Rectified Hydrocarbons by-products Limonene enriched from synthetic process

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

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.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

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.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

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.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 September 2012 - 14 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and the control were sampled.
- Sampling method: Three replicate samples were taken from each test concentration and one from the control at each analytical occasion. The samples were twofold diluted with acetonitrile before the HPLC analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (OECD medium). This mixture was shaken overnight and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium (i.e. 100 % v/v saturated solution). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain:61.81 SAG
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The preculture was incubated for four days at this test.)

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

the 48 hours are reported as well.

Test temperature:

22.3 – 23.5 °C

pH:

7.51 – 9.57

Nominal and measured concentrations:

Nominal control, 19.8, 29.6, 44.4, 66.7 and 100 % v/v saturated solution
Geometric mean measured: < LOQ (Control), 0.06, 0.07, 0.12, 0.29 and 0.28 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): In order to minimise the evaporation of the test item the test was performed in a closed system (using sealed test vessels).
- Material, size, headspace, fill volume: 200 mL fill volume
- Aeration: no
- Initial cells density:
- Control end cells density: 10**4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: artificial alagl growth medium according to OECD testguideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: start and end of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod: continuous
- Light intensity and quality: 8191 lux, fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber
- Other: Effect parameters NOEC ErC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Justification for using less concentrations than requested by guideline: NA

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: After the exposure period of 72 hours the average specific growth rates were statistically significantly different from that of the control group at the concentrations of 0.12 and 0.28 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The 72-h ErC50 was determined to be higher than 0.29 mg/L (the highest achieved concentration at 72 h). The theoretical value calculated by a Probit analysis was 1.75 mg/L with a 95 % confidence limits of 0.78 – 15.20 mg/L.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: The shape of the algal cells growing was obviously affected by the test item in the concentration range 0.12 – 0.28 mg/L after 72 hours of exposure. Pinches on the cells were observed at this concentration range. In addition, thin cells were noticed at the concentrations of 0.29 and 0.28 mg/L. .
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, potentially due to volatility
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid?
- ErC50: 1.36 mg/L, (95 % confidence limits: 1.25 – 1.49 mg/L)
- Other: The date of the last study with the reference item Potassium dichromate was: 03 – 06 September 2012.

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test (alpha = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test.
For determination of the 48-h ErC50 and EyC50 values with 95 % confidence limits, Probit analysis was performed using TOXSTAT software and for the 72-h ErC50 and EyC50 values with 95 % confidence limits, Probit analysis was performed using SPSS PC+ software.

Camphene concentrations measured during the study

Nominal concentration mg/L	Mean of the measured concentrations (mg/L) with the 95% confidence intervals  (n=3)
	September 11, 2012	September 12, 2012	September 13, 2012	September 14, 2012
19.8	0.81±0.42	0.044±0.038	0.049±0.043	b.q.l.*
29.6	1.19±0.08	0.050±0.069	0.047±0.017	b.q.l.*
44.4	1.79±0.08	0.14±0.10	0.080±0.032	b.q.l.*
66.7	3.60±0.13	0.28±0.32	0.15±0.15	0.046±0.087
100.0	5.18±0.83	0.37±0.56	0.13±0.23	0.026±0.060

*    below the quantification limit; LOQ = 0.02 mg/L

 

Because of the rapid decline of the test item concentrations during the test period (the three lowest concentrations were below the analytical quantification limit (< LOQ) at 72 h) the test item concentrations were calculated for 48-h and 72-h exposure and the endpoints of the study (NOEC, LOEC and EC values) were determined for 48-h as well as for 72-h exposure.

After the exposure period of 48 hours the average specific growth rates were statistically significantly different from that of the control group at the concentrations of 0.53 and 0.63 mg/L and after 72 hours in the concentration range of 0.12 – 0.28 mg/L. Accordingly, the 48-h NOEC related to growth rates was determined to be 0.27 mg/L and the 72-h NOEC as 0.07mg/L.

After the exposure period of 48 hours the yield was statistically significantly different from that of the control group at the concentrations of 0.53 and 0.63 mg/L and after 72 hours in the concentration range of 0.07 – 0.28 mg/L. Accordingly, the 48-h NOEC related to yield was determined to be 0.27 mg/L and the 72-h NOEC as 0.06mg/L.

 

For determination of the 48-h E_rC₅₀and E_yC₅₀values with 95 % confidence limits, Probit analysis was performed using TOXSTAT software and for the 72-h E_rC₅₀and E_yC₅₀values with 95 % confidence limits, Probit analysis was performed using SPSS PC+ software.

 

The 48-h E_rC₅₀was determined to be higher than 0.63 mg/L (the highest achieved concentration at 48 h). The theoretical value calculated by a Probit analysis was 3.51 mg/L with a 95 % confidence limits of 1.20 – 10.36 mg/L.

The 48-h E_yC₅₀was determined to be higher than 0.63 mg/L (the highest achieved concentration at 48 h). The theoretical value calculated by a Probit analysis was 0.91 mg/L with a 95 % confidence limits of 0.66 – 1.25 mg/L.

The 72-h E_rC₅₀was determined to be higher than 0.29 mg/L (the highest achieved concentration at 72 h). The theoretical value calculated by a Probit analysis was 1.75 mg/L with a 95 % confidence limits of 0.78 – 15.20 mg/L.

The 72-h E_yC₅₀vas determined as 0.23 mg/L with a 95 % confidence limits of 0.19 – 0.30 mg/L.

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 < 35%)

Conclusions:

The 72h-ErC50 of camphene to green algae Pseudokirchneriella subcapitata was calculated to be 1.75 mg/L.

Executive summary:

In the GLP compliant Klimisch 1 study from Ágh (2012), the toxicity of Camphene, technical grade, solid to Pseudokirchneriella subcapitata was determined according to OECD 201, EU method C.3 and the US EPA OPPTS 850.5400 in a static test. The test item has a purity of 82.6 % by weight and contains 12.4 % Tricyclene as impurity. A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (OECD medium). This mixture was shaken overnight and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium. This stock solution was further diluted with dilution water to result in following dilutions: 19.8, 29.6, 44.4, 66.7 and 100 % v/v saturated solution.

The concentration of Camphene was experimentally determined and following geometric mean measured concentrations were obtained: < LOQ (Control), 0.06, 0.07, 0.12, 0.29 and 0.28 mg/L. After 72 hours, 3.3, 5.5, 13.7, 17.1 and 19.4% inhibition of the growth rate relative to the control was determined at mean measured concentrations of 0.06, 0.07, 0.12, 0.29 and 0.28 mg/L. The NOEC for growth rate was determined to be 0.07 mg/L. The ErC50 was extrapolated to 1.75 mg/L (the highest mean measured concentration was 0.29 mg/L).

Overall the inhibition of the growth rate was with < 20% low and therefore extrapolation to ErC50 values is problematic. However, since several measures (sealing of test vessels, preconditioning of test vessels) were taken to avoid a loss of test material, the obtained test concentrations must be considered as the maximum of concentration which could be obtained under the conditions of the algal test. Therefore the results from the extrapolation are considered relevant.

It was tried to minimize the loss of test item by sealing the test vessels and b preconditioning the test vessels. However, the loss of test material was significant already during the first 24 hours. Calculation of the 48 hours effect concentrations (NOEC, ErC50) resulted in less conservative values and hence the 72 hour values were reported.

The results of this study are considered relevant and reliable for the risk assessment.

Description of key information

Weight of evidence: Data on short-term toxicity to aquatic algae of the main constituents are available from experimental data:

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.

Camphene: Test method according to OECD TG 201 (CABB, 2012). The 72h-ErC50 to green algae Pseudokirchneriella subcapitata was determined to be 1.75 mg/L.

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.

Based on the available data on its components, a weight of evidence approach is applied and the 72h-EC50 of the substance in green algae is calculated to be 1.62 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.62 mg/L

Additional information

Weight of evidence from experimental results with individual components:

D-limonene: 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.

Camphene:In the GLP compliant Klimisch 1 study from Ágh (2012), the toxicity of Camphene, technical grade, solid to Pseudokirchneriella subcapitata was determined according to OECD 201, EU method C.3 and the US EPA OPPTS 850.5400 in a static test. The test item has a purity of 82.6 % by weight and contains 12.4 % Tricyclene as impurity. A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (OECD medium). This mixture was shaken overnight and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium. This stock solution was further diluted with dilution water to result in following dilutions: 19.8, 29.6, 44.4, 66.7 and 100 % v/v saturated solution.

The concentration of Camphene was experimentally determined and following geometric mean measured concentrations were obtained: < LOQ (Control), 0.06, 0.07, 0.12, 0.29 and 0.28 mg/L. After 72 hours, 3.3, 5.5, 13.7, 17.1 and 19.4% inhibition of the growth rate relative to the control was determined at mean measured concentrations of 0.06, 0.07, 0.12, 0.29 and 0.28 mg/L. The NOEC for growth rate was determined to be 0.07 mg/L. The ErC50 was extrapolated to 1.75 mg/L (the highest mean measured concentration was 0.29 mg/L).

Overall the inhibition of the growth rate was with < 20% low and therefore extrapolation to ErC50 values is problematic. However, since several measures (sealing of test vessels, preconditioning of test vessels) were taken to avoid a loss of test material, the obtained test concentrations must be considered as the maximum of concentration which could be obtained under the conditions of the algal test. Therefore the results from the extrapolation are considered relevant.

It was tried to minimize the loss of test item by sealing the test vessels and b preconditioning the test vessels. However, the loss of test material was significant already during the first 24 hours. Calculation of the 48 hours effect concentrations (NOEC, ErC50) resulted in less conservative values and hence the 72 hour values were reported.

Alpha terpinene: 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.

D-alpha pinene: 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.

Conclusion on test substance: Based on the available data on its components, a weight of evidence approach is applied and the 72h-EC50 of the substance in green algae is calculated to be 1.62 mg/L.