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:
20/01-2015 - 27/01/2015
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Guaiacwood essential oil
- Physical state: solid
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAFs prepared at loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: 50 ml samples were taken after 0 and 72 hours.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis. 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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch of Guaiacwood essential oil tested was a yellowish/brown solid and a mixture of components with poor water solubility.

Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 0.32 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 and distributed over the test vessels. The final test solutions were clear and colourless.

After preparation, volumes of 110 ml were added to each vessel containing 2.2 ml of algal suspension (providing a cell density of 104 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.

- Controls: Yes, blanks

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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): 3 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 and a light intensity of 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Cells were cultured in M1 medium. 3 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:
21 - 23ºC
pH:
7.1 - 7.8
Nominal and measured concentrations:
Nominal: Control and WAFs prepared at loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
Measured (t=0h): Measured (t=72h):
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): 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: 138.3 x 10^4 cells/ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- TOC analysis: 2 replicates 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)

GROWTH MEDIUM
- Standard medium used: yes, adjusted M2 medium (according to OECD 201) 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)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH (at the start and end of the test), temperature (continuously)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 6 mmol/l HEPES-buffer added
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 78 to 79 μE.m-2.s-1.

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 at the WAF of 3.2 mg/l (i.e. concentration in which the number of cells was approximately 50% lower than in the control) to observe for any abnormal appearance of the algae.

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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 4 - 10 mg/l
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 6.8 - 13 mg/l
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 17 - 26 mg/l
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.02 - 0.31 mg/l. Indicative value obtained by extrapolation of the dose-response curve.
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
0.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.10 - 0.74 mg/l. Indicative value obtained by extrapolation of the dose-response curve.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 1.9 - 5.3 mg/l
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the end of the final test microscopic observations were performed at the WAF of 3.2 mg/l (i.e. concentration in which the number of cells was approximately 50% lower than in the control) to observe for any abnormal appearance of the algae.
- Any stimulation of growth found in any treatment: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h-ErC50: 1.5 mg/l
- 72h-EbC50: 0.41 mg/l
Reported statistics and error estimates:
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 (growth rate: Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment, yield: Williams Multiple Sequential t-test Procedure; both 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 rates of the test substance. It should be noted that the highest loading rate was not included with the analysis as it was beyond the dose-response curve.
The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Individual cell densities (x104cells/ml)

Time

Replicate

Guaiacwood essential oil, WAF prepared at (mg/l)

Control

0.32

1.0

3.2

10

32

100

0 h

1

2

3

4

5

6

1.0

1.0

1.0

1.0

1.0

1.0

1.0

 

2

1.0

1.0

1.0

1.0

1.0

1.0

1.0

 

3

1.0

1.0

1.0

1.0

1.0

1.0

1.0

 

4

1.0

 

 

 

 

 

 

 

5

1.0

 

 

 

 

 

 

 

6

1.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Std.Dev.:

 

0.0

0.0

0.0

0.0

0.0

0.0

0.0

CV:

 

0.0

0.0

0.0

0.0

0.0

0.0

0.0

 

 

 

 

 

 

 

 

24 h

1

2

3

4

5

6

8.439

10.296

11.885

10.126

11.927

7.893

2.844

 

2

4.382

9.736

9.602

8.226

14.984

8.219

4.304

 

3

7.651

12.438

7.864

6.127

9.701

6.659

4.127

 

4

9.864

 

 

 

 

 

 

 

5

8.623

 

 

 

 

 

 

 

6

10.935

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

8.3

10.8

9.8

8.2

12.2

7.6

3.8

Std.Dev.:

 

2.2

1.4

2.0

2.0

2.7

0.8

0.8

CV:

 

27.0

13.2

20.6

24.5

21.7

10.8

21.2

 

 

 

 

 

 

 

 

48 h

1

2

3

4

5

6

41.519

37.335

37.271

36.208

27.741

4.049

4.687

 

2

38.222

39.257

37.555

33.265

30.237

3.737

4.453

 

3

39.647

46.100

36.073

29.209

27.294

3.524

4.808

 

4

47.908

 

 

 

 

 

 

 

5

44.731

 

 

 

 

 

 

 

6

50.397

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

43.7

40.9

37.0

32.9

28.4

3.8

4.6

Std.Dev.:

 

4.8

4.6

0.8

3.5

1.6

0.3

0.2

CV:

 

11.0

11.3

2.1

10.7

5.6

7.0

3.9

 

 

 

 

 

 

 

 

72 h

1

2

3

4

5

6

117.863

99.887

95.200

83.201

68.820

5.992

7.708

 

2

124.720

99.490

95.270

75.131

57.765

4.957

6.006

 

3

134.010

108.957

95.001

57.857

65.487

2.787

6.871

 

4

140.137

 

 

 

 

 

 

 

5

147.717

 

 

 

 

 

 

 

6

165.190

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

138.3

102.8

95.2

72.1

64.0

4.6

6.9

Std.Dev.:

 

16.9

5.4

0.1

12.9

5.7

1.6

0.9

CV:

 

12.3

5.2

0.1

18.0

8.9

35.7

12.4

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 138. The test fulfils this validity criterion.

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

Guaiacwood essential oil WAF prepared at (mg/l)

Mean

Std. Dev.

n

%Inhibition

Control

1.641

0.0403

6

0.0

0.32

1.544

0.0171

3

5.9#

1.0

1.519

0.0005

3

7.5#

3.2

1.422

0.0625

3

13.3*

10

1.386

0.0301

3

15.6*

32

0.491

0.1329

3

70.1*

100

0.640

0.0416

3

61.0*

* - effect was statistically significant

#- effect biologically insignificant (<10%)

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

Guaiacwood essential oil WAF prepared at (mg/l)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.080

0.0

1.693

0.0

1.150

0.0

0.32

3

2.376

-14.2

1.331

21.4

0.925

19.6

1.0

3

2.267

-9.0

1.343

20.7

0.946

17.8

3.2

3

2.078

0.1

1.411

16.7

0.777

32.4

10

3

2.486

-19.5

0.860

49.2

0.810

29.5

32

3

2.023

2.8

-0.697

141.2

0.147

87.2

100

3

1.307

37.1

0.229

86.5

0.385

66.6

Percentage inhibition of yield during the final test

Guaiacwood essential oil WAF prepared at (mg/l)

Mean

Std. Dev.

n

%Decrease

Control

137.273

16.9447

6

0.0

0.32

101.778

5.3549

3

25.9*

1.0

94.157

0.1396

3

31.4*

3.2

71.063

12.9475

3

48.2*

10

63.024

5.6708

3

54.1*

32

3.579

1.6357

3

97.4*

100

5.862

0.8510

3

95.7*

* - effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Increase factor > 16 in controls, coefficient of variation of the mean specific growth rate and mean of the replicate coefficients of variation for section-by-section growth rate do not exceed validity criteria
Conclusions:
The acute toxicity (72h-ErL50) of Guaiacwood essential oil towards Pseudokirchneriella subcapitata is 21 mg/l.
Executive summary:

The toxicity of Guaiacwood essential oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs of 1.0, 3.2, 10, 32 and 100 mg/l and were observed for 72 hours. Based on nominal loading rates, the 72h-ErL10 and 72h-ErL50 were found to be 7.3 and 21 mg/l respectively.