methyl 2-hydroxybut-3-enoate

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:

2021-11-08 to 2021-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted 18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

October 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD guidance document No. 23 (ENV/JM/MONO(2000)6/REV1)

Version / remarks:

8 February 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentrations, samples were taken from the test concentration and from the concurrent vehicle control at the start and at the end of each water renewal period.
- Sample storage conditions before analysis: no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution used in the test was prepared by mechanical dispersion without using any
solubilising agent, as the substance is well water soluble. An appropriate amount of test item was dissolved in the dilution water (ISO medium) in order to obtain the nominal concentration of 6 mg/L at each water renewal period. The limit concentration was chosen based on experimental results for aquatic invertebrates and algae (please see IUCLID sections 6.1.3 and 6.1.5). After the formulation procedure the test animals were immediately introduced into the test solutions at each water renewal period.
- Eluate: no
- Controls: negative control (without test item)
- Test concentration separation factor: not applicable (limit test)
- Evidence of undissolved material: no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: not specified
- Source: Toma István e.v., 2146 Mogyoród, Gödöllői u. 41/B, Hungary
- Age at study initiation: juvenile
- Length at study initiation (length definition, mean, range and SD): 1-2 cm
- Loading: 0.8 g fish/L test medium

ACCLIMATION
- Acclimation period: held for at least 9 days before test initiation in the fish laboratory
- Acclimation conditions: same as test
- Type and amount of food during acclimation: bred in a well-known fish farm, disease- and parasite-controlled conditions
- Feeding frequency during acclimation: fed with appropriate, commercial diet for fish at least three times per week until 24-48 hours before the test start
- Health during acclimation: apparently healthy

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

post exposure observation not performed

Hardness:

249 mg/L (as CaCO3)

Test temperature:

21.7 – 22.6 °C

pH:

6.87 – 7.35

Dissolved oxygen:

69.7 – 90.9 % of air saturation

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 0 (control), 6 mg/L (limit concentration); measured: < LOQ, 5.6 mg/L (geometric mean measured); considering the concentration decline observed during that Daphnia test, the
initial test concentration was set higher in order to meet the exposure concentration
(geometric mean) of at least 2.84 mg/L. Accordingly, a limit test was performed at the nominal concentration of 6 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: aquariums
- Type: open
- Material, size, headspace, fill volume: glass, appropriate size for holding the fish up to the loading of 0.8 g fish/L
- Aeration: not necessary during the experiment
- Renewal rate of test solution: 1 exchange per 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: loading did not exceed 0.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Fish were held prior to and during the test in reconstituted water (ISO medium, prepared according to the recommendation of OECD guideline 203). Separate stock solutions of individual substances were first prepared in deionised water (BWT Christ Ministil water purification system)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 to 16 hours light daily

EFFECT PARAMETERS MEASURED: Fish were observed twice a day (in the morning and afternoon) during the exposure at approx. 2-3, 5-6, 24, 30, 48, 54, 72, 78 and 96 hours after start of the test for signs of intoxication and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Range finding study: Yes. Based on the threshold concentration derived from the alga and daphnia studies a non-GLP preliminary test was conducted at 6 mg/L nominal concentration (plus a concurrent control) in order to investigate whether the test item has toxic effect on fish up to at least this concentration level. About twice the concentration of the lowest EC50 value (2.84 mg/L in Daphnia) was chosen to cover a potential decomposition of the test item during the test, so resulting in a measured concentration clearly above 2.84 mg/L. Three fish were exposed for 96 hours in a semi-static test (with 24-h renewal periods) in the treatment and control group respectively. The test solution was prepared by dissolving an appropriate amount of test item in ISO medium (without using of any solubilising agent) resulting a test concentration of 6 mg/L for each water renewal period. The test item is assumed to be volatile, therefore the test was performed in a closed system without headspace (i.e. sealed test aquarium, filled up fully with test medium) in order to minimise the loss of test item from the test solution during the exposure period. No mortality or any sublethal effects were observed in the treatment group or in the control during the 96-h exposure period.
- Test concentrations: 6 mg/L nominal concentration
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 5.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: no
- Observations on body length and weight:
At the start of the test (on day 0) 10 fish were introduced into each aquarium, each containing ~2.3 litre testing solution. Before introduction of test animals, the total body weight of fish was measured and recorded: first the weight of a vessel filled with water was determined. Then the ten fish were selected randomly and placed into this vessel. The weight of the group of fish was registered and the average body weight calculated. On the basis of the above, the loading of the test-aquariums was calculated and was 0.29 g/L in the control and 0.27 g/L in the test item treated group. The test solutions were renewed daily. At these water renewal periods a second series of test vessels were prepared (including the fresh test medium) and fish were transferred to them. The test item is assumed to be volatile, therefore a modified exposure system according to the recommendation of OECD 23 guidance document was used in order to minimise the loss of test item from the test solution during the exposure period. In this exposure system the test aquariums were sealed by glass plates and filled up fully, thus allowing no headspace during the exposure. Water film between the cover plate and the upper edge of aquarium (filled up fully) ensured the sealing.
Total length of fish were measured prior to the initiation of the exposure in a subsample of at least 10 fish from the stock used for the test in order to check their compliance with the size range recommended for the species by the test guideline (OECD No. 203). These fish were not used in the test. Total length of each fish was measured and recorded at the end of the test. The measured values were within the range of 1.75 – 1.95 cm.
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Hatching rate control and treatment groups (embryo): no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: because of the high volatility of the substance aquaria were sealed in order to minimize evaporation
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

A limit test was carried out and no toxic effects were observed, therefore statistical analysis was not necessary.

Sublethal observations / clinical signs:

Table 1: Oxygen concentration

Test group	Dissolved Oxygen Concentration [%]
	1st renewal period		2nd renewal period		3rd renewal period		4th renewal period
	0 h	24 h	24 h	48 h	48 h	72 h	72 h	96 h
Control	90.9	71.2	89.6	73.2	88.4	73.0	89.5	69.7
5.6 mg/L (exposure concentration)	89.9	73.6	89.6	74.2	89.1	74.0	88.9	72.3

 

Table 2: Cumulative mortality data in the definitive test

Test group	Cumulative mortality (initial population = 10 fish / test group)
	2-3 h	5-6 h	24 h	30 h	48 h	54 h	72 h	78 h	96 h
Control	0	0	0	0	0	0	0	0	0
5.6 mg/L	0	0	0	0	0	0	0	0	0

 

VALIDITY OF TEST

• No mortality was observed in the control or in the treatment group during the test.


• The dissolved oxygen concentration did not fall below 60 % of air saturation value in any of the test vessels during the test.


• Test concentrations were analytically determined at the start and at the end of each water renewal period.

All validity criteria were met and therefore the study was considered to be valid.

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to OECD TG 203 with Danio rerio the 96-hour LC50 of the test item was determined to be higher than 5.6 mg/L.

Executive summary:

The acute toxicity of the test item towards fish was assessed in a study according to OECD TG 203, EU Method C.1 and US EPA OCSPP 850.1075. Fish were exposed to aqueous test media containing the test item for 96 hours in a semi-static test (24-h water renewal periods). Ten fish were exposed to limit test item concentration of 6 mg/L in nominal and control (no replicates were used). The limit concentration was chosen based on experimental results for aquatic invertebrates and algae (please see IUCLID sections 6.1.3 and 6.1.5). For determination of the test item concentrations, samples were taken from the treatment and the control group at the start and at the end of each water renewal period. The corresponding calculated exposure concentration (based on analytical measurements using a HPLC-UV method) was 5.6 mg/L. Fish were observed twice a day (in the morning and afternoon) during the exposure at approx. 2-3, 5-6, 24, 30, 48, 54, 72, 78 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen concentration and water temperature were carried out daily (at the start and end of each water renewal period). All validity criteria of the guidelines were met. No mortality or any sub-lethal effects were observed during the 96-h test period either in the exposure concentration of 5.6 mg/L or in the concurrent negative control. Accordingly, the 96-h NOEC was determined to be 5.6 mg/L. The 96-h LC50 and LOEC were determined to be higher than 5.6 mg/L.

Description of key information

In an acute toxicity test according to OECD TG 203 with Danio rerio the 96-hour LC50 of the test item was determined to be higher than 5.6 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 5.6 mg/L

Additional information

The acute toxicity of the test item towards fish was assessed in a study according to OECD TG 203, EU Method C.1 and US EPA OCSPP 850.1075. Fish were exposed to aqueous test media containing the test item for 96 hours in a semi-static test (24-h water renewal periods). Ten fish were exposed to limit test item concentration of 6 mg/L in nominal and control (no replicates were used). The limit concentration was chosen based on experimental results for aquatic invertebrates and algae (please see IUCLID sections 6.1.3 and 6.1.5). For determination of the test item concentrations, samples were taken from the treatment and the control group at the start and at the end of each water renewal period. The corresponding calculated exposure concentration (based on analytical measurements using a HPLC-UV method) was 5.6 mg/L. Fish were observed twice a day (in the morning and afternoon) during the exposure at approx. 2-3, 5-6, 24, 30, 48, 54, 72, 78 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen concentration and water temperature were carried out daily (at the start and end of each water renewal period). All validity criteria of the guidelines were met. No mortality or any sub-lethal effects were observed during the 96-h test period either in the exposure concentration of 5.6 mg/L or in the concurrent negative control. Accordingly, the 96-h NOEC was determined to be 5.6 mg/L. The 96-h LC50 and LOEC were determined to be higher than 5.6 mg/L.