Citronellyl formate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2017 - 9 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: dilutions 1:2, 1:4, and 1:8 from all sampling times; dilution 1:16, and 1:32 were not analyzed since these concentrations were below the 96-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results
- Sampling method: Duplicate samples were taken from all test media with surviving fish and the control at the start and end of the first and the last test medium renewal period (Day 0 to Day 1 and Day 3 to Day 4).
- 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 (loading rate of 100 mg/L): 258.5 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: reconstituted test water
- Evidence of undissolved material: No

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: IES Laboratories
- Length at study initiation: 2.6 ± 0.10 cm (representative sub-batch of 10 fish)
- Weight at study initiation: 0.14 ± 0.03 g (representative sub-batch of 10 fish)

ACCLIMATION
- Acclimation period: at least one week
- Acclimation conditions: same type of water (reconstituted test water) and the same temperature (22 °C) as used in the test
- Type of food during acclimation: commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49324 Melle/ Germany)
- Health during acclimation: no mortality, all fish were healthy

FEEDING DURING TEST
- The fish were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

1.25 mmol/L (125 mg/L as CaCO3)

Test temperature:

22 °C

pH:

7.2 - 7.6

Dissolved oxygen:

8.2 - 8.6 mg/L

Salinity:

NA

Conductivity:

NA

Nominal and measured concentrations:

- Nominal concentrations: undiluted filtrate (loading rate of 100 mg/L) and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 of the filtrate
- Measured concentrations (geometric mean): n.a.; n.a.; 0.72; 1.7; 1.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass bottles
- Type: closed, tightly sealed with a screw cap
- Material, size, headspace, fill volume: glass, approx. 5 L, without headspace, approx. 5 L
- Aeration: The test media and the control were not aerated during the test as a closed system was applied.
- Renewal period of test solution: 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.2 g fish per litre test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water
- Alkalinity: 0.4 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: approx.16 μmol m-2 s-1

EFFECT PARAMETERS MEASURED: number of abnormal and dead fish after 2, 14, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Concentrations: Dilution 1:4, 1:10 and 1:64 of the test item emulsion with a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI: 1.1 - 1.6 mg/L

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: dilution 1:4 (mean measured concentration 1.7 mg/L) after ≥ 72 h
- Other biological observations: apathy, tumbling during swimming

Reported statistics and error estimates:

The 96-hour LC50 at the observation times and the 95 % confidence limits were calculated as far as possible by Trimmed Spearman-Karber method for estimating median lethal concentrations, since the estimation based on the Probit or logit models was not valid (p(F) > 0.05).
Statistical analysis was performed using ToxRat Professional®.

Sublethal observations / clinical signs:

Table 1: Mean measured concentrations (geometric mean)

Treatment / Dilution°	Measured Test Item Concentrations at the Start and End of the first Renewal Period (Day 0 to Day 1)	Measured Test Item Concentrations at the Start and End of the last Renewal Period (Day 3 to Day 4)	Mean Measured Test Item Concentration over the Test Period
	[mg/L]	[mg/L]	[mg/L]
1:32	n.a.	n.a.	n.a.
1:16	n.a.	n.a.	n.a.
1:8	0.71/0.12	1.7/0.78	0.72
1:4	1.5/0.33	3.7/2.0	1.7
1:2	3.0/1.0	-/-	1.7*

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

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

--/--: All fish dead

*: As all fish were dead after 48 hours exposure, this test concentration is based on the first renewal period only where in general lower test item concentrations were found compared to the last renewal period.

 

Table 2: Mortality and Visible Abnormalities Observed in the Test Fish

Treatment / Dilution°	Mean Measured Test Item Concentration [mg/L]	Number of Abnormal and Dead Fish / Number of Dead Fish
		Observation Time [Hours]
		2	24	48	72	96
Control		0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:32	n.a.	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:16	n.a.	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:8	0.72	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
1:4	1.7	0 / 0	0 / 0	0 / 0	7 / 3 (UV)	7 / 4 (UV)
1:2	1.7#	0 / 0	7 / 0 (TS, AP)	7 / 7	--	--
LC50		n.d.	n.d.	1.7	1.4	1.3
95% C.I		n.d.	n.d.	n.d.	1.2-1.7	1.1 -1.6

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

95% C.I.: 95% confidence interval of the LC50

#: Calculation of mean measured concentration based on the results of the first renewal period only.

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

n.d.: Could not be determined

--: All fish dead

UV: Atypical behavior, i.e. fish mainly at the bottom of the aquarium

TS: Tumbling during swimming

AP: Apathy

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 of the test item was determined to be 1.3 mg/L (arithmetric mean measured concentration of two geometric means) with a 95% confidence interval of 1.1 and 1.6 mg/L.

Executive summary:

The acute toxicity of the test item to zebrafish was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008). As the test item is a volatile substance, the test was performed using 5-liter glass bottles completely filled (without headspace) with test medium that were tightly closed to avoid losses of test item by evaporation (closed system). A semi-static test design with a test medium renewal every 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 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 24 hours renewal periods the test item concentrations were in the range to 17 to 53 % of the initially measured values. Due to this decrease, the mean concentration for each analyzed test medium renewal period was calculated as geometric mean of the test item concentration measured at the start and the end of the test medium renewal period. From the values obtained, the mean measured test item concentration during the test period of 96 hours was calculated as arithmetic mean. All determined effect values are based on the measured arithmetic mean concentrations of the test item. In the control and at the test concentrations up to and including the dilution 1:8 (mean measured 0.72 mg/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the dilution 1:4 (mean measured 1.7 mg/L), all test fish showed untypical behavior caused by the test item and at the end of the exposure period 4 fish had died. At the dilution 1:2 (mean measured 1.7 mg/L) severe visible abnormalities were observed in the test fish at Day 1 resulting in the dead of all fish on Day 2. Thus, the 96-hour NOEC and LC0 of the test item to zebrafish were both determined to be 0.72 mg/L. The 96-hour LOEC was 1.7 mg/L (dilution 1:2). The 96-hour LC50 of the test item was calculated to be 1.3 mg/L with a 95% confidence interval of 1.1 and 1.6 mg/L. The 96-hour LC100 was 1.7 mg/L.

Description of key information

In a 96-h semi-static toxicity test with Zebrafish the 96-hour LC50 of the test item was determined to be 1.3 mg/L (arithmetric mean measured concentration of two geometric means).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.3 mg/L

Additional information

The acute toxicity of the test item to zebrafish was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008). As the test item is a volatile substance, the test was performed using 5-liter glass bottles completely filled (without headspace) with test medium that were tightly closed to avoid losses of test item by evaporation (closed system). A semi-static test design with a test medium renewal every 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 0.45μm membrane filter. 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 24 hours renewal periods the test item concentrations were in the range to 17 to 53 % of the initially measured values. Due to this decrease, the mean concentration for each analyzed test medium renewal period was calculated as geometric mean of the test item concentration measured at the start and the end of the test medium renewal period. From the values obtained, the mean measured test item concentration during the test period of 96 hours was calculated as arithmetic mean. All determined effect values are based on the measured arithmetic mean concentrations of the test item. In the control and at the test concentrations up to and including the dilution 1:8 (mean measured 0.72 mg/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the dilution 1:4 (mean measured 1.7 mg/L), all test fish showed untypical behavior caused by the test item and at the end of the exposure period 4 fish had died. At the dilution 1:2 (mean measured 1.7 mg/L) severe visible abnormalities were observed in the test fish at Day 1 resulting in the dead of all fish on Day 2. Thus, the 96-hour NOEC and LC0 of the test item to zebrafish were both determined to be 0.72 mg/L. The 96-hour LOEC was 1.7 mg/L (dilution 1:2). The 96-hour LC50 of the test item was calculated to be 1.3 mg/L with a 95% confidence interval of 1.1 and 1.6 mg/L. The 96-hour LC100 was 1.7 mg/L.