2,2-Difluoroethyl 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 11 January 2016 to 07 July 2016

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:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal concentrations).
- Sampling method: 10 mL samples were taken at the middle of the aquarium (in duplicate) in control and treatment groups at the beginning of the test (0 d), after 3, 9, 18, 28 and 32 days in fresh and aged medium.
- Sample storage conditions before analysis: no data available.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: A test substance stock solution (1000 mg/L) was prepared by dissolving 0.100 g test substance into 100 mL deionized water and re-prepared every day (i.e. at each renewal period) during the test period. The test solutions (1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal concentrations)) were prepared by dilution from the stock solution.
- Controls: yes, only deionized water.
- Test concentration separation factor: 2 (except for the last concentration).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: rare minnow.
- Strain: no data available.
- Source: healthy adult fish without surface damage from a breeding population in a recirculating system.
- Conditions of maintenance of the adult fish during the 14 days before the test:
Light: 16 hours photoperiod daily (light intensity: 1000 lux —1500 lux).
Temperature: (25±2) °C.
Oxygen concentration: more than 80% of the air saturation.
Feeding: The fish were fed with brine shrimp twice daily.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: The male and female fish were paired at 1:1 ratio in incubator.
- Method of collection of fertilised eggs: When the male was observed chasing the female, eggs and sperm were collected from the fish and the eggs artificially inseminated to get fertilized eggs.
- Subsequent handling of eggs: before the test, the fertilized eggs were taken at random under a microscope to confirm the batch of eggs were in the same cell stage.

POST-HATCH FEEDING
- Start date: 6 days after spawning.
- Type/source of feed:
Brood fish: brine shrimp
Newly-hatched larvae: Artemia nauplii (newly hatched)
Juvenile fish: Artemia nauplii (48 h old)
- Amount given and requency of feeding: no data available.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

Duration: hatching period 4 d, post-hatch 28 d. Thus a total of 32 d.

Hardness:

The total hardness of the test water was 145 mg (CaCO3)/L .

Test temperature:

During the test period, the temperature of the control and test solutions was between 25.0°C and 25.1°C.

pH:

During the test period, the pH values of the control and test solutions were between 7.61 and 7.85.

Dissolved oxygen:

During the test period, the Dissolved Oxygen (DO) values varied from 83% to 99% of the air saturation.

Salinity:

not concerned

Conductivity:

no data available.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L.
- Arithmetical mean of the concentrations measured in samples taken in some freshly prepared and aged test solutions (day 0, 3, 9, 18, 28 and 32): not detected, 0.962, 1.88, 3.87, 7.66 and 9.54 mg/L.
The concentrations of the test substance were stable throughout the 24-hour renewal period, with all analysed concentrations within 20% of the initial concentrations. Measured concentrations are summarised in the table 1 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM:
- Type: closed
- Material, size, headspace, fill volume: 1 L/test chamber. The test solution was completely filled the tank with zero headspace, and sealed completely.
- Renewal rate of test solution: semi-static conditions, 24 h-renewal.
- No. of fertilized eggs/embryos per vessel: 30 eggs/vessel.
- No. of vessels per concentration (replicates): two per test substance treatment (thus 60 eggs for each treatment).
- No. of vessels per control (replicates): two (thus 60 eggs for the control treatment).
- Biomass loading rate: 30 eggs/1 L.

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: Good quality tap water which had been dechlorinated for at least 24 h was used. The total hardness of the test water was 145 mg (CaCO3)/L and pH was 7.82 at room temperature.
- Total organic carbon: 1.3 mg/L.
- Particulate matter: No.
- Metals: all below the acceptable values (TG 203: "Fish, Acute Toxicity Test" (1992), TG 305: Bioaccumulation in Fish (1996)).
- Pesticides: Malathion, Methyl parathion and Parathion: all below the acceptable values.
- Chlorine: not measured (tap water dechlorinated for at least 24 h).
- Hardness: 145 mg (CaCO3)/L.
- Ca/mg ratio: no data.
- Salinity: not concerned.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: in one of the replicates of each treatment and control groups at the beginning of the test (0 d) in fresh medium and after 3, 9, 18, 28 and 32 days in fresh and aged medium (only in aged medium at 32 days).

OTHER TEST CONDITIONS:
- Adjustment of pH: none.
- Photoperiod: 16-hour light and 8-hour dark cycle daily.
- Light intensity: 1000 lux -1500 lux.

EFFECT PARAMETERS MEASURED :
- Stage of embryonic development: The embryonic stage at the beginning of exposure to the test substance was verified as precisely as possible. This was done using a representative sample of eggs suitably preserved and cleared.
- Hatching and survival: Observations on hatching and survival were made and recorded at least once daily. Dead embryos, larvae and juvenile fish were removed as soon as observed since they can decompose rapidly and may be broken up by the actions of the other fish. Extreme care was taken when removing dead individuals not to physically damage adjacent eggs/larvae, these being extremely delicate and sensitive. Criteria for death vary according to life stage: (i) for eggs: particularly in the early stages, a marked loss of translucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, (ii) for embryos: absence of body movement and/or absence of heart-beat and (iii) for larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque colouration of central nervous system and/or lack of reaction to mechanical stimulus.
- Abnormal appearance: The number of larvae or fish showing abnormality of body form was recorded and the nature of the abnormality described. It was noted that abnormal embryos and larvae occur naturally and could be of the order of several per cent in the control(s) in some species. Abnormal animals were only removed from the test vessels on death.
- Length and weight: At the end of the test, surviving fish of one of the replicates of each control and treated groups were selected to determine the length. A general anaesthetic (MS 222) was used to euthanize the fish and then the fish were preserved in ethanol. Because the test fish weight was too small at the end of the test, the whole dry weight in each concentration group was measured (dry weight, 24 hours at 60°C).

RANGE-FINDING STUDY
No. The test concentrations for this chronic toxicity study were determined based the acute toxicity data.

Reference substance (positive control):

not specified

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: all the considered endpoints (i.e. hatching, survival, weight, length, behaviour, malformations)

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

> 9.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: all the considered endpoints (i.e. hatching, survival, weight, length, behaviour, malformations)

Details on results:

- Mortality/survival at embryo, larval and juvenile stages: See table 2 in "any other information on results incl tables".
- Survival at the end of the test (post-hatch success): The survival rates after the hatching period (post-hatch success) was 100%, 98.3%, 98.2%, 100% 96.6% and 98.2% for the test concentrations 0 (control), 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal), respectively. See table 2 in "any other information on results incl tables".
- Days to hatch: Initiation of hatching on the fourth day in all treatments. See table 2 in "any other information on results incl tables".
- Numbers hatched (hatching success): The hatching rates were 95.0%, 96.7%, 93.3%, 91.7%, 96.7% and 91.7% for the test concentrations 0 (control), 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal), respectively. See table 2 in "any other information on results incl tables".
- Body length of the surviving fish at the end of the test: The mean body length were 9.49, 9.49, 9.43, 9.34, 9.31 and 9.31 mm for the test concentrations 0 (control), 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal), respectively. These results showed that there was no significant differences between the treated groups and the control group. See table 4 in "any other information on results incl tables".
- Body weight of the surviving fish at the end of the test: Because the test fish weight was too small at the end of the test, the whole dry weight in each concentration group was measured. The mean body weight (dry weight) were 0.00088, 0.00085, 0.00088, 0.00078, 0.00082 and 0.00079 g for the test concentrations 0 (control), 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (nominal), respectively.
- Type of and number with morphological abnormalities: None in any test groups and control. See table 3 in "any other information on results incl tables".
- Type of and number with behavioural abnormalities: None in any test groups and control. See table 3 in "any other information on results incl tables".
- 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:

Based on analysis of variance, the length data for each test substance concentration were compared with the control values using the Bartlett's test (ANOVA).

Table 1: Results of test substance in test medium

Nominal concentration (mg/L)	Analysed concentration (mg/L)
	Mean ^1	0 d	3 d		9 d		18 d		28 d		32 d
		fresh	aged	fresh	aged	fresh	aged	fresh	aged	fresh	aged
Blank control	ND^2	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
1.00	0.962	0.99	0.86	1.07	1.01	1.04	0.95	0.92	1.02	0.87	0.89
2.00	1.88	2.03	1.92	1.95	1.89	1.84	1.86	1.97	1.82	1.72	1.76
4.00	3.87	3.90	3.76	4.13	3.92	3.75	3.92	3.84	3.87	3.90	3.73
8.00	7.66	7.52	7.39	7.55	8.03	7.94	7.44	7.33	7.82	7.88	7.68
10.0	9.54	9.70	9.95	9.69	9.38	9.23	9.69	9.04	9.49	9.39	9.80

¹The average of the analysed concentrations on days 0, 3, 9, 18, 28 and 32.
²ND: not detected.

 

 

Table 2: Hatching and survival observed in the test fish

	Nominal concentration (mg/L)
Time (d)	Blank control		1.00		2.00		4.00		8.00		10.0
	hatching	survival^1	hatching	survival	hatching	survival	hatching	survival	hatching	survival	hatching	survival
0	0	60	0	60	0	60	0	60	0	60	0	60
1	0	60	1	59	0	60	0	60	1	59	0	57
2	0	59	0	59	0	60	0	59	0	59	0	56
32	18	58	16	59	19	59	17	58	23	59	21	56
4	57	57	58	58	56	56	55	55	58	58	55	55
5	57	57	58	58	56	56	55	55	58	58	55	55
6	57	57	58	58	56	56	55	55	58	58	55	55
7	57	57	58	58	56	55	55	55	58	57	55	55
8	57	57	58	58	56	55	55	55	58	57	55	55
9	57	57	58	58	56	55	55	55	58	57	55	55
10	57	57	58	57	56	55	55	55	58	56	55	54
11	57	57	58	57	56	55	55	55	58	56	55	54
12	57	57	58	57	56	55	55	55	58	56	55	54
13	57	57	58	57	56	55	55	55	58	56	55	54
14	57	57	58	57	56	55	55	55	58	56	55	54
15	57	57	58	57	56	55	55	55	58	56	55	54
16	57	57	58	57	56	55	55	55	58	56	55	54
17	57	57	58	57	56	55	55	55	58	56	55	54
18	57	57	58	57	56	55	55	55	58	56	55	54
19	57	57	58	57	56	55	55	55	58	56	55	54
20	57	57	58	57	56	55	55	55	58	56	55	54
21	57	57	58	57	56	55	55	55	58	56	55	54
22	57	57	58	57	56	55	55	55	58	56	55	54
23	57	57	58	57	56	55	55	55	58	56	55	54
24	57	57	58	57	56	55	55	55	58	56	55	54
25	57	57	58	57	56	55	55	55	58	56	55	54
26	57	57	58	57	56	55	55	55	58	56	55	54
27	57	57	58	57	56	55	55	55	58	56	55	54
28	57	57	58	57	56	55	55	55	58	56	55	54
29	57	57	58	57	56	55	55	55	58	56	55	54
30	57	57	58	57	56	55	55	55	58	56	55	54
31	57	57	58	57	56	55	55	55	58	56	55	54
32	57	57	58	57	56	55	55	55	58	56	55	54
Hatching rates (%)	95.0		96.7		93.3		91.7		96.7		91.7
Survival rates (%)	100		98.3		98.2		100		96.6		98.2

¹ Number of surviving larvae.

² initiation of hatching on the fourth day.

 

 

Table 3: Abnormal appearance and abnormal behaviour of test fish

Time (d)	Nominal concentration (mg/L)
	Blank control	1.00	2.00	4.00	8.00	10.0
3*	NB	NB	NB	NB	NB	NB
4	NB	NB	NB	NB	NB	NB
5	NB	NB	NB	NB	NB	NB
6	NB	NB	NB	NB	NB	NB
7	NB	NB	NB	NB	NB	NB
8	NB	NB	NB	NB	NB	NB
9	NB	NB	NB	NB	NB	NB
10	NB	NB	NB	NB	NB	NB
11	NB	NB	NB	NB	NB	NB
12	NB	NB	NB	NB	NB	NB
13	NB	NB	NB	NB	NB	NB
14	NB	NB	NB	NB	NB	NB
15	NB	NB	NB	NB	NB	NB
16	NB	NB	NB	NB	NB	NB
17	NB	NB	NB	NB	NB	NB
18	NB	NB	NB	NB	NB	NB
19	NB	NB	NB	NB	NB	NB
20	NB	NB	NB	NB	NB	NB
21	NB	NB	NB	NB	NB	NB
22	NB	NB	NB	NB	NB	NB
23	NB	NB	NB	NB	NB	NB
24	NB	NB	NB	NB	NB	NB
25	NB	NB	NB	NB	NB	NB
26	NB	NB	NB	NB	NB	NB
27	NB	NB	NB	NB	NB	NB
28	NB	NB	NB	NB	NB	NB
29	NB	NB	NB	NB	NB	NB
30	NB	NB	NB	NB	NB	NB
31	NB	NB	NB	NB	NB	NB
32	NB	NB	NB	NB	NB	NB

Note: NB: normal behaviour and appearance.

*: initiation of hatching on the fourth day.

 

 

Table 4: Individual fish length at the end of the test

	Length (mm)
Number of fish	Blank control	1.00 mg/L	2.00 mg/L	4.00 mg/L	8.00 mg/L	10.0 mg/L
1	9.76	10.55	9.67	9.26	8.60	8.87
2	8.65	9.68	8.73	9.48	9.73	9.45
3	10.11	8.42	9.52	10.19	9.55	10.08
4	9.27	9.96	9.71	9.67	8.92	10.42
5	9.58	8.72	10.45	9.72	10.41	9.57
6	9.62	10.18	10.24	8.47	9.24	8.63
7	8.94	10.59	8.66	8.79	9.38	9.75
8	10.67	8.74	9.42	9.42	9.79	9.89
9	9.95	9.22	9.79	9.79	8.06	9.74
10	9.43	8.64	8.60	10.28	9.35	9.23
11	9.26	8.89	9.61	8.94	10.26	8.65
12	8.77	9.46	10.73	9.11	8.49	8.94
13	8.52	9.39	9.22	8.65	8.18	9.71
14	9.23	9.77	8.64	9.34	9.42	9.23
15	9.48	8.43	8.99	8.79	10.43	9.73
16	8.77	9.68	9.06	10.14	9.89	8.41
17	10.24	9.74	8.87	9.48	9.80	10.29
18	9.21	10.35	9.91	9.53	8.96	8.78
19	9.86	8.92	10.32	10.72	9.73	9.27
20	9.73	9.44	8.41	9.05	9.51	9.51
21	9.52	10.26	9.38	8.77	8.64	8.39
22	10.06	8.78	9.17	9.29	9.23	9.72
23	10.41	9.89	8.96	7.96	9.58	9.05
24	9.79	9.96	9.49	8.42	7.84	8.76
25	9.82	9.27	9.27	8.99	9.26	9.14
26	8.93	9.35	9.51	10.18	9.93	9.73
27	8.65	10.01	9.66	9.64	9.16	8.48
28	9.40	9.47	10.13	/	/	/
29	/	/	/	/	/	/
30	/	/	/	/	/	/
Mean	9.49	9.49	9.43	9.34	9.31	9.31
SD	0.560	0.630	0.600	0.645	0.680	0.574
RSD (%)	5.91	6.64	6.36	6.91	7.31	6.17
NOEC (mg/L)			= 10.0 mg/L (measured concentration 9.54 mg/L)
LOEC (mg/L)			≥ 10.0 mg/L (measured concentration 9.54 mg/L)

 

Table 5: Hatching rate, survival rate and normal rate of the embryos and larvae at the end of the test

Nominal concentration (mg/L)	Measured concentration (mg/L)	Adjusted hatching rate, survival rate and normal rate (%)
		Hatching rate	Survival rate	Normal rate
Blank control	ND	95.0	100	100
1.00	0.962	96.7	98.3	100
2.00	1.88	93.3	98.2	100
4.00	3.87	91.7	100	100
8.00	7.66	96.7	96.6	100
10.0	9.54	91.7	98.2	100
EC50 (mg/L, based on measured concentration)		> 9.54	> 9.54	> 9.54
95%CI (mg/L, based on measured concentration)
NOEC (mg/L)		≥10.0 mg/L (measured concentration 9.54 mg/L)
LOEC (mg/L)		> 10.0 mg/L (measured concentration 9.54 mg/L)

 

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the OECD 210 were fulfilled. Dissolved oxygen concentration > 83%, water temperature did not differ by more than + 1.5°C and overall survival of fertilised eggs and post-hatch success in the controls were > 75%.

Conclusions:

The 32-day NOEC of 2,2-Difluoroethyl acetate to Gobiocypris rarus based on hatching, survival, abnormal appearance/behavior, length and weight was concluded to be superior or equal to the highest tested concentration of 9.54 mg/L.

Executive summary:

The chronic toxicity of 2,2-Difluoroethyl acetate to Gobiocypris rarus was investigated in a 32-day semi-static test according to the OECD test guideline 210 under GLP compliance.

Groups of 60 eggs were exposed for 32 days (i.e. 28 days post-hatching) under semi-static conditions (daily renewal) to a control treatment and to nominal concentrations of 1, 2, 4, 8 and 10 mg/L 2,2-Difluoroethyl acetate (two replicates of 30 eggs in each case). The corresponding actual concentrations were measured using GC/MS as being equal to 0.962, 1.88, 3.87, 7.66 and 9.54 mg/L, respectively (average of concentrations measured in samples taken in some freshly prepared and aged test solutions). Hatching was investigated daily until completion. Survival and abnormal appearance/behaviour of eggs, larvae and juvenile fish were also assessed daily. At the end of the test, individual length and pooled weight of surviving fish were measured.

At the end of the test, there was no statistically significant difference in any of these parameters between the control treatment and the treatments exposed to the five DFEA concentrations.

The 32-day NOEC based on hatching, survival, abnormal appearance/behavior, length and weight in G. rarus was thus concluded to be superior or equal to the highest concentration tested of 9.54 mg/L.

Description of key information

The 32-day NOEC of 2,2-Difluoroethyl acetate to Gobiocypris rarus based on hatching, survival, abnormal appearance/behavior, length and weight was concluded to be superior or equal to the highest tested concentration of 9.54 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

Based on hatching, survival, abnormal appearance/behavior, length and weight

Effect concentration:

>= 9.54 mg/L

Additional information

An experimental study performed under GLP compliance and in accordance with OECD test guideline 210 was flagged as a key study and assigned a Klimisch score of 1. It reports no effect of 2,2-Difluoroethyl acetate on the parameters investigated in Gobiocypris rarus (hatching, survival, abnormal appearance/behavior, length and weight) and the 32-day NOEC was therefore concluded to be superior or equal to the highest tested concentration of 9.54 mg/L.