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EC number: 289-904-6 | CAS number: 90045-43-5 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Citrus paradisi M., Rutaceae.
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Short-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- short-term toxicity to fish
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- 04/07/2011 - 08/07/2011
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Read-across from study according to international guideline (OECD guideline 203) under GLP. Validity criteria met. Due to the read-across purpose it was given a Reliability 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.1 (Acute Toxicity for Fish)
- 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: water accomodated fractions of 10, 18, 32, 56 and 100 mg/l
- Sampling method: Water samples were taken from the control and all surviving test groups test vessels at 0 (fresh media), 24 (old media) and 96 hours (old media) for quantitative analysis. Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (old and fresh media) were taken.
- Storage conditions of samples: at approximately -20°C. - Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (230,414, 736, 1288 and 2300 mg) were each separately added to the surface of 23 litres of dechlorinated tap water in separate stirring vessels with minimal headspace to give the 10, 18, 32, 56 and 100 mg/l loading rates respectively. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm (Considered to be sufficiently inert so as not to affect the dissolved limonene concentration obtained following siphoning of the WAF) was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing (Considered to be sufficiently inert so as not to affect the dissolved limonene concentration obtained following siphoning of the WAF) was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by middepth siphoning (the first 75-100 ml discarded) to give the 10, 18, 32, 56 and 100 mg/I loading rate WAFs.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. - Test organisms (species):
- Oncorhynchus mykiss (previous name: Salmo gairdneri)
- Details on test organisms:
- TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK. Maintained in-house since 10 March 2011.
- Length at study initiation (length definition, mean, range and SD): 4.4 +/- 0.3 cm
- Weight at study initiation (mean and range, SD): 1.07 +/- 0.19 g
- No feeding during test, starting 24h priortotest
ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: commercial trout pellets - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 96 h
- Post exposure observation period:
- Not relevant
- Hardness:
- 140 mg/l CaCO3
- Test temperature:
- 14 - 15 ºC
- pH:
- 7.5 - 7.9
- Dissolved oxygen:
- 7.4 - 10.4 mg/l
- Salinity:
- Not relevant
- Nominal and measured concentrations:
- Nominal: 10, 18, 32, 56 and 100 mg/l lime oil WAF
Measured (0h, fresh media): 0.451, 1.03/0.937, 0.401, 0.366 and 0.600 mg/l limonene
Measured (24h, old media): 0.298, 0.736/0.614, 0.278, 0.245 and 0.356 mg/l limonene
Measured (96h, old media): 0.436/- Details on test conditions:
- TEST SYSTEM
- Test vessel: round vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 20 liters, filled with 20 liters of test medium
- Aeration: none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.37 g/l
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener).
- Total organic carbon: 0.985 mg/l
- Metals: present at very low levels, not affecting test results
- Pesticides: present at very low levels, not affecting test results
- Chlorine: 22.388 mg/l
- Conductivity: 358.855 uS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 24 hours
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dawn/dusk transition periods
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): sub-lethal effects and mortality after 3, 6, 24, 48, 72 and 96 hours
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100 mg/l WAF
- Results used to determine the conditions for the definitive study: yes- Reference substance (positive control):
- no
- Duration:
- 96 h
- Dose descriptor:
- LL50
- Effect conc.:
- > 100 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: water accomodated fraction (WAF)
- Basis for effect:
- mortality (fish)
- Duration:
- 96 h
- Dose descriptor:
- EL0
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: water accomodated fraction (WAF)
- Basis for effect:
- mortality (fish)
- Details on results:
- - Other biological observations: Sub-lethal effects of exposure were observed at the 18 mg/l loading rate WAF and above. These responses were increased pigmentation, swimming at the bottom of the vessel and swollen abdomens. After approximately 70 hours exposure a single fish at 100 mg/l loading rate WAF was observed to be moribund with an extremely swollen abdomen. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) this fish was killed and classed as a mortality for the following observational time point.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none- Results with reference substance (positive control):
- Not relevant
- Reported statistics and error estimates:
- An estimate of the LL50 values was given by inspection of the mortality data.
- Sublethal observations / clinical signs:
For detailed results, see attached file "Results.doc".
- Validity criteria fulfilled:
- yes
- Remarks:
- Mortality in controls 0%, oxygen concentration above 60% of air saturation value
- Conclusions:
- The acute toxicity (96h-LL50) of mandarin oil towards Oncorhynchus mykiss is > 100 mg/l.
- Executive summary:
The acute toxicity of mandarin oil towards Oncorhynchus mykiss was investigated according to OECD guideline 203 under GLP. Fish were exposed to nominal loading rates of 10, 18, 32, 56 and 100 mg/l in a semi-static experiment and observed for 96 hours. Based on nominal loading rates the 96h-NOEL and 96h-LL50 were found to be 10 and >100 mg/l respectively.
- Endpoint:
- short-term toxicity to fish
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- 04/07/2011 - 08/07/2011
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Read-across from study according to international guideline (OECD guideline 203) under GLP. Validity criteria met. Due to the read-across purpose it was given a Reliability 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.1 (Acute Toxicity for Fish)
- 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: water accomodated fractions of 10, 18, 32, 56 and 100 mg/l
- Sampling method: Samples of each loading rate WAF were taken for chemical analysis at 0 (fresh media), 24 hours (old media) and 96 h (old media) in order to determine the stability of the component limonene over the test duration and stored at aprrox -20 degrees C for further analysis. - Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (230,414, 736, 1288 and 2300 mg) were each separately added to the surface of 23 litres of dechlorinated tap water in separate stirring vessels with minimal headspace to give the 10, 18, 32, 56 and 100 mg/l loading rates respectively. After the addition of the test item, the dechlorinated tap water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm (Considered to be sufficiently inert so as not to affect the dissolved limonene concentration obtained following siphoning of the WAF) was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing (Considered to be sufficiently inert so as not to affect the dissolved limonene concentration obtained following siphoning of the WAF) was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by middepth siphoning (the first 75-100 ml discarded) to give the 10, 18, 32, 56 and 100 mg/I loading rate WAFs.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. - Test organisms (species):
- Oncorhynchus mykiss (previous name: Salmo gairdneri)
- Details on test organisms:
- TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK. Maintained in-house since 10 March 2011.
- Length at study initiation (length definition, mean, range and SD): 4.2 +/- 0.4 cm
- Weight at study initiation (mean and range, SD): 1.09 +/- 0.33 g
ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: commercial trout pellets - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 96 h
- Post exposure observation period:
- Not relevant
- Hardness:
- 140 mg/l CaCO3
- Test temperature:
- 14 - 15 ºC
- pH:
- 7.6 - 7.9
- Dissolved oxygen:
- 5.6 - 10.2 mg/l
- Salinity:
- Not relevant
- Nominal and measured concentrations:
- Nominal: 10, 18, 32, 56 and 100 mg/l lime oil WAF
Measured (0h, fresh media): 0.2383, 0.375, 1.45, 0.272 and 0.506 mg/l limonene
Measured (24h, old media): 0.153, 0.207, 0.955, 0.171 and 0.359 mg/l limonene
Measured (96h, old media): 0.187, 0.0735, no data, 0.141 and no data mg/l limonene - Details on test conditions:
- TEST SYSTEM
- Test vessel: round vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 20 liters, filled with 20 liters of test medium
- Aeration: none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.38 g/l
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 12480 Duplex Water Softener).
- Total organic carbon: 0.985 mg/l
- Metals: present at very low levels, not affecting test results
- Pesticides: present at very low levels, not affecting test results
- Chlorine: 22.388 mg/l
- Conductivity: 358.855 uS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 24 hours
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark with 20 minute dawn/dusk transition periods
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): sub-lethal effects and mortality after 3, 6, 24, 48, 72 and 96 hours
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100 mg/l WAF
- Results used to determine the conditions for the definitive study: yes - Reference substance (positive control):
- no
- Duration:
- 96 h
- Dose descriptor:
- LL50
- Effect conc.:
- > 18 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: water accomodated fraction (WAF)
- Basis for effect:
- mortality (fish)
- Duration:
- 96 h
- Dose descriptor:
- EL0
- Effect conc.:
- 18 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: water accomodated fraction (WAF)
- Basis for effect:
- mortality (fish)
- Details on results:
- - Other biological observations: Sub-lethal effects of exposure were observed at the 32 mgtl loading rate WAF and above. These responses were increased pigmentation, swimming at the surface, lying at the bottom of the vessel and the presence of moribund fish.
After approximately 71 hours exposure two out of seven fish at 56 mgtlloading rate WAF were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none - Results with reference substance (positive control):
- Not relevant
- Reported statistics and error estimates:
- An estimate of the LL50 values was given by inspection of the mortality data.
- Sublethal observations / clinical signs:
For detailed results, see attached file "Results.doc".
- Validity criteria fulfilled:
- yes
- Remarks:
- Mortality in controls 0%, oxygen concentration above 60% of air saturation value
- Conclusions:
- The acute toxicity (96h-LL50) of lime oil towards Oncorhynchus mykiss is > 18 mg/l.
- Executive summary:
The acute toxicity of lime oil towards Oncorhynchus mykiss was investigated according to OECD guideline 203 under GLP. Fish were exposed to nominal loading rates of 10, 18, 32, 56 and 100 mg/l in a semi-static experiment and observed for 96 hours. Based on nominal loading rates the 96h-NOEL and 96h-LL50 were found to be 18 and >18 mg/l respectively.
- Endpoint:
- short-term toxicity to fish
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The read across justification is attached below
- Reason / purpose for cross-reference:
- read-across source
- 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)
- Remarks on result:
- other: 100 % mortality
- 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".
Results based on measured concentrations (Sum parameter Hydrocarbons):
96h-NOEC 2.35 mg/l
96h-LC50 2.76 mg/l, 95%-CL <2.43 - 3.07>
In this test the concentration of total hydrocarbons was measured. As the tested oil is a complex test substance that contains a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.
- Conclusions:
- Grapefruit oil is concluded to have an acute toxicity (96h-LL50) of 5.65 mg/l in Danio rerio based on the result of the read across substance Orangenöl Auro.
- Executive summary:
Grapefruit oil is concluded to have an acute toxicity (96h-LL50) of 5.65 mg/l in Danio rerio based on the result of the read across substance Orangenöl Auro. The acute toxicity of Orangenöl Auro towards Danio rerio was investigated according to OECD TG 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 closed system with additional oxygen added to keep oxygen levels sufficiently high while at the same time avoiding volatilisation of the test substance. The test media were daily renewed. The concentration of hydrocarbons was measured to ensure the stability of the exposure during the test period. The WAFs were prepared daily. The observed hydrocarbon content (IR spectrum) showed that the concentrations remained within 80% of the initial level at day 0 for the concentrations with partial effect. In the higher dose rates of 8 and 10 mg/l, where complete mortality occurred, the losses were somewhat higher.
Validity criteria for the OECD TG 203 test were met.
The acute toxicity (96h-EL50) of Orangenöl Auro towards Danio rerio in a WAF test is 5.65 mg/l and the NOEL was 4.0 mg/L.
- Endpoint:
- short-term toxicity to fish
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- 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:
- Study according to international guideline (OECD guideline 203) under GLP. Validity criteria met.
- Justification for type of information:
- Information is used for read across to Grapefruit Oil
- Reason / purpose for cross-reference:
- read-across: supporting information
- 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 temperature 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 sum of hydrocarbons (mean of geometric means): 0.49, 1.25, 2.35, 3.34 and 4.01 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
- Test concentrations (definitive test): 1, 2, 4, 8, 10 mg/l (prepared as WAF)
- Range finding study test concentration: 1 and 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)
- Remarks on result:
- other: 100 % mortality
- 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".
Results based on measured concentrations (Sum parameter Hydrocarbons):
96h-NOEC 2.35 mg/l
96h-LC50 2.76 mg/l, 95%-CL <2.43 - 3.07>
In this test the concentration of total hydrocarbons was measured. As the tested oil is a complex test substance that contains a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.
- Validity criteria fulfilled:
- yes
- Remarks:
- Mortality in controls 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.
- Executive summary:
The acute toxicity of Orangenöl Auro towards Danio rerio was investigated according to OECD TG 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 closed system with additional oxygen added to keep oxygen levels sufficiently high while at the same time avoiding volatilisation of the test substance. The test media were daily renewed. The concentration of hydrocarbons was measured to ensure the stability of the exposure during the test period. The WAFs were prepared daily. The observed hydrocarbon content (IR spectrum) showed that the concentrations remained within 80% of the initial level at day 0 for the concentrations with partial effect. In the higher dose rates of 8 and 10 mg/l, where complete mortality occurred, the losses were somewhat higher.
Validity criteria for the OECD TG 203 test were met.
The acute toxicity (96h-EL50) of Orangenöl Auro towards Danio rerio in a WAF test is 5.65 mg/l and the NOEL was 4.0 mg/L.
Referenceopen allclose all
Description of key information
The 96h-LL50 is read across from Orange Oil and is 5.65 mg/l (Loading rate, WAF study)
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Effect concentration:
- 5.65 mg/L
Additional information
The acute toxicity of grapefruit oil towards was read across from a study on orange oil 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 orange oil towards Danio rerio with a WAF test is 5.65 mg/l and the NOEL was 4.0 mg/L.
Supporting read across studies are also available for lime and mandarin oil. These studies report 96h-NOEL and 96h-LL50 values of 18 and >18 and 10 and >100 mg/l respectively.
The orange oil study was selected for read across, as the composition of orange oil most closely resembles the composition of grapefruit oil and the EL50 is the lowest value.
Remark: In the tests of Harlan (2011 and 2012) and Lebertz (2011) the concentration of limonene and total hydrocarbons, respectively, were measured. As these oils are all complex test substances that contain a range of constituents, the dose rates were prepared as Water Accommodated Fractions (WAF). The analytical measurements were used to establish the stability of the test solutions over time. They should not be used to express the toxicity of the Citrus Oils on the basis of a single substance or sum parameter.
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