Melaleuca alternifolia, 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

GLP compliance:

yes

Specific details on test material used for the study:

- Purity: 100%.
- Lot/Batch No.: 1215
- Expiration date of the lot: 30 March 2009
- Storage condition of test material: Ambient, in the dark.

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Four samples (approx. 20 ml) were taken from the freshly-prepared control and test media on each occasion (0, 24, 48 and 72 hours). Duplicate samples (approx. 20 ml) were also taken from the expired (24 hours old) medium in each vessel after 24, 48, 72 and 96 hours. Samples for chemical analysis were placed in vessels that were completely filled and sealed with no headspace.
- Sample storage conditions before analysis: On each occasion, the samples were stored in a refrigerator for up to seven days before one sample from each set was analysed. Reserve samples taken on Days 0, 1 and 2 of the test were analysed because low recoveries were obtained during the analysis of the original samples but, terpinen-4-ol was not detected in any of these samples suggesting that their integrity had been compromised by prolonged storage prior to the analysis.

Vehicle:

no

Details on test solutions:

- Method of test substance preparation: On each occasion, the test media were prepared by the direct addition of the test substance to the dilution medium. Glass aspirators (total capacity, 22.4 l) were completely filled with dilution medium and sealed with silicone bungs and laboratory film. The media were stirred at a rate capable of creating a vortex of less than 5% of the static depth of the medium in the vessels before they were sealed. The test substance (16, 32, 63, 126 or 252 μl) was injected into the stirring dilution medium in replicate (five per loading rate) aspirators to give nominal loading rates of 0.625, 1.25, 2.5, 5 and 10 mg/l (corrected for specific gravity, 0.89 g/l; HLS Study No. CSV/0010). The aspirators were covered with black bags and left to stir overnight. On cessation of stirring, the media were left to stand in the test area for approximately four hours before 20 ml of each WAF was removed and discarded. The required volume of medium (water accommodated fraction; WAF) was then siphoned mid-point from the replicate aspirators and used as the test medium. Samples were taken from the test vessels for the determination of water quality and chemical analysis. After the addition of the fish to each vessel, aliquots of the medium remaining in the aspirators were used to replace that removed from the test vessels so they remained completely filled leaving no headspace.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

- Common name: Rainbow trout
- Strain: Not specified.
- Source: The fish were supplied by a commercial fish farm in the UK and were reared at the farm from eggs which hatched in February 2007.
- Age at study initiation: Juveniles.

The size of the fish used in the definitive test was determined by weighing and measuring a sample of ten fish taken at random from the holding tank before the start of the definitive test.
- Length at study initiation: 4.9 to 6.3 cm (mean 5.63 cm).
- Weight at study initiation (mean wet weight): 1.75 g.

- Feeding during test: The fish were not fed during the 96-hour exposure period.
- Acclimation period: 14 days.
- Acclimation conditions: The fish were held in an aerated supply of diluent water under flow-through conditions until use. During the 14-day period immediately before the definitive test, temperatures remained within the range 14.2 to 14.4°C, pH values within the range 7.16 to 7.72, dissolved oxygen concentrations within the range 64 to 100% air saturation value (ASV) and total hardness within the range 140 to 160 mg/l as CaCO3.
- Type and amount of food: Commercial fish food (TROUW (UK) Ltd; Nutra Trout Fry 02), an amount equivalent to between 1 and 2% of the total wet-weight of fish in the holding tank.
- Feeding frequency: The fish were fed daily. No food was given during the 49-hour period immediately before exposure or during the exposure period itself.
- Health during acclimation: No medication was given in the 7 days before the definitive test, and mortalities were recorded as <2% in the 14 days before the definitive test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

The total hardness of the dilution water measured in the first and last vessel at the start of the test ranged from 162 to 174 mg/l as CaCO3.

Test temperature:

Treatment and control groups were maintained at 13.8 to 16.9°C throughout the exposure period. The temperature of the water in a satellite vessel was continuously monitored during the study. The protocol stated that the temperature of the test dilutions should be maintained between 12 and 15°C, remain constant to within ± 1°C and would not vary by more than 1°C during any 24-hour period during the study. However, these limits were not achieved at nominal loading rates of 0.625, 2.5, 5.0 or 10 mg/l. This deviation from the protocol does not affect the integrity or validity of the study as similar temperature ranges were measured at levels where no effect (0.625 mg/l) and significant effects (5.0 mg/l) on the fish were observed confirming that the effects on the fish were due to the toxicity of the test substance.

pH:

The pH range of each vessel (V) throughout the exposure period is stated below:
Control (V1): 7.14 - 7.30; Control (V2): 7.18 - 7.34.
0.625 mg/l - (V3): 7.24 - 7.45; (V4): 7.24 - 7.48.
1.25 mg/l - (V5): 7.24 - 7.50; (V6): 7.24 - 7.51.
2.5 mg/l - (V7): 7.23 - 7.50; (V8): 7.21 - 7.51.
5.0 mg/l - (V9): 7.21 - 7.52; (V10): 7.21 - 7.52.
10.0 mg/l - (V11): 7.23 - 7.53; (V12): 7.24 - 7.54.

Dissolved oxygen:

The range of dissolved oxygen concentrations of each vessel (V) throughout the exposure period is stated below (% air saturation value):
Control (V1): 71 - 91; Control (V2): 69 - 94.
0.625 mg/l - (V3): 68 - 99; (V4): 67 - 98.
1.25 mg/l - (V5): 69 - 98; (V6): 72 - 99.
2.5 mg/l - (V7): 66 - 100; (V8): 69 - 100.
5.0 mg/l - (V9): 62 - 102; (V10): 73 - 102.
10.0 mg/l - (V11): 65 - 104; (V12): 62 - 101.
The protocol stated that the dissolved oxygen concentration of the water in the holding tanks during the pre-exposure period should remain >80% of the air saturation value (ASV). However, the dissolved oxygen was measured to be 64 and 73% ASV on separate occasions during this period. This deviation from the protocol does not affect the integrity or validity of the study as in the five days preceding the definitive test the dissolved oxygen concentrations were in the range 94 to 100% ASV. In addition, mortalities in the holding tank were recorded as <2% in the 14 days before the definitive confirming the suitability of the fish for testing.

Salinity:

No data provided.

Nominal and measured concentrations:

The nominal concentrations of terpinen-4-ol were 0.516, 1.03, 2.07 and 4.13 mg/l at the following respective nominal Tea Tree Oil loading rates: 1.25, 2.50, 5.00 and 10.0 mg/l. The mean measured concentrations at each loading rate were as follows (V = vessel no.):
1.25 mg/l - (V5): 0.516 mg/l; (V6): 0.582 mg/l
2.50 mg/l - (V7): 1.08 mg/l; (V8): 1.12 mg/l
5.00 mg/l - (V9): 2.29 mg/l; (V10): 2.31 mg/l
10.0 mg/l - (V11): 4.47 mg/l; (V12): 4.52 mg/l

Details on test conditions:

- Type of vessel: Closed.
- Material, size, headspace, fill volume: Glass, 40 litre capacity, no headspace, completely filled and sealed.
- Aeration: No supplementary aeration was provided.
- Renewal rate of test solution: Daily batch renewal of the media.
- No. of organisms per vessel: 10 fish/vessel.
- No. of vessels per concentration: Two vessels/concentration.
- No. of vessels per control: Two control vessels were used.
- Loading rates: 0.625, 1.25, 2.5, 5 and 10 mg/l.

- Source/preparation of dilution water: The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through a water purification system. It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated tap water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.
- Chlorine: The levels of total and free chlorine in the diluent water remained at <0.05 mg/l for the duration of the definitive test.
- Intervals of water quality measurement: Daily records of temperature, pH and dissolved oxygen were kept for each control and test vessel together with measurements of total hardness for selected vessels at 0 hours. The concentration of total and free chlorine in the diluent water was determined on each day of the test.
- Other: The test media were colourless.

- Photoperiod: A photoperiod of 16 hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase.
- Light intensity: Ranged from 341 to 413 lux.

- Criteria of effect: The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to physical stimulation of the caudal peduncle. In addition to observations on mortality at approximately 3, 6, 24, 48, 72 and 96 hours, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish.

- Range finding study: A range finding test was conducted under semi-static conditions at nominal loading rates of 1, 10 and 100 mg/l. However, the test was invalid due to unacceptably low levels of dissolved oxygen in the test media. Consequently, a formulation trial was conducted that confirmed that satisfactory environmental conditions could be maintained if a larger volume of media were prepared. As a result, a second range finding test was conducted at nominal loading rates of 0.01, 0.1, 1 and 10 mg/l using the conditions employed in the formulation trial. At the end of the test, all of the fish at 10 mg/l had died; no significant mortality or sub-lethal effects were observed at remaining levels. Before the start of the definitive test, a trial was conducted to confirm that satisfactory environmental conditions could be maintained under the conditions proposed for the test. Based on the results of the second range finding test, the definitive test employed nominal loading rates of 0.625, 1.25, 2.5, 5 and 10 mg/l. In the definitive test, ten fish were placed at random into replicate test vessels (40 litre total capacity glass tanks) which were completely filled with the prepared control or test media and sealed with lids to provide an initial static loading of 0.44 g bodyweight/litre.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

7.75 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL = 5.81 & 12.2 mg/l

Details on results:

- Mortality: After 48 hours, one fish at 10 mg/l was accidentally lost whilst the fish were being transferred to the fresh medium. As sufficient numbers of fish remained to provide a reliable estimate of toxicity, this had no effect on the integrity or validity of the study. The highest concentration resulting in no significant mortality was 2.5 mg/l. At the highest loading rate (10 mg/l) 75% mortality was observed. See Table 2 in 'Any other information on results' and attached Fig. 2.
- Abnormalities and observations: No abnormalities were detected in controls or fish exposed to 0.625, 1.25 or 2.50 mg/l of test substance. Treatment-related effects were observed at 5 and 10 mg/l. Darkened pigmentation (body and eye orbits), hyperventilation and loss of orientation (swimming at the top of the vessel) were observed at 5 mg/l. Darkened pigmentation (body and eye orbits), quiescence and loss of orientation (swimming at the top of the vessel) were observed at 10 mg/l.

Reported statistics and error estimates:

LL50 values were calculated using the SAFEStat LD50 application (SAS 8.2.) and the mean measured concentrations. The "no observed effect loading rate" (NOELR) was derived by direct inspection of the data. An incidence rate of more than 10% of the fish affected per group is considered to be significant.

Sublethal observations / clinical signs:

Table 2. Cumulative mortality.

Nominal Tea Tree Oil loading rate (mg/l)	Vessel No.	Cumulative mortality (initial population = 10 fish/vessel)							96 h% mortality
		0.25 h	3 h	6 h	24 h	48 h	72 h	96 h
Control	1	0	0	0	1	1	1	1	10
	2	0	0	0	0	0	1	1	10
0.625	3	0	0	0	0	0	0	0	0
	4	0	0	0	0	0	1	1	10
1.25	5	0	0	0	0	0	0	0	0
	6	0	0	0	0	0	0	0	0
2.5	7	0	0	0	0	0	1	1	10
	8	0	0	0	0	0	1	1	10
5.0	9	0	0	0	0	0	0	0	0
	10	0	0	0	0	2	2	3	30
10.0	11	0	0	0	0	4	7	10	100
	12	0	0	0	0*	2	4	4	44

*  One fish accidentally lost during transfer to the fresh medium; not included in the calculation of the results.

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LL50 value for Tea Tree Oil with rainbow trout was 7.75 mg/l, with 95% confidence limits of 5.81 and 12.2 mg/l. The "no observed effect loading rate" was 2.50 mg/l.

Executive summary:

This study was conducted in accordance with the OECD Guideline for Testing of Chemicals No. 203 and assessed the acute toxicity of tea tree oil to rainbow trout (Oncorhynchus mykiss) under semi-static exposure conditions.  Duplicate groups of ten juvenile fish were exposed to Water Accommodated Fractions (WAFs) prepared from aqueous mixtures of tea tree oil, at nominal loading rates of 0.625, 1.25, 2.5, 5 and 10 mg/l.  The test was conducted in completely filled and sealed vessels.  Quantification of the test concentrations was based on the measured concentration of terpinen-4-ol.  At levels of 2.5 mg/l and above, the mean measured concentrations of terpinen-4-ol ranged between 88 and 129% of their nominal values and remained within 84 to 118% of their initial values during the test.  This excludes the results for a replicate sample of freshly prepared medium taken on Day 2 at 5 mg/l and the results of analysis of all expired samples taken on Day 2 where the results of analysis are considered to be erroneous.  In accordance with the recommendation of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Number 23), the test results have been expressed in terms of the nominal loading rates.  After 96 hours, the highest loading rate at which no significant mortality had occurred (NOELR) was 2.50 mg/l.  The 96-hour LL50 was 7.75 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

7.75 mg/L

Additional information

In accordance with the recommendation of the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Number23), the test results have been expressed in terms of the nominal loading rates (i.e. the nominal weights of the test substance used to prepare the aqueous mixtures from which theWAFs were removed), the LL50.