Orange, sweet, 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:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value

Justification for type of information:

See QMRF document "QMRF iSafeRat holistic approach v1.4.pdf" attached to this record.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The QSAR is based on data from studies performed at between 20 - 23 °C.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

The QSAR is based on data from studies performed on freshwater species.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

(QSAR model)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Descriptor domain: The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87.
- Mechanistic domain, if relevant: Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the acute aquatic toxicity for chemicals generally classified as MOA 1 substances (non-polar narcotics). Citral as an aldehyde is expected to fall on the aldehydes regression line. However it is assumed here that the acute toxicity of citral to daphnid can be determined using MOA 1 regression.
- Considerations on structural analogues: iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water solubility and daphnia acute aquatic toxicity for 58 substances with MOA 1.
- conclusion: Citral, assumed to act by non-polar narcosis, falls within the applicability domain of the model and can therefore be considered a reliably prediction for acute toxicity (48h-EC50) to daphnid for sole use within iSafeRat® calculation method in order to determine toxicity of multiconstituent substance where citral is a minor substance.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

no data

Validity criteria fulfilled:

yes

Remarks:

yes (For this purpose, it was assumed that the substance act by non-polar narcosis and falls into the applicability domains of the QSAR model.)

Conclusions:

48h-EC50 for citral = 16 mg test item/L.

Executive summary:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to daphnia of citral has been investigated using a QSAR model that predicts daphnia mortality in an OECD 202 study. Citral as an aldehyde and a minor constituent of Orange oil is here assumed to act by non-polar narcosis.

The 48-h EC50 based on mobility was 16 mg test material/L. 

This toxicity study is acceptable and can be used for purpose of iSafeRat® calculation method where citral is a minor constituent of multiconstituent substance. 

Results Synopsis

Test Type: QSAR model

EC50: 16 mg test material/L

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value

Justification for type of information:

See QMRF document "QMRF iSafeRat holistic approach v1.4.pdf " attached to this record.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The QSAR is based on data from studies performed at between 20 - 23 °C.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

The QSAR is based on data from studies performed on freshwater species.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

(QSAR model)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Descriptor domain: The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87.
- Mechanistic domain, if relevant: Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the acute aquatic toxicity for chemicals generally classified as MOA 1 substances (non-polar narcotics). Decanal as an aldehyde is expected to fall on the aldehydes regression line. However it is assumed and verified here that the acute toxicity of decanal to daphnid can be determined using MOA 1 regression. Indeed, the iSafeRat® prediction of decanal for acute toxicity to daphnid is 1.7 mg/L which is very close to the experimentally measured value of 1.17 mg/L (ECHA disseminated dossier, 2010). The value of 1.7 mg/L can thus be used for purpose of iSafeRat® calculation method where decanal is a minor constituent of multiconstituent substance.
- Considerations on structural analogues: iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water solubility and daphnia acute aquatic toxicity for 58 substances with MOA 1.
- conclusion: Decanal, assumed to act by non-polar narcosis, falls within the applicability domain of the model and can therefore be considered a reliably prediction for acute toxicity (48h-EC50) to daphnid for sole use within iSafeRat® calculation method in order to determine toxicity of multiconstituent substance where decanal is a minor substance.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

no data

Validity criteria fulfilled:

yes

Remarks:

(For this purpose, it was assumed that the substance act by non-polar narcosis and falls into the applicability domains of the QSAR model.)

Conclusions:

48h-EC50 for decanal = 1.7 mg test item/L.

Executive summary:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to daphnia of decanal has been investigated using a QSAR model that predicts daphnia mortality in an OECD 202 study. Decanal as an aldehyde and a minor constituent of Orange oil is here assumed to act by non-polar narcosis. The prediction of the acute toxicity of decanal to daphnid (1.7 mg/L) is very close to the experimentally measured value of 1.17 mg/L (ECHA disseminated dossier, 2010).

The 48-h EC50 based on mobility was 1.7 mg test material/L.

This toxicity study is acceptable and can be used for purpose of iSafeRat® calculation method where decanal is a minor constituent of multiconstituent substance. 

Results Synopsis

Test Type: QSAR model

EC50: 1.7 mg test material/L

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value

Justification for type of information:

See QMRF document "QMRF iSafeRat holistic approach v1.4.pdf" attached to this record.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The QSAR is based on data from studies performed at between 20 - 23 °C.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

The QSAR is based on data from studies performed on freshwater species.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

(QSAR model)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.62 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Descriptor domain: The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87.
- Mechanistic domain, if relevant: Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the acute aquatic toxicity for chemicals generally classified as MOA 1 substances (non-polar narcotics). Based on the chemical structure of alkenes, these substances are expected to fall on the MOA 1 regression line. dl-limonene as an alkene, is attributed to the class of non-polar narcotic compounds (MOA 1).
- Considerations on structural analogues: iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water solubility and fish acute aquatic toxicity for 58 substances with MOA 1.
- conclusion: dl-limonene falls within the applicability domain of the model and can therefore be considered a reliably prediction for acute toxicity (48h-EC50) to daphnid. According to the principles laid down by the OECD and cited under the REACH regulation, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

no data

Validity criteria fulfilled:

yes

Remarks:

yes (The substance falls into the applicability domains of the QSAR model.)

Conclusions:

48h-EC50 for dl-limonene = 0.62 mg test item/L.

Executive summary:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to daphnia of dl-limonene has been investigated using a QSAR model that predicts daphnia mortality in an OECD 202 study. dl-limonene falls within the applicability domain of the model as demonstrated in the QPRF.

The 48-h EC50 based on mobility was 0.62 mg test material/L. 

This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

EC50: 0.62 mg test material/L

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29/08/2007 - 29/09/2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Performed under under GLP, validity criteria met. Report contains errors; was clarified by e-mail contacts

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

Vehicle:

yes

Details on test solutions:

The test item was applied as follows: the test item was tested for toxicity towards daphnia as aqueous extracts ("WAF") from up to 100 mg test item/L (final concentration). Different amounts of the test item were dispersed in dilution water. The elution of the water soluble part (WAF) of the test item took place on a shaking machine over night (18h) at room temperature. During that time an equilibration between the test item and the water was considered to be achieved. Thereafter, the suspensions were filtered through glass fibre filters in order to eliminate non-dissolved parts of the test item. The eluates were used for the test without further dilution steps.
Four parallels were prepared for each concentration and the control without any test item, respectively, using 5 animals each parallel (main test). Two parallels were prepared for each concentration and the control without any test item, respectively, using 5 animals each parallel in the screening test. As the test item is partially volatile, the test solutions were filled into 100 mL carboys with glass stoppers in a manner, that no gas phase was present in the vessel. By that method, volatility was minimized. As the test item may adsorb to glass surfaces, a first proportion of the test solutions was filled into the beakers, and after standing for approx. 1 hour, the test solution was renewed by a second one. By that measure, adsorption of the test item was minimized. As an additional test solution, a reagents blank was prepared using the highest amount of the solvent used within the treatments with the test item. Further measures were the same as described before.

The main test was performed using the "WAFs" from 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, and 10 mg/L in the same way as described above. For this purpose, 4, 8, 16, 32, 40 mg of the test item was weighed directly into the vessels used for preparation of the WAFs. Then, 4000 mL dilution water was added to each vessel, and after closing the vessels the solutions were shaken on the shaking machine for 18h. Thereafter, the suspensions were filtered through glass fibre filters in order to eliminate non-dissolved parts of the test item. The eluates were used for the test without further dilution steps.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (clone 5) of laboratory bred (derived from Dr. M. Bergtold, BASF, D-67117 Limburgerhof; the strain originally derived from the Institut National de Recherche Chimique'Appliquee, France in 1978 and cultivated since then at BASF using medium M4 acc. Elendt) were used for the test. In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this bred was tested prior to beginning of the test. The value of the EC5o (24h) in this period of time was 1.04 mg/L potassium dichromate. This value is in accordance with the recommendations given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test). The cultivation of the daphnia is performed in a way that the animals are transferred in new test medium at an interval of 1 to 3 days followed by feeding with Desmodesmus subspicatus- and/or Chlorella-cells. Daphnia used for the test are not older than 24h. Therefore, in every case, daphnia are transferred into fresh medium one day before starting of the daphnia test in order to get daphnia for the test younger than 24h. During the test the daphnia were not fed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Nominal and measured concentrations:

Test concentrations reached on day 0 were < 10% of nominal concentrations. After 48 hours the concentrations were 80.3 to 85.9% of the concentrations at day 0. Thus the concentrations remained stable during the test.

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

10.218 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.:

8.577 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

5.626 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

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

Validity criteria fulfilled:

yes

Remarks:

No mortality in controls, oxygen saturation > 60%

Conclusions:

The acute toxicity (48h-EL50) of Orangenöl WMB towards Daphnia magna is 8.577 mg/l (Water Accomodated Fraction).

Executive summary:

The toxicity of Orangenöl WMB towards Daphnia magna was investigated according to OECD guideline 202 under GLP.

Daphnids wree exposed to nominal (WAF) concentrations of 1, 2, 4, 8 and 10 mg/l and observed for 48 hours. Validity criteria for the test were met. The 48h-EL50 were found to be 8 and 8.577 mg/l respectively.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value

Justification for type of information:

See QMRF document "QMRF iSafeRat holistic approach v1.4.pdf" attached to this record.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The QSAR is based on data from studies performed at between 20 - 23 °C.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

The QSAR is based on data from studies performed on freshwater species.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

(QSAR model)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Descriptor domain: The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HAQSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87.
- Mechanistic domain, if relevant: Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the acute aquatic toxicity for chemicals generally classified as MOA 1 substances (non-polar narcotics). Based on the chemical structure of alcohols, these substances are expected to fall on the MOA 1 regression line. Linalool as an alcohol, is attributed to the class of non-polar narcotic compounds (MOA 1).
- Considerations on structural analogues: iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water solubility and fish acute aquatic toxicity for 58 substances with MOA 1.
- conclusion: Linalool falls within the applicability domain of the model and can therefore be considered a reliably prediction for acute toxicity (48h-EC50) to daphnid. According to the principles laid down by the OECD and cited under the REACH regulation, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

no data

Validity criteria fulfilled:

yes

Remarks:

yes (The substance falls into the applicability domains of the QSAR model.)

Conclusions:

48h-EC50 for linalool = 45 mg test item/L.

Executive summary:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to daphnia of linalool has been investigated using a QSAR model that predicts daphnia mortality in an OECD 202 study. Linalool falls within the applicability domain of the model as demonstrated in the QPRF.

The 48-h EC50 based on mobility was 45 mg test material/L. 

This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

EC50: 45 mg test material/L

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value

Justification for type of information:

See QMRF document "QMRF iSafeRat holistic approach v1.4.pdf" attached to this record.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action of non-polar narcosis (MOA1). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The QSAR is based on data from studies performed at between 20 - 23 °C.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

The QSAR is based on data from studies performed on freshwater species.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

(QSAR model)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.45 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Descriptor domain: The water solubility value given as input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87.
- Mechanistic domain, if relevant: Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the acute aquatic toxicity for chemicals generally classified as MOA 1 substances (non-polar narcotics). Based on the chemical structure of alkenes, these substances are expected to fall on the MOA 1 regression line. Myrcene as an alkene, is attributed to the class of non-polar narcotic compounds (MOA 1).
- Considerations on structural analogues: iSafeRat® Holistic HA-QSAR is mainly based on the thermodynamic relationship between water solubility and the aquatic toxicity. This model was constructed using high quality data for water solubility and fish acute aquatic toxicity for 58 substances with MOA 1.
- conclusion: Myrcene falls within the applicability domain of the model and can therefore be considered a reliably prediction for acute toxicity (48h-EC50) to daphnid. According to the principles laid down by the OECD and cited under the REACH regulation, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

no data

Validity criteria fulfilled:

yes

Remarks:

yes (The substance falls into the applicability domains of the QSAR model.)

Conclusions:

48h-EC50 for myrcene = 0.45 mg test item/L.

Executive summary:

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to daphnia of myrcene has been investigated using a QSAR model that predicts daphnia mortality in an OECD 202 study. Myrcene falls within the applicability domain of the model as demonstrated in the QPRF.

The 48-h EC50 based on mobility was 0.45 mg test material/L. 

This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

EC50: 0.45 mg test material/L

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

supporting study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Calculation method is used ; calculation method applicable for the endpoint.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(calculation method)

Principles of method if other than guideline:

The acute toxicity to daphnids was determined using a validated QSAR for the Mode of Action in question. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading of the WAF. The method has been validated using data derived from 48-hour EC50 tests on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction. It should be noted that the KREATiS WAF calculation methodology is proprietary information but full details will be provided to the Competent Authorities upon request further to common accord on the terms of a mutually acceptable non-disclosure agreement.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material: not applicable

Analytical monitoring:

not required

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

48-EL50 (effective loading rate of WAF)

Post exposure observation period:

not applicable

Hardness:

Hardness is not a necessary component of the WAF calculation.

Test temperature:

The temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine EL50s for daphnids between 20 and 23°C.

pH:

The pH is not a necessary component of the WAF calculation.

Dissolved oxygen:

The oxygen concentration is not a necessary component of the WAF calculation.

Salinity:

Salinity is not a necessary component of the WAF calculation. However as the daphnid QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater.

Nominal and measured concentrations:

The calculation determines measured concentrations.

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.3 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: composition 1 (Orange oil cold pressed 10-fold)

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.1 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: composition 2 (Orange essence oil 7-fold)

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

3.9 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: composition 3 (Orange oil decolorised 1-fold)

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

At the 48-hour EL₅₀the expected concentrations of each constituent in the mixture according to the composition 1 (Orange oil cold pressed 10-fold) (based on thermodynamic calculation) are as follows:

constituents	concentration in the WAF (mg.L^-1)
dl-limonene	3.3
linalool	0.22
decanal	0.14
citral	0.11
sum of other aldehyde constituents (<1%)	0.097
sum of other constituents (<1%)	0.0041

At the 48-hour EL₅₀the expected concentrations of each constituent in the mixture according to the composition 2 (Orange essence oil 7-fold) (based on thermodynamic calculation) are as follows:

constituents	concentration in the WAF (mg.L^-1)
dl-limonene	3.6
linalool	0.83
sum of other aldehyde constituents (<1%)	0.065
sum of other constituents (<1%)	0.0033

At the 48-hour EL₅₀the expected concentrations of each constituent in the mixture according to the composition 3 (Orange oil decolorised 1 -fold) (based on thermodynamic calculation) are as follows:

constituents	concentration in the WAF (mg.L^-1)
dl-limonene	3.7
myrcene	0.057
sum of other aldehyde constituents (<1%)	0.016
sum of other constituents (<1%)	0.00089

Validity criteria fulfilled:

yes

Conclusions:

48h-EL50 for composition 1 of Orange oil (Orange oil cold pressed 10-fold) = 4.3 mg test item/L; 48h-EL50 for composition 2 of Orange oil (Orange essence oil 7-fold) = 4.1 mg/L and 48h-EL50 for composition 3 of Orange oil (Orange oil decolorised 1-fold) = 3.9 mg/L.

Executive summary:

Orange oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage and its reported variation of each constituent is known.

Its acute toxicity to daphnids property has been investigated using an in-house calculation method that replaces an OECD 202 study and considers the recommendations in the OECD Serie on Testing and Assessment no 23 (i.e. WAF conditions).

Three compositions (or qualities) have been investigated : Orange oil cold pressed 10-fold, Orange essence oil 7-fold and Orange oil decolorised 1-fold provided from data supplied in the SIP.

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading of the WAF.

The 48h-EL50 was 4.3 mg test material/L for the composition of Orange oil cold pressed 10-fold, the 48h-EL50 was 4.1 mg test material/L for the composition of Orange essence oil 7-fold and the 48h-EL50 was 3.9 mg test material/L for Orange oil decolorised 1-fold. EL50 were based on mobility. 

Based on the results of this study, Orange oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: Calculation method

EL50: 4.3 mg test material/L for composition 1 (Orange oil cold pressed 10-fold)

EL50: 4.1 mg test material/L for composition 2 (Orange essence oil 7-fold)

EL50: 3.9 mg test material/L for composition 3 (Orange oil decolorised 1-fold)

Reference 8

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

Remarks:

Performed under GLP, validity criteria met.

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:

- Loading rates in the WAF: 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 also used 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 its 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 Orange oil/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
- At the EL50 level, the Limonene concentration was 0.67 mg/L

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.

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

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 Orange oil/l respectively.

Description of key information

The result from the key study using WAFs was a 48h-EL50 = 1.1 mg/l (loading rate). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.1 mg/L

Additional information

Three experimental toxicity studies were performed with Daphnia magna and the toxicity was also investigated using a validated QSAR calculation method.

Endpoint	Result	Remarks	Reference
48h-EL50	1.1 mg/l (WAF Orange oil, essential oil (1 -folded CP), Limonene content 95.1%)	OECD 202, GLP, Rel. 1, KEY	Vinken and Wydra 2007a
48h-EL50	8.577 mg/l (WAF Orange oil (Orange Terpenes, Limonene content 93 -95% W/W)	OECD 202, GLP, Rel. 2	Lebertz 2007
48h-EC50	34.1 mg/l (Orange oil with solvent)	OECD 202, GLP, Rel. 3	Barth 2001
48h-EL50	3.9 - 4.3 mg/l (QSAR for WAF)	~ OECD 202	Thomas 2015

Three toxicity studies are available with Orange oil, see table. One is not reliable for many reasons including the use of a solvent to obtain the high test concentrations far above the water solubility limits (Barth 2001, Rel. 3). The key study was carried out using Water Accommodated Fractions (WAFs) where undissolved test substance was removed from the water phase. The median effective loading rate 48h-EL50 = 1.1 mg/l (Vinken and Wydra 2007a).

The toxicity of Orange oil to daphnids has also been investigated using the QSAR calculation method iSafeRat that replaces an OECD 202 study and takes into consideration the OECD Series on Testing and Assessment no 23 (i.e. WAF conditions). This method takes into account the bioavailable fraction per constituent in the WAF. The EC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study summary in IUCLID 6.1.3. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading of the WAF of the test material. The 48h-EL50 ranged from 3.9 to 4.3 mg /L (Rel. 2).