Cinnamomum zeylanicum, 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:

01/03/2011 - 04/03/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to international guideline (OECD guideline 201) under GLP. No deviations from guideline reported.

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)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: Samples of the uninoculated control and each loading rate WAF test group were taken for analysis at 0 and 72 hours. Duplicate samples were taken.
- Storage: At -20 ºC

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 74, 23, 74 and 230 mg) were each separately added to the surface of 23, 23, 2.3, 2.3 and 2.3 litres of culture medium in stirring vessels with minimal headspace 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 water surface. The
stirring was stopped after 23 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/I loading rate WAFs.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseukirchneriella 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
- Method of cultivation: master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: yes, but no time is specified
- Culturing media and conditions (same as test or not): yes, except for addition of 500 mg/l sodium bicarbonate to conteract pH increase in test solutions

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 +/- 1 ºC

pH:

7.9 - 9.6

Dissolved oxygen:

Not relevant

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal loading rates: 1.0, 3.2, 10, 32 and 100 mg/l
Measured loading rates (t=0h): 3.3, 2.8, 4.9, 21 and 61mg C/l
Measured loading rates (t=72h):

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250ml filled with 250 ml medium
- Aeration: None
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 4.03 - 5.5 x 10^3 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/I
MgCI2.6H20 12.164 mg/I
CaC12.2H20 4.41 mg/l
MgS04.7H20 14.7 mg/l
K2HPO4 1.044 mg/I
NaHCO3 15.0 mg/I
H3B03 0.1855 mg/l
MnCl2.4H20 0.415 mg/l
ZnCI2 0.00327 mg/I
FeCI3.6H20 0.159 mg/l
CoCI2.6H20 0.00143 mg/l
Na2MoO4.2H20 0.00726 mg/I
CuCI2.2H20 0.000012 mg/I
Na2EDTA.2H20 0.30 mg/l
Na2Se03.5H2O 0.000010 mg/I

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/I) was added to the prepared culture medium prior to use.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water
- Total organic carbon: - Culture medium different from test medium: No, except for sodium bicarbonate to counteract pH increase during test. Costantly shaken at 150 rpm
- Intervals of water quality measurement: at 0 and 72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- 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: Coulter Multisizer Particle Counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accomodated Fraction (WAF)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

7.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 10 - 14 mg/l

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water accomodated Fraction (WAF)

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): cell debris was observed to be present in the test cultures at 32 and 100 mg/l loading rate WAF.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:At the start of stirring all loading rate WAFs were observed to have formed clear colourless media columns with test item floating at the media surface and settled on the bottom of the mixing vessel. After the 23-Hour stirring and 1-Hour standing periods all loading rate WAFs were observed to have formed clear colourless media columns with test item floating at the media surface. No test item was visible on the bottom of the stirring vessels. Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.2 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).

For detailed results, see attached file "Results.doc".

Validity criteria fulfilled:

yes

Remarks:

Mean coefficient of variation for specific growth <35%, >16-fold increase of cell density over test period, coefficient of variation for average specific growth <7%

Conclusions:

The acute toxicity (72h-ErL50) of cinnamon leaf oil towards Pseudokirchneriella subcapitata in a WAF test is 17 mg/l.

Executive summary:

The acute toxicity of cinnamon leaf oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algae were exposed to nominal loading rates (WAF) of 1.0, 3.2, 10, 32 and 100 mg/l and observed for 72h hours. Based on nominal loading rates the 72h-NOErL and 72h-ErL50 were found to be 3.2 and 17 mg/l respectively.

Description of key information

The acute toxicity of cinnamon leaf oil towards Pseudokirchneriella subcapitata in a WAF study resulted in a 72h-ErL50 of 17 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

17 mg/L

Additional information

The acute toxicity of cinnamon leaf oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algae were exposed to nominal loading rates (WAF) of 1.0, 3.2, 10, 32 and 100 mg/l and observed for 72h hours. Based on nominal loading rates the 72h-NOErL and 72h-ErL50 were found to be 3.2 and 17 mg/l respectively.