Orange, sweet, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18/04/2011 - 16/05/2011

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

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:1, 2, 4, 8 and 10 mg/l WAF
- Sampling method: Water samples were collected during the first exposure at t=0h and t = 24h, and during the last exposure at incubation time t=72 h before and after the exposure. As the concentration of the test item was very low, the complete test solution was poured into a 10-litre-separation funnel together with 700 g NaCl, 50 mL of 1 M HCl, and 25 mL trichlorotrifluoroethane (in total, respectively). The separation funnel was closed with a glass stopper, and then, the funnel was shaken manually for approximately 1-3 min. After shaking the phases of the liquid were allowed to separate, and the organic phase was collected in a screw-cap flask made of glass containing sodium sulphate for drying purpose.
- Sample storage conditions before analysis: The flasks were stored at +4°C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because of the limited water-solubility and the volatile properties of the test item, special measures had to be regarded: The test item was tested as extracts from up to 10 mg per litre of dilution water. Higher concentrations were not tested as there was a 100% mortality at 10 mg/L in the screening-test. The extract is called „water accomodated fraction (abbreviated as „WAF“ hereafter). For this purpose, as a first step, a suspension of the test item was prepared in drinking water, as follows: the test item was weighed directly into glass bottles filled with 5000 mL of drinking water, respectively. The space above the liquids was saturized with molecular oxygen in order to facilitate oxygen saturation in the liquids. The preparation of the suspensions with drinking water was necessary for physiological conditions for the fish in the test solution. The drinking water used for the test refers to the OECD Guideline 203 and is controlled regularly (monthly). The test item was introduced into the water whilst incubation on a magnetic stirrer (main test) at room temperatu¬re for 16-24h. It is assumed that during that time equilibration between the test item and the water was achieved. Thereafter, stirring was stopped, and the solutions were filtered through glass fibre filters filters. The filtrates were used directly without any further dilution steps. Thus, the animals were exposed to the water-dissolved part of the test item. After temperature equilibration, the test animals were transferred into the prepared test solutions. After introducing the fish into the test solutions, the space above the liquids was saturized with molecular oxygen using oxygen from the central oxygen supply of the test facility in order to facilitate oxygen saturation in the liquids without further external aeration measures which normally would lead to loss of the volatile test item from the liquid. pH of the test solutions was not adjusted. As the test item exhibits volatile properties the test was conducted semi-statically in order to assure a constant test concentration. For this purpose, the test solutions were prepared daily and the test animals were transferred into the new test solutions daily, respectively.
- Controls: yes

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Zierfischgroßhandel Willibald Müller, Ascheberg
- Length at study initiation (length definition, mean, range and SD): 2.0 +/- 1.0 cm
- Feeding: fish were not fed 24h prior to testing and during the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not relevant

Hardness:

No data

Test temperature:

21.1 - 22.9 ºC

pH:

7.91 - 8.29

Dissolved oxygen:

9.3 - 23.6 mg/l

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: 1.0, 2.0, 4.0, 8.0 and 10.0 mg/l WAF
Measured (hydrocarbons, mean of geometric means): 0.49, 1.25, 2.35, 3.34 and 4.01 mg/l
Concentration at level of the EL50 was 2.76 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: “Weck” preserving jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 5 liter volume filled with 5 liter of medium, equipped with a special sealing for aeration device
- Aeration: no
- Renewal rate of test solution (frequency): once per day
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates):1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: drinking water
- Composition:
Ca2+: 2.02 mmol/l
Mg2+: 0.71 mmol/l
Na+: 0.65 mmol/l
K+: 0.06 mg/l
HCO3-: 4.04 mmol/l
Cu2+: 0.0008 mmol/l
- Culture medium different from test medium: No
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality: daily; behaviour: at t = 1, 2, 4-6, 18-20, 24, 28-30, 42-44, 48, 48-66, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations:1.25 - 2
- Range finding study
- Test concentrations: 10 mg/l WAF
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

5.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL10

Effect conc.:

5.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
Time (h) Effects / Observations
1 At 8 mg/L all fish very nervous swimming at the surface of the water; at 10 mg/L same appearance as at 8 mg/L but 3 fish at the ground
2 At 10 mg/L some fish lying laterally on the ground, only one fish swimming at the surface, but gasping for breath; at 8 mg/L all fish at the ground
4 – 6 At 8 and 10 mg/L all fish at the ground; at 10 mg/L two fish desoriented
21.5*) At 4 mg/L, 8 mg/L and 10 mg/L all fish at the bottom of the vessels, partially in lateral position
24 At 8 mg/L most of the fish apathically sitting on the ground; at 10 mg/L 3 fish motionless on the groud, two fish very nervous
26 – 30 At 10 mg/L 3 further fish dead, two apathically on the ground; at 8 mg/L four fish on the ground
42 – 44 At 10 mg/L all fish dead; at 8 mg/L remaining fish on the ground
48 At 8 mg/L 1 fish disoriented and nervous, remaining fish at the ground; at 4 mg/L all fish almost on the ground
48-66 At 8 mg/L 1 fish dead and remaining fish apathic; at 4 mg/L all fish at the ground
72 At 8 mg/L all remaining fish dead; at 4 mg/L most fish at the ground
96 At 4 mg/L fish at the ground

- Mortality of control: 0%

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

Statistical procedures for calculation of EC-values, as indicated in the study plan, were done upon receipt of the final mortalities using the commercial computer program ToxRatPro, Version 2.10.

Sublethal observations / clinical signs:

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

Validity criteria fulfilled:

yes

Remarks:

Mortality in controls at 0%, oygen saturation above 60%

Conclusions:

The acute toxicity (96h-EL50) of Orangenöl Auro towards Danio rerio with a WAF test is 5.65 mg/l. The 96h-LC50 is 2.76 mg/L (range 2.43-3.07) based on measured concentration of hydrocarbons.

Executive summary:

The acute toxicity of Orangenöl Auro towards Danio rerio was investigated according to OECD guideline 203 under GLP. Fish were exposed to nominal WAFs of 1.0, 2.0, 4.0, 8.0 and 10.0 mg/l in a semi-static setup and observed for 96 hours. The concentration of hydrocarbons was measured. Validity criteria for the test were met.

The acute toxicity (96h-EL50) of Orangenöl Auro towards Danio rerio with a WAF test is 5.65 mg/l and the NOEL was 4.0 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, 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):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

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 14 - 25 °C (depending on the species considered).

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:

96 h

Dose descriptor:

LC50

Effect conc.:

16 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 -4.63 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, here it is assumed that the acute toxicity of citral to fish 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 fish acute aquatic toxicity for 67 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 (96h-LC50) to fish 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

Sublethal observations / clinical signs:

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:

96h-LC50 for citral = 16 mg test item/L.

Executive summary:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to fish of citral has been investigated using a QSAR model that predicts fish mortality in an OECD 203 study. Citral as an aldehyde and a minor constituent of Orange oil is here assumed to act by non-polar narcosis. The 96-h LC50 based mortality 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

LC50: 16 mg test material/L.

Reference 3

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, 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):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

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 14 - 25 °C (depending on the species considered).

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:

96 h

Dose descriptor:

LC50

Effect conc.:

1.4 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 -4.63 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 here it is assumed and verified that the acute toxicity of decanal to fish can be determined using MOA 1 regression. Indeed, the iSafeRat® prediction of decanal for acute toxicity to fish is 1.4 mg/L which is very close to the experimentally measured value of 1.45 mg/L (ECHA disseminated dossier, 2010). The value of 1.4 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 fish acute aquatic toxicity for 67 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 (96h-LC50) to fish 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

Sublethal observations / clinical signs:

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:

96h-LC50 for decanal = 1.4 mg test item/L.

Executive summary:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to fish of decanal has been investigated using a QSAR model that predicts fish mortality in an OECD 203 study. Decanal as an aldehyde and a minor constituent of Orange oil is here assumed to act by non-polar narcosis. The 96-h LC50 based mortality was 1.4 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

LC50: 1.4 mg test material/L.

Reference 4

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, 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):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

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 14 - 25 °C (depending on the species considered).

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:

96 h

Dose descriptor:

LC50

Effect conc.:

0.46 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 -4.63 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 67 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 (96h-LC50) to fish. 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

Sublethal observations / clinical signs:

no data

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domains of the QSAR model.

Conclusions:

96h-LC50 for dl-limonene = 0.46 mg test item/L.

Executive summary:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to fish of dl-limonene has been investigated using a QSAR model that predicts fish mortality in an OECD 203 study. dl-limonene falls within the applicability domain of the model as demonstrated in the QPRF. The 96-h LC50 based mortality was 0.46 mg test material/L.This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

LC50: 0.46 mg test material/L.

Reference 5

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, 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):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

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 14 - 25 °C (depending on the species considered).

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:

96 h

Dose descriptor:

LC50

Effect conc.:

50 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 -4.63 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 67 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 (96h-LC50) to fish. 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

Sublethal observations / clinical signs:

no data

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domains of the QSAR model.

Conclusions:

96h-LC50 for linalool = 50 mg test item/L.

Executive summary:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to fish of linalool has been investigated using a QSAR model that predicts fish mortality in an OECD 203 study. Linalool falls within the applicability domain of the model as demonstrated in the QPRF. The 96-h LC50 based mortality was 50 mg test material/L.This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

LC50: 50 mg test material/L.

Reference 6

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, 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):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

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 14 - 25 °C (depending on the species considered).

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:

96 h

Dose descriptor:

LC50

Effect conc.:

0.32 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 -4.63 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 67 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 (96h-LC50) to fish. 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

Sublethal observations / clinical signs:

no data

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domains of the QSAR model.

Conclusions:

96h-LC50 for myrcene = 0.32 mg test item/L.

Executive summary:

The acute toxicity to fish 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 67 chemicals derived from 96-hour test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The acute toxicity to fish of myrcene has been investigated using a QSAR model that predicts fish mortality in an OECD 203 study. Myrcene falls within the applicability domain of the model as demonstrated in the QPRF. The 96-h LC50 based mortality was 0.32 mg test material/L.This toxicity study is acceptable and can be used for that endpoint.

Results Synopsis

Test Type: QSAR model

LC50: 0.32 mg test material/L.

Reference 7

Endpoint:

short-term toxicity to fish

Type of information:

calculation (if not (Q)SAR)

Remarks:

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 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

calculation method

Principles of method if other than guideline:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question (MOA 1, non-polar narcosis). 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 LC50s 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 Lethal Loading of the WAF. The method has been validated using data derived from 96-hour LC50 tests on fish, 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 (migrated information):
not applicable

Analytical monitoring:

not required

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LL50 (lethal 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 LL50s for fish between 12 and 28°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 fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater fish.

Nominal and measured concentrations:

The calculation determines measured concentrations.

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

3.1 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

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

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

2.9 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

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

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

2.8 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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

Sublethal observations / clinical signs:

At the 96 -hour LL50 the 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	2.4
linalool	0.16
myrcene	0.10
decanal	0.075
citral	2.4
sum of other aldehyde constituents (<1%)	0.097
sum of other constituents (<1%)	0.0041

At the 96 -hour LL50 the 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	2.6
linalool	0.59
sum of other aldehyde constituents (<1%)	0.59
sum of other constituents (<1%)	0.0033

At the 96 -hour LL50 the 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	2.7
myrcene	0.40
sum of other aldehyde constituents (<1%)	0.12
sum of other constituents (<1%)	0.00089

*: referred to as AMAP, the concentration Analytically Measurable in the Aqueous Phase (Water Accommodated Fraction). See attachment report in section 6.0 for more details.

Validity criteria fulfilled:

yes

Conclusions:

96h-LL50 for composition 1 of Orange oil (Orange oil cold pressed 10-fold) = 3.1 mg test item/L; 96h-LL50 for composition 2 of Orange oil (Orange essence oil 7-fold) = 2.9 mg/L and 96h-LL50 for composition 3 of Orange oil (Orange oil decolorised 1-fold) = 2.8 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 fish has been investigated using an in-house calculation method that replaces an OECD 203 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 LC50s 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 Lethal Loading of the WAF.

The 96h-LL50 was 3.1 mg test material/L for the composition of Orange oil cold pressed 10-fold, the 96h-LL50 was 2.9 mg test material/L for the composition of Orange essence oil 7-fold and the 96h-LL50 was 2.8 mg test material/L for Orange oil decolorised 1-fold. LL50 were based on fish mortality. 

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

LL50: 3.1 mg test material/L for composition 1 (Orange oil cold pressed 10-fold)

LL50: 2.9 mg test material/L for composition 2 (Orange essence oil 7-fold)

LL50: 2.8 mg test material/L for composition 3 (Orange oil decolorised 1-fold)

Description of key information

The results from the key study using WAF was 96h-EL50: 5.65 mg/l (WAF loading rate)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5.65 mg/L

Additional information

The acute toxicity of Orange oil towards fish was assessed in a WAF test and the toxicity was also investigated using a validated QSAR calculation method.

The acute toxicity of Orange oil (Orange terpenes, Limonene content 98.4 area%) towards Danio rerio was investigated according to OECD guideline 203 under GLP (Lebertz 2011, GLP, rel. 1). Fish were exposed to nominal WAFs of 1.0, 2.0, 4.0, 8.0 and 10.0 mg/l in a semi-static setup and observed for 96 hours. The concentration of hydrocarbons was measured. The acute toxicity (96h-EL50) of Orange Oil towards Danio rerio in a WAF test is 5.65 mg/l and the NOEL was 4.0 mg/L.

The toxicity of Orange Oil to fish was also investigated using the QSAR calculation method iSafeRat that replaces an OECD 203 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 LC50s 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 6.1.1. 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 96h-LL50 ranged from 2.8 to 3.1 mg /L (Thomas 2015, Rel. 2).