Hex-3-en-1-yl acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 - 18 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: each test concentration and the control
- Sampling method: control and all test item concentrations at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours)
- Sample storage conditions before analysis: samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: stock solution (100 mg/L) was stirred at 1100 rpm for 30 minutes at room temperature; test solutions in glass flasks sealed with screw caps (made from polypropylene) filled up with the test solutions having nearly no headspace.
- Controls: yes
- Evidence of undissolved material:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: 2 - 24 hours old daphnids, juvenile daphnids were removed from culture vessels latest 22 hours before the start of the exposure. The juveniles born within this period of max. 22 hours were used for the test after an acclimatisation phase in the dilution water of at least 2 hours. No first brood progeny was used for the test
- Method of breeding: culture medium was Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture was grown in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max. 1500 lux. Daphnids were fed with ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL, at least 5 times per week.
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 2 hours in dilution water

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

161 mg CaCO3/L (start of exposure, 0 h)
170 mg CaCO3/L (renewal, 24 h)

Test temperature:

20.8 - 20.9 °C

pH:

7.52 - 8.80 (Control)
7.72 - 8.55 (test concentrations)

Dissolved oxygen:

8.04 - 9.52 mg O2/L (Control)
7.83 - 9.10 mg O2/L (test concentrations)

Conductivity:

420 - 421 µS/cm

Nominal and measured concentrations:

4.27, 9.39, 20.7, 45.5 and 100 mg/L (nomminal)
3.15, 7.65, 17.1, 39.8 and 91.3 mg/L (based on the geometric mean measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL test volume without headspace per concentration and control) with screw caps made from polypropylene
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Concentrations of reference substance: 0.500; 1.00; 2.00; 4.00 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no, Same composition as the culture medium Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaC03/L, is used.
- Intervals of water quality measurement: at the start (0 and 24 h) and at the end (24 and 48 h) of the exposure time

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: diffuse light, intensity max. 1500 lux

EFFECT PARAMETERS MEASURED: Mobility

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Range finding study: yes (non-GLP)
- Test concentrations: 1.0; 10 and 100 mg/L (nominal concentrations)
- Results used to determine the conditions for the definitive study: see table 1 in `any other information on materials and methods´

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits

Remarks:

4.43 - 15.9 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

3.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

17.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

7.65 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: no
- Other: Biological and Analytical results are summarized within section "Any other infformation on results incl. tables"

Results with reference substance (positive control):

- Dose-response test: 0.500; 1.00; 2.00 and 4.00 mg/L
- EC50: 2.05 mg/L

Reported statistics and error estimates:

The EC10- and EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression. 95 % confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly.
The EC100-values (after 24 and 48 hours) and the NOEC (after 48 hours) were deduced directly from the observation data, without mathematical calculation. All effect levels (EC50 / 0 / 10 / 100 and NOEC) were based on the geometric mean measured concentrations of the test item, as measured test item concentrations did not remain within ± 20 % of the nominal concentrations.

Results of non-GLP range-finding test

Table 1: Immobilization Rates in the non-GLP Preliminary Range Finding Test (n = 20, divided into 2 replicates with 10 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	20	20	20	30	70	50
10	0	0	0	0	0	0
1	0	0	0	0	0	0
Control	0	0	0	0	0	0

Biological Results of the definitive test

Table 2: EC10 -, EC50- (with 95 % Confidence Limits), EC100-values, NOEC and LOEC

Effect concentrations	Test duration [hours]	Geometric mean measured test item concentrations [mg/L]
EC0	24	7.65
	48	3.15
EC10 (with 95% confidence limits)	24	8.12 (Cl: 5.65-11.0)
	48	7.83 (Cl: 4.19-15.7)
EC50 (with 95% confidence limits)	24	14.5 (Cl: 11.8-17.2)
	48	8.37 (Cl: 4.43-15.9)
EC100	24	91.3
	48	17.1
NOEC	24	7.65
	48	7.65

 

Table 3: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration of the test item [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
91.3	100	100	100	100	100	100% mortality after 24 hours
39.8	60	80	80	80	75	100	100	100	100	100
17.1	60	80	60	80	70	100	100	100	100	100
7.65	0	0	0	0	0	20	0	0	0	5
3.15	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Analytical Results:

Table 4: Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2017-03-15 0 hours Start of the exposure interval		2017-03-16 24 hours End of the exposure interval		2017-03-16 24 hours Start of the exposure interval		2017-03-17 48 hours End of the exposure interval		Geometric mean measured test item concentration [mg/L]
Start of analysis	2017-03-15		2017-03-16 1)		2017-03-16 1)		2017-03-17 1)
Nominal test item concentration [mg/L]	Test Item
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	98.9	99	84.4	84	93.1	93	n.a.	n.a.	91.3
45.5	44.4	98	38.7	85	43.6	96	33.7	74	39.8
20.7	19.9	96	14.7	71	19.0	92	15.5	75	17.1
9.39	9.31	99	6.46	69	9.02	96	6.30	67	7.65
4.27	4.23	99	2.46	58	4.06	95	2.32	54	3.15
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, single determinations, dilution factors taken into account

% = percentage of the initially measured concentration of the test item

n.a. = not analyzed

LOQ = limit of quantification of the analytical method (0.001 mg test item/L)

1) = measured on 2017-03-18

Validity criteria fulfilled:

yes

Conclusions:

The substance had acute effects on the mobility of Daphnia magna after 48 h of exposure. The test was conducted according to the OECD guideline 202 (GLP). The EC50-value was found to be 8.37 mg/L based on geometric mean measured concentrations.

Description of key information

EC50 (48h) = 8.37 mg/L (geometric mean measured, OECD 202, Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.37 mg/L

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates. The study was performed according to the OECD 202 (GLP) with the water flea Daphnia magna. Five nominal test item concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/L (corresponding to 3.15, 7.65, 17.1, 39.8 and 91.3 mg/L based on the geometric mean measured concentrations) were tested under semi-static conditions. The test was conducted in a closed system without headspace. The analytical samples were analysed at the start of the exposure intervals (0 and 24 hours) and at test end (24 and 48 hours) in all concentrations levels and the control. The measured concentrations of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) were in the range of 92 to 99% of the nominal values. The measured concentrations in old media at renewal and at the end of the test (24 and 48 hours) were in the range of 54 to 85% of nominal values. Since the measured test item concentrations did not remain within ± 20 % of the nominal concentrations, the effect values are based on the geometric mean measured concentrations of the test. Based on the geometric mean measured concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 8.37 mg/L (95 % confidence limits: 4.43 – 15.9 mg/L).