α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

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 December 17th, 2015 to January 14th, 2016.

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)

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)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-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 filtration. Such prepared sludge was washed in the medium and was placed in laboratory-scale unit where the aerobic conditions were maintained by means of an intense aeration with compressor and aerators. Inoculum was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge (in mineral medium) at the test temperature of 22ºC. For the test the activated sludge, inoculum, was taken after 2 days from this pre-conditioning. 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 A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.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

Details on study design:

TEST CONDITIONS
- 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.
- Additional substrate: no.
- Solubilising agent (type and concentration if used): no.
- Test temperature: 22 ± 2°C
- pH: 7.4
- pH adjusted: no.
- 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
- Volume of test solution in flask, V: 0.164 L

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #1, 2, 3 containing test item (105.9 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.
- Test performed in open system: no.
- Details of trap for CO2 and volatile organics if used: Three flakes of potassium hydroxide were added to each of the CO2-absorber compartments.

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #4, 5, 6, containing only inoculum 30mg/L SS.
- Procedure control: flasks #7, 8, 9 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:

% degradation (O2 consumption)

Value:

15

Sampling time:

28 d

Details on results:

At the 28th day of the test the aerobic biodegradation of the testing:
- the test item attained 15.0% of biodegradation,
- the reference item reached 82.9% of biodegradation,
- in the toxicity test the biodegradation was equal to 30.5%, and therefore, the test item is not inhibitory,
- the oxygen uptake of the inoculum blank was equal to 34.0 mg/l in 28 days,
- the pH values of all flasks were inside the range 7.29-8.98.

Results with reference substance:

The reference item reached 82.9% biodegradation by day 28.

Table 2. Sample oxygen uptake: biodegradability

		time, days
		3	5	7	9	12	14	16	18	21	23	25	28
Test item O2uptake, mg/l	a1	10.1	13.8	16.7	20.3	22.5	25.2	26.0	28.1	32.7	34.0	36.6	39.4
	a2	12.0	16.6	21.7	25.7	32.5	37.2	39.4	45.2	50.8	53.8	56.5	60.5
	a3	5.9	14.4	20.5	26.3	34.2	40.1	45.4	50.4	56.9	59.8	62.7	68.9
	am. avg	9.0	15.5	21.1	26.0	33.4	38.7	42.4	47.8	53.9	56.8	59.6	64.7
Blank test O2uptake. mg/l	b1	3.5	7.3	12.1	16.9	25.2	28.9	33.8	38.4	44.7	47.8	51.7	57.9
	b2	9.4	14.0	17.6	20.5	22.7	24.9	25.7	27.2	30.0	32.4	33.1	33.7
	b3	10.0	12.8	15.3	17.4	19.7	24.3	25.8	26.3	30.9	31.3	33.5	34.2
	bm. avg	9.7	13.4	16.5	18.9	21.2	24.6	25.8	26.7	30.5	31.8	33.3	34.0
Reference item O2uptake. mg/l	w1	44.9	57.5	67.3	73.8	81.6	86.6	90.7	93.1	95.7	98.5	101.1	103.7
	w2	47.2	59.3	68.0	73.9	80.5	84.9	86.4	87.2	90.0	90.3	92.8	96.4
	w3	44.4	55.8	63.2	71.0	76.3	81.0	84.9	88.0	92.4	92.8	95.4	95.7
	wm. avg	45.5	57.5	66.1	72.9	79.4	84.2	87.4	89.4	92.7	93.9	96.4	98.6
Toxicity control O2uptake. mg/l	a4tox1	56.8	59.8	65.5	70.7	76.7	81.7	85.1	88.9	94.7	95.1	97.7	99.4
	a5tox2	55.1	59.7	67.6	70.9	79.2	81.6	85.1	88.2	92.8	95.7	98.5	100.9
	a6tox3	55.6	62.9	72.5	80.2	88.4	93.4	99.2	101.4	110.0	113.3	115.2	121.4
	toxm. avg	55.8	60.8	68.5	73.9	81.4	85.6	89.8	92.8	99.2	101.4	103.8	107.2
Corrected test item O2uptake, mg/l	(a1-bm)	2.4	2.4	1.6	2.0	-0.1	-0.8	-2.5	-2.5	-2.5	-3.1	-2.9	-2.6
	(a2-bm)	2.3	3.1	5.3	6.8	11.3	12.6	13.6	18.4	20.4	21.9	23.2	26.6
	(a3-bm)	-3.8	1.0	4.0	7.4	13.0	15.6	19.6	23.6	26.4	27.9	29.4	35.0
Reference item % degradation ThOD = 0.78 mgO2/mg C = 100 mg/l	R1(w1)	45.2	56.5	65.2	70.4	77.4	79.6	83.3	85.0	83.6	85.5	86.8	89.4
	R1(w2)	48.1	58.8	66.0	70.6	75.9	77.3	77.8	77.5	76.3	75.0	76.3	80.0
	R3(w3)	44.5	54.3	59.9	66.8	70.6	72.3	75.8	78.5	79.5	78.1	79.6	79.1
	Rtoxavg	45.9	56.5	63.7	69.2	74.6	76.4	78.9	80.4	79.8	79.5	80.9	82.9

The results a₁and b₁are not taken into average values calculations, as they are distinct greater than other test item O2 uptake and blank test O2 uptake, respectively.

Table 3. Toxicity test.

days	0			28			difference
1) Concentration of nitrate (mg N-NO3/l)	#10	#11	#12	#10	#11	#12	#10	#11	#12
	0.118	0.138	0.142	1.64	1.85	1.91	1.52	1.71	1.77
							1.67 ± 0.13
2) Oxygen equivalent (4.57× N-NO3) (mg/l)							7.63
3) concentration of nitrite  (mg N-NO2/l)	#10	#11	#12	#10	#11	#12	#10	#11	#12
	0.029	0.037	0.032	0.014	0.019	0.015	-0.015	-0.018	-0.017
							-0.017 ± 0.002
4) Oxygen equivalent (3.43×N-NO2) (mg/l)							-0.06
5) total oxygen equivalent 2) + 4)							7.57

Table 4. pH values of the test flasks (no adjustment of pH was conducted).

flask #	1	2	3	4	5	6	7	8	9	10	11	12
	Test item			Control			Reference item			Toxicity test
initial	7.42	7.44	7.45	7.35	7.47	7.48	7.43	7.41	7.30	7.46	7.36	7.45
final	7.75	7.80	7.56	7.35	7.29	7.62	8.30	8.54	8.58	8.75	8.94	8.98

As the test substance contains nitrogen, the observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate.

Table 5. Correction for oxygen uptake for interference by nitrification:

days	0			28			difference
1) Concentration of nitrate (mg N-NO3/l)	#7	#8	#9	#7	#8	#9	#7	#8	#9
	0.124	0.131	0.127	2.16	2.54	2.31	2.04	2.41	2.18
							2.21 ± 0.19
2) Oxygen equivalent (4.57× N-NO3) (mg/l)							10.10
3) concentration of nitrite  (mg N-NO2/l)	#7	#8	#9	#7	#8	#9	#7	#8	#9
	0.033	0.025	0.020	0.020	0.018	0.015	-0.013	-0.007	-0.005
							-0.008 ± 0.004
4) Oxygen equivalent (3.43×N-NO2) (mg/l)							-0.027
5) total oxygen equivalent 2) + 4)							10.07

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks, attachments' section.

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance reached 15.0% biodegradation after 28 days. Thus, it is not redily biodegradable.

Executive summary:

The study of ready biodegradability of the test item in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4 Method (GLP study). 30 mg/L of activated sludge were exposed to 100 mg/L of test item during 28 days under aerobic conditions at 22ºC. The blank tests were run in parallel with only inoculum but without test item. A reference item (sodium acetate) was run in parallel to check the operation of the procedures. To check the possible inhibitory effect of the test item, a toxicity test was run in parallel. The degradation was followed by the determination of oxygen uptake and measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. As the test substance contains nitrogen, the observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. After the correction for oxygen uptake for interference for interference by nitrification and by the blank inoculum control, the final biodegradation of the test item on the 28th test day was 15.0%. Therefore, the substance is not readily biodegradable under test conditions. In the toxicity test, the biodegradation attained a 30.5% and therefore, the test item is not inhibitory.

Description of key information

Key study. Method according to OECD 301F and EU C.4 Method (GLP study). The final biodegradation of the test item on the 28th test day was 15.0%. Therefore, the substance is not readily biodegradable under test conditions.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The study of ready biodegradability of the test item in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4 Method (GLP study). 30 mg/L of activated sludge were exposed to 100 mg/L of test item during 28 days under aerobic conditions at 22ºC. A blank control, reference item (sodium acetate) control, and a toxicity control were run in parallel. The degradation was followed by the determination of oxygen uptake and measurements were taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. As the test substance contains nitrogen, the observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate, as well as for the blank inoculum control. The final biodegradation of the test item on the 28th test day was 15.0%. Therefore, the substance is not readily biodegradable under test conditions. In the toxicity test, the biodegradation attained a 30.5% and therefore, the test item is not inhibitory.