Melaleuca alternifolia, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Principles of method if other than guideline:

In error, the protocol stated the test would be conducted in accordance with, amongst others, the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test" (1984), however this test followed the principles outlined in the most recently updated version of this guideline "Freshwater alga and Cyanobacteria, Growth Inhibition Test" (2006). This deviation from the protocol had no impact on either the integrity or validity of the study.

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Liquid.
- Purity: 100%.
- Lot/Batch No.: 1215
- Expiration date of the batch: 30 March 2009 (the substance was used within three months of opening the sealed container).
- 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 at the start of the test. After 96 hours, the contents of the replicate flasks from each group were pooled and four samples (approx. 20 ml) were taken from each level for analysis. Additional samples were also taken from flasks containing Tea Tree Oil at 5 and 80 mg/l but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells. Samples for chemical analysis were placed in vessels that were completely filled and sealed with no headspace.

- Sample storage conditions before analysis: The samples were either analysed immediately or stored in a refrigerator for up to three days until duplicate samples from each set were analysed. The remaining samples were held refrigerated in case further analysis was required.

Vehicle:

no

Details on test solutions:

As the test substance was a complex mixture comprising many components with a range of different molecular weights and solubilities, the algae were exposed to Water Accomodated Fractions (WAFs) of the test substance.

- Preparation of test substance: The method of preparation used during the definitive test was based on the results of the range finding tests. Glass bottles (total capacity, ca. 2.68 l) were completely filled with algal culture medium and sealed using a screw cap fitted with a silicone septum. The contents of the vessels were stirred at a rate capable of creating a vortex of approximately 5% of the static depth of the medium in each vessel before they were sealed. The test substance (15, 30, 60, 120 or 241 μl) was injected through the silicone septum into stirring dilution medium to give media with nominal Tea Tree Oil loading rates of 5, 10, 20, 40 and 80 mg/l (corrected for specific gravity, 0.89 g/l; HLS Study No. CSV/0010). The preparation vessels 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 water accommodated fraction (WAF) was removed and discarded. The required volume of medium (WAF) was then siphoned from mid-point in each bottle and used in the test.

An aliquot (8.49 ml) of the secondary algal inoculum was added to a portion (900 ml) of the test medium at each concentration to give an initial cell density of 1 x 10^4 cells/ml. An aliquot (60 ml) of the appropriate inoculated test medium was added to each of the test vessels, which were completely filled with no headspace and sealed.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Strain: CCAP 278/4
- Source: Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 1 May 2007.
- Pre-culture: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 ml) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 1.06 x 10^6 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

No data provided.

Test temperature:

The temperature of the prepared control and test medium at the start of the test and of the media remaining in each vessel at the end of the test were recorded. The minimum and maximum temperature was determined each day. The temperature remained within acceptable limits throughout the study. The temperature of the incubator ranged between 23.0 and 24.2°C.

pH:

The pH of the prepared control and test medium at the start of the test and of the media remaining in each vessel at the end of the test were recorded. The pH remained within acceptable limits throughout the study. The pH of the medium in the control and test groups up to 20 mg/l increased by more than 1.5 units during the test (range 6.99 - 7.08 at test start, 9.09 - 9.82 at termination). This increase in pH was attributed to the growth of the algal cells at these levels under the exposure conditions employed in this test. As the objectives of the test were met, this had no impact on the validity or integrity of the study.

Dissolved oxygen:

No data provided.

Nominal and measured concentrations:

The nominal Tea Tree Oil loading rates used in this study were 5, 10, 20, 40 and 80 mg/l. Replicate vessels containing 5 and 80 mg/l had culture medium incubated under test conditions without algal cells. The mean measured concentrations of terpinen-4-ol at nominal Tea Tree Oil concentrations of 40 and 80 mg/l were 7.75 and 23.0 mg/l, respectively.

Details on test conditions:

- Material, size, headspace, fill volume: Glass reaction vials, 60 ml total capacity, completely filled and sealed, with no headspace.
- Test vessel preparation: Before the start of the test, the required number of empty test vessels were covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). Twenty-four flasks were established for the control group and twelve flasks for each test group, plus two additional flasks at nominal concentrations of 5 and 80 mg/l, which contained test medium but no algal cells. After each vessel was completely filled with the inoculated test medium, they were sealed using aluminium caps fitted with Teflon coated seals. All of the control and test flasks were incubated. The media remaining in the preparation flasks were used for water quality measurements at the start. The control cultures were prepared as for the test medium except that no test substance was added and a larger volume (1.6 litres) of medium was made.
- Initial cells density: 1 x 10^4 cells/ml.
- Other: Suspension of the algal cells was ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute.

- Standard medium used: Yes, sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201. Because the test was conducted in completely filled and sealed vessels, with no headspace for gaseous exchange, the concentration of sodium bicarbonate in the culture medium was increased to 300 mg/l. The pH of the culture medium was also adjusted to approximately 7.0 to reduce the impact of increasing pH caused by cell growth under sealed conditions.
- Other: At the start of the test, the test media were colourless.

- Photoperiod, light intensity and quality: Glass vials, each containing control or test culture (60 ml), were placed in an illuminated orbital incubator according to a random number sequence; the position of the test vessels was randomised each day of the test. The cultures were incubated, without renewal of medium for 96 hours under continuous illumination provided by 30 W "cool white" 1 metre fluorescent tubes. The light intensity at each position in the incubator was determined daily and ranged from 3260 to 4580 lux over the test period (mean 3963 lux). The minimum light intensity differed from the mean value by up to 18% during the test. This did not affect the validity of the study as the position of individual cultures in the incubator was randomised on each sampling occasion.

- Determination of cell concentrations: Samples were taken from control and test flasks at 24, 48, 72 and 96 hours and the cell densities measured using a Coulter counter. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, conducted in duplicate. Six control and three test flasks were removed from the incubator each day and were used for cell counting. These cultures were then discarded. The presence of any abnormal cells was also noted.

- Range finding study: The study comprised three range finding tests, a main test (originally planned as the definitive test) and the definitive test. A range finding test was conducted at nominal loading rates of 0.01, 0.1, 1 and 10 mg/l in which no inhibition of growth of the algal cells was observed. As a result, the test was repeated at a single loading rate of 100 mg/l; growth of the algal cells at this level was inhibited by 100% after 72 hours when compared to the control group. Based on the results of these tests, a main test (originally planned as the definitive test) was conducted at nominal concentrations of 5.6 (originally intended as 5 mg/l however a slightly higher level was prepared in error), 10, 20, 40 and 80 mg/l plus a dilution medium control group. However, this test was terminated after 72 hours as growth of the algal cells was inhibited at the lowest concentration. Consequently, an additional range finding test was conducted which confirmed that inhibition observed in the main test was not attributed to the test substance. Consequently, the main test was repeated (as the definitive test); the results of this test were in line with those of range finding tests.

Reference substance (positive control):

no

Remarks:

Sensitivity of Pseudokirchneriella subcapitata cultured in this lab is periodically assessed using ref. substance potassium dichromate. The most recent result (72-hour EbC50 0.63 mg/l), was in line with typical toxicity data for this substance.

Duration:

96 h

Dose descriptor:

EL10

Effect conc.:

25.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

biomass

Remarks on result:

other: 95% CL = 22.6 & 27.8 mg/l

Duration:

96 h

Dose descriptor:

EL10

Effect conc.:

29.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 25.2 & 30.2

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

30.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

biomass

Remarks on result:

other: 95% CL = 27.1 & 33.4 mg/l

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

35.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

growth rate

Remarks on result:

other: 95% CL = 33.7 & 36.2 mg/l

Duration:

96 h

Dose descriptor:

other: LOAELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

other: NOAELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

other: water-accommodated fraction

Basis for effect:

growth rate

Details on results:

The cell density in one of the replicate flasks in the control group was erroneously low after 48 and 72 hours. As a result, this replicate has been excluded from the statistical analysis.

Biomass:
The calculated area under the growth curve values (biomass) are shown in Table 2 and are expressed in terms of percentage inhibition by comparing the test group values with that of the control curve. Algal growth was significantly inhibited at 5 mg/l and significantly enhanced at 10 and 20 mg/l. These results are not attributed to the test substance as they are considered to be the result of variable cell growth in replicate vessels at these levels after 48 and 72 hours. At the end of the test, the cell densities at these levels were comparable to the control group showing no adverse effects on growth.
As a result of apparent stimulation of the growth of algae at 10 and 20 mg/l, the No Observed Adverse Effect Loading Rate (NOAELR) and Lowest Observed Adverse Effect Loading Rate (LOAELR) have been used in the calculation of the test results for biomass (the area under the curve). The NOAELR and LOAELR are defined as the highest concentration that caused no adverse effect and lowest concentration that caused an adverse effect (i.e. reduction) in growth of the algae.

Attached Figure 2 shows % growth inhibition against nominal concentration after 96 hours.

Growth rate:
The calculated average specific growth rate values are shown in Table 3 and are expressed in terms of percentage inhibition by comparing the test group value with that of the control curve over each time specific growth period (0 - 24, 24 - 48 hours etc) and over the entire test period (0 - 96 hours).
The mean coefficient of variation for the daily change in specific growth rates (0-24 hours, 24-48 hours, 48-72 hours and 72-96 hours) in the control cultures was < 35%.
The coefficient of variation of average specific growth rates over the 96 hour test period in replicate control cultures was < 7%.

- Observation of abnormalities: No microscopic abnormalities of the cells were detected.

Reported statistics and error estimates:

The data were compiled in an Excel spreadsheet and analysed using SAS 8.2 (SAS Institute 1999). All 95% confidence intervals for EC50 were calculated using the likelihood ratio method (Donaldson and Schnabel, 1985). For Area Under Curve (AUC), each flask at each time was a separate observation in the analysis. These were analysed by mixed linear model with treatment by time as a fixed factor, separate variance for each time, all observations uncorrelated. The area under curve for each treatment was estimated as AUC0-96h = 24mean24 + 24mean48 + 24mean72 16mean96 - 60 x time 0 value. The significance of the difference between treatment and control was determined as the significance of the contrast of the difference between the treated and control AUCs. For growth rate, Williams' test (1971, 1972) and Dunnetts test (1955, 1964) were also used to compare each treated group with the control.

Table 2. Inhibition of growth - Area under curve (96 hours).

Nominal tea tree oil loading rate (mg/l)	AUC	% inhibition	p
Control	31.2	0.0
5 mg/l	27.5	12.0	<0.001***^$
10 mg/l	34.5	-10.6	<0.001***^$
20 mg/l	35.6	-13.9	<0.001***^$
40 mg/l	1.2	96.0	<0.001***
80 mg/l	0.5	98.3	<0.001***

p values by contrasts from a mixed model

* p < 0.05, ** p < 0.01, *** p < 0.001

^$ not attributed to the test substance

Table 3. Inhibition of growth - average specific growth rate.

Parameter	Nominal tea tree oil loading rate (mg/l)	Sample size	Mean	% Inhibition	p
Growth rate to 96 hours	Control	5	0.0472	0.0	-
	5	3	0.0483	-2.3	>0.999^w
	10	3	0.0479	-1.4	>0.999^w
	20	3	0.0479	-1.5	>0.999^w
	40	3	0.0104	78.1	<0.001***^w
	80	3	0.0025	94.6	<0.001***^w
0 to 24 hours	Control	5	0.0575	0.0	-
	5	3	0.0490	14.9	0.160^w
	10	3	0.0466	19.0	0.160^w
	20	3	0.0518	10.0	0.160^w
	40	3	0.0344	40.2	<0.001***^w
	80	3	0.0247	57.1	<0.001***^w
24 to 48 hours	Control	5	0.0699	0.0	-
	5	3	0.0558	20.2	0.159^D
	10	3	0.0791	-13.2	0.512^D
	20	3	0.0781	-11.7	0.615^D
	40	3	0.0033	95.2	<0.001***^D
	80	3	-0.0050	107.2	<0.001***^D
48 to 72 hours	Control	5	0.0441	0.0	-
	5	3	0.0587	-33.0	>0.999^w
	10	3	0.0536	-21.5	>0.999^w
	20	3	0.0504	-14.2	>0.999^w
	40	3	0.0008	98.1	<0.001***^w
	80	3	0.0016	96.3	<0.001***^w
72 to 96 hours	Control	5	0.0172	0.0	-
	5	3	0.0297	-72.5	>0.999^w
	10	3	0.0121	29.6	0.229^w
	20	3	0.0113	34.6	0.161^w
	40	3	0.0029	83.4	0.002**^w
	80	3	-0.0111	164.3	<0.001***^w

p values are for the comparison with Control using Williams' test (W) and Dunnett's test (D)

* p < 0.05, ** p < 0.01, *** p < 0.001

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour EbC50 and ErC50 of Tea Tree Oil for the inhibition of algal growth when tested as water accommodated fractions were 30.8 and 35.9 mg/l respectively. The "no observed adverse effect loading rate" (NOAELR) for area under the growth curve and the "no observed effect loading rate" (NOELR) for growth rate was 20 mg/l.

Executive summary:

This study was conducted in accordance with the OECD Guideline for Testing of Chemicals No. 201 and assessed the effect of tea tree oil on the growth of the unicellular green alga Pseudokirchneriella subcapitata under non-axenic conditions.  The design of the study intentionally deviated from the guidelines in that the composition of the OECD medium and its pH were altered to suit the exposure regime employed.  Algal cultures, with an initial cell density of 1 x 10^4/ml, were exposed for 96 hours to Water Accommodated Fractions (WAFs) prepared from aqueous mixtures of tea tree oil, at nominal loading rates of 5, 10, 20, 40 and 80 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 the start of the test, the mean measured levels of terpinen-4-ol in samples of freshly prepared medium ranged between 76 and 99% of their nominal values except at 40 mg/l where the mean measured concentration was 69% of its nominal value.  In samples of expired (96-hours old) media, at 40 and 80 mg/l, the mean measured concentrations of terpinen-4-ol were 46% and 81% of their initial values respectively.  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.  The 96-hour EL50 for area under the growth curve was 30.8 mg/l, and the EL50 for the average specific growth rate (0 – 96 h) was 35.9 mg/l.  The NOAELR for area under the growth curve and the NOELR for growth rate was 20 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

35.9 mg/L

EC10 or NOEC for freshwater algae:

29.9 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 EL50.