2-(2-ethoxyethoxy)ethyl acetate

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:

From August 05, 2020 to January 25, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26th July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120
- Expiration date of the lot/batch: 2023-04-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0, 0.2, 0.6, 1.5, 3.8 and 10.0 mg/L (nominal)
- Sampling method: On Day 0 (start of exposure), 6, 12, 18 and 27 a 10 mL sample from each replicate of freshly prepared medium was drawn and mixed together for each treatment group. This procedure was repeated 72 hours after these samples were taken (exhausted medium).
- Sample storage conditions before analysis: Collected samples were divided into two portions. One portion was analysed immediately and the second stored as back up samples at -20 ± 5 ºC.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dilution of stock solution
- Eluate: Water
- Controls: Water alone
- Chemical name of vehicle: None used
- Concentration of vehicle in test medium: N/A
- Evidence of undissolved material: No

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Laboratory bred at Jai Research Foundation

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: For breeding, male to female ratio was 2:1. Fish having ≥ 96.5% fertilisation rate were selected to produce eggs. Nine sets of male and female were kept for breeding in breeding tank, a day prior to treatment
- Method of collection of fertilised eggs: Collected into a petri dish and washed in order to remove debris and dead eggs.
- Subsequent handling of eggs: Embryos were selected from fish having ≥99% of fertilization ratio. Embryos were observed under stereoscope microscope. Embryos in same stage were used for study

POST-HATCH FEEDING
- Start date: 2 days post-hatch
- Type/source of feed: Larvae were fed with commercial larvae food (Gemma Micro, Skretting, France). During juvenile stage, brine shrimp nauplii (48 hours old) (INVE Aquaculture, USA) were provided as a food.
- Amount given: ad libitum whilst minimizing surplus. Surplus food and faeces were removed to avoid accumulation of waste.
- Frequency of feeding: Twice daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Hardness:

145.6 - 156.8 mg/L as CaCO3

Test temperature:

25.3 - 26.0 °C

pH:

7.11 - 7.82

Dissolved oxygen:

≥ 86.1%

Nominal and measured concentrations:

Nominal: 0.0, 0.2, 0.6, 1.5, 3.8 and 10.0 mg/L
Measured: 0.0, 0.21, 0.60, 1.55, 3.98 and 10.41 mg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups: Petrii dish, at least 25 mL fill volume
- Test vessel: Glass beaker, at least 400 mL fill volume
- Type: Open
- Aeration: No
- Renewal rate of test solution: Semi-static system, medium renewed avery 72 hours
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 24
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis water
- Total organic carbon: Not detected
- Particulate matter: Not detected
- Metals: Not detected
- Pesticides: Not detected
- Chlorine: Not detected
- Ca/mg ratio: Ca 30.8 mg/L, Mg 17.04 mg/L, Ca/Mg ratio 1.81
- Culture medium different from test medium: No
- Intervals of water quality measurement: 6 month intervals

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 610 - 722 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Stage of embyronic development
Hatching, survival, abormal appearance and behaviour observed daily
Body length measured on study termination
Body weight measured on study termination

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: 0.0, 0.1, 1.0, 5.0, 10.0 and 50.0 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: No mortality was observed during the embryo, larvae, and juvenile stages in any treatment group
- Days to hatch or time to release of young: Hatching completed by Day 3
- Numbers hatched: 80 / 80 (100% hatching)
- Observations on body length and weight of young and/or exposed parents at one or more time periods: At end of exposure period, no difference in body length or body weight of treated fish compared with controls
- Number of healthy fish at end of test: 80 / 80 (100% normal)
- Type of and number with morphological abnormalities: None
- Type of and number with behavioural abnormalities: None
- Type and number of developmental effects: None
- Type and magnitude of hormonal changes: N/A
- Other biological observations: None
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: None

Reported statistics and error estimates:

NOEC and LOEC were determined by statistical procedure for multi sample comparison. Data of body length and body weight of juvenile fish were subjected to Bartlett’s test for homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s Test

Survival/mortality - Embryo Stage

 

Test Concentration (mg/L)	Mortality N° (Day)						Cumulative Mortality (%) up to Day 5
	0	1	2	3	4	5
0.0 (Control)	0	0	0	0	0	0	0
0.2	0	0	0	0	0	0	0
0.6	0	0	0	0	0	0	0
1.5	0	0	0	0	0	0	0
3.8	0	0	0	0	0	0	0
10.0	0	0	0	0	0	0	0

 

 

Survival/mortality - Larvae Stage

 

Test Concentration (mg/L)	Mortality N° (Day)
	6	7	8	9	10	11	12	13	14	15
0.0 (Control)	0	0	0	0	0	0	0	0	0	0
0.2	0	0	0	0	0	0	0	0	0	0
0.6	0	0	0	0	0	0	0	0	0	0
1.5	0	0	0	0	0	0	0	0	0	0
3.8	0	0	0	0	0	0	0	0	0	0
10.0	0	0	0	0	0	0	0	0	0	0

 

Test Concentration (mg/L)	Mortality N° (Day)
	16	17	18	19	20	21	22	23	24	25
0.0 (Control)	0	0	0	0	0	0	0	0	0	0
0.2	0	0	0	0	0	0	0	0	0	0
0.6	0	0	0	0	0	0	0	0	0	0
1.5	0	0	0	0	0	0	0	0	0	0
3.8	0	0	0	0	0	0	0	0	0	0
10.0	0	0	0	0	0	0	0	0	0	0

 

Test Concentration (mg/L)	Mortality N° (Day)				Cumulative Mortality (%) between Days 0 - 29
	26	27	28	29	N°	%
0.0 (Control)	0	0	0	0	0	0
0.2	0	0	0	0	0	0
0.6	0	0	0	0	0	0
1.5	0	0	0	0	0	0
3.8	0	0	0	0	0	0
10.0	0	0	0	0	0	0

 

 

Survival/mortality - Juvenile Stage                                                                      

 

Test Concentration (mg/L)	Mortality N° (Day)				Cumulative Mortality (%) Between Days 30 - 33
	30	31	32	33	N°	%
0.0 (Control)	0	0	0	0	0	0
0.2	0	0	0	0	0	0
0.6	0	0	0	0	0	0
1.5	0	0	0	0	0	0
3.8	0	0	0	0	0	0
10.0	0	0	0	0	0	0

 

 

Body Length of Juvenile Fish

 

Test Concentration (mg/L)	Body Length (mm)		% Inhibition in Length
	Mean	SD
0.0 (Control)	11.2	0.4	-
0.2	11.2	0.2	0.00
0.6	11.2	0.1	0.00
1.5	11.1	0.3	0.89
3.8	11.2	0.2	0.00
10.0	11.1	0.1	0.89

Key: SD = Standard deviation, - = Not applicable

 

 

Body Weight of Juvenile Fish

 

Test Concentration (mg/L)	Body weight (mg)		% Inhibition in Body Weight
	Mean	SD
0.0 (Control)	6.2	0.3	-
0.2	6.2	0.2	0.00
0.6	6.1	0.2	1.61
1.5	6.1	0.2	1.61
3.8	6.1	0.1	1.61
10.0	6.1	0.2	1.61

Key: SD = Standard deviation, - = Not applicable

 

Validity criteria fulfilled:

yes

Conclusions:

Exposure to 2-(2-ethoxyethoxy)ethyl acetate caused no effect on the embryo, larvae, and juvenile stages of zebrafish. Hatch time and hatching success were not affected. Body length and body weight of juvenile fish were unaffected by treatment. The No Observed Effect Concentration (NOEC) was 10.0 mg/L, the highest concentration investigated.

Executive summary:

Potential effects of long-term exosure of 2 -(2 -ethoxyethoxy)ethyl acetate to fish has been investigated using methods described by OECD TG 210. No effect on the embryo, larvae, and juvenile stages of exposed zebrafish was observed. Hatch time and hatching success were not affected. Body length and body weight of juvenile fish were unaffected by treatment. The No Observed Effect Concentration (NOEC) was 10.0 mg/L, the highest concentration investigated.

Description of key information

Exposure to 2-(2-ethoxyethoxy)ethyl acetate caused no effect on the embryo, larvae, and juvenile stages of zebrafish. Hatch time and hatching success were not affected. Body length and body weight of juvenile fish were unaffected by treatment. The No Observed Effect Concentration (NOEC) was 10.0 mg/L, the highest concentration investigated.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

10 mg/L