Melaleuca alternifolia, ext.

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Melaleuca alternifolia, ext.

EC Number:

285-377-1

EC Name:

Melaleuca alternifolia, ext.

Cas Number:

85085-48-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

Essential oil of melaleuca alternifolia

Test material form:

liquid

Specific details on test material used for the study:

- Physical state: Liquid.
- Purity: 100%
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil).
- Lot No.: 1215

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: A sample of activated sludge was obtained from the aeration tank of Worlingworth sewage treatment works, which treats predominantly domestic waste, three days before the start of the test. On the day of collection, the sample was passed through a sieve (mesh ca. 1 mm²), transferred to the laboratory then aerated until used.

- Preparation of inoculum for exposure: A sub-sample (ca. 300 ml) was taken and washed three times in mineral salts medium (MSM) by centrifugation (ca. 3000 rpm for ca. 1 minute), the supernatant removed and the volume replenished. The final sample was made up to volume with MSM and aerated until used. Aliquots (10 ml) of a homogenised sample of the washed activated sludge were filtered through dried (approximately 105°C) and pre-weighed Whatman GF/C filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed to determine the solids level in the sludge. An appropriate volume of the washed sludge was used to inoculate vessels containing MSM to give a final suspended solids concentration of 4mg/l. This inoculated medium was aerated ("aged") for three days prior to test initiation, with oil free air treated to remove carbon dioxide (Carbosorb AS) and dried (fused CaCl2).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mgC/l

Based on:

ThIC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

- Test temperature: 22 ± 2°C.
- pH: At test initiation (Day 0) inoculated medium that had been aerated for three days was pH adjusted to 7.4 ± 0.2 with 5M HCl then used to prepare test and control mixtures. The reference substance was added from an aqueous stock solution to a separate vessel of pH adjusted medium to give a final concentration of 10 mgC/l. Preliminary work to establish whether the test substance adversely affected the pH of the mineral salts medium confirmed that pH compensation was unnecessary.
- Suspended solids concentration: 4 mg/l
- Continuous darkness: Yes.
- Number of culture flasks/concentration: Three test cultures were analysed on each of the following sampling occasions: Day 0, Day 5, Day 7, Day 11, Day 12, Day 13, Day 14 and Day 22. Three control cultures were also analysed on each of these sampling occasions. Five test cultures and five control cultures were sampled on Day 28.
- Test performed in closed vessels due to significant volatility of test substance: Aliquots (100 ml) of medium from the designated vessels were transferred to numbered and appropriately labelled vials (nominal 160 ml capacity). Each vial was sealed immediately after the addition of the appropriate medium, and in the case of the test mixtures, the addition of the test substance (1.4 μl, equating to 10 mg of carbon/litre), with aluminium coated septa and crimp sealed. The cultures were incubated until the appropriate sampling occasion on an orbital shaker at a speed of 150 - 200 rpm in darkness at a temperature of 22 ± 2°C and inorganic carbon concentrations determined at intervals in three mixtures from each group (five cultures on the final analysis occasion).

Reference substance

Reference substance:

other: sodium benzoate (AR grade)

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

43.8

Sampling time:

5 d

Remarks on result:

other: mean

Remarks:

tea tree oil

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

60.7

Sampling time:

13 d

Remarks on result:

other: mean

Remarks:

tea tree oil

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Value:

82.8

Sampling time:

28 d

Remarks on result:

other: mean

Remarks:

tea tree oil

Details on results:

The levels of CO2 produced in control, test and reference mixtures and percentage degradation at intervals during the test are given in Tables 1-4 and 5, respectively (see 'Any other information on results'). Biodegradation of the test and reference substances is illustrated in the attached Figure 1.

The pH of MSM at the start of the test was 7.5. The temperature of a 100 ml volume of water held under test conditions ranged from 20.0°C to 22.3°C during the test period.

Sodium benzoate biodegradation had achieved 63% by Day 5, 77% by Day 14 and 87% by Day 28. In the presence of Tea Tree Oil, sodium benzoate had been degraded by 62% after 14 days. The production of CO2 in the control cultures, expressed as a percentage of the nominal organic carbon load as test substance in the test system (at most, 5.7% on Day 28) was acceptable for this assay system (recommended maximum, 15%). These results confirm that the inoculum was viable, the test was valid and that the test substance had not adversely affected the degradative activity of the inoculum at the test concentration.

The production of CO2 by mixtures containing Tea Tree Oil was equivalent to 10% of the theoretical maximum by approximately Day 3, assuming a lag period of three days, and 60% by approximately Day 13; degradation had reached 83% by the end of the test on Day 28.

BOD5 / COD results

Results with reference substance:

The ThOD value and carbon content of sodium benzoate were calculated from its empirical formula to be:
Sodium benzoate - C7H5O2Na : MW = 144.
Carbon content = 58.3%
ThOD = 1.67 mgO2/mg
The biodegradation of sodium benzoate in the presence of the test substance was monitored in the sealed-vessel CO2 evolution test in order to assess whether inhibitory effects were exerted on the activity of the inoculum. None were observed.

Any other information on results incl. tables

Elemental (C, H, N) analysis of the test substance was performed as part of the study at MEDAC Ltd. The measured carbon content (81.4%) was used in calculations of the dose quantity in the sealed-vessel CO₂ evolution test, for a volume equating to 10 mg C/l.

The results obtained in this study fulfilled validity citeria for assay precision and degradation of the positive control substance.

Table 1. Inorganic carbon measurements in the control cultures in the sealed-vessel CO₂ evolution test.

Day	Analysis Replicate	Control culture (mg IC/l)
		1	2	3	4	5
0	1	1.14501	1.10299	1.11388	-	-
	2	1.14969	1.09055	1.11232	-	-
5	1	1.31729	1.26987	1.31570	-	-
	2	1.30622	1.26513	1.29357	-	-
7	1	1.23267	1.41009	1.31072	-	-
	2	1.32576	1.40707	1.31072	-	-
11	1	1.33731	1.32652	1.55076	-	-
	2	1.37895	1.33422	1.49493	-	-
12	1	1.42995	1.51759	1.41185	-	-
	2	1.37418	1.25244	1.28546	-	-
13	1	1.37230	1.52187	1.52490	-	-
	2	1.30756	1.37682	1.32862	-	-
14	1	1.50299	1.39929	1.27774	-	-
	2	1.18539	1.29139	1.37190	-	-
22	1	1.400	1.515	1.543	-	-
	2	1.462	1.511	1.544	-	-
28	1	1.756	1.605	1.722	1.578	1.486
	2	1.716	1.632	1.707	1.926	1.797

Samples analysed using the OI Model 1010 carbon analyser were reported to three decimal places and samples analysed using the OI Model 700 carbon analyser were reported to five decimal places.

Table 2. Inorganic carbon measurements in the cultures containing the reference substance in the sealed-vessel CO₂ evolution test.

Day	Analysis Replicate	Reference culture (mg IC/l)
		1	2	3	4	5
0	1	1.04860	1.03618	1.04394	-	-
	2	1.05636	1.03773	1.04083	-	-
5	1	7.61427	7.55124	7.56078	-	-
	2	7.61236	7.5436	7.54170	-	-
7	1	7.79767	7.85488	8.41532	-	-
	2	8.30849	7.97328	8.40218	-	-
11	1	8.79882	8.93642	8.76015	-	-
	2	9.38190	9.21518	8.81042	-	-
14	1	8.83873	8.89430	9.20246	-	-
	2	9.48014	8.87896	8.94227	-	-
22	1	9.887	10.118	10.015	-	-
	2	9.821	10.066	9.862	-	-
28	1	10.771	10.501	10.375	10.386	10.361
	2	10.343	10.321	10.332	10.198	10.204

Samples analysed using the OI Model 1010 carbon analyser were reported to three decimal places and samples analysed using the OI Model 700 carbon analyser were reported to five decimal places.

Table 3. Inorganic carbon measurements in the cultures containing the test substance in the sealed-vessel CO₂ evolution test.

Day	Analysis Replicate	Test culture (mg IC/l)
		1	2	3	4	5
0	1	1.12166	1.10766	1.21518	-	-
	2	1.13256	1.10766	1.26987	-	-
5	1	5.55568	5.68567	5.80345	-	-
	2	5.55388	5.70195	5.76173	-	-
7	1	5.81943	5.64091	5.74305	-	-
	2	5.89947	5.71532	5.90296	-	-
11	1	6.49825	7.15650	6.85323	-	-
	2	6.51637	7.06431	6.97789	-	-
12	1	7.29730	6.62462	7.14504	-	-
	2	7.26280	7.06195	7.13058	-	-
13	1	7.62514	7.14034	8.03322	-	-
	2	7.54813	6.72134	7.79443	-	-
14	1	7.44233	7.05637	7.23875	-	-
	2	8.41809	7.11463	7.31564	-	-
22	1	9.935	9.566	8.755	-	-
	2	10.033	9.423	8.781	-	-
28	1	9.356	10.102	10.548	9.840	9.986
	2	9.217	10.249	10.514	9.997	9.941

Samples analysed using the OI Model 1010 carbon analyser were reported to three decimal places and samples analysed using the OI Model 700 carbon analyser were reported to five decimal places.

Table 4. Inorganic carbon measurements in the test plus reference cultures in the sealed-vessel CO₂ evolution test.

Day	Analysis Replicate	Test plus reference culture (mg IC/l)
		1	2	3
0	1	1.06723	1.02997	1.05791
	2	1.07811	1.03773	1.06102
5	1	8.75441	9.57644	9.42945
	2	9.28505	9.50791	9.56837
14	1	13.6865	13.1911	13.0344
	2	13.8938	13.3591	12.7039

Table 5. Percentage biodegradation of the test and reference substances in the sealed-vessel CO₂ evolution test.

Group	Culture replicate	Day number and percentage biodegradation
		5	7	11	12	13	14	22	28
		% deg	%deg	%deg	%deg	%deg	% deg	% deg	% deg
Reference substance - Sodium benzoate (10 mgC/l)	1	63.2	67.2	76.9	-	-	78.2	83.6	88.6
	2	62.5	65.8	76.7	-	-	75.5	86.0	87.2
	3	62.6	70.8	73.8	-	-	77.3	84.4	86.6
	4	-	-	-	-	-	-	-	86.0
	5	-	-	-	-	-	-	-	85.9
	Mean % deg	62.8	67.9	75.8	-	-	77.0	84.7	86.9
Test substance - Tea tree oil (10 mgC/l)	1	42.6	45.3	51.0	59.0	61.8	65.9	84.9	75.9
	2	44.0	43.5	57.1	54.6	55.3	57.5	80.0	84.8
	3	44.9	44.9	55.1	57.6	65.1	59.4	72.7	88.4
	4	-	-	-	-	-	-	-	82.3
	5	-	-	-	-	-	-	-	82.7
	Mean % deg	43.8	44.5	54.4	57.1	60.7	60.9	79.2	82.8
Inhibition assay - Sodium benzoate (10 mgC/l)	1	33.4	-	-	-	-	67.0	-	-
	2	38.7	-	-	-	-	61.9	-	-
	3	38.2	-	-	-	-	57.8	-	-
	4	-	-	-	-	-	-	-	-
	5	-	-	-	-	-	-	-	-
	Mean % deg	36.8	-	-	-	-	62.2	-	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The production of CO2 by mixtures containing tea tree oil was equivalent to 60% by approximately Day 13 and 83% by the end of the test on Day 28. Tea tree oil was considered to be readily biodegradable under this test condition, assuming a lag period equating to 3 days of incubation.

Executive summary:

The ready biodegradability of tea tree oil was assessed using the Sealed-Vessel CO2 Evolution test based on International Standard ISO 14593 and draft Test Guideline OECD 310 (CO2 in sealed vessels (Headspace Test)) 2003.  Inoculated mineral salts medium (100 ml), aged for three days with CO2-free air in the laboratory, was added to a series of 160 ml vials and the test and reference substances added to appropriate bottles (nominally, 10 mgC/l).  The vials were sealed then shaken until destructively analysed for inorganic carbon following the addition of concentrated sodium hydroxide to selected cultures at intervals.  The reference substance, sodium benzoate, was degraded by 62% after 14 days in the presence of tea tree oil, so the material was not considered inhibitory to the activity of the inoculum.  CO2 evolution in control cultures, as a percentage of the nominal organic carbon load as test substance, was acceptable for this assay system.  The production of CO2 by mixtures containing tea tree oil was equivalent to 60% by approximately Day 13 and 83% by the end of the test on Day 28.  Tea tree oil was considered readily biodegradable under this test condition, assuming a lag period equating to 3 days of incubation.