Lime (Citrus aurantifolia), ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04/07/2011 - 08/07/2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Performed under GLP. Chemical identity not reported. Despite WAF preparation remaining consistent, the concentration of limonene in the WAFs varied throughout the tests between both preparation timings and loading rates.

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.

Description of key information

The 96h-LL50 is >18 mg/L (Loading rate, WAF study). 

Additional information

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.