Lime (Citrus aurantifolia), 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:

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.

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

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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.

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

Results based on measured concentrations (limonene):

48h-NOEC 0.15 mg/l

48h-EC50 0.42 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 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 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key 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.

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

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.

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

Results based on measured concentrations (limonene):

48h-NOEC 0.021 mg/l

48h-EC50 0.39 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 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.

Description of key information

The 48h-EL50 is 5.0 mg/L (Loading rate, WAF study).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

5 mg/L

Additional information

For Lime oil, two valid WAF studies on the aquatic toxicity are available for Daphnia. The studies are conducted according to OECD 202 and are both validated with reliability 1, see table below. The study with the lowest WAF toxicity value (ErL50) is selected as the key study for lime oil. The other study is used as supporting study.

Summary of Daphnia studies

Endpoint	Result	Remarks	Reference
48h-EL50	5.0 mg/l Lime oil distilled	OECD 201, GLP, Rel. 1, KEY study	Harlan 2011
48h-EL50	12 mg/l Lime oil CP	OECD 201, GLP, Rel. 1, supporting study	Harlan 2011

 

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