Citronellyl butyrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June - 27 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office for the Environment

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test concentrations
- Sampling method: duplicate samples without algae were taken from the test media of all test concentrations and the control at the start of the test and duplicate stability samples with algae were taken, after 24 and 48 hours and at the end of the test (72 hours)
- Sample storage conditions before analysis: storage at -20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- 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

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater microalga
- Strain: Strain No. 61.81 SAG
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae were cultivated under standardized conditions according to the test guidelines

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L as CaCO3

Test temperature:

23 °C

pH:

7.9 - 8.2 (control)
7.9 - 8.2 (test concentrations)

Nominal and measured concentrations:

nominal: control, 1:22 dilution, 1:10 dilution, 1:4.6 dilution, 1:2.2 dilution, undiluted filtrate (loading rate of 100 mg/L)
geometric mean measured: < 0.069 mg/L, n/a, 0.043 mg/L, 0.052 mg/L, 0.063 mg/L, 0.16 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks nearly completely filled (without headspace) with 60 mL test medium and tightly sealed with
glass stoppers
- Aeration: no
- Initial cells density: 5000 cells/mL (corresponding to 1.19 x 10^4 relative fluorescence units)
- Control end cells density: 119.0 x 10^4 relative fluorescence units
- No. of organisms per vessel: 5000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared according to the test guidelines, but modified according to the International Standard ISO 14442 [2] as a closed test system was applied. The concentration of NaHCO3 was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the pH of the test media as constant as possible.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: LED light; intensity approximately 67 μE s-1m-2 (range: 64 to 69 μE s-1m-2)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control, dilution 1:20, dilution 1:5, undiluted filtrate
- Results used to determine the conditions for the definitive study: EC50 > Undiluted Filtrate in range finding study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 0.09 - 0.10

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.11 - 0.12

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.062 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 0.059 - 0.065

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.083 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.076 - 0.088

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 0.045 - 0.052

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.043 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.043 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.052 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.052 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Other: Biological and analytical results are summarized within the section "Any other information on results incl.tables"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (72 h) for growth rate: 1.0 mg/L (range recommended by the guideline: 0.92-1.46 mg/L)

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test. Statistical analysis was performed using ToxRat Professional®.

Table 1: Biomass of Algae

Treatment/ Dilution	Mean Measured Test Item Concentration [mg/L]	Repl. No.	Biomass of Algae*
			24 Hours	48 Hours	72 Hours
Control		1	4.2	27.1	113.1
		2	4.6	26.2	122.6
		3	4.0	24.1	119.4
		4	3.9	27.2	114.1
		5	4.1	27.6	121.3
		6	3.7	25.3	123.6
		Mean	4.1	26.3	119.0
		SD	0.3	1.3	4.4
1:22	n.a.	1	3.6	27.9	116.5
		2	4.0	29.1	119.0
		3	2.9	24.5	130.1
		Mean	3.5	27.2	121.9
		SD	0.6	2.4	7.2
1:10	0.043	1	3.7	25.2	119.8
		2	4.4	25.4	122.9
		3	3.3	25.8	122.4
		Mean	3.8	25.4	121.7
		SD	0.6	0.3	1.7
1:4.6	0.052	1	3.8	26.0	110.7
		2	3.0	24.5	97.2
		3	3.7	22.6	96.7
		Mean	3.5	24.4	101.5
		SD	0.4	1.7	7.9
1:2.2	0.063	1	3.9	20.9	90.1
		2	2.6	21.7	93.4
		3	2.9	21.7	91.5
		Mean	3.1	21.4	91.7
		SD	0.6	0.5	1.7
Undiluted Filtrate°	0.16	1	1.7	4.2	23.5
		2	1.1	5.0	27.8
		3	1.0	3.9	21.6
		Mean	1.3	4.4	24.3
		SD	0.4	0.6	3.2

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

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 1.19 x 104 relative fluorescence units.

Table 2: Analytical Results for Test Samples

Sampling Day / Age of Sample [d/h]	Dilution Factor of Filtrate of Loading Rate 100 mg Test Item /L	Measured Concentration of Test Item x [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item c [d/h] [mg/L]
0/0	Control	n.d.	1.667	<LOQ
(fresh)	1:10-Diluted Filtrate	0.0484	1.667	0.0807
	1:4.6-Diluted Filtrate	0.108	1.667	0.180
	1:2.2-Diluted Filtrate	0.223	1.667	0.372
	Undiluted Filtrate	0.460	1.667	0.767
1/24	Control	n.d.	1.667	<LOQ
(aged)	1:10-Diluted Filtrate	n.d.	1.667	<LOQ
	1:4.6-Diluted Filtrate	n.d.	1.667	<LOQ
	1:2.2-Diluted Filtrate	n.d.	1.667	<LOQ
	Undiluted Filtrate	0.462	1.667	0.770
2/48	Control	n.d.	1.667	<LOQ
(aged)	1:10-Diluted Filtrate	n.d.	1.667	<LOQ
	1:4.6-Diluted Filtrate	n.d.	1.667	<LOQ
	1:2.2-Diluted Filtrate	n.d.	1.667	<LOQ
	Undiluted Filtrate	n.d.	1.667	<LOQ
3/72	Control	n.d.	1.667	<LOQ
(aged)	1:10-Diluted Filtrate	n.d.	1.667	<LOQ
	1:4.6-Diluted Filtrate	n.d.	1.667	<LOQ
	1:2.2-Diluted Filtrate	n.d.	1.667	<LOQ
	Undiluted Filtrate	n.d.	1.667	<LOQ

  LOQ: 0.069 mg/L

n.d. = not detected

n.a. = not applicable

Table 3: Mean measured Concentrations based on the Analytical Results

Treatment Dilution	Analytical Measured Concentration of the Test Item [mg/L]				Mean Measured Concentration (Geometric Mean)*
	0 hours	24 hours	48 hours	72 hours	[mg/L]
Dilution 1:22	n.a.	n.a.	n.a.	n.a.	n.a.
Dilution 1:10	0.081	<LOQ	<LOQ	<LOQ	0.043
Dilution 1:4.6	0.18	<LOQ	<LOQ	<LOQ	0.052
Dilution 1:2.2	0.37	<LOQ	<LOQ	<LOQ	0.063
Undiluted Filtrate°	0.77	0.77	<LOQ	<LOQ	0.16

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

LOQ: Limit of quantification (LOQ = 0.069 mg test item/L)

*: If the measured concentration was below LOQ, half of the LOQ (½ LOQ =0.035 mg/L) was used to calculate the mean measured concentration

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

Validity criteria fulfilled:

yes

Conclusions:

The study with the green algae Pseudokirchneriella subcapitata resulted in an measured ErC50 (72 h) of > 0.16 mg/L and an ErC10 value 0.083 mg/L. The NOEC was determined to be 0.043 mg/L.
The results are based on geometric mean measured concentrations of the test item.

Description of key information

ErC50 (72 h) > 0.16 mg/L (geometric mean measured concentration , OECD 201, Pseudokirchneriella subcapitata)

ErC10 (72 h) = 0.083 mg/L (geometric mean measured concentration, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.083 mg/L

Additional information

There is one GLP guideline study available, which determined the effects of the freshwater green algal species Pseudokirchneriella subcapitata in a 72-hour static test according to the OECD Guideline 201. As the test item is a volatile substance, the test was performed using glass tubes completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation. Due to the low water solubility of the test item, the test media were prepared following the filtration method. Therefore 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. The concentrations of the test item were analytically verified by HPLC-UV analysis. The analytical measurements from test media at the start of the test show the correct preparation of the test media. During the first 24 and 48 hours test period the test item concentrations in the test media decreased below the Limit of Quantification. Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations. The mean measured concentrations were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48 and 72 hours). Since the inhibition of growth rate was only 34.6 % at the highest test item concentration of 0.16 mg/L (undiluted filtrate) the ErC50 (72 h) was > 0.16 mg/L and the corresponding NOErC (72 h) was 0.043 mg/L. The ErC10 (72 h) was at 0.083 mg/L.