p-mentha-1,3-diene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2014 - December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Commercial name: Terpinene Alpha
Givaudan Code: 9202001
Chemical Nature: 1-methyl-4-(1-methylethyl)-1,3-cyclohexadiene
Empirical Formula: C10H16
Molecular Weight: 136.2 g/mol
CAS No.: 99-86-5
Lot No.: VE00294368
Purity: 94.1%
Expiration Date: 2015-04-06

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

Source of inoculum
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland).

Storage and treatment
The sludge was kept aerobic and used on the same day as collection. Sludge was washed with mineral medium (centrifuging at 1000 g for 10 mins, discarding the supernatent and resuspending in the mineral medium). The dry weight and suspended solids of the sludge was determined by taking two 50 ml samples of sludge, evaporating the water off in a steam bath and drying in an oven at 105 - 110 °C for two hours and weighing the residue. The dry weight of suspended solids was 4.2 g/l which was diluted to 2.62 g/l during the test. To obtain a concentration of 30 mg/l (dry weight ) in 437 ml total volume, 5.00 ml of sludge was added to 432 ml of mineral medium.

Duration of test (contact time):

70 d

Initial conc.:

15.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Method is the one described in 301F in the OECD guidelines for testing of chemicals.

Test Conditions
- Composition of mineral medium:
Four stock solutions were prepared.
Solution A, dissolved into 1 litre of water:
KH2PO4: 85 g
K2HPO4: 21.75 g
Na2HPO4.2 H2O: 33.4 g
NH4Cl: 0.5 g
Solution B, dissolved into 1 litre of water:
CaCl2: 27.5 g
Solution C, dissolved into 1 litre of water:
MgSO4.7 H2O: 22.5 g
Solution D, dissolved into 1 litre of water:
FeCl3.6 H2O: 0.25 g
HCl Conc.: one drop

The mineral medium was prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if neccessary adjusted to 7.4 +- 0.2 with phosphoric acid of potassium hydroxide.
- Test temperature: 20.8-21.9°C
- pH: 7.6-8.2
- Suspended solids concentration: 4.2 g/l, diluted to 2.62 g/l
- Continuous darkness: Not reported

Test system
- Number of culture flasks/concentration: Two replicates each of the test substance at 15.2 mg/l, Reference substance (sodium benzoate at 100 mg/l) and controls (total six flasks).
- Measuring equipment: Oxitop Control system, Wissenschaftlich-Technische Werkstatten (WTW) Weilhiem, Germnay.

Test substance samples (6.65mg, corresponding to 15.2 mg/l in 437 ml of test medium) were added to the test flasks. Flasks were filled with 432 ml of mineral medium. Samples of test/reference substance are added. 5.00ml of suspended sludge diluted to a concentration of 2.62 g/l dry matter was added. Every day oxygen consumption of each flask is recorded by the Oxitop test flasks, and the temperature and stirring were checked. At the end of the test period, the pH of each flask was measured again.
Sodium hydroxide pellets were placed in the quivers at the top of the bottle and the flasks closed tightly with the measuring heads. The flasks were allowed to equilibrate to the test temperature. The measurement was started by programming the measuring unit of the oxitop test flasks, and the test flasks are placed in the temperature controlled unit of the Oxitop test flasks. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment). Every day the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.

The duration of the test was extended passed the 28 day mark for ready biodegradation to 70 days. The OECD TG 301 state "Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau
has not been reached by day 28, but in such cases the chemical would not be classed as readily biodegradable."

The optional toxicity control as suggested by OECD guideline 301F was not performed. The validity of the study was not adversely affected.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

30

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

34

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

40

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

48

Sampling time:

42 d

Parameter:

% degradation (O2 consumption)

Value:

60

Sampling time:

56 d

Parameter:

% degradation (O2 consumption)

Value:

62

Sampling time:

60 d

Parameter:

% degradation (O2 consumption)

Value:

66

Sampling time:

70 d

Details on results:

ThOD for test substance = 3.29 mg O2/mg.

Oxygen uptakes, as read on the Oxytop controller, are corrected to account for the small differences between actual and nominal concentrations of the test and reference substances. The repeatability validity criterion (not more than 20% difference between replicates) was fullfilled. Therefore, the test is considered valid. The test substance did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.
It was shown that alpha-Terpinene undergoes 40% biodegradation after 28 days under the test conditions which is below the 60% required in the guidelines to classify this substance as "readily biodegradable". However, there was 62% degradation after 60 days and 66% degradation at 70 days. As such, alpha-terpinene should be regarded as not readily biodegradable but is inherently and ultimately biodegradable.

Results with reference substance:

87% biodegredation in 28d with reference substance sodium benzoate

Theoretical Oxygen Deman (ThOD) of test sustance was calulated to be 3.29 mg O₂/mg

Table 2. O₂ Uptake (mg O₂/l adjusted to nominal concentrations):

	Days:		14	21	28	42	56	60	70
O2 Uptake of Sludge (inoculum blank)	3	B1	19.2	23.4	23.4	25.6	25.6	27.7	27.7
	4	B2	19.0	23.3	25.4	27.5	27.5	27.5	27.5
	mean	B	19.1	23.4	24.4	26.6	26.6	27.6	27.6
O2 Uptake of Test Subst. + sludge	15	C1	33.0	39.2	43.3	49.5	55.8	57.9	59.9
	16	C2	35.0	41.1	45.2	51.4	57.7	59.7	61.8
O2 Uptake of Test Substance		C1 -B	13.9	15.9	18.9	23.0	29.2	30.3	32.3
		C2 -B	15.9	17.8	20.8	24.9	31.1	32.1	34.2
% biodegradation of test substance		D1	28	32	38	46	58	61	65
		D2	32	36	42	50	62	64	68
	mean	D	30	34	40	48	60	62	66

Calculations:

B1, B2, C1, C2, A1, A2, E1: Experimental O2 uptake values.

B = (B1 + B2)/2

D1 = 100 * (C1 -B)/ThOD * [S]

D2 = 100 * (C2 -B)/ThOD * [S]

D=(D1 +D2)/2

where [S]: Initial test substance concentration (mg/l)

Validity criteria fulfilled:

yes

Remarks:

The optional toxicity control recommended in OECD 301F guidelines was not performed. The validity of the study is not adversely affected.

Interpretation of results:

inherently biodegradable

Conclusions:

It was shown that alpha-terpinene undergoes 40% biodegradation after 28 days under the test conditions which is below the 60% required in the guidelines to classify this substance as "readily biodegradable". However, there was 62% degradation after 60 days and 66% degradation at 70 days. As such, alpha-terpinene should be regarded as not readily biodegradable but is inherently and ultimately biodegradable.

Executive summary:

The biodegradation of the test item (alpha-terpinene) was assessed according to the OECD TG 301F. It was shown that alpha-terpinene undergoes 40% biodegradation after 28 days under the test conditions which is below the 60% required in the guidelines to classify this substance as "readily biodegradable". However, there was 62% degradation after 60 days and 66% degradation at 70 days. As such, alpha-terpinene should be regarded as not readily biodegradable but is inherently and ultimately biodegradable.

Description of key information

Key study: Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. Alpha-terpinene underwent 40% biodegradation after 28 days under the test conditions and should be regarded as not readily biodegradable although inherently and ultimately biodegradable since there was 62% degradation after 60 days and 66% degradation at 70 days.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater