Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
8/12/2014 - 22/01/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study according to international guideline (OECD guideline 201) under GLP. No deviations from guideline reported.
Reason / purpose:
reference to same study
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
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes (incl. certificate)
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 water accommodated fractions (WAF) of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: 50 ml samples were taken at t=0 and t=72 hours. It should be noted that samples were taken from solutions incubated without algae and HEPES buffer.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Spearmint Essential oil, ex Mentha cardiaca is a clear colourless to yellow liquid 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.

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 of were added to each vessel 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, test medium without test substance or other additives
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. 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not relevant
Hardness:
24 mg/l CaCO3
Test temperature:
22 - 23ºC
pH:
7.1 - 7.7
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal: WAFs of control, 0.32, 1.0, 3.2, 10, 32 and 100 mg/l
Measured (t=0h, TOC): -0.2293, 0.01529, -0.03736, 1.883, 6.00, 21.44 and 62.61 mg/l
Measured (t=72h, TOC): -0.6087, -0.498, -0.2505, 1.283, 5.316, 19.22 and 57.43 mg/l
Concentrations below 1.0 mg TOC/l cannot be reliably quantified
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: 112.1 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
- Intervals of water quality measurement:
- 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: t the end of the final test microscopic observations were performed at the WAF of 32 mg/l to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 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:
EL10
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 14-27 mg/l
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 23 - 38 mg/l
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 56 - 76 mg/l
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.57 - 6.9 mg/l
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
6.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Cl: 1.9 - 12 mg/l
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Cl: 13 - 49 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: 1.5 mg/l; 82hEbC50: 0.41 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).


Growth rates were in the range of the controls at the loading rates ranging from 0.32 to 10 mg/l during the 72-hour test period, whereas the growth rate of algae exposed to WAFs prepared at 32 and 100 mg/l were increasingly reduced. Due to mathematical reasons statistical analysis could not be performed for the data gathered after 72 hours of exposure. Therefore, the NOELR for growth rate inhibition could not be determined. Instead, the EL10was calculated as an equivalent alternative.

 

Inhibition of yield increased with increasing loading rate of Spearmint Essential oil, ex Mentha cardiaca from 3.2 mg/l upwards resulting in 97% inhibition at the highest loading rate tested. Statistically significant inhibition of yield was found at the two highest WAFs. 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

Spearmint Essential oil,

ex Mentha cardiaca

WAF prepared at (mg/l)

Mean

Std. Dev.

n

%Inhibition

Control

1.570

0.0539

6

0.0

0.32

1.472

0.0180

3

6.2

1.0

1.483

0.0278

3

5.5

3.2

1.560

0.0413

3

0.6

10

1.538

0.0275

3

2.0

32

1.244

0.2231

3

20.8

100

0.486

0.0117

3

69.0

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

Spearmint Essential oil,

 ex Mentha cardiaca

WAF prepared at (mg/l) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.950

0.0

1.542

0.0

1.217

0.0

0.32

3

2.068

-6.1

1.490

3.4

0.858

29.5

1.0

3

2.129

-9.2

1.394

9.6

0.927

23.8

3.2

3

2.052

-5.2

1.473

4.5

1.157

4.9

10

3

2.160

-10.8

1.282

16.9

1.173

3.6

32

3

2.009

-3.1

0.845

45.2

0.877

27.9

100

3

1.773

9.0

0.115

92.5

-0.430

135.3

Percentage inhibition of yield during the final test

Spearmint Essential oil,

ex Mentha cardiaca

WAF prepared at (mg/l)

Mean

Std. Dev.

n

%Decrease

Control

111.059

16.3530

6

0.0

0.32

81.852

4.5147

3

26.3*

1.0

84.823

7.3310

3

23.6

3.2

107.456

12.9902

3

3.2

10

100.243

8.4980

3

9.7

32

46.443

24.9677

3

58.2*

100

3.304

0.1495

3

97.0*

* - effect was statistically significant

Individual cell densities (x104cells/ml)

Time

Replicate

Spearmint Essential oil, ex Mentha cardiaca

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

6.813

8.486

8.581

8.339

8.142

9.319

5.575

 

2

8.559

7.472

8.987

7.804

8.705

6.442

5.462

 

3

5.912

7.810

7.714

7.241

9.206

6.909

6.712

 

4

7.512

 

 

 

 

 

 

 

5

6.932

 

 

 

 

 

 

 

6

6.706

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

7.1

7.9

8.4

7.8

8.7

7.6

5.9

Std.Dev.:

 

0.9

0.5

0.7

0.5

0.5

1.5

0.7

CV:

 

12.6

6.5

7.7

7.0

6.1

20.4

11.7

 

 

 

 

 

 

 

 

48 h

1

2

3

4

5

6

31.206

35.013

33.870

29.967

27.901

25.170

6.588

 

2

31.673

33.684

38.025

37.152

33.487

10.254

7.545

 

3

32.135

36.696

30.232

35.125

32.642

20.294

5.811

 

4

34.399

 

 

 

 

 

 

 

5

36.020

 

 

 

 

 

 

 

6

31.961

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

32.9

35.1

34.0

34.1

31.3

18.6

6.6

Std.Dev.:

 

1.9

1.5

3.9

3.7

3.0

7.6

0.9

CV:

 

5.7

4.3

11.5

10.9

9.6

41.0

13.1

 

 

 

 

 

 

 

 

72 h

1

2

3

4

5

6

110.460

81.760

81.551

93.686

94.463

67.339

4.431

 

2

122.353

78.984

94.288

118.107

98.489

19.426

4.341

 

3

111.626

87.813

81.630

113.574

110.776

55.565

4.139

 

4

119.881

 

 

 

 

 

 

 

5

126.795

 

 

 

 

 

 

 

6

81.236

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n:

 

6

3

3

3

3

3

3

Mean:

 

112.1

82.9

85.8

108.5

101.2

47.4

4.3

Std.Dev.:

 

16.4

4.5

7.3

13.0

8.5

25.0

0.1

CV:

 

14.6

5.4

8.5

12.0

8.4

52.6

3.5

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 112. The test fulfils this validity criterion.

Validity criteria fulfilled:
yes
Remarks:
Control cell density increased by a factor > 15, coefficients of variation did not exceed guideline limits
Conclusions:
The acute toxicity (72h-ErL50) of Spearmint oil towards Pseudokirchneriella subcapitata is 65 mg/l.
Executive summary:

The acute toxicity of Spearmint oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l and were observed 72 hours. Based on nominal concentrations the 72h-ErL10 and 72h-ErL50 were found to be 21 and 65 mg/l respectively.

Description of key information

The 72h-ErL10 and 72h-ErL50 of Spearmint oil towards Pseudokirchneriella subcapitata are 21 and 65 mg/l respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
65 mg/L
EC10 or NOEC for freshwater algae:
21 mg/L

Additional information

The acute toxicity of Spearmint oil towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. Algal cells were exposed to nominal WAFs with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/l and were observed 72 hours. Based on nominal concentrations the 72h-ErL10 and 72h-ErL50 were found to be 21 and 65 mg/l respectively