Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl-1H-imidazole-1-ethanol and sodium hydroxide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Testing was initiated as requested in ECHA decision TPE-D-2114359620-51-01/F.
The rationale to read across the data is attached in section 13.

Reason / purpose for cross-reference:

read-across source

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.

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

Duration:

33 d

Dose descriptor:

LOEC

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:

morphology

Remarks:

Malformation of caudal fin

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. 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

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. Malformations of the caudal fin were observed in the highest test concentration of 1.6 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. This result is read across to the registered substance, the rationale is attached in Section 13.

Executive summary:

The test was performed with a substance analogue. The rationale to read across the data is attached in section 13.

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.

Reference 2

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

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

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

The test was performed with a substance analogue. The rationale to read across the data is attached in section 13.

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 for weight and length was 0.61 mg solids/L.

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

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

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.