Citronellyl 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:

24 April 2017 - 23 June 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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: undiluted filtrate (loading rate 100 mg/L) and the dilutions 1:2, 1:4 and 1:8 of the filtrate, dilutions 1:16 and 1:32 were not analysed since these concentrations were below the 48-hour NOEC
- Sampling method: Duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods. For the 24-hour stability samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: acetonitrile was added (6 mL acetonitrile per 9 mL sample volume), frozen (at -20 ±5 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Highest test concentration (100 mg/L): 258 μL of the test item in 2320 mL test water, intensive stirring for 3 h in a closed vessel, after stirring filtered through a 0.45 μm membrane filter (pre-conditioned with 200 mL filtrate, negative pressure of the filtration unit reduced to a minimum)
Lower test concentrations: highest test concentration further diluted with test water
- Controls: synthetic test water (AAP Medium)
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Source: Daphnia Collection of the University of Basel/Switzerland, bred in the testing laboratory under temperature and light conditions identical to those of the tests
- Age at the start of the test: 6-24 hours old
- Feeding during test: no
- Food type: algal suspension of the green algae Desmodesmus subspicatus
- Frequency: three times a week

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (250 as CaCO3)

Test temperature:

21 - 22 °C.

pH:

7.6 - 8.0

Dissolved oxygen:

≥ 8.1 mg/L

Salinity:

NA

Conductivity:

NA

Nominal and measured concentrations:

- Nominal concentrations: undiluted filtrate (loading rate 100 mg/L) and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the filtrate
- Measured concentrations (arithmetic mean): 12; 5.6; 2.4; 1.2 mg/L; n.a. and n.a

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL test tubes
- Type: closed with glass stoppers
- Material, size, headspace, fill volume: filled with 60 mL test medium; without headspace
- Aeration: prior to the start of the study until oxygen saturation was reached
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: 19 - 20 μmol m-2 s-1

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: undiluted filtrate (loading rate: 100 mg/L) and the dilutions 1:5 and 1:20 of the filtrate

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: at the highest concentration all remaining daphnia showed slower swimming activity compared to the control animals
- Mortality of control: none
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 24-hour EC50: 1.2 mg/L (March 2017)

Reported statistics and error estimates:

The 48-hour EC50 and the 95 % confidence limits were calculated by Trimmed Spearman- Karber method for estimating median lethal concentrations, since the estimation based on the Probit or logit models was not be valid (p(F) > 0.05).
Statistical analysis was performed using ToxRat Professional®.
The 24-hour and 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

Table 1: Mean measured concentrations (geometric mean)

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]
1:32	n.a.	n.a.	n.a.
1:16	n.a.	n.a.	n.a.
1:8	2.20 / 2.02	0.785 / 0.566	1.2
1:4	4.06 / 3.46	1.77/1.37	2.4
1:2	7.89 / 7.96	3.99/3.95	5.6
Undiluted Filtrate°	15.8/15.7	9.47/9.10	12

°: 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.

Table 2: Effect of the 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
1:32	n.a.	20	0	0	0	0
1:16	n.a.	20	0	0	0	0
1:8	1.2	20	0	0	0	0
1:4	2.4	20	0	0	1 (19F)	5
1:2	5.6	20	0	0	1 (19F)	5
Undiluted filtrate°	12	20	7 (13F)	35	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

Values in parenthesis: number of mobile test animals with adverse effects:

F = slower swimming activity

 

Table 3: Analytic results for test samples

Sampling Day / Sample Age  [d/h]	Loading Rate of 100 mg Test Item/L	Measured Concentration of Test Item x [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [mg/L]	% of Initially Measured   [%]
0/0 (fresh)	Control Dilution 1:8 Dilution 1:4 Dilution 1:2 Undiluted Filtrate°	n.d. 1.32 2.44 4.74 9.51	1.67 1.67 1.67 1.67 1.67	<LOQ 2.20 4.06 7.89 15.8	- - - - -
1/24 (aged)	Control Dilution 1:8 Dilution 1:4 Dilution 1:2 Undiluted Filtrate°	n.d. 0.471 1.06 2.39 5.68	1.67 1.67 1.67 1.67 1.67	<LOQ 0.785 1.77 3.99 9.47	n.a. 36 44 51 60
1/0 fresh)	Control Dilution 1:8 Dilution 1:4 Dilution 1:2 Undiluted Filtrate°	n.d. 1.21 2.08 4.78 9.45	1.67 1.67 1.67 1.67 1.67	<LOQ 2.02 3.46 7.96 15.7	- - - - -
2/24 (aged)	Control Dilution 1:8 Dilution 1:4 Dilution 1:2 Undiluted Filtrate°	n.d. 0.340 0.822 2.37 5.46	1.67 1.67 1.67 1.67 1.67	<LOQ 0.566 1.37 3.95 9.10	n.a. 28 40 50 58

Remark: The samples of the lower test concentrations (dilution 1:16, and 1:32) were not analyzed since these concentrations were below the 48-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.

LOQ= 0.118 mg test item /L

n.a. = not applicable

n.d. = no test item detected

° =undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:

yes

Conclusions:

The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test. The 48-hour EC50 was calculated to be 7.6 mg/L with 95%-confidence limits of 6.8 and 8.4 mg/L.

Executive summary:

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour semi-static test with a test medium renewal after 24 hours according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. 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 following the stirring/filtration 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, 1:4, 1:8, 1:16 and 1:32 of the filtrate were used as test media. Additionally, a control (test water without test item) was tested in parallel. At the end of the two renewal periods, the test item concentrations were in the range of 28 to 60 % of the initially measured values, demonstrating a decrease of the test item concentrations during the 24-hour renewal periods. The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the two geometric means which were determined from the test item concentrations measured at the start and end of each of the two test medium renewal periods. Due to the decrease of the test item concentrations during the 24-hour renewal periods, the biological results were based on the mean measured test item concentrations resulting in a 48-hour EC0 and 48-hour NOEC of 1.2 mg/L, a 48-hour EC100 of 12 mg/L and a 48-hour EC50 of 7.6 (95 % CI: 6.8 – 8.4 mg/L).

Description of key information

The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test. The 48-hour EC50 was calculated to be 7.6 mg/L with 95%-confidence limits of 6.8 and 8.4 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

7.6 mg/L

Additional information

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour semi-static test with a test medium renewal after 24 hours according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. 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 following the stirring/filtration 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, 1:4, 1:8, 1:16 and 1:32 of the filtrate were used as test media. Additionally, a control (test water without test item) was tested in parallel. At the end of the two renewal periods, the test item concentrations were in the range of 28 to 60 % of the initially measured values, demonstrating a decrease of the test item concentrations during the 24-hour renewal periods. The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the two geometric means which were determined from the test item concentrations measured at the start and end of each of the two test medium renewal periods. Due to the decrease of the test item concentrations during the 24-hour renewal periods, the biological results were based on the mean measured test item concentrations resulting in a48-hour EC0 and 48-hour NOEC of 1.2 mg/L, a 48-hour EC100 of 12 mg/L and a 48-hour EC50 of 7.6 (95 % CI: 6.8 – 8.4 mg/L).