Orange, sweet, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, 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: not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErC50

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 - 25°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:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 -4.38 to 0.49.
- 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 toxicity of citral to algae 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 algal growth inhibition test for 40 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 toxicity (72h-ErC50) to algae 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:

(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:

72h-ErC50 for citral = 15 mg test item/L.

Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of citral has been investigated using a QSAR model that predicts algae growth rate inhibition in an OECD 201 study. Citral as an aldehyde and a minor constituent of Orange oil is here assumed to act by non-polar narcosis.

The 72-h ErC50 based on growth rate was 15 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

ErC50: 15 mg test material/L

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, 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: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErC50

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 - 25°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:

72 h

Dose descriptor:

EC50

Effect conc.:

1.5 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 -4.38 to 0.49.
- 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 here that the toxicity of decanal to algae 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 algal growth inhibition test for 40 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 toxicity (72h-ErC50) to algae 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:

72h-ErC50 for decanal = 1.5 mg test item/L.

Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of decanal has been investigated using a QSAR model that predicts algae growth rate inhibition in an OECD 201 study. Decanal 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 growth rate was 1.5 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

ErC50: 1.5 mg test material/L

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, 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: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErC50

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 - 25°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:

72 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 -4.38 to 0.49.
- 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 MOA1 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 algal growth inhibition test for 40 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 (72h-ErC50) to algae. 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:

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

Conclusions:

72h-ErC50 for dl-limonene = 0.50 mg test item/L.

Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of dl-limonene has been investigated using a QSAR model that predicts algae growth rate inhibition in an OECD 201 study. dl-limonene falls within the applicability domain of the model as demonstrated in the QPRF.

The 72-h ErC50 based on growth rate was 0.50 mg test material/L. 

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

 

Results Synopsis

Test Type: QSAR model

ErC50: 0.50 mg test material/L.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/09/2007 - 14/09/2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.5, 25, 50, 100 and 200 mg/l
- Sampling method:
The quantification of the test item (fraction of dissolved material of the test item) in the test solutions (*) was performed according to the method given in CEC L-33-A-93, DIN 51828-2, as agreed with the Sponsor: The use of the specific peak of the IR at 2930 ± 10 cm-1 for hydrocarbons is comparable with a common parameter like TOC or COD.

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

For quantification of any test item possibly adsorbed to the glass walls of the test vessels within the 72h of incubation, a proportion of 25 mL trichlorotrifluoroethane was given into the empty test vessels which then were thoroughly rinsed with the solvent. The solvent was collected in a glass flask. This procedure was repeated in the same way with all test vessels. The organic phases were unified and then reduced to a final volume of 5 mL and then analysed by IR-spectroscopy using the specific peak at 2930 ± 10 cm-1 for hydrocarbons.

Samples were taken after 0 and 48 hours.
(*): The analytical section in the test report erroneously mentions that test solutions were from the Daphnia test. The study on algae was performed in the same lab in the same period as the one on daphnia with the same analytical procedure.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Initial algal cell concentration of 1.65*10^4 cells/mL (screening test) and 1.61 x10^4 cells/ml (main test) in the test solutions.
The determination of cell concentration was performed by photometrical measurement using a calibration curve prepared by using a spectrophotometer at 578 nm.
- Test substance was dissolved in DCM and specific amounts for each test concentration (according to a table) were brought into vessels. DCM was allowed to evaporate - in this way the test item was distributed in the flask as a fine layer. Then a defined water volume was added(as in table). After shaking for 18h at 130 RPM, the solution was filtered through a filter papere previously rinsed with ultrapure water. Algal suspensions where then added to obtain algal solution at the appropriate test substance concentration levels.
- Controls: Yes
- Solvent: dichloromethane is used and evaporated. Thus DSM was used only to "coat" the vessels with the defined amount of test substance, but it was not present in the water medium. Therefore the preparation can be considered as a WAF - water accomodated fraction.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

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

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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not relevant

Hardness:

No data

Test temperature:

23.2 - 24.0 degrees Celsius

pH:

7.17 - 10.00

Dissolved oxygen:

No data

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal (WAF): 12.5, 25, 50, 100 and 200 mg/l
Measured: 0.62, 1.31, 2.62, 5.31 and 10.88 mg/l (total hydrocarbons, geometric mean of t0 and t72h))

Details on test conditions:

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

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

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

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

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

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

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

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

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

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

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

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

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data, Report states wrong value

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Interpretation of data; deviates from report

Details on results:

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

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

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

Summary of results

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

See also attached file "results.doc".

Validity criteria fulfilled:

yes

Remarks:

Increase of more than 16X in control cell density

Conclusions:

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

Executive summary:

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

The test report shows some deficiencies:

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

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

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

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

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

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, 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: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErC50

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 - 25°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:

72 h

Dose descriptor:

EC50

Effect conc.:

46 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 -4.38 to 0.49.
- 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 MOA1 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 algal growth inhibition test for 40 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 (72h-ErC50) to algae. 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:

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

Conclusions:

72h-ErC50 for linalool = 46 mg test item/L.

Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of linalool has been investigated using a QSAR model that predicts algae growth rate inhibition in an OECD 201 study. Linalool falls within the applicability domain of the model as demonstrated in the QPRF.

The 72-h ErC50 based on growth rate was 46 mg test material/L. 

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

 

Results Synopsis

Test Type: QSAR model

ErC50: 46 mg test material/L.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(QSAR model)

Principles of method if other than guideline:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, 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: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErC50

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 - 25°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:

72 h

Dose descriptor:

EC50

Effect conc.:

0.36 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 -4.38 to 0.49.
- 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 MOA1 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 algal growth inhibition test for 40 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 (72h-ErC50) to algae. 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:

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

Conclusions:

72h-ErC50 for myrcene = 0.36 mg test item/L.

Executive summary:

The growth inhibition of algae was determined using a validated QSAR for the Mode of Action non-polar narcosis (MOA 1). The QSAR is based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The toxicity to algae of myrcene has been investigated using a QSAR model that predicts algae growth rate inhibition in an OECD 201 study. Myrcene falls within the applicability domain of the model as demonstrated in the QPRF.

The 72-h ErC50 based on growth rate was 0.36 mg test material/L. 

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

 

Results Synopsis

Test Type: QSAR model

ErC50: 0.36 mg test material/L.

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

key study

Study period:

2015-08-03 to 2015-08-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Remarks:

calculation method applicable for the endpoint.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(calculation method)

Principles of method if other than guideline:

The toxicity to algae 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 ErC50s 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 72-hour EC50 tests on algae. 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: algae spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72h-ErL50 (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 algae between 20 and 25°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 algae QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater organisms.

Nominal and measured concentrations:

The calculation determines measured concentrations.

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.8 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.5 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.3 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

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 72 -hour ErL₅₀the 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	3.7
linalool	0.25
decanal	0.16
citral	0.12
sum of other aldehyde constituents (<1%)	0.097
sum of other constituents (<1%)	0.0041

At the 72 -hour ErL₅₀the expected concentrations* of each constituent in the mixture according to the composition 1 (Orange essence oil 7-fold) (based on thermodynamic calculation) are as follows:

constituents	concentration in the WAF (mg.L^-1)
dl-limonene	4.0
linalool	0.92
sum of other aldehyde constituents (<1%)	0.065
sum of other constituents (<1%)	0.0033

At the 72 -hour ErL₅₀the expected concentrations* of each constituent in the mixture according to the composition 1 (Orange oil decolorised 1-fold) (based on thermodynamic calculation) are as follows:

constituents	concentration in the WAF (mg.L^-1)
dl-limonene	4.2
myrcene	0.57
sum of other aldehyde constituents (<1%)	0.16
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:

72h-ErL50 for composition 1 of Orange oil (Orange oil cold pressed 10-fold) = 4.8 mg test item/L; 72h-ErL50 for composition 2 of Orange oil (Orange essence oil 7-fold) = 4.5 mg/L and 72h-ErL50 for composition 3 of Orange oil (Orange oil decolorised 1-fold) = 4.3 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 toxicity to algae has been investigated using an in-house calculation method that replaces an OECD 201 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 72h-ErL50 was 4.8 mg test material/L for the composition of Orange oil cold pressed 10-fold, the 72h-ErL50 was 4.5 mg test material/L for the composition of Orange essence oil 7-fold and the 72h-ErL50 was 4.3 mg test material/L for Orange oil decolorised 1-fold. ErL50 were based on algae growth rate. 

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

ErL50: 4.8 mg test material/L for composition 1 (Orange oil cold pressed 10-fold)

ErL50: 4.5 mg test material/L for composition 2 (Orange essence oil 7-fold)

ErL50: 4.3 mg test material/L for composition 3 (Orange oil decolorised 1-fold)

Description of key information

72h-NOELr was 50 mg/l and 72h-ELr50 was 150 mg/l for Desmodesmus subspicatus using WAFs of Orange oil. Using a QSAR approach for 3 qualities of Orange oil, the 72h-ErL50 ranged from4.3 to 4.8 mg/L. As a conservative approach, the value of 4.3 mg/L was selected (loading rate). As the QSAR approach does not provide NOEL or EL10 values, the experimental NOEC is entered. 

Key value for chemical safety assessment

EC50 for freshwater algae:

4.3 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

Two very different key studies were identified.

The acute toxicity of orangenöl WMB (Orange Terpenes, Limonene content 93 -95% W/W) towards Desmodesmus subspicatus was investigated according to OECD guideline 201 using WAFs, under GLP. Algal cells were exposed to nominal concentrations from 12.5 up to 200 mg/l. The test report shows some deficiencies: (1) Chemical identity of test substance not reported, but it was confirmed to be a sample representative of the registered substance; and (2) the statistical details are not reported and some corrections were made by the reviewer based on reported data. Yet the biological effects and the analytical results of the test are clear and support the presented conclusion. The 72h-NOELr and 72h-ELr50 were found to be circa 50 and circa 150 mg/l, respectively (Lebertz, 2007, Rel. 2).

The toxicity of Orange oil to algae was also investigated using a validated QSAR calculation method iSafeRat that replaces an OECD 201 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 6.1.5. 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 72h-ErL50 ranged from 4.3 to 4.8 mg /L (Thomas 2015, Rel. 2).

It is recognised that the results from the experimental WAF study with an ErL50 are significantly higher than that of the QSAR, and far above the water solubility of many of the constituents. The high value may be related to volatility during the preparation of the WAF. The lowest EL50 of the QSAR-WAF is taken into account to confirm that algae are not significantly more sensitive than daphnia and fish.