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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:
supporting study
Study period:
14/03/2011 - 26/03/2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP. Validity criteria met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The oil is poorly soluble in water. It was tested as a Water Accomodated Fraction (OECD Series on Testing and Assessment: Ecotoxicity Testing No 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
The oil is poorly soluble in water. It was tested as a Water Accomodated Fraction (OECD Series on Testing and Assessment: Ecotoxicity Testing No 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)
Principles of method if other than guideline:
Instead of true solutions of the test substance, Water Accomodated Fractions were prepared and tested for their toxicity.
GLP compliance:
yes (incl. QA statement)
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 loading rates of 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: Samples were taken from the control (replicates R1 - R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- All samples taken for analysis were done so in inert airtight glassware with minimal headspace and analysed immediately.
- Duplicate samples were taken at each occasion and stored at -20 degrees Celsius.
- Given the volatile nature of the test item an additional test replicate was prepared at each test concentration and incubated alongside the test remaining unopened until the end of the test. A sample of each of these analytical test replicates was taken for chemical analysis at 72 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 74, 23, 74 and 230 mg) were each separately added to the surface of 23, 23, 2.3, 2.3 and 2.3 litres of culture medium in stirring vessels with minimal headspace to give the 1.0, 3.2, 10, 32 and 100 mg/I loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm* was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing* was inserted into the glass tube and pushed through the Nescofilm seal. Both materials were considered to sufficiently inert as not to affect the dissolved limonene concentration obtained following siphoning of the WAF. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. An aliquot (2 litres) of each of the loading rate WAFs was separately inoculated with algal suspension (27 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/I loading rate WAF.
- Differential loading: test solutions at different loading rates were prepared separately, no dilution was used.
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture
Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.


ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 -150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 -10^5 cells/ml.
- Culturing media and conditions (same as test or not): No. Additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use to counteract the pH-increase due to algal growth in an enclosed system.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not relevant
Hardness:
No data
Test temperature:
24 +/- 1 degrees Celsius
pH:
At day 0: 7.8 - 8.1
After 72 h: 7.9 - 8.0 in 10, 32 and 100 mg/l
After 72h: 9.7 in control, 1 and 3.2 mg/l
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal loading rates: 1.0, 3.2, 10, 32 and 100 mg/l
Measured (t=0h, limonene): 0.0510, 0.143, 0.820, 0.970 and 0.919 mg/l
Measured (t=72h, limonene): 0.0276, 0.0493, 0.428, 0.582 and 0.472 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 ml filled with 250 ml medium
- Aeration: No
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 5.52 x 10^5 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Substance Concentration
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2H PO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3QO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/i
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/I
CuCI2.2H2O 0.000012 mg/I
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/I) was added to the prepared culture medium prior to use.

- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was recorded daily, pH was measured after 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: warm white lighting (380-730 nm) at 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer Particle Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as WAF
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 8.8 - 14 mg/l
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as WAF
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/I loading rate WAFs, however no intact cells were observed to be present in the test cultures at 100 mg/l loading rate WAF.
- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAFs. At both the start and end of the mixing period and following the 1-Hour settlement period all loading rate WAFs were observed to have formed clear colourless media columns with an oily slick of test item floating at the media surface. Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present. At the start of the test all control and test cultures were observed to be clear colourless
solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/I loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mg/I loading rate WAF test preparations were observed to be clear colourless solutions.



Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 0-72h-ECr50: 1.2 mg/l
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

For detailed results, see attached file "results.doc".

Results based on measured concentrations (limonene):

72h-NOEC 0.085 mg/l

72h-EC50 0.44 mg/l (95% CL: 0.39 - 0.51)

In this test the concentration of limonene was measured. As the tested oil is a complex test substance that contains a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.

Validity criteria fulfilled:
yes
Remarks:
Exponential growth in controls, mean coefficient of variation for section by section specific growth rate < 35%, coefficient of variation for average specific growth rate <7%
Conclusions:
The toxicity (72h-ErL50) of Lime Oil Cold-Pressed 1-Fold, Lime (Citrus aurantifolia), ext. towards Pseudokirchneriella subcapitata is 11 mg/l. The 72h-ErL10 is 2.2 mg/l, whereas the 72h-NOEL is 3.2 mg/l.
Executive summary:

The toxicity of Lime Oil Cold-Pressed 1-Fold, Lime (Citrus aurantifolia), ext. towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Algae were exposed to nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l of the test substance and observed for 72 hours. Based on growth rate the 72h-ErL50 was 11 mg/l, the 72h-ErL10 was 2.2 mg/l, whereas the 72h-NOErL was 3.2 mg/l.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14/03/2011 - 02/04/2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP. Validity criteria met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The oil is poorly soluble in water. It was tested as a Water Accomodated Fraction (OECD Series on Testing and Assessment: Ecotoxicity Testing No 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
The oil is poorly soluble in water. It was tested as a Water Accomodated Fraction (OECD Series on Testing and Assessment: Ecotoxicity Testing No 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)
Principles of method if other than guideline:
Instead of true solutions of the test substance, Water Accomodated Fractions were prepared and tested for their toxicity.
GLP compliance:
yes (incl. QA statement)
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 loading rates of 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: Samples were taken from the control (replicates R1 - R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- All samples taken for analysis were done so in inert airtight glassware with minimal headspace and analysed immediately.
- Duplicate samples were taken at each occasion and stored at -20 degrees Celsius.
- Given the volatile nature of the test item an additional test replicate was prepared at each test concentration and incubated alongside the test remaining unopened until the end of the test. A sample of each of these analytical test replicates was taken for chemical analysis at 72 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 74, 23, 74 and 230 mg) were each separately added to the surface of 23, 23, 2.3, 2.3 and 2.3 litres of culture medium in stirring vessels with minimal headspace to give the 1.0, 3.2, 10, 32 and 100 mg/I loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex (depth ~0.2 cm) was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Both materials were considered to be sufficiently inert so as not to affect the dissolved limonene concentration obtained following siphoning of the WAF. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. An aliquot (2 litres) of each of the loading rate WAFs was separately inoculated with algal suspension (27 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/I loading rate WAF.
- Differential loading: test solutions at different loading rates were prepared separately, no dilution was used.
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture
Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/ml. The flasks were plugged with polyurethane foam stoppers
and kept under constant agitation by orbital shaker (100 -150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 -10^5 cells/ml.
- Culturing media and conditions (same as test or not): No. Additional sodium bicarbonate (500 mg/I) was added to the prepared culture medium prior to use to counteract the pH-increase due to algal growth in an enclosed system.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not relevant
Hardness:
No data
Test temperature:
24 +/- 1 degrees Celsius
pH:
At day 0: pH 7.6 - 8.0
After 72h: in 10, 32 and 100 mg/l: pH 8.0
in Control, 1 and 3.2 mg/l: pH 9.6 - 9.8
Dissolved oxygen:
Not relevant
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal loading rates: 1.0, 3.2, 10, 32 and 100 mg/l
Measured (t=0h, limonene): 0.0286, 0.0699, 0.659, 0.815 and 1.21 mg/l
Measured (t=72h, limonene): 0.0199, 0.0309, 0.436, 0.571 and 0.442 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 ml filled with 250 ml medium
- Aeration: No
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 5.52 x 10^5 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Substance Concentration
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2H PO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3QO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/i
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/I
CuCI2.2H2O 0.000012 mg/I
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/I) was added to the prepared culture medium prior to use.

- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was recorded daily, pH was measured after 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: warm white lighting (380-730 nm) at 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer Particle Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as a WAF
Basis for effect:
growth rate
Remarks on result:
other: 95%CL: 7.3 - 8.8 mg/l
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
5.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as a WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tested as a WAF
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/l loading rate WAF, however few intact cells were observed to be present in the test cultures at 32 and 100 mg/l loading rate WAF.
- Effect concentrations exceeding solubility of substance in test medium: Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of the mixing period all loading rate WAFs were observed to have formed clear colourless media columns with an oily slick of test item floating at the media surface. After both the 23-Hour stirring and 1-Hour standing periods the 1.0 and 3.2 mg/l loading rate WAFs were observed to have formed clear colourless media columns with an oily slick of test item floating at the media surface whilst the 10, 32 and 100 mg/l
loading rate WAFs were observed to have formed clear colourless media columns with an oily slick and white flakes of test item floating at the media surface. Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0 and 3.2 mg/I loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mg/I loading rate WAF test preparations were observed to be clear colourless solutions.





Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 0-72h-ECr50: 1.2 mg/l
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

For detailed results, see attached file "results.doc".

Results based on measured concentrations (limonene):

48h-NOEC 0.046 mg/l

48h-EC50 0.4 mg/l (95% CL: 0.35 - 0.45)

In this test the concentration of limonene was measured. As the tested oil is a complex test substance that contains a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.

Validity criteria fulfilled:
yes
Remarks:
Exponential growth in controls, mean coefficient of variation for section by section specific growth rate < 35%, coefficient of variation for average specific growth rate <7%
Conclusions:
The toxicity (72h-ErL50) of Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. towards Pseudokirchneriella subcapitata is 8.0 mg/l. The 72h-ErL10 is 5.1 mg/l, whereas the 72h-NOEL is 3.2 mg/l .
Executive summary:

The toxicity of Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. towards Pseudokirchneriella subcapitata was investigated according to OECD guideline 201 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Algae were exposed to nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l of the test substance and observed for 72 hours. Based on growth rate the 72h-ErL50 was 8.0 mg/l, the 72h-ErL10 was 5.1 mg/l, whereas the 72h-NOErL was 3.2 mg/l .

Description of key information

The 72h-EL50 is 8.0 mg/L (Loading rate, WAF study).

Key value for chemical safety assessment

EC50 for freshwater algae:
8 mg/L

Additional information

For Lime oil, two valid WAF studies on the aquatic toxicity are available for algae. The studies are conducted according to OECD 201 and are both validated with reliability 1, see table below. The study with the lowest WAF toxicity value (ErL50) is selected as the key study for lime oil. The other study is used as supporting study.

Summary of Algae studies

Endpoint

Result

Remarks

Reference

72h-EL50

8.0 mg/l Lime oil distilled

OECD 201, GLP, Rel. 1,

KEY study

Harlan 2011

 72h-EL50

 11 mg/l Lime oil CP

OECD 201, GLP, Rel. 1,

supporting study

Harlan 2011

 

Remark: In the above-mentioned tests the concentration of limonene was measured. As the tested oil is a complex test substance that contains a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.