Litsea cubeba, ext.

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:

2/7/2014 - 7/7/2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples were taken from the uninoculated control and the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test groups at 0 and 72 hours for analysis.
- Sample storage conditions before analysis: stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (22.5, 72, 23.5, 75.2 and 235 mg) were each separately added to the surface of 22.5, 22.5, 2.35, 2.35 and 2.35 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the media surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Due to the light sensitive nature of the test item all test item preparation was perfumed under non-actinic light. An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (10.3 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.
- Controls: Yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations on the test media were carried out during the mixing and testing of the WAFs. At both the start and end of stirring, and following a 1-hour standing period, the 1.0 3.2, 10, 32 and 100 mg/L loading rate WAFs were observed to be clear colorless media columns with oily globules of test item floating at the media surface. Microscopic observations of the WAFs after siphoning showed no microscopic particles to be present. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 32 and 100 mg/L loading rate WAF test cultures were observed to be clear colorless solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

ACCLIMATION
- Culturing media and conditions (same as test or not): Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10^4 – 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not relevant

Hardness:

No data

Test temperature:

24 +/- 1ºC

pH:

7.9 - 9.6 in controls; 7.8 - 9.6 in treated vessels (maximum increase of 1.7 during test)

Dissolved oxygen:

Not relevant

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured (t=0h): Measured (t=72h):

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 300 mL volume filled with 300 mL test medium
- Initial cells density: 3.67*10^3 cell/mL
- Control end cells density: 5.61x10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water*
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: continuous
- Light intensity and quality: warm white lighting (380 – 730 nm) at 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: range finding test 1: 10 and 100 mg/L; range finding test 2: 1.0, 10 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CI: 22 - 28 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/L loading rate WAFs. Swollen cells and cell debris were observed to be present in the test cultures at 32 mg/L loading rate WAF whilst no intact cells were observed to be present in the 100 mg/L loading rate WAF test cultures
- Any stimulation of growth found in any treatment: yes, in two 1.0 mg/L vessels and in one 10 mg/L vessel.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations on the test media were carried out during the mixing and testing of the WAFs. At both the start and end of stirring, and following a 1-hour standing period, the 1.0 3.2, 10, 32 and 100 mg/L loading rate WAFs were observed to be clear colorless media columns with oily globules of test item floating at the media surface. Microscopic observations of the WAFs after siphoning showed no microscopic particles to be present. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 32 and 100 mg/L loading rate WAF test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.1 mg/L
- EbC50: 0.51 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

The effects observed at 3.2 mg/L loading rate WAF were considered to be erroneous given that no significant effects were observed at the higher test concentration of 10 mg/L loading rate WAF. Given this the "No Observed Effect Loading Rate" (NOEL) based on growth rate was considered to be 10 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 32 mg/L loading rate WAF.

Cell densities and pH-values:

Nominal Loading Rate (mg/L)		pH	Cell Densities*(cells per mL)				pH
		0 h	0 h	27 h	46 h	72 h	72 h
Control	R1	7.9	4.78E+03	3.24E+04	1.48E+05	5.32E+05	9.6
	R2		3.52E+03	3.34E+04	1.47E+05	5.93E+05
	R3		2.32E+03	3.55E+04	1.31E+05	6.02E+05
	R4		4.14E+03	2.40E+04	1.24E+05	5.41E+05
	R5		3.34E+03	3.12E+04	1.39E+05	5.49E+05
	R6		3.93E+03	3.37E+04	1.20E+05	5.49E+05
	Mean		3.67E+03	3.17E+04	1.35E+05	5.61E+05
1.0	R1	7.8	2.90E+03	2.65E+04	1.12E+05	6.47E+05	9.5
	R2		2.20E+03	3.43E+04	1.38E+05	7.45E+05
	R3		3.73E+03	2.72E+04	1.03E+05	5.57E+05
	Mean		2.94E+03	2.93E+04	1.18E+05	6.50E+05
3.2	R1	8.2	3.67E+03	3.17E+04	1.03E+05	3.55E+05	9.4
	R2		2.96E+03	2.67E+04	9.10E+04	2.64E+05
	R3		3.05E+03	2.98E+04	9.18E+04	3.49E+05
	Mean		3.23E+03	2.94E+04	9.53E+04	3.22E+05
10	R1	8.0	4.05E+03	2.53E+04	1.23E+05	6.49E+05	9.6
	R2		2.61E+03	2.95E+04	1.07E+05	6.00E+05
	R3		2.87E+03	2.59E+04	9.47E+04	5.15E+05
	Mean		3.18E+03	2.69E+04	1.08E+05	5.88E+05
32	R1	8.0	2.79E+03	1.20E+04	1.62E+04	2.17E+04	7.9
	R2		2.90E+03	9.30E+03	1.38E+04	1.89E+04
	R3		6.04E+03	1.04E+04	1.25E+04	1.47E+04
	Mean		3.91E+03	1.06E+04	1.41E+04	1.85E+04
100	R1	7.9	4.11E+03	1.21E+04	1.31E+04	1.77E+04	7.9
	R2		4.99E+03	8.92E+03	7.83E+03	1.15E+04
	R3		3.81E+03	1.14E+04	1.02E+04	1.62E+04
	Mean		4.30E+03	1.08E+04	1.04E+04	1.51E+04

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Daily specific growth rates in controls:

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.069	0.080	0.049
	R2	0.070	0.078	0.054
	R3	0.073	0.069	0.059
	R4	0.058	0.086	0.057
	R5	0.068	0.079	0.053
	R6	0.071	0.067	0.058
	Mean	0.068	0.077	0.055

R₁- R₆= Replicates 1 to 6

Inhibition of growth rate and yield:

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.065		5.27E+05
	R2	0.066		5.89E+05
	R3	0.067		5.99E+05
	R4	0.065	-	5.37E+05	-
	R5	0.065		5.46E+05
	R6	0.065		5.45E+05
	Mean	0.066		5.57E+05
	SD	0.001		2.97E+04
1.0	R1	0.068	[3]	6.44E+05
	R2	0.069	[5]	7.43E+05
	R3	0.065	2	5.54E+05
	Mean	0.067	[2]	6.47E+05	[16]
	SD	0.002		9.46E+04
3.2	R1	0.059	11	3.51E+05
	R2	0.055	17	2.61E+05
	R3	0.059	11	3.45E+05
	Mean	0.058	13	3.19E+05	43
	SD	0.002		5.06E+04
10	R1	0.068	[3]	6.45E+05
	R2	0.066	0	5.97E+05
	R3	0.064	3	5.12E+05
	Mean	0.066	0	5.85E+05	[5]
	SD	0.002		6.73E+04
32	R1	0.020	70	1.89E+04
	R2	0.019	71	1.60E+04
	R3	0.015	77	8.68E+03
	Mean	0.018	73	1.45E+04	97
	SD	0.003		5.28E+03
100	R1	0.018	73	1.36E+04
	R2	0.012	82	6.54E+03
	R3	0.016	76	1.24E+04
	Mean	0.015	77	1.08E+04	98
	SD	0.003		3.77E+03

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Growth curves are shown below.

Validity criteria fulfilled:

yes

Remarks:

> 16-fold increase in controls, mean of coefficients of variation < 35%, specific coefficient of variation < 7%

Conclusions:

The ecotoxicity (72h-NOErL and 72h-ErL50) of Litsea cubeba essential oil towards Pseudokirchnerella subcapitata is 10 and 25 mg/L respectively.

Executive summary:

The ecotoxicity of Litsea cubeba essential oil towards Pseudokirchnerella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to WAFs with nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L and were observed for 72 hours. The 72h-NOErL and 72h-ErL50 were found to be 10 and 25 mg/L respectively based on nominal concentrations.

Description of key information

The ecotoxicity of Litsea cubeba essential oil towards Pseudokirchnerella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to WAFs with nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L and were observed for 72 hours. The 72h-NOErL and 72h-ErL50 were found to be 10 and 25 mg/L respectively based on nominal concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

25 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L