Registration Dossier

Ecotoxicological information

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:
24/11/2014 - 16/12/2014
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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cinnamon Bark Oil (CBO)
- Physical state: Liquid
- Storage condition of test material: ambient (+5 to +30°C), dark, dry
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:
- Concentrations: nominal Water accomodated fractions (WAF) of 20.0; 8.00; 3.20; 1.28 and 0.512 mg/L.
- Sampling method: The content of total organic carbon (TOC) was measured. Furthermore the total organic carbon in the test item itself was analysed in order to quantify the dissolved content of carbon in the test solution compared to the nominal loading rate. For the calculation of the content of test item in the test solution the blank value of the control was subtracted from the measured value at each loading rate. Analytical samples were taken from each loading rate and control at 0 d (initial value) and after 3 d from aged test solutions. All analytical samples taken were analysed by TOC analysis. These samples were taken from a separate vessel without algae.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on a non-GLP range-finding test the GLP test was performed with the following loading rates (spaced by a geometric factor of 2.5): 20.0; 8.00; 3.20; 1.28 and 0.512 mg/L. Due to low water solubility of the test item two WAFs with nominal loading rates of 20.0 and 8.00 mg/L were prepared. 20.0 mg/L (S1) and 8.00 mg/L (S2) were prepared by weighing the required amount of test item on a weighing scoop and transfer it to a volumetric flask. Test medium was added up to the bench mark. These solutions were stirred by a magnetic stirrer in a closed vessel at room temperature in the dark over 23 hours to dissolve a maximum amount of the test item. Subsequently the undissolved test item was allowed to sediment and/or float for a period of one hour until the phases had separated. The test item solutions were withdrawn from the middle of the preparation vessel by suction (according to OECD Series on Testing and Assessment No. 23). Transfer of undissolved test item was avoided. This was checked by microscopic observation. The test item solutions 3.20, 1.28 and 0.512 mg/L were prepared by dilution of the withdrawn phase of 8.00 mg/L test item stock solution with test medium. Algae cells were added and the solutions were transferred into serum bottles which were filled up to the brim and sealed with crimp caps.

Deviation from protocol: The test item is classified as CMR substance as mentioned in the MSDS. Therefore as a deviation from the original study plan due to technical and security reasons the loading rates 3.20 and 1.28 were not prepared by direct weighting but by dilution of the WAF with 8 mg/l. For safe handling of this CMR substance, all WAF preparations need to be done in a fume cupboard. This has an impact for small amounts of test substances. In addition the maximum solvent volume was reduced to 1 L and 2 L because larger volumes could not be handled in the fume cupboard. Based on these facts the weighing procedure was performed as described in the deviation.
- Controls: yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Transfer of undissolved test item was avoided. This was checked by microscopic observation.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany.
- Method of cultivation: The algae are grown semi-continuously in sterile liquid cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are also kept on slanted agar tubes and are ordered regularly from the culture collection. Culture conditions were as follows:
- Illumination: continuously (approx. 60 – 120 μE m^-2 s^-1)
- Temperature: 21 – 24 °C
- Culture flasks: 100 mL Erlenmeyer flasks
- CO2 supply: by shaking on a rotating shaker, approximately 105 rpm. Cells from this semi-continuous liquid stock culture will be used for the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
No data
Test temperature:
21.6 - 23.9°C
pH:
7.40 - 9.33
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal (WAF, t=0h): 0.512, 1.28, 3.20, 8.00 and 20.00 mg/L.
Measured (TOC, control corrected, t=0h): 3.30, 6.30, 9.90. 15.9 and 16.9 mg/L
Measured (TOC, control corrected, t=72h): 1.87, 3.78, 6.64, 10.2 and 16.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Serum bottle
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 120 mL serum bottles with crimp caps
- CO2-supply: by shaking on a rotating shaker, 105 rpm
- Initial cells density: 0.55 × 10^4 cells/mL
- Control end cells density: 34.01 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, AAP medium (Annex 3 of OECD 201)
- Details: 300 mg NaHCO3 were added per 1 L test medium as CO2 supply for the algae in the closed test system.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0d and t = 3d. The temperature was measured at day 0, 1, 2 and 3 within a separate vessel. The light intensity at 30 different places at test start.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuously
- Light intensity and quality: 4700 – 7100 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At defined dates (1, 2 and 3 days), the number of cells in each replicate was determined in duplicate. The determination was performed by means of a calibration curves, where cell numbers (y axis) were plotted versus fluorescence signals (x axis). To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture.

The daily fluorescence measurements were performed with a Tecan Reader (infinite 200Pro; serialnumber 1305001441) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.10.4.0).

Samples for determination of biomass were taken using a syringe via the septum in the crimp cap. Withdrawn sample volume was replaced with medium which was added via the septum using another syringe.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 0.01, 0.1, 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 concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
8.51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 7.29 - 10.5 mg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2.12 - 3.56 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
4.71 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 4.06 - 5.40 mg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.945 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.659 - 1.24 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
20 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
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: ErC50: 0.84 mg/L, ErB50: 0.39 mg/L
Reported statistics and error estimates:
The statistical evaluation for day 3 was performed for growth rate and yield. The NOEL and LOEL were determined by using a multiple comparison method (Welch Bonferroni-Holms corrected for growth rate and Jonckheere-Terpstra for yield). A test for normality of the data was performed by using the Shapiro-Wilk’s statistic and homogeneity of variance was performed according to Levene. The values for ErL10, ErL50 (growth rate) and EyL10, EyL50 (yield) were determined by probit analysis following the Gompertz distribution. The evaluation of data was performed by SAS® (2002-2010). Negative values were set to zero and values above 100 were set to 100.

Any other information on results incl. tables

Individual cell numbers (x 10^4)

Test Item Loading Rate [mg/L] Cells/mL) Yield
0d 1d 2d 3d 0d-3d
Control 0.61 1.48 5.30 27.76 27.15
0.52 1.60 7.23 38.56 38.04
0.58 1.53 7.27 39.13 38.55
0.55 1.48 6.18 34.65 34.10
0.49 0.98 6.67 30.59 30.10
0.57 1.32 4.65 33.37 32.80
Mean 0.55 1.40 6.22 34.01 33.46
0,512 0.67 1.36 6.02 32.12 31.45
0.64 1.30 7.89 34.68 34.04
0.62 1.58 6.04 39.31 38.69
Mean 0.64 1.41 6.65 35.37 34.73
128 0.63 1.42 6.63 26.26 25.63
0.54 1.35 5.81 39.63 39.09
0.61 1.51 6.28 -1.57 -2.18
Mean 0.59 1.43 6.24 21.44 20.85
320 0.54 0.85 3.19 35.41 34.87
0.61 0.85 4.25 19.20 18.59
0.59 0.86 4.20 33.52 32.93
Mean 0.58 0.85 3.88 29.38 28.80
800 0.53 0.67 0.77 27.00 26.47
0.50 0.74 0.79 2.21 1.71
0.62 0.75 0.69 1.39 0.77
Mean 0.55 0.72 0.75 10.20 9.65
200 0.48 0.22 0.15 1.15 0.67
0.56 0.44 0.24 0.19 -0.37
0.48 0.29 0.12 0.13 -0.35
Mean 0.51 0.32 0.17 0.49 -0.02

Control growth rates

  Growth rates, µ [d-1]
0d – 1d 0d - 2d 0d - 3d
Control 0.88634

1.08100

1.27263
1.12393 131,608 143,538
0.96999 1.26424 1.40387
0.98988 1.20958 1.38104
0.69315 1.30548 1.37801
0.83975 1.04949 1.35659
Mean Std. Dev. CV [%] 0.91717 1.20431 1.37125
0.14694 0.11447 0.05524
16 95 40

Control daily growth rates

  Growth rates, µ [d-1]
0d – 1d 1d – 2d 2d – 3d
Control 0.88634 1.27566 1.65589
1.12393 1.50824 1.67398
0.96999 1.55849 1.68313
0.98988 1.42928 1.72398
0.69315 1.91782 1.52305
0.83975 1.25924 1.97079
Mean Std. Dev. CV [%] 0.91717 1.49146 1.70514
0.14694 0.24121 0.14694
16 162 86

Coefficient of variation (CV) of control daily growth rates

Mean daily growth rates µ [d-1] Standard deviation CV [%]
1.27263 0.38478 30
1.43538 0.28217 20
1.40387 0.38088 27
1.38105 0.36942 27
1.37801 0.62509 45
1.35659 0.57177 42
  Mean CV 32

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control cell number increase and coefficients of variation meet validity criteria
Conclusions:
The acute toxicity (72h-ErL50) of Cinnamon bark oil towards Pseudokirchneriella subcapitata is 8.51 mg/L.
Executive summary:

The acute toxicity of Cinnamon bark oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs of 0.512, 1.28, 3.2, 8 and 20 mg/L and were observed for 72 hours. Based on the nominal loading rates the 72h-ErL50 and 72h-ErL10 were found to 8.51 and 2.91 mg/L respectively.