Eucalyptus maculata citriodora, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Rationale & experimental data: For fish, key WAF studies are available for Citronella oil and Litsea oil. These studies are conducted according to OECD 203 and both evaluated as Klimisch 1 studies. For the data gap of Eucalyptus oil concerning fish toxicity, the studies are considered suitable for read-across. An overview of these studies is provided in the table below. The lowest toxicity value (EL50) for fish is found in the test with Litsea oil and is 4.2 mg/L. It is considered justified to read across this result to Eucalyptus oil for the reasons mentioned above. Thus, for Eucalyptus oil an EL50 of 4.2 mg/l is expected.

Results considered (nominal, WAF tests):
- Litsea cubeba oil (Harlan 2013): EL50 = 4.2 mg/L
- Citronella oil (Harlan 2013): EL50 = 7.5 mg/L

Conclusion and dose descriptor: the toxicity towards Daphnia for Eucalyptus oil is read across from the group of citral oils in particular Litsea oil. For Eucalyptus oil an EL50 of 4.2 mg/L for fish is expected.

Reason / purpose for cross-reference:

read-across source

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 5.6 - 10 mg/L

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 5.6 - 10 mg/L

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 5.6 - 10 mg/L

Duration:

86 h

Dose descriptor:

NOELR

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: 0%
- Effect concentrations exceeding solubility of substance in test medium: At the start and end of each mixing period, and after the 1-Hour settlement period the loading rates were observed to be clear colorless water columns with test item floating on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

The LL50 values and associated confidence limits were calculated using the geometric mean method.

Sublethal observations / clinical signs:

Cumulative mortality

Nominal loading rate (mg/L)	Cumulative mortality (initial population is 7)						% Mortality
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours	96 hours
Control	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0
5.6	0	0	0	0	0	0	0
10.0	0	0	0	7	7	7	100

Sub-lethal effects

Nominal loading rate (mg/L)	Sub-lethal effects	Time
		3 hours	6 hours	24 hours	48 hours	72 hours	96 hours
Control	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
1.0	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
1.8	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
3.2	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
5.6	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
10.0	No abnormalities detected	7/7	7/7	7/7
	Moribund				7/7	A/D

Graphs are presented below.

Validity criteria fulfilled:

yes

Remarks:

Mortality 0% in controls, physical-chemical parameters within acceptable limits.

Conclusions:

The acute toxicity (96h-LL50) of Cymbopogon winterianus towards Oncorhynchus mykiss is 7.5 mg/L, which is read-across to Eucalyptus citriodora.

Executive summary:

The acute toxicity of Cymbopogon winterianus towards Oncorhynchus mykiss was investigated according to OECD guideline 203 under GLP. Fish were exposed to nominal WAFs of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L in a semi-static setup and were observed for 96 hours. The 96h-NOELR and 96h-LL50 were found to be 5.6 and 7.5 mg/L respectively. These results are read-across to Eucalyptus citriodora.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7/1/2013 - 10/1/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/L WAF
- Sampling method: Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each surviving test group at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media).
- Sample storage conditions before analysis: stored at -20ºC.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (22.5, 40.5, 72, 126 and 225 mg) were each separately added to the surface of 22.5 liters of dechlorinated tap water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively and the vessels were completely filled and sealed. 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 47 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm 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 was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no microdispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Due to the light-sensitive nature of the test item, all test preparation was performed under non-actinic light conditions and the stirring, test and sampling vessels were shielded from light.
- Controls: Yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of each mixing period the loading rates were observed to be clear colorless water columns with globules of test item floating on the surface. At the end of each mixing period, and after the 1-Hour settlement period the loading rates were observed to be clear colorless water columns with an oily layer of test item floating on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the loading rates were observed to be clear, colorless solutions.

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 March 7th 2013
- Length at study initiation (length definition, mean, range and SD): 4.6 (=/- 0.3) cm
- Weight at study initiation (mean and range, SD): 0.81 (+/- 0.14) g
- Feeding during test: no

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same
- Type and amount of food: The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): There was no mortality in the 7 days prior to the start of 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:

140 mg/L CaCO3

Test temperature:

15ºC

pH:

7.8 - 8.1

Dissolved oxygen:

8.6 - 10.6 mg/L (85 - 105% of ASV)

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L
0 hours (fresh media): 0.85, 1.67, 2.96, 1.88 and 4.96 mg/L
96 hours (old media): 1.15, 1.59 and 2.69 mg/L (no old media for 5.6 and 10 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 20 L filled
- 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: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite
Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener).
- Total organic carbon: 0.92 mg/L
- Metals: all < LoD
- Pesticides: all < LoD
- Chlorine: 0.27 mg/L
- Conductivity: 419 uS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality, sub-lethal effects (24 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Initial study, test concentration 10 mg/L
- Range finding study
- Test concentrations: 1.0 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 3.2 - 5.6 mg/l

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 3.2 - 5.6 mg/l

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 3.2 - 5.6 mg/l

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI: 3.2 - 5.6 mg/l

Duration:

86 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: 0%
- Effect concentrations exceeding solubility of substance in test medium: At the start of each mixing period the loading rates were observed to be clear colorless water columns with globules of test item floating on the surface. At the end of each mixing period, and after the 1-Hour settlement period the loading rates were observed to be clear colorless water columns with an oily layer of test item floating on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

The LL50 values and associated confidence limits were calculated using the geometric mean method.

Sublethal observations / clinical signs:

Cumulative mortality

Nominal loading rate (mg/L)	Cumulative mortality (initial population is 7)						% Mortality
	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours	96 hours
Control	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	0
5.6	0	0	7	7	7	7	100
10.0	7	7	7	7	7	7	100

Sub-lethal effects

Nominal loading rate (mg/L)	Sub-lethal effects	Time
		3 hours	6 hours	24 hours	48 hours	72 hours	96 hours
Control	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
1.0	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
1.8	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
3.2	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7
5.6	Loss of equilibrium	7/7	7/7	A/D
10.0		A/D

Graphs are presented below.

Validity criteria fulfilled:

yes

Remarks:

Mortality 0% in controls, physical-chemical parameters within acceptable limits.

Conclusions:

The acute toxicity (96h-LL50) of Litsea cubeba towards Oncorhynchus mykiss is 4.2 mg/L.

Executive summary:

The acute toxicity of Litsea cubeba towards Oncorhynchus mykiss was investigated according to OECD guideline 203 under GLP. Fish were exposed to nominal WAFs of 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L in a semi-static setup and were observed for 96 hours. The 96h-NOELR and 96h-LL50 were found to be 3.2 and 4.2 mg/L respectively.

Description of key information

96h-LL50: 4.2 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.2 mg/L

Additional information

For Eucalyptus citriodora, ext., no studies on the aquatic toxicity are available for fish. For this endpoint, read-across is conducted to Litsea cubeba and Cymbopogon winterianus.

Summary of fish studies for the citral/citronellal group

Endpoint	Result	Remarks	Reference
96h-LL50	4.2 mg/l (WAF Litsea cubeba, ext.)	OECD 203, GLP, Rel. 2	Harlan Laboratories, 2014
96h-LL50	7.5 mg/l (WAF Cymbopogon winterianus, ext.)	OECD 203, GLP, Rel. 2	Harlan Laboratories, 2014

Two toxicity studies are available with other oils of the citral/citronellal group, see table. Read across is conducted to these studies. The justification of this read across is described in detail in the read across justification document included in the endpoint study records.

The highest toxicity (lowest WAF value) was observed in the study with Litsea cubeba, ext.. This key study was carried out using water accommodated fractions (WAFs). The nominal effective loading rate 96h-LL50 = 4.2 mg/l (Harlan Laboratories, 2014). Supporting evidence is obtained from the other citral/citronellal oil Cymbopogon winterianus, ext..