Tangor, Murcote, ext.

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:

supporting study

Study period:

14/03/2011 - 08/04/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to international guidance (OECD Guideline 201) under GLP. Validity criteria met.

Reason / purpose for cross-reference:

read-across: supporting information

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, 3.2 and 100 mgtl 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 as not to affect the dissolved limonene concentration obtained after siphoninh 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, 3.2 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/l loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Differential loading: test solutions at different loading rates were prepared seperately, 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):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 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
- Age of inoculum (at test initiation): cells in log phase
- 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 addded to the prepared culture medium prior to use to counteract the pH-increase due to algal growth in an enclosed medium.

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°C

pH:

At day 0: 7.8 - 8.5
After 72h at 10, 32 and 100 mg/l: 8.0 - 8.1
After 72h at 1 and 3.2 mg/l and control: 9.6 - 9.7

Dissolved oxygen:

Not relevant

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32, 100 mg/L
Measured concentration of Limonene found (mg/L)
0 hours: 0.0794, 0.199, 0.899, 1.43, 2.05 mg/L
72 hours: 0.0445, 0.102, 0.482, 0.776, 1.39 mg/L
72 hours (unopend vessel): 0.0414, 0.108, 0.295, 0.957, 1.75 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml containing 250 ml medium
- Aeration: No
- Initial cells density: 5.14 x 10^3 cells/ml
- Control end cells density: 4.93 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
NaN03 25.5 mg/l
MgCb.6H20 12.164 mg/l
CaCI2.2H20 4.41 mg/l
MgS04.7H20 14.7 mg/l
K2HP04 1.044 mg/l
NaHC03 15.0 mg/l
H3B03 0.1855 mg/l
MnCI2.4H20 0.415 mg/l
ZnCb0.00327 mg.l
FeCb.6H20 0.159 mg/l
CoCI2.6H20 0.00143 mg/l
Na2Mo04.2H20 0.00726 mg/l
CuCI2.2H20 0.000012 mg/l
Na2EDTA.2H20 0.30 mg/l
Na2Se03.5H20 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/l) was added to the prepared culture medium prior to use.
- Culture medium different from test medium: additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium
- 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, pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI
- Photoperiod: continuous illumination
- 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: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a
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

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Tested as WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Tested as WAF

Basis for effect:

growth rate

Key result

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, and 3.2 mg/I loading rate WAF, however cell debris was observed to be present in the test cultures at 10, 32 and 100 mg/l loading rate WAF.
- Colour differences: 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 mgtl loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mgtl loading rate WAF test preparations were observed to be clear colourless solutions.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Due to the volatile nature of the test item, an additional test replicate was prepared for each test concentration at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours. Analysis of these preparations showed measured test concentrations to range from 0.041 to 1.8 mg/l (33% to 85% of the 0-Hour measured test concentrations). Given that a decline in measured concentrations was observed in both the vessels which had remained unopened throughout the test duration and those that had been opened daily for cell density determination it was considered that the decline observed was due to a combination of volatility and adsorption of the test item to the algal cells present.
- 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 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/l loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mg/lloading rate WAF test preparations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the results from the positive control with potassium dichromate were within the normal
ranges for this reference item.
- EC50: 1.2 mg/l

Reported statistics and error estimates:

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (lDBS). ELx values were then determined from the equation for the fitted line. It was not possible to calculate 95% confidence limits for the EL50 values as the data generated did not fit the models available.

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).

Validity criteria fulfilled:

yes

Remarks:

(Exponential growth in controls, mean coefficient of varaition for control <35%, specific growth rate variation coefficient < 7 %)

Conclusions:

The toxicity (72h-ErL50) of Mandarin oil (Citrus Reticulata Blanco) ext. towards Pseudikirchneriella subcapitata is 9.7 mg/l. The 72h-ErL10 is 7.8 mg/l, whereas the 72h-NOELR is 3.2 mg/l.

Executive summary:

The acute toxicity of Mandarin oil (Citrus Reticulata Blanco) ext. towards Pseudikirchneriella 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 (Water Accomodated Fractions (WAFs))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 9.7 mg/l, the 72h-ErL10 was 7.8 mg/l, whereas the 72h-NOErL was 3.2 mg/l.

 

Reference 2

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.

Reason / purpose for cross-reference:

read-across: supporting information

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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

Key result

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

Key result

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

Key result

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).

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.

Reference 3

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.

Reason / purpose for cross-reference:

read-across: supporting information

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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

Key result

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

Key result

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

Key result

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".

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 .

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03/09/2007 - 14/09/2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study according to international guideline (OECD guideline 201) under GLP, validity criteria met. Chemical identity of test substance not reported, but it was confirmed to be a sample representative of the registered substance. Statistical treatment not reported. Corrections made by the reviewer based on reported data .

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not included

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.5, 25, 50, 100 and 200 mg/l
- Sampling method:
The quantification of the test item (fraction of dissolved material of the test item) in the test solutions of the daphnia-test was performed according to the method given in CEC L-33-A-93, DIN 51828-2, as agreed with the Sponsor: The use of the specific peak of the IR at 2930 ± 10 cm' for hydrocarbons is comparable with a common parameter like TOC or COD.

For this purpose, subsequent amounts of the test solutions of the daphnia-test were poured into a separation funnel, 30 g sodium chloride (for each 100 mL of specimen), 1 mL (for each 100 mL of specimen) of I M hydrochloric acid, and 25 mL trichlorotrifluoroethane were added followed by shaking for approximately 1-3 min. After shaking the phases were allowed to separate. The organic phase was passed through sodium sulphate, and the collected organic phase was analysed by IR-spectroscopy using the specific peak at 2930 ± 10 cm-1 for hydrocarbons. The extraction procedure was performed in every case on the same day of sample collection. The organic solvents with the extracts were stored at 4-8°C until the date of analyses, if required.

For quantification of any test item possibly adsorbed to the glass walls of the test vessels within the 72h
of incubation, a proportion of 25 mL trichlorotrifluoroethane was given into the empty test vessels which
then were thoroughly rinsed with the solvent. The solvent was collected in a glass flask. This procedure
was repeated in the same way with all test vessels. The organic phases were unified and then reduced to
a final volume of 5 mL and then analysed by IR-spectroscopy using the specific peak at 2930 ± 10 cm-'
for hydrocarbons.

Samples were atken after 0 and 48 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Initial algal cell concentration of 1.65*10^4 cells/mL (screening test) and 1.61 x10^4 cells/ml (main test) in the test solutions.
The determination of cell concentration was performed by photometrical measurement using a calibration curve prepared by using a spectrophotometer at 578 nm.
- Test substance was dissolved in DCM and brought into vessel. After evaporation of the DCM, water was added. After shaking for 18h at 130 RPM, the solution was filtered through a filter papere and rinsed with ultrapure water. Algal suspensions where then added to obtain algal solution at the appropriate test substance concentration levels.
- Controls: Yes
- Solvent: dichloromethane is used and evaporated

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysologisches Institut der Universitët Göttingen (SAG), Strain-No. 86.81
- Age of inoculum (at test initiation):
- Method of cultivation: under aseptic conditions

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions (same as test or not): not the same as test: This culture was prepared in „pre-culture medium".

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:

23.2 - 24.0 degrees Celsius

pH:

7.17 - 10.00

Dissolved oxygen:

No data

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal (WAF): 12.5, 25, 50, 100 and 200 mg/l
Measured: 0.62, 1.31, 2.62, 5.31 and 10.88 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: cylinder
- Material, size, headspace, fill volume: glass, 50 ml
- Initial cells density: 1.61x10^4 cells/ml
- Control end cells density: 1.55x10^6 cells/ml
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Detailed composition if non-standard medium was used:
Mineral Nutrient Salts
1.5 g NH4Cl
1.2 g MgCl2 x 6 H2O
1.8 g CaCl2 x 2 H2O
1-59 MgSO4 x 7 H2O
0.16 g KH2PO4
were dissolved in 1 L deionised water and autoclaved.

Fe-Complex
0.08 g FeCl3 x 6 H2O
0.10g Na2EDTA x 2 H2O
were dissolved in 1 L delonised water and autoclaved.

TraceElements
185 mg H3BO3
415 mg MnCl2 x 4 H2O
3 mg ZnCl2
1.5 mg CoC12 x 6 H2O
0.01 mg CuCl2 x 2 H2O
7 mg Na2MoO4 x 2 H2O

Sodium Hydrogen Carbonate
1.00 g/L NaHCO3 (twice as high as indicated in OECD 201)

pre-culture medium: 1 ml of each of the solutions mentioned above in 88 ml of ultrapure water, then diluted at 50/500 ml ultrapure water.

Test medium: 1 ml of each of the solutions mentioned above in 88 ml of ultrapure water, then used at 15.65 - 500 mg/l

Algal stock solution was diluted with test medium to reach the desired initial concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra pure water
- Culture medium different from test medium: Yes, see above
- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Sterile test conditions: culturing conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: >=120 uE/m^2/s (+/- 8000 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: spectrophotometer at 578 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: 13% growth rate inhibition at 100 mg/l

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data, Report states wrong value

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data; deviates from report

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes, at 25 and 50 mg/l treatments
- Effect concentrations exceeding solubility of substance in test medium: Water accomodated fractions were used to formulate the test solutions

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

The inhibitions of the biomass production and of the growth rate of the algae were calculated according to the Guideline. The inhibitions of biomass production and of the growth rate were calculated using a validated computer program on the basis of EXCEL 5.0 (which was re-validated using EXCEL 97 under Windows NT), the EC50-value was determined as well as the EC100 and the NOEC together with the 95 percent confidence limits.

Summary of results

Nominal conc., mg/l	Geom. mean measured conc. (IR: hydrocarbons), mg/l	Growth rate Inhibition %	Biomass Yield Inhibition %
Control		-	-
12.5	0.62	5.8	0.6
25	1.31	-1.8	-29.2
50	2.62	-2.0	-29.6
100	5.31	25.7	73.8
200	10.88	66.5	95.3

Validity criteria fulfilled:

yes

Remarks:

Increase of more than 16X in control cell density

Conclusions:

The 72h-ELr50 of orangenöl WMB towards Desmodesmus subspicatus is 150 mg/l (WAF). The 72h-NOECr was circa 50 mg/l (WAF).

Executive summary:

The acute toxicity of orangenöl WMB towards Desmodesmus subspicatus was investigated according to OECD guideline 201 using WAFs, under GLP. Algal cells were exposed to nominal concentrations of 12.5, 25, 50, 100 and 200 mg/l and observed for 72 hours. The test solutions were prepared as a WAF.

The test report shows some deficiencies:

- Chemical identity of test substance not reported, but it was confirmed to be a sample representative of the registered substance.

- The statistical details are not reported and some corrections were made by the reviewer based on reported data.

Moreover, the value of 150 mg/L is far above the water solubility of many of the constituents. The high value may be related to volatility during the preparation of the WAF. During the test the concentration of total hydrocarbons remained above 80% of the initial concentration, showing constant exposure.

The biological effects and the analytical results of the test are clear and support the presented conclusion.

The 72h-NOELr and 72h-ELr50 were found to be circa 50 and circa 150 mg/l, respectively. These are corrected results based on the data in the report.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guidance (OECD Guideline 201) under GLP. Validity criteria met. Due to the read-across purpose it was given a Reliability 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Justification for type of information:

The read across justification is attached below

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Tested as WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Tested as WAF

Basis for effect:

growth rate

Key result

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, and 3.2 mg/I loading rate WAF, however cell debris was observed to be present in the test cultures at 10, 32 and 100 mg/l loading rate WAF.
- Colour differences: 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 mgtl loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mgtl loading rate WAF test preparations were observed to be clear colourless solutions.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Due to the volatile nature of the test item, an additional test replicate was prepared for each test concentration at 0 hours and incubated alongside the test to provide samples for unopened vessel analysis at 72 hours. Analysis of these preparations showed measured test concentrations to range from 0.041 to 1.8 mg/l (33% to 85% of the 0-Hour measured test concentrations). Given that a decline in measured concentrations was observed in both the vessels which had remained unopened throughout the test duration and those that had been opened daily for cell density determination it was considered that the decline observed was due to a combination of volatility and adsorption of the test item to the algal cells present.
- 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 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/l loading rate WAF test preparations were observed to be green dispersions whilst the 10, 32 and 100 mg/lloading rate WAF test preparations were observed to be clear colourless solutions.

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

Conclusions:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 9.7 mg/l in Pseudokirchneriella subcapitata based on the result of the read across substance Mandarin oil (Citrus Reticulata Blanco) ext. The 72h-ErL10 is 7.8 mg/l, whereas the 72h-NOELR is 3.2 mg/l.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 9.7 mg/l  in Pseudokirchneriella subcapitata based on the result of the read across substance Mandarin oil (Citrus Reticulata Blanco) ext. The acute toxicity of Mandarin oil (Citrus Reticulata Blanco) ext. towards Pseudikirchneriella 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 (Water Accomodated Fractions (WAFs))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 9.7 mg/l, the 72h-ErL10 was 7.8 mg/l, whereas the 72h-NOErL was 3.2 mg/l.

 

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guideline (OECD Guideline 201) under GLP. Validity criteria met. Due to the read-across purpose it was given a Reliability 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Justification for type of information:

The read across justification is attached below

Reason / purpose for cross-reference:

read-across source

Key result

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

Key result

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

Key result

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.

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

Conclusions:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 11 mg/l in Pseudokirchneriella subcapitata based on the result of the read across substanceLime Oil Cold-Pressed 1-Fold, Lime (Citrus aurantifolia), ext. The 72h-ErL10 is 2.2 mg/l, whereas the 72h-NOEL is 3.2 mg/l.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 11 mg/l  in Pseudokirchneriella subcapitata based on the result of the read across substance Lime Oil Cold-Pressed 1-Fold, Lime (Citrus aurantifolia), ext. 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.

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guideline (OECD Guideline 201) under GLP. Validity criteria met. Due to the read-across purpose it was given a Reliability 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Justification for type of information:

The read across justification is attached below

Reason / purpose for cross-reference:

read-across source

Key result

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

Key result

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

Key result

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.

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

Conclusions:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 8.0 mg/l in Pseudokirchneriella subcapitata based on the result of the read across substance Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. The 72h-ErL10 is 5.1 mg/l, whereas the 72h-NOEL is 3.2 mg/l .

Executive summary:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 8.0 mg/l  in Pseudokirchneriella subcapitata based on the result of the read across substance Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. 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 .

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guideline (OECD guideline 201) under GLP, validity criteria met. Chemical identity of test substance not reported. Unclear how NOECs were derived, corrections made by the reviewer based on reported data. The justification for read across is provided in the attached background material of the chapter summary.

Justification for type of information:

The read across justification is attached below

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data, Report states wrong value

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data; deviates from report

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes, at 25 and 50 mg/l treatments
- Effect concentrations exceeding solubility of substance in test medium: Water accomodated fractions were used to formulate the test solutions

See also attached file "results.doc".

Conclusions:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 150 mg/l in Desmodesmus subspicatus based on the result of the read across substance orangenöl WMB. The 72h-NOECr was circa 50 mg/l (WAF).

Executive summary:

Tangerine oil is concluded to have an acute toxicity (72h-ErL50) of 150 mg/l  in Desmodesmus subspicatus based on the result of the read across substance orangenöl WMB. The acute toxicity of orangenöl WMB towards Desmodesmus subspicatus was investigated according to OECD guideline 201 using WAFs, under GLP. Algal cells were exposed to nominal concentrations of 12.5, 25, 50, 100 and 200 mg/l and observed for 72 hours. The test solutions were prepared as a WAF.

The test report shows some deficiencies:

- Chemical identity of test substance not reported, but it was confirmed to be a sample representative of the registered substance.

- The statistical details are not reported and some corrections were made by the reviewer based on reported data.

Moreover, the value of 150 mg/L is far above the water solubility of many of the constituents. The high value may be related to volatility during the preparation of the WAF. During the test the concentration of total hydrocarbons remained above 80% of the initial concentration, showing constant exposure.

The biological effects and the analytical results of the test are clear and support the presented conclusion.

The 72h-NOELr and 72h-ELr50 were found to be circa 50 and circa 150 mg/l, respectively. These are corrected results based on the data in the report.

Description of key information

The 72h-EL50 is read across from Lime oil and is 8.0 mg/L and the EL10 is 5.1 mg/l  (Loading rates, WAF study)

Key value for chemical safety assessment

EC50 for freshwater algae:

8 mg/L

EC10 or NOEC for freshwater algae:

5.1 mg/L

Additional information

For Tangerine oil, no studies on the aquatic toxicity are available for algae.For this endpoint, read-across is conducted to the citrus group. Key WAF studies are available for Orange oil, Mandarin oil and Lime oil, see summary in the table.

Test substance (Read across)	Endpoint (growth)	Remarks	Reference
Lime Oil Distilled	72h-EL50 = 8 mg/l (WAF) 72h-EL10 = 5.1 mg/l (WAF)	OECD 201, GLP, Rel. 2, KEY	Harlan 2011
Mandarin Oil	72h-EL50 = 9.7 mg/l (WAF) 72h-EL10 = 7.8 mg/l (WAF)	OECD 201, GLP, Rel. 2,	Harlan 2011
Lime Oil Cold Pressed	72h-EL50 = 11 mg/l (WAF) 72h-EL10 = 2.2 mg/l (WAF)	OECD 201, GLP, Rel. 2,	Harlan 2011
Orange Oil	72h-EL50 = 150 mg/l 72h-NOELr = 50 mg/l	OECD 201, GLP, Rel. 2,	Lebertz 2007

 

For the data gap of Tangerine oil these studies are considered suitable for read-across and therefore given Reliability 2. The WAF toxicity values (ErL50) for Mandarin oil and both Lime oils are very similar, while for orange oil the WAF result showed an ErL50 10 times higher (less toxic).

The study with Lime oil distilled constitutes the lowest toxicity value (lowest WAF value, highest toxicity) for this category and is selected as key study.The values for Orange oil, Mandarin oil and the other quality of Lime oil are used as supporting studies.

Remark:

In the tests of Harlan (2011) and Lebertz (2007) the concentration of d-limonene and total hydrocarbons, respectively, were measured. As these oils are all complex test substances that contain a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were be 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.