2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 05, 2020 - July 23, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Composition of test material: Sodium-2-ethylhexyliminomonopropionate and Sodium-2-ethylhexyliminodipropionate
- Analytical purity: 98.5% (after freeze-drying)
- Lot/batch No.: WI7L16X01
- Expiration date of the lot/batch: 11 June 2020
- Appearance: White paste (after freeze-drying)
- Storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At t = 0h and t = 96h from all test concentrations and the control
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis

Vehicle:

no

Details on test solutions:

The test item was a pale yellow paste and was completely soluble in test medium at the test concentrations. Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15-minute period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp (Cyprinus carpio, Teleostei Cyprinidae)
- Source: ‘De Haar Vissen’, Zodiac proefacc. Wageningen University & Research, The Netherlands
- Length at study initiation (length definition, mean, range and SD): 3.7 ± 0.2 cm (range-finding test) / 3.2 ± 0.4 cm (final test)
- Weight at study initiation (mean and range, SD): 0.78 ± 0.16 g (range-finding test) / 0.49 ± 0.23 g (final test)

ACCLIMATION
- Acclimation period: at least 12 days after delivery.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: pelleted fish food (F-0.5 GR Pro Aqua Brut, Skretting, Fontaine-lès-Vervins, France)
- Feeding frequency during acclimation: daily until at the latest 24 hours before the start of exposure
- Health during acclimation (any mortality observed): mortality during the seven days prior to the start of the test was less than 5%

FEEDING DURING TEST
- No feeding from 24 hours prior to the test and during the total exposure period

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg/L (expressed as CaCO3)

Test temperature:

22°C

pH:

7.3-8.0

Dissolved oxygen:

4.5-9.3 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/L (final test)

Details on test conditions:

TEST SYSTEM
- Test vessels: 10 liters, all-glass, containing 8 liters of test solution.
- Aeration: introduced on Day 2 of exposure.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.43 g fish/liter

TEST MEDIUM / WATER PARAMETERS: the test medium was prepared by adding 211.5 mg CaCl2.2H2O/L, 88.8 mg MgSO4.7H2O/L, 46.7 mg NaHCO3/L and 4.2 mg KCl/L to tap water purified by reverse osmosis.

OTHER TEST CONDITIONS
- Adjustment of pH: yes (from 9.4 to 7.9 with 1 mol HCl/L in the range-finding test; from 9.4 to 7.5 with 1 mol HCl/L in the final test)
- Photoperiod: 16 hours photoperiod daily
- Light intensity: 573-612 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality was recorded at 4, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from Day 0, observations were made for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8 (final test)
- Range finding study : yes (control, 0.10, 1, 10 and 100 mg/L)

Reference substance (positive control):

not specified

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on analytically confirmed nominal concentrations

Remarks:

95%-CI = 72-108 mg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on analytically confirmed nominal concentrations

Remarks:

95%-CI = 50-80 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on analytically confirmed nominal concentrations

Remarks:

95%-CI = 50-80 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on analytically confirmed nominal concentrations

Remarks:

95%-CI = 32-56 mg/L

Details on results:

- Mortality and clinical effects: no mortality was observed in the control and the three lowest test concentrations throughout the exposure period. At the two highest test concentrations, mortality increased with increasing concentration and increasing exposure duration, resulting in 100% mortality at the end of the test in both concentrations. Clinical effects were observed at the three highest test concentration ranging from loss of buoyancy and orientation to discoloration and fraying of skins. The severity of observed effects increased with increasing concentration and increasing exposure duration.

- Validity of the test:
1. No mortality was observed in the control throughout the test.
2. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/L at 22°C).
3. Test concentrations have been analytically measured.
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Reported statistics and error estimates:

The 24h-EC50 could not be determined using a regression method. Instead, the 24h-EC50-value was calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the percentages of affected daphnids and the logarithms of the corresponding nominal test item concentrations. The 48h- and 72h-LC50-values were calculated from the weibits of the percentages of dead fish and the logarithms of the corresponding test item concentrations (analytically confirmed nominal) using the maximum likelihood estimation method. Since there was no concentration between the highest concentration (A) at which 0% mortality had occurred and the lowest concentration (B) at which 100% mortality had occurred at the end of the test, the 96h-LC50 was calculated as (AB)½, where A and B are limits of the 95% confidence interval. ToxRat Professional v 3.2 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Sublethal observations / clinical signs:

Incidence of Mortality and Total Mortality in the Final Test

Test item Nominal conc. (mg/L)	Initial Number of fish	Cumulative mortality					Total Mortality (%)
		4h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
10	7	0	0	0	0	0	0
18	7	0	0	0	0	0	0
32	7	0	0	0	0	0	0
56	7	0	0	2	2	7	100
100	7	0	5	7	7	7	100

Clinical Effects Observed in the Final Test

Test item Nominal conc. (mg/L)	Time of recording (hours)	Specification of effects	Relative number
32	72 96	Fraying of fins Fraying of fins	7/7 7/7
56	24 48 72	Discoloured and corkscrew swimming Fraying of fins Fraying of fins and corkscrew swimming	4/7 4/5 5/5
100	4     24	Discoloured and corkscrew swimming Immobile and floating at surface Swimming at bottom Hypoactive/corkscrew swimming, floating/swimming at surface	3/7 1/7 3/7 2/2

Validity criteria fulfilled:

yes

Remarks:

The study met the acceptability criteria prescribed by the study plan and was considered valid

Conclusions:

The 96h-LC50 for carp (Cyprinus carpio) exposed to the test item was 42 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 32 and 56 mg/L).

Executive summary:

The toxicity of the test item to freshwater fish Cyprinus carpio was determined in a GLP-compliant study performed in accordance with OECD Guideline No. 203. A 96-hour 50% lethal concentration LC50 of 42 mg/L (95% CL 32-56 mg/L) was obtained based on analytically confirmed nominal concentrations.

Description of key information

The toxicity to fish of 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) was investigated in a GLP-compliant study performed in accordance with OECD Guideline 203. A 96-hour 50% lethal concentrations LC50 of 42 mg/L (95% CL 32-56 mg/L) was obtained based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

42 mg/L

Additional information

The toxicity to fish of 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) was investigated in a GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.