Cedrus atlantica, ext.

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2015 - 2 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Test and Assessment Number 23

Test guideline 4

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cedrus atlantica, ext.

EC Number:

295-985-9

EC Name:

Cedrus atlantica, ext.

Cas Number:

92201-55-3

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

Essential oil of Cedarwood Atlas obtained from the wood of Cedrus atlantica by steam distillation

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: CEDAR ATLANTICA OIL

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1.0, 3.2, 10, 32, 100 mg/L
- Sampling method: 50 mL samples for analysis were taken from all test groups at t=0 and t = 48h; Reserve samples of 50 mL were taken for possible analysis. Samples were stored in a freezer. At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Additionally, reserve samples of 50 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
- Sample storage conditions before analysis: Stored in refrigerator until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs). Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 1.0 to 100 mg/L depending on the test . Exact amounts of test substance were added to test medium and magnetically stirred for 2 days in closed vessels with minimal headspace. Thereafter, all solutions were left to stabilise overnight. Subsequently, the WAFs were collected by siphoning the water phase and distributed over the test vessels. The final test solutions were clear and colourless.
- Differential loading: Combined limit/range-finding test: Control, 1.0, 10 and 100 mg/L; First full test: Control, 4.6, 10, 22, 46, 100 mg/L; Second full test (Final test): Control, 1.0, 3.2, 10, 32 and 100 mg/L
- Controls: 1 per test run
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved material was detected after the collection of the WAFs.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history
- Age at study initiation: < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Food type during breeding: A suspension of fresh water algae
- Frequency during breeding: Daily
- Feeding during test: No

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:

180 mg/L CaCO3

Test temperature:

20 - 22°C

pH:

7.6 - 8.2

Dissolved oxygen:

7.2 - 8.7 mg/L

Salinity:

not relevant

Nominal and measured concentrations:

Nominal WAF: Control, 1.0, 3.2, 10, 32 and 100 mg/L
Measured (TOC, t=0h), corrected for control: -, 0.27, 0.65, 1.2, 4.1, 7.9 mg/l
Measured (TOC, t=48h), corrected for control: -, 0.09, 0.55, 1.3, 4.1, 7.9 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass
- Type: closed
- Material, size, headspace, fill volume: 60 mL, closed airtight with headspace reduced to minimum
- Aeration: No aeration of the test solutions
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Medium composition:
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements: B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients: Na2SiO3. 9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/l

- Culture medium different from test medium: No
- Intervals of water quality measurement: pH and dissolved oxygen are measured at the beginning and at the end of the test, for all concentrations and the control. Temperature is monitored continually.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light / 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility (including mortality) recorded at 24 hours and at 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations of the final test: 3.2
A spacing factor of 3.2 was used because the preceding tests produced unequivocal results and therefore wider range needed to be tested.
- Range finding study, first full test and second full test (Final test)
- Test concentrations of range finding test: 1, 10 and 100 mg/L
- Test concentrations of first full test: 4.6, 10, 22, 46 and 100 mg/L
- Test concentrations of second full test (Final test): 1.0, 3.2, 10, 32 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.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%-CL: 11-22 mg/L

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%-CL: 1.0-3.2 mg/L

Details on results:

- Mortality of control: 0%

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: The 24h-EC50 was 0.72 mg/L with a 95% confidence interval between 0.64 and 0.82 mg/L. The 48h-EC50 was estimated to correspond with 0.36 mg/L, with 10% effect at 0.32 mg/L and 100% effect at 0.56 mg/L.

Reported statistics and error estimates:

The 24h-EL50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test substance loading rates using the maximum likelihood estimation method (Weibull analysis).
The 48h EL50 could not be determined with above described method due to too high variation in data. Therefore, this value was estimated using the binomial distribution.

Any other information on results incl. tables

Number of introduced daphnids and incidence of immobility in the second full test

Time (h)	Replicate	Cedar Atlantica Oil WAF prepared at (mg/L)
		Control	1.0	3.2	10	32	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0 (2)	2 (1)	5 (5)	5
	B	0	0	0 (1)	3 (1)	3 (5)	5 (5)
	C	0	0	0 (4)	2 (1)	3 (4)	5 (2)
	D	0	0	0 (5)	2 (1)	5 (5)	5 (5)
	Total immobilised	0	0	0	9	16	20
	Effect %	0	0	0	45	80	100
48	A	0	1	5 (1)	2	5	5
	B	0	0	4 (1)	5 (2)	5	5
	C	0	0	2 (1)	2	5	5
	D	0	1 (1)	4 (2)	3	5 (2)	5
	Total immobilised	0	2	15	12	20	20
	Effect %	0	10	75	60	100	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. This organism was reimmersed into the respective solutions before recording of mobility.

pH and oxygen concentrations during the second full test

Cedar Atlantica Oil	Start (t = 0h)		End (t = 48h)
Loading rate (mg/L)	pH	O2	pH	O2
Control	7.6	8.7	8.2	7.6
1.0	7.7	8.7	8.2	7.4
3.2	7.7	8.6	8.1	7.2
10	7.7	8.6	8.1	7.3
32	7.8	8.7	8.1	7.3
100	7.8	8.7	8.1	7.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Mortality in control < 10%, dissolved oxygen > 3 mg/l

Conclusions:

The acute toxicity (48h-EL50) of Cedarwood Atlas oil towards Daphnia magna is 2.0 mg/l.

Executive summary:

The acute toxicity of Cedarwood Atlas oil towards Daphnia magna was investigated according to OECD guideline 202 under GLP, the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000. Cedarwood Atlas oil is a liquid mixture of components with poor water solubility. A full test was performed based on the results obtained in preceding combined limit/range-finding test and a first full test. Water Accommodated Fractions (WAFs) were prepared at individual loading rates of 1.0, 3.2, 10, 32 and 100 mg/L and used as test concentrations for the final test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the WAFs. The total exposure period was 48 hours and was performed in airtight closed vessels with headspace reduced to minimum. Samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. The measured TOC concentrations increased with applied dose showing proper preparation of test solutions. The study met the acceptability criteria prescribed by the protocol and was considered valid. The 48h-EL50 was 2.0 with the 95% confidence interval between 1.0 and 3.2 mg/L.