of the fathead minnow (Pimephales promelas). The test item was a clear light yellow viscous liquid UVCB substance consisting of 39.6% solids in water. A correction factor of 2.525 was used to correct for the water content of the test item. Concentrations are expressed based on solids.

The study was conducted in accordance with OECD 210 and in compliance with GLP. Fertilized eggs were exposed to an untreated control and the test item at mean measured concentrations of 0.035, 0.090, 0.22, 0.61 and 1.6 mg solids/L (80 eggs per group, divided into four replicates).

Embryonic and larval survival was not affected at concentrations up to and including 1.6 mg solids/L; EC10 values were >1.6 mg solids/L. Consistent malformations of the caudal fin were observed in the highest test concentration of 1.6 mg solids/L, and thus the NOEC was considered to be 0.61 mg solids/L. EC10 values for growth reduction based on weight and length were 0.80 and 0.79 mg solids/L, respectively. The NOEC based on weight and length was 0.61 mg solids/L.

The study met all validity criteria and is considered valid without restriction.

Description of key information

A long-term toxicity test for freshwater fish was performed with a substance analogue. The NOEC based on malformations (0.61 mg solids/L) is used as worst-case key value for chemical safety assessment of the registered substance.

Key value for chemical safety assessment

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

Additional information

Embryonic and larval survival was not affected at concentrations up to and including 1.6 mg solids/L; EC10 values were >1.6 mg solids/L. Consistent malformations of the caudal fin were observed in the highest test concentration of 1.6 mg solids/L, and thus the NOEC was considered to be 0.61 mg solids/L. EC10 values for growth reduction based on weight and length were 0.80 and 0.79 mg solids/L, respectively. The NOEC for weight and length was 0.61 mg solids/L.

The NOEC based on malformations (0.61 mg solids/L) is used as worst-case key value for chemical safety assessment.