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:
06/09/2012 - 21/09/2012
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 for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Reference substance name:
Cassia oil
IUPAC Name:
Cassia oil
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cassia oil
- Physical state: liquid
- Storage condition of test material: At room temperature in the dark
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: 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: Samples were taken from the uninoculated control and each loading rate WAF test group at 0 and 72 hours for this analysis. Duplicate samples were taken.
- Sample storage conditions before analysis: stored at approximately -20ºC for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (20, 32 and 20 mg) were each separately added to the surface of 20, 10 and 2 liters of culture medium to give the 1.0, 3.2 and 10 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 and 10 mg/L loading rate WAFs. A series of dilutions was made from the 1.0 mg/L loading rate WAF stock solution to give further stock solutions of 0.32 and 0.10 mg/L loading rate WAF.
- 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

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella 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. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

ACCLIMATION
- Culturing media and conditions (same as test or not): Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10^4 – 10^5 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not relevant

Test conditions

Hardness:
No data
Test temperature:
24 +/- 1ºC
pH:
7.5 - 8.1
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal: 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Measured (t=0h): Measured (t=72h):
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL volume filled with 100 mL test medium
- Initial cells density: 5.37*10^3 cell/mL
- Control end cells density: 7.79x10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water*
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- 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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations:10 and 100 mg/L; 0.010, 0.10 and 1.0 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95%CI: 0.96 - 1.0 mg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.83 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.32 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
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.10, 0.32 and 1.0 mg/L loading rate WAFs, however cell debris was observed to be present in the test cultures at 3.2 and 10 mg/L loading rate WAF.
- Any stimulation of growth found in any treatment: yes, in the 0.32 mg/L test vessels.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the mixing period the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with thin, colorless globules of test item floating on the surface. The 3.2 and 10 mg/L loading rate WAFs were observed to have formed clear colorless media columns with thin colorless globules of test item floating on the surface and dispersed throughout the media column. After both the 23-Hour stirring period and 1-Hour standing period the 1.0, 3.2 and 10 mg/L loading rate WAFs were observed to have formed clear colorless solutions. Microscopic examination of the WAFs showed there to be no microscopic particles or micro-dispersions of test item present. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.10, 0.32 and 1.0 mg/L loading rate WAF test cultures were observed oto be green dispersions whilst the 3.2 and 10 mg/L loading rate WAF test cultures were observed to be extremely pale green dispersions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.1 mg/L
- EbC50: 0.73 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).

Any other information on results incl. tables

Cell densities and pH-values:

Nominal Loading Rate

(mg/L)

pH

Cell Densities*(cells per mL)

pH

0 h

0 h

23 h

46 h

72 h

72 h

Control

R1

7.7

5.19E+03

1.31E+04

8.98E+04

6.44E+05

8.1

 

R2

6.39E+03

1.43E+04

9.43E+04

7.18E+05

 

R3

5.16E+03

2.04E+04

1.23E+05

9.42E+05

 

R4

5.69E+03

1.47E+04

1.09E+05

7.69E+05

 

R5

4.58E+03

1.53E+04

1.19E+05

8.46E+05

 

R6

5.19E+03

1.40E+04

9.87E+04

7.53E+05

 

Mean

5.37E+03

1.53E+04

1.06E+05

7.79E+05

0.10

R1

7.6

6.63E+03

1.38E+04

8.17E+04

6.74E+05

8.1

 

R2

4.81E+03

1.73E+04

9.45E+04

5.62E+05

 

R3

4.63E+03

2.14E+04

1.00E+05

8.67E+05

 

Mean

5.36E+03

1.75E+04

9.21E+04

7.01E+05

0.32

R1

7.5

6.83E+03

2.21E+04

1.22E+05

9.34E+05

8.0

 

R2

6.66E+03

2.23E+04

8.45E+04

7.46E+05

 

R3

5.69E+03

2.27E+04

9.85E+04

7.93E+05

 

Mean

6.39E+03

2.24E+04

1.02E+05

8.24E+05

1.0

R1

7.5

5.49E+03

2.04E+04

**

**

8.0

 

R2

6.22E+03

2.09E+04

7.82E+04

3.98E+05

 

R3

9.21E+03

1.68E+04

7.92E+04

4.04E+05

 

Mean

6.97E+03

1.94E+04

7.87E+04

4.01E+05

3.2

R1

7.5

5.34E+03

8.04E+03

1.65E+04

4.60E+04

7.8

 

R2

4.63E+03

9.33E+03

1.68E+04

4.63E+04

 

R3

5.69E+03

7.98E+03

1.46E+04

3.78E+04

 

Mean

5.22E+03

8.45E+03

1.60E+04

4.34E+04

10

R1

7.5

6.22E+03

6.78E+03

9.12E+03

2.49E+04

7.8

 

R2

8.51E+03

7.19E+03

7.89E+03

2.19E+04

 

R3

5.98E+03

5.84E+03

7.04E+03

1.73E+04

 

Mean

6.90E+03

6.60E+03

8.02E+03

2.14E+04

*Cell densities represent thean number of cells per mL calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

** No values available due to breakage of replicate prior to sampling at 48 hours.

R1- R6= Replicates 1 to 6

Daily specific growth rates in controls:

 

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.042

0.084

0.076

R2

0.046

0.082

0.078

R3

0.061

0.078

0.078

R4

0.047

0.087

0.075

R5

0.049

0.089

0.075

R6

0.045

0.085

0.078

Mean

0.048

0.084

0.077

Inhibition of growth rate and yield:

Nominal Loading Rate
(mg/L)

Growth Rate

(cells/mL/hour)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.067

 

6.39E+05

 

 

R2

0.069

 

7.12E+05

 

 

R3

0.073

 

9.37E+05

 

 

R4

0.070

-

7.63E+05

-

 

R5

0.071

 

8.42E+05

 

 

R6

0.070

 

7.48E+05

 

 

Mean

0.070

 

7.74E+05

 

 

SD

0.002

 

1.04E+05

 

0.10

R1

0.068

3

6.67E+05

 

 

R2

0.066

6

5.58E+05

 

 

R3

0.072

[3]

8.62E+05

 

 

Mean

0.069

2

6.96E+05

10

 

SD

0.003

 

1.54E+05

 

0.32

R1

0.073

[4]

9.27E+05

 

 

R2

0.070

0

7.39E+05

 

 

R3

0.070

0

7.88E+05

 

 

Mean

0.071

[1]

8.18E+05

[6]

 

SD

0.002

 

9.75E+04

 

1.0

R1

**

**

**

 

 

R2

0.061

13

3.92E+05

 

 

R3

0.061

13

3.95E+05

 

 

Mean

0.061

13

3.94E+05

49

 

SD

0.000

 

2.12E+03

 

3.2

R1

0.031

56

4.07E+04

 

 

R2

0.031

56

4.16E+04

 

 

R3

0.028

60

3.21E+04

 

 

Mean

0.030

57

3.81E+04

95

 

SD

0.002

 

5.23E+03

 

10

R1

0.022

69

1.87E+04

 

 

R2

0.021

70

1.34E+04

 

 

R3

0.017

76

1.13E+04

 

 

Mean

0.020

72

1.45E+04

98

 

SD

0.003

 

3.79E+03

 

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

** No values available due to breakage of replicate prior to sampling at 48 hours.

R1– R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Growth curves are shown below.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
> 16-fold increase in controls, mean of coefficients of variation < 35%, specific coefficient of variation < 7%
Conclusions:
The ecotoxicity (72h-ErL10 and 72h-ErL50) of Cassia oil towards pseudokirchnerella subcapitata is 0.55 and 3.3 mg/L respectvely.
Executive summary:

The ecotoxicity of Cassia oil towards Pseudokirchnerella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to WAFs with loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L and were observed for 72 hours. The 72h-ErL10 and 72h-ErL50 were found to be 0.55 and 3.3 mg/L respectively.