Tangor, Murcote, ext.

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14/11/2006 - 03/04/2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 relevant

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.094, 0.21, 0.48, 1.0 and 2.2 mg/l
- Sampling method: duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test. Furthermore, the stability of the test item under the test conditions and the maintenance of the test item concentrations during the test period were determined. For this purpose two replicates of each treatment group (closed Erlenmeyer flasks) were given to the analytical department.
- Sample storage: 0-8 degrees Celsius

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test media for the highest two concentrations were prepared by suspending 5.97 ul and 5.43 ul of the test item in 2300 ml and 4600 ml of test water respectively. The suspensions were stirred for 24 hours to dissolve as much test item as possible. Then, undissolved fractions of the test item, possibly existent as second invisible thin layer at the surface, were removed by extracting the upper 5 ml of the suspension with a pipette. The solution with the concentration of 1 mg/l was as stock solution to prepare the test media of the lower concentrations. Media were prepared just before the start of the test.

The test item and it's main component, d-limonene, respectively, are highly volatile. To keep the test item concentrations during the preparation of the solutions, during the course of the biological incubation and in the analytical part as constant as possible, the test vessels containing the test media were only opened as often and as long as it was necessary during the whole period of study.

Furthermore the test item is not well soluble in test water. To avoid physical effects of non-dissolved test item on Daphnia no concentrations above the solubility limit of the test item in test water were tested.
- Controls: Yes

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, clone 5
- Source: ECT Oekotoxicologie GmbH, Flörsheim am Main, Germany
- Age at study initiation (mean and range, SD): 6-23 hours
- Method of breeding: The Daphnia were bred in the laboratories of IBACON under similar temperature and light conditions as in the test. The cultivation of the parental Daphnia was performed in reconstituted water of a similar quality regarding pH, components of the main ions and total hardness as the test water used in the test. The test organisms were not first brood progeny.
- Food type: Desmodesmus subspicatus algal suspension + tetramin extract
- Frequency: algal suspension: on weekdays, Tetramin extract: at least once a week

ACCLIMATION
- Acclimation period: 6 hours
- Acclimation conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Hardness:

2.5 mmol/l (=250 mg/l) as CaCO3

Test temperature:

20 degrees Celsius

pH:

6.8 - 6.9

Dissolved oxygen:

8.7 - 9.7 mg/l

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal (loading rate): 0.094, 0.21, 0.48, 1.0 and 2.2 mg/l
Measured(lowest and highest measured concentration at 0 and 48 hours): 0.048-0.062, 0.084-0.094, 0.182-0.211, 0.551-0.626 and 1.693-1.899 mg limonene/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 300 ml, filled with 350 ml test medium
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water containing:
CaCl2 x 2H2O - 2.0 mmol/l
MgSO4 x 7H2O - 0.5 mmol/l
NaHCO3 - 0.75 mmol/l
KCl - 0.075 mmol/l
- Alkalinity: 0.8 mmol/l
- Ca/mg ratio: 4:1
- Conductivity: < 5 uS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 48h

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light / 8h dark
- Light intensity: 0-1200 lux (average 320-420 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility (24 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

- Mortality of control: 0
- Effect concentrations exceeding solubility of substance in test medium: WAF-test, no test concentrations above limit of water solubility

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

The 24-hour and 48-hour EL50 and where possible their 95% confidence intervals were calculated by Probit analysis and the NOEL and LOEL were calculated by the Williams t-Test after arcsine-transformation of the data. The EL0 and EL100 were determined directly from the raw data.

The software used to perform the statistical analysis was Toxrat Professional, Version 2.09, Toxrat(R) Solutions GmbH, 2005.

Results based on the mean levels of limonene measured in the WAF:

the 48h-EC50 was estimated at 0.67 mg/l, with 95% confidence levels 0.52 - 0.87 mg/l

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:

Mortality in controls < 10%, oxygen saturation > 60%

Conclusions:

The acute toxicity (48h-EL50) of orange oil towards Daphnia magna established with a WAF test is 1.1 mg/l.

Executive summary:

The acute toxicity of orange oil towards Daphnia magna was determined according to OECD guideline 202 under GLP. Daphnids were exposed to nominal (WAF) concentrations of 0.94, 0.21, 0.48, 1.0 and 2.2 mg/l and were observed for 48 hours. Validity criteria for the test were met. The 48h-NOEL and 48h-EL50 were found to be 0.48 and 1.1 mg/l respectively.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14/03/2011- 20/04/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to international guideline (OECD guideline 202) under GLP. Validation criteria met, no deviations from guideline reported

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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.2 (Acute Toxicity for Daphnia)

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:

water organisms are exposed to a Water Accomodated Fraction (WAF) of the test item, because of poor water soluble test item

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, 2.2, 4.8, 11 and 24 mg/l
- Sampling method: Samples were taken from the control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken at each occasion. All samples taken for analysis were done do in inert airtight glassware with minimal headspace and analysed immediately.
- Duplicate samples were stored at -20 degrees celsius.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 51 and 110 mg) were added separately to the surface of 23 1itres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 1.0, 2.2 and 4.8 mg/1. Similarly, amounts of test item (25 and 55 mg) were added separately to the surface of 2.3 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 11 and
24 mg/1. After the addition of the test item, the reconstituted water 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 mixture 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 following siphoninh of the WAF. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 2.2, 4.8, 11 and 24 mg/I loading rate WAF.
- Differential loading: The solution for each test solution was prepared seperately. no dilution was used.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAF showed no microdispersions
or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 ml glass beakers containing approximately 100 ml of Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Hardness:

250 mg/l as CaC03.

Test temperature:

21 to 22 degrees Celsius

pH:

7.8 +- 0.2

Dissolved oxygen:

8.2-8.9 mg/l

Salinity:

not relevant (freshwater used)

Nominal and measured concentrations:

nominal loading rate: 1.0, 2.2, 4.8, 11 and 24 mg/l mandarin oil
measured (limonene, 0 hours): 0.0358, 0.167, 0.242, 0.530 and 0.896 mg/l
measured (limonene, 48 hours): 0.0186, 0.0413, 0.136, 0.455 and 0.731 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, containing 250 ml medium
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Solution & Concentration of stock solution (mg/l)
H3B03 51790
MnCI2.4H20 7210
LiCI 6120
RbCI 1420
SrCI2.6H20 3040
NaBr 320
Na2Mo04.2H20 1260
CuCI2.2H20 335
ZnCI2 260
CoCb.6H2O 200
KI 65
Na2Se03 43.8
NH4V03 11.5
Na2EDTA.2H20 5000
FeS04.7H20 1991

An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21°C.

Macro Nutrient Stock Solutions
Solution Concentration of stock solution (gIl)
CaCI2.2H20 293.80
NaHC03 64.80
MgS04.7H20 246.60
Na2Si03.9H20 50.00
KCI 58.00
NaN03 2.74
K2HP04 1.84
KH2P04 1.43

Vitamin Nutrients
Solution Concentration of stock solution (mg/l)
Thiamine hydrochloride 750
Cyanocobalamine (vitamin B12) 100
D(+) biotin (vitamin H) 75

The final medium was prepared by adding an aliquot of stock solution 2 along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21°C.

- Culture medium different from test medium: No
- Intervals of water quality measurement: pH and oxygen at 0 and 48 hours, temperature every 24 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dusk/dawn intervals
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilsation (24 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100 mg/l loading rate WAF
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

8.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 7.1 - 11 mg/l

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 16 - 19 mg/l

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 48h EC50: 1.5 mg/l (1.3-1.7 mg/l)

Reported statistics and error estimates:

The EL50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 48 hours the EL50 value and associated confidence limit, slope of the response curve and its standard error by the maximumlikelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

Probit analysis is used where two or more partial responses to exposure are shown.

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Validity criteria fulfilled:

yes

Remarks:

<10% immobilisation of daphnids, dissolved O2 at end >= 3 mg/L

Conclusions:

The acute toxicity (48h-EL50 based on nominal loading rates) of Mandarin Oil (Citrus Reticulata Blanco) ext., towards Daphnia magna is 8.9 mg/l, with a 95% confidence interval of 7.1 - 11 mg/l.

Executive summary:

The acute toxicity of Mandarin Oil (Citrus Reticulata Blanco) ext. towards Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOEL and 48h-EL50 were found to be 2.2 and 8.9 mg/l respectively.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15/03/2011 - 14/04/2011

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 202 (Daphnia sp. Acute Immobilisation 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.2 (Acute Toxicity for Daphnia)

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, 2.2, 4.8, 11 and 24 mg/l
- Sampling method: Samples were taken from the control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken at each occasion. All samples taken for analysis were done do in inert airtight glassware with minimal headspace and analysed immediately.
- Duplicate samples were stored at -20 degrees celsius.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 51 and 110 mg) were added separately to the surface of 23 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 1.0, 2.2 and 4.8 mg/l. Similarly, amounts of test item (25 and 55 mg) were added separately to the surface of 2.3 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 11 and 24 mg/l. After the addition of the test item, the reconstituted water 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 mixture 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 ontained following siphoning of the WAF. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 1.0, 2.2, 4.8, 11 and 24 mg/I loading rate WAFs.
- Differential loading: The solution for each test concentration was prepared seperately, no dilution was used.
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAF showed no microdispersions or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source:In-house laboratory culture
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 ml glass beakers containing approximately 100 ml of Elendt M7 medium in a temperature controlled room at. approximately 21-22ºC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Hardness:

250 mg/l CaCO3

Test temperature:

21 - 22 degrees Celsius

pH:

6.9 - 7.7

Dissolved oxygen:

8.5 - 9.1 mg/l

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal loading rates: 1.0, 2.2, 4.8, 11 and 24 mg/l
Measured (limonene, 0 hours): 0.0401, 0.140, 0.170, 0.749, 0.567 mg/l
Measured (limonene, 48 hours):

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250ml, containing 250 ml medium
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Solution Concentration of
stock solution (mg/l)
H3BO3 57190
MnCI2.4H2O 7210
LiCI 6120
RbCI 1420
SrC12.6H2O 3040
NaBr 320
Na2MoO4.2H2O 1260
CuCI2.2H2O 335
ZnC12 260
CoCI2.6H2O 200
KI 65
Na2SeO3 43.8
NH4VO3 11.5
Na2EDTA.2H2O 5000
FeSO4.7H20 1991

An aliquot (dependant on the volume of medium required) of each stock solution was
added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21 °C.

Macro Nutrient Stock Solutions

Solution Concentration of
stock solution (g/l)
CaCI2.2H2O 293.80
NaHCO3 64.80
MgSO4.7H2O 246.60
Na2SiO3.9H2O 50.00
KCI 58.00
NaNO3 2.74
K2HPO4 1.84
KH2PO4 1.43

Vitamin Nutrients

Solution Concentration of
stock solution (mg/1)
Thiamine hydrochloride 750
Cyanocobalamine (vitamin 100
B12)
D(+) biotin (vitamin H) 75

The final medium was prepared by adding an aliquot of stock solution 2 along with
aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21°C.

- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and oxygen at 0 and 48 hours, temperature every 24 hours

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dusk/dawn intervals

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation (24 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- 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:

48 h

Dose descriptor:

EL50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 48h-EC50: 1.5 mg/l (1.3 - 1.7 mg/l)

Reported statistics and error estimates:

At 24 and 48 hours the EL50 value, slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).

It was not possible to calculate associated 95% confidence limits as the data generated did not fit the models available.

Validity criteria fulfilled:

yes

Remarks:

Mortality in controls < 10%, oxygen concentration > 3 mg/l

Conclusions:

The acute toxicity (48h-EL50 based on nominal loading rates) of Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna is 12 mg/l.

Executive summary:

The acute toxicity of Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor water solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOELR and 48h-EL50 were found to be 4.8 and 12 mg/l repectively.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15/03/2011 - 07/04/2011

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 202 (Daphnia sp. Acute Immobilisation 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.2 (Acute Toxicity for Daphnia)

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, 2.2, 4.8, 11 and 24 mg/l
- Sampling method: Samples were taken from the control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken at each occasion. All samples taken for analysis were done do in inert airtight glassware with minimal headspace and analysed immediately.
- Duplicate samples were stored at -20 degrees celsius.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (23, 51 and 110 mg) were added separately to the surface of 23 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 1.0, 2.2 and 4.8 mg/l. Similarly, amounts of test item (25 and 55 mg) were added separately to the surface of 2.3 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 11 and 24 mg/l. After the addition of the test item, the reconstituted water 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 mixture 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 approximate 75-100 ml discarded) to give the 1.0, 2.2, 4.8, 11 and 24 mg/I loading rate WAFs.
- Differential loading: The solution for each test concentration was prepared seperately, no dilution was used
- Controls: Yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAF showed no microdispersions or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory culture
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 ml glass beakers containing approximately 100 ml of Elendt M7 medium in a temperature controlled room at. approximately 21-22ºC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Hardness:

250 mg/l CaCO3

Test temperature:

21 - 22 ºCelsius

pH:

7.8 - 8.1

Dissolved oxygen:

6.9 - 9.0 mg/l

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal loading rates: 1.0, 2.2, 4.8, 11 and 24 mg/l
Measured (limonene, 0 hours): 0.0748, 0.121, 0.500, 0.444, 1.25 mg/l
Measured (limonene, 48 hours):

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 100ml, containing 100 ml medium
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Solution Concentration of
stock solution (mg/l)
H3BO3 57190
MnCI2.4H2O 7210
LiCI 6120
RbCI 1420
SrC12.6H2O 3040
NaBr 320
Na2MoO4.2H2O 1260
CuCI2.2H2O 335
ZnC12 260
CoCI2.6H2O 200
KI 65
Na2SeO3 43.8
NH4VO3 11.5
Na2EDTA.2H2O 5000
FeSO4.7H20 1991

An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21 °C.

Macro Nutrient Stock Solutions

Solution Concentration of
stock solution (g/l)
CaCI2.2H2O 293.80
NaHCO3 64.80
MgSO4.7H2O 246.60
Na2SiO3.9H2O 50.00
KCI 58.00
NaNO3 2.74
K2HPO4 1.84
KH2PO4 1.43

Vitamin Nutrients

Solution Concentration of
stock solution (mg/1)
Thiamine hydrochloride 750
Cyanocobalamine (vitamin 100
B12)
D(+) biotin (vitamin H) 75

The final medium was prepared by adding an aliquot of stock solution 2 along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21°C.

- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and oxygen at 0 and 48 hours, temperature every 24 hours

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dusk/dawn intervals

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation (24 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- 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:

48 h

Dose descriptor:

EL50

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 0.79 - 38 mg/l

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rate

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 13 - 17 mg/l

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 48h-EC50: 1.5 mg/l (1.3 - 1.7 mg/l)

Reported statistics and error estimates:

The EL50 value (=Effective Loading Rate) and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 48 hours the EL50 value and associated confidence limit, slope of the response curve and its standard error by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999) .

Probit analysis is used where two or more partial responses to exposure are shown.

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Validity criteria fulfilled:

yes

Remarks:

Mortality in controls < 10%, oxygen concentration > 3 mg/l

Conclusions:

The acute toxicity (48h-EL50 based on nominal loading rates) of Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna is 5 mg/l, with 95% confidence interval 0.79 - 38 mg/l.

Executive summary:

The acute toxicity of Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOEL and 48h-EL50 were found to be 2.2 and 5 mg/l respectively.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

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 202) 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:

48 h

Dose descriptor:

EL50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and 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 (48h-EL50 based on nominal loading rates) of 12 mg/l in Daphnia magna based on the result of the read across substance Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (48h-EL50 based on nominal loading rates) of 12 mg/l  in Daphnia magna based on the result of the read across substance Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext. The acute toxicity of Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor water solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOELR and 48h-EL50 were found to be 4.8 and 12 mg/l repectively.

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

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 202) 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

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

- Mortality of control: 0
- Effect concentrations exceeding solubility of substance in test medium: WAF-test, no test concentrations above limit of water solubility

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

Conclusions:

Tangerine oil is concluded to have an acute toxicity (48h-EL50) of 1.1 mg/l in Daphnia magna based on the result of the read across substance orange oil.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (48h-EL50) of 1.1 mg/l  in Daphnia magna based on the result of the read across substance orange oil. The acute toxicity of orange oil towards Daphnia magna was determined according to OECD guideline 202 under GLP. Daphnids were exposed to nominal (WAF) concentrations of 0.94, 0.21, 0.48, 1.0 and 2.2 mg/l and were observed for 48 hours. Validity criteria for the test were met. The 48h-NOEL and 48h-EL50 were found to be 0.48 and 1.1 mg/l respectively.

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

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 202) under GLP. Validation criteria met, no deviations from guideline reported. 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

Vehicle:

no

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

8.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 7.1 - 11 mg/l

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

tested as WAF

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 16 - 19 mg/l

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and 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 (48h-EL50 based on nominal loading rates) of 8.9 mg/l in Daphnia magna based on the result of the read across substance Mandarin Oil (Citrus Reticulata Blanco) ext.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (48h-EL50 based on nominal loading rates) of 8.9 mg/l  in Daphnia magna 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 Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOEL and 48h-EL50 were found to be 2.2 and 8.9 mg/l respectively.

Reference 8

Endpoint:

short-term toxicity to aquatic invertebrates

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 202) 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:

48 h

Dose descriptor:

EL50

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 0.79 - 38 mg/l

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rate

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 13 - 17 mg/l

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and throughout the duration of the test all control and 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 (48h-EL50 based on nominal loading rates) of 5 mg/l in Daphnia magna based on the result of the read across substance Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext.

Executive summary:

Tangerine oil is concluded to have an acute toxicity (48h-EL50 based on nominal loading rates) of 5 mg/l  in Daphnia magna based on the result of the read across substance Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. The acute toxicity of Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext. towards Daphnia magna was investigated according to OECD guideline 202 under GLP. In view of the poor solubility in water, Water Accomodated Fractions were prepared. Daphnids were exposed to nominal loading rates of 1.0, 2.2, 4.8, 11 and 24 mg/l of the test substance and observed for 48 hours. Based on nominal loading rates, the 48h-NOEL and 48h-EL50 were found to be 2.2 and 5 mg/l respectively.

Description of key information

The 48h-EL50 is read across from Orange Oil and is 1.1 mg/L (Loading rate, WAF study).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.1 mg/L

Additional information

For Tangerine oil, no studies on the aquatic toxicity are available for aquatic invertebrates. For this endpoint, read-across is conducted to the citrus group.

Summary of Daphnia studies for the Citrus group

Endpoint	Result	Remarks	Reference
48h-EL50	1.1 mg/l (WAF Orange Oil)	OECD 202, GLP, Rel. 2	Vinken and Wydra 2007
48h-EL50	8.9 mg/l (WAF Mandarin oil)	OECD 202, GLP, Rel. 2	Harlan 2011
48h-EL50	5.0 mg/l (WAF Lime oil distilled)	OECD 202, GLP, Rel. 2	Harlan 2011
48h-EL50	12 mg/l (WAF Lime oil CP)	OECD 202, GLP, Rel. 2	Harlan 2011

 

Four toxicity studies are available with other oils of the Citrus group, see table. Read across is conducted to these studies. The justification of this read across is described in detail in the read across justification document.

The highest toxicity (lowest WAF value) was observed in the study with Orange oil. This key study was carried out using water accommodated fractions (WAFs). The median effective loading rate 48h-EL50 = 1.1 mg/l (Vinken and Wydra 2007). Supporting evidence is obtained from the other citrus oils Mandarin oil and Lime oil.

Remark:

In the tests of Vinken and Wydra (2007) and Harlan (2011) the concentration of d-limonene was 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.