Theobromine

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:

From March 3rd to March 31st, 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to EU Method C.4-D. GLP study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No M15015C
- Manufacture date of the lot/batch: 01/2015
- Expiration date of the lot/batch: 01/2020
- Analytical purity: 99.8
OTHER SPECIFICS:
- Physical state: solid

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source: A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw, receiving predominantly domestic sewage. - Preparation of inoculum: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated, at the test temperature of 22 °C, until application next day. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Amount of inoculum suspended: 30 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

(BOD)

Details on study design:

TEST CONDITIONS
- pH: 7.4 ± 0.2
- Test temperature: 22 ± 2°C
- Volume of test solution in flask, V: 0.164 L
- Initial test item concentration: 100 mg/L (51.3 mg/L of organic carbon)
- Composition of medium: 10ml of solution (A) in 800 ml water, plus 1 ml solutions (B), (C), (D) and 11mL water. The following stock solutions were used, prepared with analytical grade reagents:
- Solution (A) contains: 8.50 g monopotassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium monohydrogen orthophosphate (K2HPO4), 33.40 g disodium monohydrogen orthophosphate dihydrate (Na2HPO4·2H2O), 0.50 g ammonium chloride (NH4Cl), in 1 L double-distilled water.
- Solution (B) contains: 27.50 g calcium chloride, anhidrous (CaCl2) in 1L water.- Solution (C) contains: 22.50 g magnesium sulphate heptahydrate (MgSO4·7H2O) in 1L water.
- Solution (D) contains: 0.25 g iron(III) chloride hexahydrate (FeCl3·6H2O) in 1L water.
- The water used is double-distilled, containing 3mg/L of organic carbon (< 10% of the organic carbon content introduced by the test item), checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.- Suspended solids concentration: 30 mg/L.

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #7, 8, 9, containing test item (100 mg/l) and inoculum 30mg/L SS.
- Measuring equipment: O2 uptake was measured by a closed WTW OxiTop OC 110 repirometer. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #1, 2, 3, containing only inoculum 30mg/L SS.
- Procedure control: flasks #4, 5, 6, containing reference item (sodium acetate 100 mg/l) and inoculum 30mg/L SS.
- Toxicity control: flasks #10, 11, 12, containing test item, reference item and inoculum 30mg/L SS.

STATISTICAL METHODS: The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.

Reference substance:

acetic acid, sodium salt

Remarks:

CAS No: 127-09-3, purity ≥ 98.5%, source: Eurochem.

Key result

Parameter:

other: BOD/ThOD

Value:

100

Sampling time:

28 d

Details on results:

After correction for oxygen uptake for nitrification and for the blank inoculum control, a 100 % of degradation was calculated on day 28.

Results with reference substance:

The reference item reached 75.5% biodegradation after 28d.

Table 1. Sample oxygen uptake: biodegradability

		time, days
		3	5	7	9	12	14	16	18	21	23	25	28
Test item O2uptake, mg/l	a1	22.1	135.6	204.7	231.8	242.0	247.1	250.3	251.7	255.9	256.0	259.1	260.8
	a2	17.1	135.3	201.5	229.7	245.0	249.7	253.7	256.4	260.1	262.0	264.2	270.7
	a3	22.0	111.6	152.9	190.5	227.4	236.8	239.2	241.3	244.9	245.0	245.8	248.0
	am. avg	20.4	127.5	186.3	217.4	238.1	244.5	247.7	249.8	253.6	254.3	256.4	259.9
Blank test O2uptake. mg/l	b1	17.1	20.3	25.6	28.4	32.8	34.5	37.4	38.2	40.2	42.9	43.4	45.0
	b2	12.1	16.6	20.7	23.3	27.1	28.1	30.7	31.9	34.2	33.8	35.5	36.6
	b3	13.3	16.1	19.7	22.1	24.6	26.9	28.9	30.8	32.6	34.3	36.7	37.6
	bmavg	14.2	17.7	22.0	24.6	28.2	29.8	32.3	33.7	35.7	37.0	38.5	39.7
Reference item O2uptake. mg/l	w1	36.5	37.3	42.6	49.3	57.9	61.8	65.6	69.4	73.5	76.0	77.7	82.2
	w2	51.2	63.7	73.8	80.2	87.8	90.6	92.9	96.1	99.0	99.3	101.4	101.6
	w3	49.1	61.8	70.8	76.5	82.3	87.1	87.2	90.1	93.0	92.8	96.1	95.7
	wm. avg	50.1	62.7	72.3	78.3	85.0	88.9	90.0	93.1	96.0	96.1	98.8	98.6
Toxicity control O2uptake. mg/l	a4tox1	80.7	184.2	242.8	289.7	301.6	304.9	308.0	310.6	315.9	318.1	317.5	324.0
	a5tox2	76.4	181.1	253.8	291.9	308.1	316.5	321.8	324.8	333.5	335.7	338.6	343.8
	a6tox3	96.3	182.3	241.3	282.1	293.6	298.1	0.0	0.0	0.0	0.0	0.0	0.0
	toxm. avg	78.6	182.6	248.3	290.8	304.9	310.7	314.9	317.7	324.7	326.9	328.1	333.9
Corrected test item O2uptake, mg/l	a1- bm	7.9	118.0	182.8	207.3	213.8	217.3	218.0	218.0	220.2	219.0	220.6	221.1
	a2- bm	2.9	117.7	179.5	205.2	216.8	219.8	221.3	222.8	224.4	225.0	225.7	231.0
	a3-bm	7.8	94.0	130.9	165.9	199.3	207.0	206.8	207.6	209.2	208.0	207.3	208.3
Reference item % degradation ThOD = 0.78 mgO2/mg C = 100 mg/l	R1(w2)	47.4	59.0	66.4	71.3	76.5	77.9	77.6	80.0	81.1	79.9	80.6	79.3
	R3(w3)	44.7	56.6	62.5	66.6	69.4	73.5	70.3	72.4	73.5	71.5	73.8	71.8
	Rtoxavg	46.1	57.8	64.5	68.9	72.9	75.7	74.0	76.2	77.3	75.7	77.2	75.5

The result w₁is not taken into average value calculations, as it is distinctly lower than other reference item O₂uptakes.

The a₆tox3 result from flask #12 is not taken into average value calculation, as this flask was  withdrawn from test after 14 day incubation to nitrate and nitrite analysis

Validity criteria fulfilled:

yes

Remarks:

See overall remarks.

Interpretation of results:

readily biodegradable

Conclusions:

Theobromine was completely degraded (100%) within 28 days based on the BOD and ThOD. The test substance can be considered readily biodegradable.

Executive summary:

A 28-day ready aerobic biodegradability test through the manometric respirometry method was performed with theobromine according to OECD 301F / EC C.4 – D under GLP conditions. 100 mg/L of test item was inoculated with adapted activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature 22ºC(± 2) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. The validity criteria was considered fulfilled. The substance was completely degraded (100%) within the 28 -day period based on BOD (based on measurements of oxygen uptake) and ThOD. Theobromine was considered readily biodegradable.

Description of key information

Key study: OECD Guideline 301F. GLP study. The substance was completely degraded within a 28-day period. Theobromine is regarded as ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Key study: A 28-day ready aerobic biodegradability test was conducted through the manometric respirometry method with theobromine, according to OECD 301F and EC C.4 – D, under GLP conditions. At the 28th day of the test, the degradation of the test item attained 100% based on oxygen uptake and the ThOD. Therefore, the test substance was considered readily biodegradable.