Cedrus atlantica, ext.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31/03/2015 - 30/06/2015

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

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: nominal water accommodated fractions (WAF) of 0.20, 0.80, 4.0, 20 and 100 mg/l
- Sampling method: 50 ml samples were taken at t=0 and t=72 hours.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Cedar Atlantica Oil is a clear yellow liquid and a UVCB substance.

Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 0.20 to 100 mg/l. Exact amounts of test substance were added to test medium containing no HEPES buffer 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.

After preparation, volumes of 110 ml were added to each vessel containing 2.2 ml of an algal suspension (providing a cell density of 10^4 cells/ml) and HEPES buffer (0.66 ml) was spiked to each vessel.
Solutions for analysis of TOC concentrations:
• At the start of the test samples were taken from freshly prepared solutions (before preparation of the exposure vessels).
• At the end of the exposure period: volumes of 110 ml solutions of the same WAFs added at t=0 to the vessels containing no algae. These vessels were not spiked with HEPES buffer.
This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.
- Controls: Yes, test medium without test substance or other additives (6 replicates)
- Other checks: 1 replicate of each group without algae for sampling purposes (these replicates contained no HEPES buffer but were incubated at the same conditions as the other replicates) and 1 replicate of each concentration without algae but containing HEPES as a turbidity control.


• At the end of the exposure period: volumes of 110 ml solutions of the same WAFs added at t=0 to the vessels containing no algae. These vessels were not spiked with HEPES buffer.
This was done to prevent that the carbon originating from the buffer will obscure the results of TOC analysis.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/l CaCO3

Test temperature:

22 - 24ºC

pH:

7.4 - 7.7

Nominal and measured concentrations:

Nominal: WAFs of control, 0.20, 0.80, 4.0, 20, and 100 mg/l
Measured (t=0h, TOC): n.d., n.d., n.d., 0.3382, 2.227 and 6.622 mg/l
Measured (t=72h, TOC): n.d., n.d., n.d., 0.25, 1.907 and 6.29 mg/l
n.d. = not detected
Thus the concentrations were maintained at a level of 74 to 95% of the initial TOC level.

Details on test conditions:

TEST SYSTEM
- Type: closed
- Material, size, headspace, fill volume: 120 ml, all-glass, containing 110 ml of test solution, closed airtight.
- Aeration: none
- Initial cells density: 1 x 10^4 cells/ml
- Control end cells density: 121.8 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, adjusted M2 medium
- Composition of the medium: adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 300 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
HEPES 6 mmol/l
pH 7.1 ± 0.3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: No
- pH: At the beginning and at the end of the test. The pH of the control solutions should preferably not deviate by more than 1.5 units during the test.
- Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps, 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- Other: At the end of the final test microscopic observations were performed on the two highest concentrations to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5
- Justification for using less concentrations than requested by guideline: A spacing factor of 5 was used because the results of the combined limit/range-finding test showed very shallow dose-response relationship.
Range finding study
- Test concentrations: 1, 10 and 100 mg/l with 3, 3 and 6 replicates, respectively
- 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

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 5.4 - 14 mg/l

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determinded

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.054 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.015 - 0.13 mg/l

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Cl: 0.22 - 0.88 mg/l

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% Cl: 21 - 57 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Any stimulation of growth found in any treatment: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 72hErC50: 1.3 mg/l ; 72hEyC50: 0.46 mg/l

Reported statistics and error estimates:

The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments. The average growth rate at each test substance concentration is then compared with the control value and the percentage inhibition.

For determination of the NOELR and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rate of the test substance.

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Growth rates were in the range of those for the controls at the lowest loading rate during the 72-hour test period, whereas the growth rate of algae exposed to the loading rate of 0.80 mg/l and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at the loading rate of 0.80 mg/l and higher.

 

Inhibition of yield increased with increasing concentration of the test substance from the loading rate of 0.80 mg/l upwards resulting in 56% inhibition at the highest loading rate. Statistically significant inhibition of yield was found at the loading rate of 0.80 mg/l and higher.

It should be noted that at the two lowest WAFs an inhibition of 26 and 24% was observed. However, it was assumed to be not related to the test substance as the TOC concentrations measured in these WAFs were below the concentrations measured in the higher loading rates.

Percentage inhibition of growth rate (total test period) during the final test

Cedar Atlantic Oil Loading rate (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	1.688	0.0185	6
0.20	1.699	0.0377	3	-0.6
0.80	1.563	0.0154	3	7.4*
4.0	1.519	0.0179	3	10.0*
20	1.497	0.0391	3	11.3*
100	1.415	0.0362	3	16.2*

* - effect was statistically significant

Percentage inhibition of growth rate at different time intervals during the final test

Cedar Atlantica Oil Loading rate (mg/l)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.130	0.0	1.640	0.0	1.294	0.0
0.20	3	2.161	-1.5	1.672	-1.9	1.264	2.3
0.80	3	2.196	-3.1	1.552	5.4	0.942	27.1
4.0	3	2.107	1.1	1.519	7.4	0.931	28.0
20	3	1.934	9.2	1.320	19.5	1.238	4.3
100	3	1.462	31.4	1.602	2.3	1.179	8.8

 

Percentage inhibition of yield during the final test

Cedar Atlantica Oil Loading rate (mg/l)	Mean	Std. Dev.	n	%Inhibition
Control	157.4	8.63	6
0.20	163.1	18.26	3	-3.7
0.80	108.0	4.98	3	31.4*
4.0	94.3	5.18	3	40.0*
20	88.7	10.83	3	43.6*
100	68.9	7.81	3	56.2*

* - effect was statistically significant

  

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 158. The test fulfils this validity criterion.

Conclusions:

The 72h-EL10 (growth rate) was 9.1 mg/L whereas the 72h-NOEL (growth rate) was 0.80 mg/l. The 72h-EL50 (growth rate) was > 100 mg/L

Executive summary:

The toxicity of Cedarwood Atlas oil on the freshwater alga Pseudokirchneriella subcapitata was determined according to OECD TG 201 (2006; Annex 5 corrected 28 July 2011), meeting also the test method of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000. The study was in compliance with the principles for GLP.

Cedarwood Atlas oil is a liquid mixture of components with poor water solubility (UVCB). Therefore Water Accommodated Fractions (WAFs) were prepared at loading rates of 0.20, 0.80, 4.0, 20 and 100 mg/l and used as test concentrations.

Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 0.20, 0.80, 4.0, 20 and 100 mg Cedarwood Atlas oil per litre. Initial cell density was 10⁴cells/ml. The total exposure period was 72 hours and the test was performed in airtight closed vessels. Samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure.

 

At the two lowest loading rates the Total Organic Carbon concentrations could not be measured during the exposure. In the WAFs prepared at 4.0, 20 and 100 mg/l the measured TOC concentrations increased with the loading rate at the start of the test indicating proper preparation of WAFs. At the end of the test the measured TOC concentrations were at the level of 74-95% of initial.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

The 72h-EL10 (growth rate) was 9.1 mg/L whereas the 72h-NOEL (growth rate) was 0.80 mg/l. The 72h-EL50 (growth rate) was > 100 mg/L. The 72h-EL50 (growth rate) was beyond the range tested, i.e. exceeded the concentration obtained in the WAF prepared at a loading rate of 100 mg/l.

The 72h-EL₅₀ for yield inhibition was 33 mg/l with a 95% confidence interval ranging from 21 to 57 mg/l.