2-ethylhexanoic acid, monoester with propane-1,2-diol

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2020 to 22 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study undertaken to GLP certification, based on OECD guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Guideline study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
RM1007030 RL750/18
- Expiration date of the lot/batch:
24/10/2020
- Purity: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Closed vessel at room temperature (17.3 – 23.0 °C).
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Solubility H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: > 1 g/L; CH3CN: > 1 g/L; DMSO: unknown; Stability: H2O: unknown; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: unknown

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Nominal concentrations were 0.25 / 0.5 / 1 / 1.5 mg/L
Time weighted mean concentrations were 0.24 / 0.44 / 0.93 / 1.33 mg/L

- Sampling method: On day 0, the content of the test item in the aquaria was measured in one replicate per concentration and in the inlet of the aquaria, also in one replicate per concentration. On day 1, two further measurements (morning and afternoon in one replicate per concentration) were performed. In the ongoing test, the content of the test item in one replicate per concentration was measured once a week changing systematically amongst replicates.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Following OECD (2019)Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, stock solutions containing 15 mg/L,10 mg/L, 5 mg/L and 2.5 mg/L in dilution water were prepared. For this, the loading rates of 16.0 µL/L, 10.6 µL/L, 5.3 µ/L and 2.7 µ/L (based on a density of 0.94 g/mL, stated in the SDS) was mixed with dilution water and shaken on an orbital shaker for 30 minutes in order to achieve complete dissolution. For each concentration, 9 L stock solution was fresh prepared daily. The stock solutions were mixed with dilution water. The mixture flows directly into the aquaria. Flow of stock solution and dilution water was adjusted to reach the planned concentration of 1.5 mg/L, 1 mg/L, 0.5 mg/L and 0.25 mg/L in the treatments. The concentrations of the stock solutions were 10-fold higher than the nominal concentrations in the treatments.

- Controls:
Negative and positive (potassium dichromate) controls used

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name:
Zebra fish
- Source:
Offspring form original University Heidelberg, 2019 source

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Two males and one female
- Method of collection of fertilised eggs: Spawn dishes
- Subsequent handling of eggs: Fertilised eggs identified and transferred to a glass beaker filled with dilution water

POST-HATCH FEEDING
After hatching, around day 5, Powdered flake food 1 - 2 times per day
From day 19 until the end oft he test, Artemia nauplii, once a working day

Test type:

flow-through

Water media type:

freshwater

Remarks:

Reconstituted tap water

Limit test:

no

Total exposure duration:

35 d

Remarks on exposure duration:

17/04/2020 to 21/05/2020

Post exposure observation period:

None

Hardness:

Measured at the start and end of the test - 35.6 and 35.65 mg/L CaCO3

Test temperature:

26 (+/-1.5 °C) across the test concentrations, replicates and test duration.

pH:

7.1 to 7.4 across the test concentrations, replicates and test duration.

Dissolved oxygen:

6.1 to 8.2 mg/l across the test concentrations, replicates and test duration.

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations were 0.25 / 0.5 / 1 / 1.5 mg/L
Time weighted mean concentrtions were 0.24 / 0.44 / 0.93 / 1.33 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria made of stainless-steel
- Size of vessel: 12 L
- Material, size, headspace, fill volume: 12L stainless steel
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic
- Renewal rate of test solution (frequency/flow rate): Adjusted to meet concentration requirements
- No. of organisms per vessel: 30 eggs
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted freshwater is used in the test. Tap water is desalinated by reverse osmosis. The conductivity and pH value are then adjusted by adding electrolyte solutions (sea salt and sodium bicarbonate).

Reference substance (positive control):

no

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post hatch survival

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

0.93 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post hatch survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

1.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

- Mortality/survival at embryo and larval stages:
- Overall mortality/survival:
120 eggs per concentration (pooled replicates): 0 mg/l - 98 hatched / 96 survived; 0.25 mg/l - 100 hatched / 100 survived; 0.5 mg/l - 97 hatched / 93 survived; 1 mg/l - 91 hatched / 87 survived; 1.5 mg/l - 87 hatched / 80 survived
- Data for length and weight of surviving fish:
Total mean fish length (pooled replicates): 0 mg/l - 10.56 mm; 0.25 mg/l - 10.05 mm; 0.5 mg/l - 10.32; 1 mg/l - 9.99 mm; 1.5 mg/l - 8.83 mg
Total mean fish weight (pooled replicates): 0 mg/l - 13.3 mg; 0.25 mg/l - 14.43 mg; 0.5 mg/l - 13.33 mg; 1 mg/l - 12.94 mg; 1.5 mg/l - 11.24 mg

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Analysis of hatchability and post hatch survival data carried out using Bonferroni Chi Squared; length and weight data carried out using Williams. All statistical analysis was carried out using the ToxRat Professional computer software package.

Endpoint	NOEC	LOEC
Hatchability	≥ 1.33 mg/L	> 1.33 mg/L
Post hatch survival	≥ 1.33 mg/L	> 1.33 mg/L
Wet Weight	≥ 1.33 mg/L	> 1.33 mg/L
Length	0.93 mg/L	1.33 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item in the Early-Life Stage Toxicity Test (OECD Guideline 210) for Danio rerio was tested under flow-through conditions and resulted in 35-day NOECs for hatchability and post-hatch survival of >1.33 mg/l (measured twa).

Executive summary:

The toxicity of the test item in the Early-Life Stage Toxicity Test (OECD Guideline 210) for Danio rerio was tested under flow-through conditions. Four concentrations ranging from 0.25 mg/L to 1.5 mg/L test item was exposed to 30 fertilised eggs for each replicate of the control and each replicate of the treatments. Slight toxicity was observed only for the endpoint fish body length in the highest tested concentration 1.5 mg/L (nominal). For the other endpoints no toxicity was observed. Test substance concentrations were measured at intervals and reported to be between 71 % and 135 % of the nominal concentration. Therefore, the determination of the results was based on the time-weighted mean of the measured concentrations (see annex 2 OECD guidance document No. 23).

The 35-day NOECs for hatchability and post-hatch survival were both >1.33 mg/l (measured twa).

Description of key information

 The 35d NOEC (hatchability) for D. rerio in the reliable OECD 210 FELS test was >=1.33 mg/l (Muckle, 2020).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.33 mg/L