Essential oil obtained from the leaves of Cymbopogon flexuosus, gramineae by distillation

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

14/1/2013 - 16/1/2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to study according to international guideline (OECD guideline 202) under GLP. No deviations from guideline reported.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Litsea cubeba

IUPAC Name:

Litsea cubeba

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Litsea cubeba
- Physical state: liquid

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Not relevant
- Concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/L WAF
- Sampling method: Samples were taken from the control and each test group (replicates R1 - R4 pooled) at 0 and 48 hours.
- Sample storage conditions before analysis: stored at -20ºC

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the light-sensitive nature of the test item, all test preparation was performed under non-actinic light conditions and the stirring and test vessels were shielded from light. Amounts of test item (23.2, 41.4, 17.9, 13.5 and 23 mg) were each separately added to the surface of 23.2, 23, 5.6, 2.4 and 2.3 liters of reconstituted water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 4 7 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. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or W AF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs.
- Controls: Yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the mixing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear colorless water columns with oily globules of test item floating on the surface. After 47 hours stirring and a 1-Hour standing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to remain clear colorless water columns with oily globules of test item floating on the surface. Microscopic inspection of the W AFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear, colorless solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory strain
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: no

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Test conditions

Hardness:

250 mg/L CaCO3

Test temperature:

20 - 22ºC

pH:

7.7 - 8.0

Dissolved oxygen:

8.0 - 9.1 mg/L (88 - 105% of ASV)

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal loading rates: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
TOC (0h): 1.44, 1.92, 3.26, 6.20 and 6.15 mg/L
TOC (48h): 3.85, 4.61, 6.21, 11.56, 10.34 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: test vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 130 mL filled with 130 mL test medium
- 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: reconstituted water
- Culture medium different from test medium: No (Elendt M7)
- Conductivity: <5 uS/cm^-1
- Intervals of water quality measurement: 24 hours

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark with 20 minute transition period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilisation (24 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 3.2 -5.6 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 3.2 - 5.6 mg/L

Effect concentrations 3

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: At the start of the mixing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear colorless water columns with oily globules of test item floating on the surface. After 47 hours stirring and a 1-Hour standing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to remain clear colorless water columns with oily globules of test item floating on the surface. Microscopic inspection of the W AFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.45 mg/L

Reported statistics and error estimates:

The EL 50 values and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
If there is no immobilization between 0% and 100% immobilization, then the geometric mean of the highest loading rate showing no lethality and the lowest loading rate showing 100% lethality is calculated. The concentrations resulting in 0% and 100% immobilization will be the 95%
confidence limits.

Any other information on results incl. tables

Cumulative immobility:

Nominal loading rate (mg/L)	Cumulative immobilized Daphnia (initial population: 5 per replicate)
	24 hours						48 hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0	0	0
32	5	5	5	5	20	100	5	5	5	5	20	100
100	5	5	5	5	20	100	5	5	5	5	20	100

Graphs are presented below.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

No mortality in controls, physical-chemical parameters within acceptable limits

Conclusions:

The acute toxicity (48h-EL50) of Litsea cubeba towards Daphnia magna is 4.2 mg/L.

Executive summary:

The acute toxicity of Litsea cubeba towards Daphnia magna was investigated according to OECD guideline 202 under GLP. Daphnids were exposed to nominal WAF's of 1.0, 1.8, 3.2, 5.6 and 10 mg/L and observed for 48 hours. The 48h-EL50 was found to be 4.2 mg/L based on nominal concentrations.