Geranyl formate

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:

21 Sep - 23 Nov 2016

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken from an additional vessel (identical conditions) since the test vessels had to remain completly filled (closed system); duplicate samples were taken (9 mL sample to 6 mL acetonitrile) at test start and after 24, 48 and 72 h.
- Sample storage conditions before analysis: deep-frozen (at about -20 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 of the filtrate were used as test media.
- Controls: blank water control
- Differential loading: no
- Eluate: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Feeding during test: no feeding
- Food type: with an algal suspension (Desmodesmus subspicatus supplied by the Collection of Algal Cultures, SAG, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany) or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, Melle, Germany).
- Frequency: fed three times a week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: organisms used in the test were 6-24 hours old and were not first brood progeny

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L or 250 as CaCO3 mg/L

Test temperature:

20 °C

pH:

7.7 - 7.9 (control)
7.6 - 7.9 (test concentrations)

Dissolved oxygen:

8.2 - 8.5 (control)
8.0 - 8.6 (test concentrations)

Nominal and measured concentrations:

nominal: 1:3.2 dilution, 1:10 dilution, 1:32 dilution, 1:100 dilution and undiluted filtrate (loading rate of 100 mg/L)
measured (arithmetic mean): not analyzed, 0.35 mg/L, 1.5 mg/L, 5.9 mg/L and 22 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel
- Type: closed
- Material, size, headspace, fill volume: glass vessels, completely filled (without headspace) with 60 mL test medium and tightly sealed with glass stoppers
- Aeration: test medium was aerated prior to test start until oxygen saturation was reached
- Renewal rate of test solution: two 24-hour test medium renewal periods; test organisms were transferred by wide-bore pipette in freshly prepared test medium of the corresponding concentration
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: test medium provided for each daphnid was 12 mL (60 mL per replicate)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
- Ca/Mg ratio: 4:1
- Na/K ratio: 10:1
- Alkalinity: 0.8 mmol/L
- Culture medium different from test medium: yes (Elendt M7 Medium was used for cultivation and ISO medium was used for the test)

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: 13 - 18 μmol m-2 s-1

EFFECT PARAMETERS MEASURED: immobility at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: control, 1:100 dilution, 1:10 dilution and undiluted filtrate
- Results used to determine the conditions for the definitive study: The effects were observed in the undiluted filtrate after 24 h. One Daphnia was observed as immobile in the dilution 1:10 after 48 h. No effect was observed in the dilution of 1:100.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL

Remarks:

1.6 - 3.3 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: no immobilized test organisms in the control and up to and including 0.35 mg/L (dilution 1:32). At 1.5 mg/L and 5.9 mg/L, 20% and 100% of the daphnids were found to be immobile, respectively.
- Mortality of control: none
- Other adverse effects control: none
- Any observations that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.
- Other: Biological and analytical results are summarized within the section "Any other information on results incl.tables"

Results with reference substance (positive control):

- ECx: 24-h EC50: 1.0 mg/L (October 2016, IES Laboratories Study 20160278)

Reported statistics and error estimates:

The 24- and 48-h EC50 and the 95% confidence limits were calculated by Weibull Analysis. Statistical analysis was performed using ToxRat Professional®. The 24-h and 48-h NOEC, EC0 and EC100 were determined directly from the raw data.

Validity criteria:

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress. Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Table 1: Effect of test item on the mobility of Daphnia magna

Treatment/Dilution	Mean Measured Concentration [mg/L]	No. of Daphnids Tested	Immobilized Daphnids after 24 Hours		Immobilized Daphnids after 48 Hours
			No.	[%]	No.	[%]
Control	---	20	0	0	0	0
1:100	n.a.	20	0	0	0	0
1:32	0.35	20	0	0	0	0
1:10	1.5	20	0	0	4	20
1:3.2	5.9	20	10	50	20	100
Undiluted Filtrate*	22	20	20	100	--	--

n.a.: Not analyzed since below the NOEC determined in this test.

*: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

--: Test medium no longer prepared since all daphnids were dead after 24 h exposure.

Table 2: Analytical Results for Test Samples

Sampling Day / Age of Sample	Dilution Factor of Filtrate of Loading Rate 100 mg Test Item /L	Measured Concentration of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item c	% of Initially Measured Concentration
[d/h]		[mg/L]		[mg/L]
0/0	Control	n.d.	1.667	<LOQ
(fresh)	1:32	0.515	1.667	0.859
	1:10	1.91	1.667	3.18
	1:3.2	1.21	8.333	10.1
	Undiluted filtrate	1.77	16.67	29.5
1/24	Control	n.d.	1.667	<LOQ
(aged)	1:32	0.0502	1.667	0.0836	10
	1:10	0.324	1.667	0.541	17
	1:3.2	0.357	8.333	2.98	30
	Undiluted filtrate	0.954	16.67	15.9	54
1/0	Control	n.d.	1.667	<LOQ
(fresh)	1:32	0.605	1.667	1.01
	1:10	1.98	1.667	3.30
	1:3.2	1.24	8.333	10.3
	Undiluted filtrate	1.79	16.67	29.9
2/24	Control	n.d.	1.667	<LOQ
(aged)	1:32	0.111	1.667	0.184	18
	1:10	0.495	1.667	0.824	25
	1:3.2	0.457	8.333	3.81	37

n.d. = no test item detected

LOQ: 0.056 mg/L

Table 3: Mean measured concentration based on the Analytical Results

Treatment / Dilution	Measured Test Item Concentrations at the Start of the Renewal Periods (Day 0 / Day 1)	Measured Test Item Concentrations at the End of the Renewal Periods (Day 1 / Day 2)	Mean Measured Concentration of the Test Item (arithmetic mean)
	[mg/L]	[mg/L]	[mg/L]
Dilution 1:100	n.a.	n.a.	n.a.
Dilution 1:32	0.86 / 1.0	0.084 / 0.18	0.35
Dilution 1:10	3.2 / 3.3	0.54 / 0.82	1.5
Dilution 1:3.2	10 / 10	3.0 / 3.8	5.9
Undiluted Filtrate*	30 / 30	16 / --	22

°: Undiluted filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

n.a.: Not analyzed since below the NOEC determined in this test.

--: Not analyzed since all daphnids were dead after 24 h test duration.

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD 202 and resulted in an EC50 (48 h) of 2.3 mg/L (arithmetic mean measured concentration) based on immobility of Daphnia magna (OECD 202, GLP).

Description of key information

EC50 (48 h) = 2.3 mg/L (Daphnia magna, OECD 202, arithmetic mean measured concentration)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.3 mg/L

Additional information

There is one GLP guideline study available, which assessed the effects of substance towards aquatic invertebrates (Daphnia magna) according to the OECD guideline 202. As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). A semi-static test design with a test medium renewal after 24 hours was used. Due to the low water solubility of the test item, the test media were prepared as saturated solution with a loading rate of 100 mg/L. Therefore 100 mg/L test item was mixed in test water, and was intensively stirred for 3 hours. After stirring the emulsion was filtered through a 0.45 μm membrane filter. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32, and 1:100 of the filtrate were used as test media and analytically verified by HPLC-UV analysis. At the end of the two renewal periods, the test item concentrations were in the range of 10 to 54% of the initially measured values, demonstrating a decrease of the test item concentrations during the 24-h renewal period. The mean measured test item concentration were calculated as the arithmetic mean of the two geometric means measured at test start and end of each medium renewal period. An EC50 (48 h) of 2.3 mg/L based on immobilisation was calculated (arithmetic mean measured concentration).