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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 December 2018 – 02 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.55, 1.11, 2.3, 4.8, 10.1, 21.3 and 44.9 mg/L.
- Sampling method: During Range finding study and main study, TOC was analyzed at 0 hour (Day 0) and at 72 hour (Day 3) from samples of all test nominal loading rates.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is a complex mixture (UVCB substance) which was found to be partially miscible in test medium (OECD Alga Medium) according to a lab internal method. Therefore, the Algae Test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in test medium.
In order to determine the optimum mixing duration to be used for WAF preparation, a water accommodated fraction (WAF) equilibration trail was performed prior to exposure: One individual WAF was prepared at each of three loading levels 1.0, 50.0 and 100 mg/L. At 2, 4, 6 and 24 hours after initiation of mixing, mixing was stopped at respective time points and the solutions were allowed to settle for one hour. A sample of WAF was removed from each loading level and mixing was resumed. The concentration of organic carbon that had solubilized into the WAF from the test item was measured using the TOC analyzer described above.
The test item equilibrium was attained at 24 hour mixing according to the equilibration trail, hence mixing for 24 hours was followed during the test media preparation.
Test media preparation: For every test item concentration, individual WAF was prepared by adding the appropriate amount of test item to the test medium. Each mixing vessel was filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers. The mixture was stirred for 24 h (based on equilibration trail) using a magnetic stirrer at minimum of 100 rpm. The mixtures were allowed to settle and equilibrate to test temperature (before removing the test solution). The aqueous portions of WAFs were collected using steri-pipette and transferred to test vessels.
- Controls: OECD Alga Medium only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test substance was not fully soluble at the tested loading rate. However the method of preparing the WAFs was designed to mimimise exposure to undissolved test material.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection.
- Method of cultivation: Alga stock culture (maintained in OECD alga medium) is periodically subcultured at least once in a week and maintained with the illumination and temperature of 4440 to 8880 lux and 21 to 24°C, respectively. From healthy axenic culture of alga, pre-culture was prepared of desired cell density to provide the inoculum for test cultures.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same conditions as test. The algal cell density of pre-cultures (acclimatation period) for range finding study and main study was respectively 40650 and 43050 cells/mL at start.
- Any deformed or abnormal cells observed: Not reported.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
22.1ºC - 22.9ºC
pH:
8.00 - 8.16
Nominal and measured concentrations:
Nominal loading rates: 0 (control), 0.55, 1.11, 2.3, 4.8, 10.1, 21.3 and 44.9 mg/L WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Not specified.
- Initial cells density: 5858 cells/mL
- Control end cells density: 950684 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature and light intensity was monitored once in a day during pre-culture and exposure. The pH of the test nominal loading rates was measured at the beginning (0 h) and at the end (72 h) of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 5218 to 5826 lux was maintained with a universal white type fluorescent lamp

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: counting chamber (observation times: 24, 48 and 72 hours): Samples collected were loaded on a haemocytometer and cells were counted manually using microscope. Cell density was obtained by calculating the average cell count (by summating the number of cells in all the four large corner squares and divide the sum by 4) and multiplied with chamber conversion factor (CCF) and dilution factor: Cell Density (cells/mL) = Average cell count × 10000 (CCF) × Dilution factor.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.1
- Justification for using less concentrations than requested by guideline: Not applicable; 7 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L, the inhibition growth rate was calculated to be 1.1, 2.8, 5.2, 35.2, 100 and 100% respectively.
Reference substance (positive control):
yes
Remarks:
(3, 5-Dichlorophenol )
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
26.61 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = [23.69 – 35.89]
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
4.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = [2.70 – 7.22]
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
5.93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval = [5.20 – 6.75]
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
1.79 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval = [1.48 – 2.16]
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1.11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.

Results with reference substance (positive control):
- Results with reference substance valid? Yes, the 72 hours growth rate inhibition and percent inhibition in yield lie within the validity criteria acceptance range and establish the acceptability of test system response and test procedures followed.
- ErC50-72 h=1.60 mg/L and EyC50-72 h=1.28 mg/L.


Reported statistics and error estimates:
The 72 hours EL10 and EL50 values were calculated from the responses (growth rate and yield) data against log nominal loading rates using the Probit analysis method (Finney, 1971). NOELR and LOELR were determined by SPSS statistical procedure (t-test) for multiple sample comparison using individual replicate values of the specific growth rate.

Table 1: Mean cell density after exposure during range finding study

Group

Nominal Loading Rate
 (mg/L)

R

Mean Cell Density (104Cells/mL)

Observation Periods

24 h

48 h

72 h

G1

0.0

R1

4.13

35.20

92.95

R2

3.30

32.73

95.43

R3

3.58

29.98

97.90

G2

0.00897

R1

2.75

33.83

89.10

R2

1.65

34.38

91.58

G3

0.0895

R1

2.48

33.28

84.15

R2

2.48

34.10

81.13

G4

0.897

R1

3.58

26.13

75.63

R2

1.93

25.85

70.68

G5

9.1

R1

1.65

13.20

14.85

R2

2.20

12.93

16.78

G6

44.9

R1

0.55

0.28

0.28

R2

0.55

0.28

0.28

G7

89.7

R1

0.28

0.00

0.00

R2

0.28

0.00

0.00

Table 2. Percent inhibition in cell density, growth rate and yield during range finding study

Group

Nominal Loading Rate
(mg/L)

72h % Inhibition

 in Cell Density

72h % Inhibition

in Growth Rate

72h % Inhibition
in Yield

G1

0

-

-

-

G2

0.00897

5.3

1.1

5.4

G3

0.0895

13.4

2.8

13.5

G4

0.897

23.3

5.2

23.5

G5

9.1

83.4

35.2

83.9

G6

44.9

99.7

100.0

100.0

G7

89.7

100.0

100.0

100.0

Table 3. Mean cell density after exposure during main study

Group

Nominal Loading Rate
(mg/L)

R

Mean Cell Density (104Cells/mL)

Observation Periods

24 h

48 h

72 h

G1

0.0

1

4.95

30.80

95.98

2

6.33

32.73

94.60

3

6.05

35.20

97.35

4

4.95

31.08

98.18

5

5.50

33.83

97.63

6

5.78

31.63

90.20

G2

0.55

1

5.23

30.53

96.25

2

5.50

34.65

92.68

3

5.23

34.10

97.08

G3

1.11

1

4.40

30.25

88.83

2

4.68

31.90

90.48

3

3.58

29.43

89.10

G4

2.3

1

3.85

25.85

78.93

2

3.03

27.50

75.90

3

3.58

25.85

80.30

G5

4.8

1

2.75

21.73

56.65

2

3.03

25.03

52.53

3

2.48

23.38

52.80

G6

10.1

1

2.48

19.25

32.18

2

1.65

18.98

33.83

3

2.20

18.70

29.43

G7

21.3

1

1.38

14.85

18.70

2

1.93

15.95

20.90

3

1.38

14.03

19.80

G8

44.9

1

0.83

0.83

1.38

2

0.55

0.55

0.83

3

0.28

0.55

1.10

Table 4. Average specific growth rate during main study

Group

Nominal Loading Rates
(mg/L)

R

Growth Rate

Mean Growth
Rate

Observation Intervals (Days)

0 to 1

1 to 2

2 to 3

0 to 3

G1

0

1

2.1

1.83

1.14

1.70

1.70

2

2.4

1.64

1.06

1.69

3

2.3

1.76

1.02

1.70

4

2.1

1.84

1.15

1.71

5

2.2

1.82

1.06

1.71

6

2.3

1.70

1.05

1.68

Mean

2.25

1.76

1.08

1.70

±SD

0.10

0.08

0.05

0.01

CV

4.54

4.44

4.88

0.62

G2

0.55

1

2.2

1.77

1.15

1.70

1.70

2

2.2

1.84

0.98

1.69

3

2.2

1.88

1.05

1.70

Mean

2.21

1.83

1.06

1.70

±SD

0.03

0.06

0.08

0.01

CV

1.34

3.09

7.84

0.48

G3

1.11

1

2.0

1.93

1.08

1.67

1.68

2

2.1

1.92

1.04

1.68

3

1.8

2.11

1.11

1.67

Mean

1.97

1.99

1.08

1.68

±SD

0.14

0.11

0.03

0.00

CV

7.15

5.35

3.04

0.2

G4

2.3

1

1.9

1.90

1.12

1.63

*1.63

2

1.6

2.21

1.02

1.62

3

1.8

1.98

1.13

1.64

Mean

1.78

2.03

1.1

1.63

±SD

0.12

0.16

0.06

0.01

CV

6.95

7.78

5.87

0.59

G5

4.8

1

1.5

2.07

0.96

1.52

*1.51

2

1.6

2.11

0.74

1.50

3

1.4

2.25

0.81

1.50

Mean

1.54

2.14

0.84

1.51

±SD

0.10

0.09

0.11

0.01

CV

6.51

4.33

13.17

0.93

G6

10.1

1

1.4

2.05

0.51

1.34

*1.33

2

1.0

2.44

0.58

1.35

3

1.3

2.14

0.45

1.31

Mean

1.27

2.21

0.52

1.33

±SD

0.21

0.20

0.06

0.02

CV

16.47

9.27

12.12

1.77

G7

21.3

1

0.9

2.38

0.23

1.15

*1.17

2

1.2

2.11

0.27

1.19

3

0.9

2.32

0.34

1.17

Mean

0.97

2.27

0.28

1.17

±SD

0.19

0.14

0.06

0.02

CV

20.12

6.14

20.58

1.58

G8

44.9

1

0.3

0.00

0.51

0.28

*0.20

2

-0.1

0.00

0.41

0.11

3

-0.8

0.69

0.69

0.21

Mean

-0.16

0.23

0.54

0.20

±SD

0.56

0.40

0.15

0.09

CV

-349.69

173.21

27.12

42.07

±SD: Standard deviation; CV: Coefficient of Variation; R: Replicate;*: Statistical significance of >0.05

Table 5. Yield of alga during main study

Group

Nominal Loading Rate
(mg/L)

R

Cell density (Cells/mL)

Yield
(72 hours)

0 h

72 h

G1

0

1

5858

959750

953893

2

5858

946000

940143

3

5858

973500

967643

4

5858

981750

975893

5

5858

976250

970393

6

5858

902000

896143

 

Mean

956542

950684

 

±SD

29691

29691

G2

0.55

1

5858

962500

956643

2

5858

926750

920893

3

5858

970750

964893

 

Mean

953333

947476

 

±SD

23388

23388

G3

1.11

1

5858

888250

882393

2

5858

904750

898893

3

5858

891000

885143

 

Mean

894667

888809

 

±SD

8840

8840

G4

2.3

1

5858

789250

783393

2

5858

759000

753143

3

5858

803000

797143

 

Mean

783750

777893

 

±SD

22510

22510

G5

4.8

1

5858

566500

560643

2

5858

525250

519393

3

5858

528000

522143

 

Mean

539917

534059

 

±SD

23063

23063

G6

10.1

1

5858

321750

315893

2

5858

338250

332393

3

5858

294250

288393

 

Mean

318083

312226

 

±SD

22228

22228

G7

21.3

1

5858

187000

181143

2

5858

209000

203143

3

5858

198000

192143

 

Mean

198000

192143

 

±SD

11000

11000

G8

44.9

1

5858

13750

7893

2

5858

8250

2393

3

5858

11000

5143

 

Mean

11000

5143

 

±SD

2750

2750

Table 6. Percent inhibition in cell density, growth rate and yield during main study

Group

Nominal Loading
(mg/L)

72h % Inhibition in
 Cell Density

72h % Inhibition
in Growth Rate

72h % Inhibition
in Yield

G1

0

-

-

-

G2

0.55

0.3

0.1

0.3

G3

1.11

6.5

1.3

6.5

G4

2.3

18.1

3.9

18.2

G5

4.8

43.6

11.2

43.8

G6

10.1

66.7

21.6

67.2

G7

21.3

79.3

30.9

79.8

G8

44.9

98.9

88.1

99.5

Table 7. Acceptance criteria for control during main study

0 - 72 Hours Mean Coefficient of Variation of Section-by-Section Average Specific Growth Rate

Daily average growth rate

SD

% CV out of the daily growth rates

1.70

0.51

30.07

1.69

0.66

38.97

1.70

0.66

38.76

1.71

0.50

29.56

1.71

0.60

35.05

1.68

0.62

36.95

                                                       Mean % of CV 0 - 72 hours

34.89

Biomass increase during the whole test period

Initial biomass (cells/mL)

 Cell density on day 3 (cells/mL)

Biomass increase Factor

5858

959750

163.85

5858

946000

161.50

5858

973500

166.20

5858

981750

167.61

5858

976250

166.67

5858

902000

153.99

Mean

956541.67

163.30

Table 8. TOC analysis during exposure - main study

 

 

TOC During Exposure
(mg)

Group

Nominal Loading rate (mg/L)

Day 0
(Fresh)

Day 3
(Aged)

G1

0.0

-

-

G2

0.55

0.047

0.0452

G3

1.11

0.063

0.058

G4

2.3

0.89

0.73

G5

4.8

1.03

0.98

G6

10.1

3.26

2.98

G7

21.3

16.18

15.23

G8

44.9

26.61

25.89

Table 9. Temperature and light intensity during pre-culture and exposure main study

Day

Temperature of the surrogate

(°C)

Light intensity (Lux)

Revolutions per minute (rpm)

1

2

3

4

0

22.9

5458

5612

5826

5381

100

1

22.6

5612

5813

5718

5638

100

2

22.1

5536

5439

5482

5438

100

3

22.3

5316

5777

5682

5781

100

4

22.4

5439

5459

5538

5328

100

5

22.6

5614

5361

5416

5218

100

6

22.3

5382

5385

5518

5429

100

Mean

22.5

5480

5549

5597

5459

-

±SD

0.3

113.4

186.2

147.2

190.8

-

Min

22.1

5218

Max

22.9

5826

Table 10. pH of the test medium during main study

Group 

Nominal Loading Rate (mg/L)

pH

0 hours

72 hours

R1

R2

R3

R4

R5

R6

R1

R2

R3

R4

R5

R6

G1

0

8.06

8.09

8.02

8.00

8.08

8.09

8.11

8.13

8.16

8.09

8.12

8.14

G2

0.6

8.10

8.08

8.12

-

-

-

8.13

8.12

8.14

-

-

-

G3

1.2

8.11

8.16

8.10

-

-

-

8.14

8.13

8.12

-

-

-

G4

2.6

8.09

8.08

8.11

-

-

-

8.08

8.14

8.13

-

-

-

G5

5.4

8.12

8.10

8.11

-

-

-

8.14

8.12

8.15

-

-

-

G6

11.3

8.10

8.09

8.08

-

-

-

8.11

8.08

8.12

-

-

-

G7

23.8

8.09

8.10

8.13

-

-

-

8.08

8.09

8.16

-

-

-

G8

50.0

8.06

8.11

8.12

 

 

 

8.12

8.14

8.15

 

 

 

Min

8.00

8.08

Max

8.16

8.16

Validity criteria fulfilled:
yes
Remarks:
(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)
Conclusions:
In an Alga Growth Inhibition Test with Pseudokirchneriella subcapitata green algae, the growth rate inhibition effect concentrations were determined to be 72 h-ErL50 = 26.61 mg/L and 72 h-ErL10 = 4.42 mg/L.


Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on test item PINE OIL 50% over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. The test item is a UVCB substance which was found to be partially miscible in OECD Alga Medium which was used as test medium. Therefore, the test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in the test medium. Based on a preliminary WAF equilibration trail, individual WAF solutions for every test item concentration were prepared in closed vessels with minimal headspace and stirred for 24 h. Then, the mixtures were allowed to settle and equilibrate to test temperature and aqueous portions of WAFs were transferred to the test vessels. In a preliminary range finding study the average growth inhibition rates were 1.1, 2.8, 5.2, 35.2, 100 and 100% at the tested nominal loading rates of 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L, respectively. Based on these results, the main test was conducted at nominal loading rates of 0.55, 1.11, 2.3, 4.8, 10.1, 21.3 and 44.9 mg/L along with control group. The algal growth (cell density) was assessed at 24, 48 and 72 hours post-exposure. Exponentially growing alga cells (5858 cells/mL) were exposed to the selected nominal loading rates in 3 replicates for the treatment groups and 6 replicates for the control group. Negative control consisted of test medium alone and a solution of 3, 5-Dichlorophenol was used as reference substance. A growth rate inhibition of 0.1, 1.3, 3.9, 11.2, 21.6, 30.9 and 88.1 % was observed at the nominal loading rates 0.55, 1.11, 2.3, 4.8, 10.1, 21.3 and 44.9 mg/L respectively as compared with the control. Similarly, inhibition in yield of 0.3, 6.5, 18.2, 43.8, 67.2, 79.8 and 99.5% was observed at the nominal loading rates 0.55, 1.11, 2.3, 4.8, 10.1, 21.3 and 44.9 mg/L respectively as compared with the control. All validity criteria were fulfilled. TOC measurements for each nominal loading rate were maintained within ± 20% during the test period. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErL10 = 4.42 mg/L (95% CL: 2.70 – 7.22) and 72 h-ErL50 = 26.61 mg/L (95% CL: 23.69 – 35.89), and the yield inhibition effect concentrations were 72h-EyL10 = 1.79 mg/L (95% CL: 1.48 – 2.16) and 72h-EyL50 = 5.93 mg/L (95% CL: 5.20 – 6.75). Finally, the 72 hours LOELR and NOELR were determined to be 2.3 and 1.11 mg/L respectively based on growth rate.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ASTM methods (ASTM, 1988)
Version / remarks:
The inhibition concentration (IC50), the concentration at which there was a 50 percent growth inhibition was calculated using a linear interpolation program (Marcus and Holtzman, 1988; Norberg-King, 1988).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 494, 988, 1975 and 3950 μg/L
- Sampling time: 0, 24, and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Seventy-five milliliters of test solution were placed in 125 ml volumetric flasks. This was done to minimize headspace. Dilution water was deionized then filtered through a 0.22 micron filter. After this procedure, micro and macronutrients were added to the dilution water.
Stock solutions were also supplied with the necessary nutrients to make concentrations compatible to the dilution water. Five concentrations of stock were used: 100%, 50%, 25%, 12.5% and 0%.
- Test cell concentrations were approximately 1 x10^4 cells/ml. A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume
- Controls: Yes, dilution water
- Chemical name of vehicle: water
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
ACCLIMATION
No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0, 494, 988, 1975 and 3950 μg/L
Measured concentrations: No available
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 ml volumetric flasks
- Material, size, headspace, fill volume: glass, 125 ml, 75 milliliters of test solution were placed in the flask to minimize headspace.
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
According to ASTM.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised.
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 96 hrs (the 96 hr sample was done only if the chemical was present at the 24 hr sampling)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
- Test concentrations: 0, 494, 988, 1975 and 3950 μg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 3 950 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth inhibition
Remarks on result:
other: No significant inhibition observed up to the highest concentration tested
Results with reference substance (positive control):
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The 96h-EC50 of alpha terpinene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 3950 μg/L based on growth inhibition.
Executive summary:

A toxicity test was conducted on alpha terpinene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 494, 988, 1975 and 3950 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 3950 μg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ASTM methods (ASTM, 1988)
Version / remarks:
The inhibition concentration (IC50), the concentration at which there was a 50 percent growth inhibition was calculated using a linear interpolation program (Marcus and Holtzman, 1988; Norberg-King, 1988).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 88, 175, 350 and 700 μg/L
- Sampling time: 0, 24, and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Seventy-five milliliters of test solution were placed in 125 ml volumetric flasks. This was done to minimize headspace. Dilution water was deionized then filtered through a 0.22 micron filter. After this procedure, micro and macronutrients were added to the dilution water.
Stock solutions were also supplied with the necessary nutrients to make concentrations compatible to the dilution water. Five concentrations of stock were used: 100%, 50%, 25%, 12.5% and 0%.
- Test cell concentrations were approximately 1 x10^4 cells/ml. A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume
- Controls: Yes, dilution water
- Chemical name of vehicle: water
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
ACCLIMATION
No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0, 88, 175, 350 and 700 μg/L
Measured concentrations: No available
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 ml volumetric flasks
- Material, size, headspace, fill volume: glass, 125 ml, 75 milliliters of test solution were placed in the flask to minimize headspace.
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
According to ASTM.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised.
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 96 hrs (the 96 hr sample was done only if the chemical was present at the 24 hr sampling)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
- Test concentrations: 0, 88, 175, 350 and 700 μg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 700 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth inhibition
Remarks on result:
other: No significant inhibition observed up to the highest concentration tested
Results with reference substance (positive control):
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The 96h-EC50 of d-alpha pinene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 700 μg/L based on growth inhibition.
Executive summary:

A toxicity test was conducted on d-alpha pinene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 88, 175, 350 and 700 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 700 μg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ASTM methods (ASTM, 1988)
Version / remarks:
The inhibition concentration (IC50), the concentration at which there was a 50 percent growth inhibition was calculated using a linear interpolation program (Marcus and Holtzman, 1988; Norberg-King, 1988).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 188, 375, 750 and 1500 μg/L
- Sampling time: 0, 24, and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Seventy-five milliliters of test solution were placed in 125 ml volumetric flasks. This was done to minimize headspace. Dilution water was deionized then filtered through a 0.22 micron filter. After this procedure, micro and macronutrients were added to the dilution water.
Stock solutions were also supplied with the necessary nutrients to make concentrations compatible to the dilution water. Five concentrations of stock were used: 100%, 50%, 25%, 12.5% and 0%.
- Test cell concentrations were approximately 1 x10^4 cells/ml. A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume
- Controls: Yes, dilution water
- Chemical name of vehicle: water
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
ACCLIMATION
No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0, 188, 375, 750 and 1500 μg/L
Measured concentrations: No available
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 ml volumetric flasks
- Material, size, headspace, fill volume: glass, 125 ml, 75 milliliters of test solution were placed in the flask to minimize headspace.
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
According to ASTM.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised.
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 96 hrs (the 96 hr sample was done only if the chemical was present at the 24 hr sampling)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
- Test concentrations: 0, 188, 375, 750 and 1500 μg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 500 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth inhibition
Remarks on result:
other: No significant inhibition observed up to the highest concentration tested
Results with reference substance (positive control):
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The 96h-EC50 of d-limonene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 1500 μg/L based on growth inhibition.
Executive summary:

A toxicity test was conducted on d-limonene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 188, 375, 750 and 1500 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 1500 μg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: ASTM methods (ASTM, 1988)
Version / remarks:
The inhibition concentration (IC50), the concentration at which there was a 50 percent growth inhibition was calculated using a linear interpolation program (Marcus and Holtzman, 1988; Norberg-King, 1988).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 438, 875, 1750 and 3500 μg/L
- Sampling time: 0, 24, and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Seventy-five milliliters of test solution were placed in 125 ml volumetric flasks. This was done to minimize headspace. Dilution water was deionized then filtered through a 0.22 micron filter. After this procedure, micro and macronutrients were added to the dilution water.
Stock solutions were also supplied with the necessary nutrients to make concentrations compatible to the dilution water. Five concentrations of stock were used: 100%, 50%, 25%, 12.5% and 0%.
- Test cell concentrations were approximately 1 x10^4 cells/ml. A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume
- Controls: Yes, dilution water
- Chemical name of vehicle: water
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
ACCLIMATION
No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0, 438, 875, 1750 and 3500 μg/L
Measured concentrations: No available
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 ml volumetric flasks
- Material, size, headspace, fill volume: glass, 125 ml, 75 milliliters of test solution were placed in the flask to minimize headspace.
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
According to ASTM.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised.
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 96 hrs (the 96 hr sample was done only if the chemical was present at the 24 hr sampling)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: A coulter counter, an electronic particle counter, was used to count cells and determine a mean cell volume

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
- Test concentrations: 0, 438, 875, 1750 and 3380 μg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 3 500 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth inhibition
Remarks on result:
other: No significant inhibition observed up to the highest concentration tested
Results with reference substance (positive control):
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The 96h-EC50 of terpinolene to green algae Pseudokirchneriella subcapitata was calculated to be higher than 3500 μg/L based on growth inhibition.
Executive summary:

A toxicity test was conducted on terpinolene using green algae (Selenastrum capricornutum) according to ASTM methods for conducting static 96 hour toxicity tests with microalgae (ASTM, 1988). Deionised water was used to formulate the test solutions. The test was performed in nominal concentrations of 0, 438, 875, 1750 and 3500 μg/L. Flasks were set up in replicates of four at each concentration and shaken continuously. GC analysis was performed at 0, 24 and 96 hrs. No significant effects based on growth inhibition were observed at any concentration tested. Thus, the 96h-NOEC and 96h-EC50 were found to be higher than 3500 μg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Study conducted according to OECD guideline 201
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared by mixing a stock solution of the test substance, which was prepared by adding and stirring the substance under the water surface of the Erlenmeyer flask filled with medium
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
22.3-22.6ºC
Nominal and measured concentrations:
Nominal concentrations: 0.0 (control), 5, 16, 50, 160 and 500 mg/L (geometric ratio; square root of 10)
Details on test conditions:
TEST SYSTEM
- Test vessel: rotary shaking culture (ca. 100 rpm)
- Material, size, headspace, fill volume: test solution volume; 600 mL/control (100 mL/vessel), 300 mL/concentration (100 mL/vessel)
- No. of organisms per vessel: 6896 (at 0h)
- No. of vessels per concentration (replicates): 3 (Separately from that, another vessel for analysis at 48 hours was prepared.)
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Photoperiod: Constant illumination
- Light intensity and quality: 98 - 99 microE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Measurement of biomass: cell concentration

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 220-270 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be 250 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP.The test concentrations selected for the study were 0.0 (control), 5, 16, 50, 160 and 500 mg/L (geometric ratio; square root of 10) (nominal). Three replicates were conducted for each concentration of the test item and six replicates for the control group. An analytical method based on gas chromatography was used to monitor the concentration of cineole in the test solution at the start, after 48 h and at the end of exposure. The effect concentration at 50% inhibition of growth rate was calculated to be 72h-ErC50 = 250 (95% CL: 220 – 270) mg/L. Also, the NOEC (72h) for growth rate was determined to be 16 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Study conducted according to OECD guideline 201
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: analyses of the test substance were performed at beginning and at end of exposure time.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Method of cultivation: Shaking culture method (100 rpm)
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 2ºC
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L
Measured concentrations: Since the measured concentrations of the test substance exceeded the nominal concentrations by ± 20%, the results were calculated from the measured concentrations.
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 mL/vessel
- Initial cells density: 1×104 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

OTHER TEST CONDITIONS
- Photoperiod: Constant illumination
- Light intensity and quality: 4000-5000 lx in the vicinity of the liquid surface of the flask
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 5.6-8.2 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 5.1-5.8 mg/L)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (95% CL: 3.1-4.2 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
EbC50, ErC50 and 95% CL were calculated by linear regression analysis method.
NOEC: Dunnett's multiple comparison test
Validity criteria fulfilled:
not specified
Conclusions:
In the Pseudokirchneriella subpicata green algae toxicity test the 72h-ErC50 of the test substance was determined to be 6.7 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata green algae on the test substance over a period of 72 h according to OECD Guideline 201, following GLP. The test concentrations selected for the study were 0.0 (control), 1.0, 2.2, 4.6, 10, 22, 46 and 100 mg/L (nominal). Three replicates were conducted for each concentration of the test item and 1replicate for the control group. An analytical method based on gas chromatography mass spectrometry was used to monitor the concentration of p-cymene in the test solution at the start and at the end of exposure. The effect concentration at 50% inhibition of growth rate was calculated to be 72h-ErC50 = 6.7 (95% CL: 5.6 – 8.2) mg/L. Also, the NOEC (72h) for growth rate was determined to be 2.7 mg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations in Pseudokirchneriella subcapitata green algae were determined to be 72 h-ErL50 = 26.61 mg/L (95% CL: 23.69 – 35.89) and 72 h-ErL10 = 4.42 mg/L (95% CL: 2.70 – 7.22).

Key value for chemical safety assessment

EC50 for freshwater algae:
26.61 mg/L
EC10 or NOEC for freshwater algae:
4.42 mg/L

Additional information

Supporting studies with individual components: Data on short-term toxicity to aquatic algae of the main constituents are available from experimental data:

Terpinolene: Test method according to ASTM methods (similar to OECD Guideline 201) (Broderius S, 1990). The 96h-EC50 to green algae Pseudokirchneriella subcapitata was determined to be higher than 3.50 mg/L based on growth inhibition.

Cineole: Test method according to OECD TG 201 (NITE, 2010). The 72h-ErC50 to green algae Pseudokirchneriella subcapitata was determined to be 250 mg/L.

D-Limonene: Test method according to ASTM methods (similar to OECD Guideline 201) (Broderius S, 1990). The 96h-EC50 to green algae Pseudokirchneriella subcapitata was determined to be higher than 1.50 mg/L based on growth inhibition.

Alpha terpinene: Test method according to ASTM methods (similar to OECD Guideline 201) (Broderius S, 1990). The 96h-EC50 to green algae Pseudokirchneriella subcapitata was determined to be higher than 3.95 mg/L based on growth inhibition.

D-alpha pinene: Test method according to ASTM methods (similar to OECD Guideline 201) (Broderius S, 1990). The 96h-EC50 to green algae Pseudokirchneriella subcapitata was determined to be higher than 0.7 mg/L based on growth inhibition.

p-cymene: Test method according to OECD TG 201 (MITI, 1998). The 72h-ErC50 to green algae Pseudokirchneriella subcapitata was determined to be 6.7 mg/L.