P-1401

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:

2 July 2015 - 30 July 2015

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 of inoculum/activated sludge: Aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw. 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.
- Preparation of inoculum for exposure: The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated until application. After complete re-suspension was achieved, a sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Pretreatment: Inocula was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge in mineral medium for 2 days at the test temperature of 22 ºC
- Concentration of sludge: 30 mg/litre SS.

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:
The following stock solutions were prepared using analytical grade reagents:
(a) Monopotassium dihydrogen orthophosphate, KH2PO4 8.50 g
Dipotassium monohydrogen orthophosphate, K2HPO4 21.75 g
Disodium monohydrogen orthophosphate dihydrate Na2HPO4 · 2 H2O 33.40 g
Ammonium chloride, NH4Cl 0.50 g
Dissolve in water and make up to 1 litre The pH of the solution should be 7.4.
(b) Calcium chloride, anhydrous, CaCl2 27.50 g
Dissolve in water and make up to 1 litre
(c) Magnesium sulphate heptahydrate, MgSO4 · 7 H2O 22.50 g
Dissolve in water and make up to 1 litre.
(d) Iron(III) chloride hexahydrate, FeC13 · 6H2O 0.25 g
Dissolve in water and make up to 1 litre.
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 11 with dilution water.
- Test temperature: 22 ±2ºC
- pH: 7.4 ±0.2ºC
- pH adjusted: no
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: thermo-cabinet WTW TS 606 CZ-G/3-VAR
- Number of culture flasks/concentration:
Flasks 1, 2, 3: Test suspension containing test item (100 mg/l) and inoculum 30 mg/l SS.
Flasks 4, 5 and 6: Inoculum blank containing only inoculum 30 mg/l SS.
Flask 7, 8 and 9: Procedure control containing reference item (sodium acetate 100 mg/l) ) and inoculum30 mg/SS.
Flask 10, 11 and 12: Toxicity control: containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30 mg/l SS.
- Measuring equipment:
Consumption of oxygen: Closed WTW OxiTop OC 110 respirometer for BOD determination. The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide.
Determination of nitrates and nitrites: Spectrophotometer Hach DR 3900 and 45600 reactor COD for nitrate and nitrite concentration measurements. Nitrates were determined using LANGE Cuvette Test method 8039 ( and nitrites were determined using LANGE Cuvette Test LCK341 (ISO 8466-1 and DIN 38402).

SAMPLING
The oxygen uptake from the readings taken at regular and frequent intervals was calculated, using the methods given by the manufacturer of the equipment. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recoded and stored in the measuring heads of the sample bottles. These collected data, using the controller, were infrared read out from the heads and stored in the controller.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 flasks containing only inoculum 30 mg/l SS.
- Procedure control: 3 flasks containing reference item (sodium acetate 100 mg/l) ) and inoculum 30 mg/SS.
- Toxicity control: 3 flasks containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30 mg/l SS.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

23.8

Sampling time:

28 d

Details on results:

At the 28th day of the test the aerobic biodegradation of the testing:
The test item attained 23.8% of biodegradability,
In the toxicity test the biodegradation at 14th day was equal to 38.9%,
The oxygen uptake of the inoculum blank was equal to 34.8 mg/l in 28 days,
The pH values of all flasks were inside the range 6.90-8.86.

Results with reference substance:

At the 28th day of the test the aerobic biodegradation of the testing, the reference item reached 76.4% of biodegradability.

The mineral medium solution pH was equal to 7.57.

The preconditioned suspension of inoculum pH was equal to 7.23.

The initial concentration of test item in test flasks was equal to 110.9 mg/l.

Correction for oxygen uptake for interference by nitrification:

Test item

days	0			28				difference
1) Concentration of nitrate (mg N/l)	#1	#2	#3	#1		#2	#3	#1	#2	#3
	8.46	8.51	8.49	11.8		11.8	11.6	3.34	3.29	3.11
								3.25 ± 0.12
2) Oxygen equivalent (4.57× N) (mg/l)								14.85
3) concentration of nitrite (mg N/l)	#1	#2	#3		#1	#2	#3	#1	#2	#3
	0.099	0.101	0.092		0.156	0.150	0.154	0.057	0.049	0.062
								0.056 ± 0.007
4) Oxygen equivalent (3.43×N) (mg/l)								0.192
5) total oxygen equivalent 2) + 4)								15.04

Toxicity test

days	0			28						difference
1) Concentration of nitrate (mg N/l)	#10	#11	#12	#10		#11		#12		#10	#11	#12
	8.28	8.26	8.30	11.7		11.7		11.7		3.42	3.44	3.40
										3.42 ± 0.02
2) Oxygen equivalent (4.57× N) (mg/l)										15.63
3) concentration of nitrite (mg N/l)	#10	#11	#12		#10		#11		#12	#10	#11	#12
	0.092	0.094	0.090		0.141		0.123		0.132	0.049	0.029	0.042
										0.04 ± 0.01
4) Oxygen equivalent (3.43×N) (mg/l)										0.137
5) total oxygen equivalent 2) + 4)										15.77

The pH values of test flask

flask #	1	2	3	4	5	6	7	8	9	10	11	12
	Test item			Control			Reference item			Toxicity test
initial	7.25	7.35	7.37	7.42	7.52	7.56	7.54	7.64	7.65	7.32	7.40	7.42
final	6.96	6.92	6.90	7.48	7.53	7.56	10.98	8.86	8.87	7.94	7.20	7.92

The recorded measurement temperature didn’t exceed - by 0.45 °C - the expected 22 °C.

Sample oxygen uptake: biodegradability

		time, days
		3	5	7	9	12	14	16	18	21	23	25	28
Test item O2uptake, mg/l	a1	35,9	46,7	55,1	62,3	67,7	70,6	72,3	74,9	76,0	77,6	79,0	80,1
	a2	35,9	49,5	56,5	62,1	69,9	74,4	75,7	77,5	80,9	82,0	82,9	86,6
	a3	42,1	50,5	57,7	66,3	71,9	76,9	78,4	80,8	83,9	84,4	86,0	88,5
	am. avg	38,0	48,9	56,4	63,6	69,8	74,0	75,5	77,8	80,3	81,3	82,6	85,1
Blank test O2uptake. mg/l	b1	7,8	11,7	15,5	19,2	22,5	23,9	25,5	26,4	28,3	28,1	30,1	30,9
	b2	10,6	17,4	22,2	26,0	28,1	31,1	33,9	34,3	36,6	37,3	39,4	41,8
	b3	6,3	11,7	14,1	17,0	22,1	22,6	26,1	25,3	27,8	27,9	31,3	31,7
	bm. avg	8,3	13,6	17,3	20,7	24,2	25,8	28,5	28,7	30,9	31,1	33,6	34,8
Reference item O2uptake. mg/l	w1	0,0	0,0	0,0	3,2	16,8	29,8	57,0	63,5	68,2	68,5	70,5	73,4
	w2	45,3	59,1	68,6	75,9	82,1	85,9	88,9	90,1	93,0	95,7	95,7	98,5
	w3	45,3	59,1	68,1	74,0	82,3	84,9	87,9	87,2	90,5	90,0	90,4	93,0
	wm. avg	45,3	59,1	68,3	74,9	82,2	85,4	88,4	88,7	91,8	92,9	93,0	95,8
Toxicity control O2uptake. mg/l	a4tox1	84,2	99,1	110,4	120,0	129,3	130,5	133,9	136,4	140,0	141,3	142,1	144,3
	a5tox2	85,4	99,4	110,8	120,6	128,7	132,3	134,7	136,4	140,3	140,9	141,2	143,0
	a6tox3	85,0	100,6	110,5	120,1	128,2	129,0	129,0	129,0	129,4	127,7	127,0	128,4
	toxm. avg	84,9	99,7	110,6	120,3	128,7	130,6	132,5	133,9	136,6	136,6	136,8	138,6
Corrected test item O2uptake, mg/l	(a1-bm)	27,6	33,1	37,8	41,6	43,4	44,8	43,9	46,3	45,1	46,5	45,4	45,2
	(a2-bm)	27,6	35,9	39,2	41,4	45,7	48,6	47,3	48,8	50,0	50,9	49,3	51,8
	(a3-bm)	33,8	36,9	40,4	45,6	47,6	51,0	49,9	52,2	53,0	53,3	52,4	53,7
Reference item % degradation ThOD = 0.78 mgO2/mg C = 100 mg/l	R1(w1)	-	-	-	-	-	-	-	-	-	-	-	-
	R1(w2)	47,4	58,3	65,8	70,8	74,1	77,0	77,5	78,8	79,6	82,9	79,6	81,6
	R3(w3)	47,5	58,4	65,1	68,3	74,4	75,7	76,2	75,0	76,4	75,6	72,8	74,6
	Rtoxavg	47,5	58,3	65,5	69,5	74,3	76,4	76,8	76,9	78,0	79,2	76,2	78,1

The results of w1 from reference item are not taken into average test item degradation calculations, as they are distinct lower than w2 and w3 and pH of it is 10.98. Probably, some of potassium hydroxide fell to this w1 bottle.

After correction for oxygen uptake for nitrification and for the blank inoculum control, the biodegradation of the test item after 28 days was calculated to be 23.8%.

More than 25% of biodegradability (based on ThODNH4) occurred in 14 days and the test items can not be assumed to be inhibitory.

Validity criteria fulfilled:

yes

Remarks:

(no difference of extremes of replicates (<20%), reference item reached pass level of 60% on day 7, the substance was not toxic to microorganisms (38.9% on day 14, pH between 6-8)

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

At the 28th day of the test the aerobic biodegradation of the testing item was 23.8%. The substance cannot be deemed as readily biodegradable undertest conditions.

Executive summary:

The study on ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4-D 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 22 ºC. 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 the 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 correction, for oxygen uptake for interference by nitrification and by the blank inoculum control, the final biodegradation of test item on 28th test day was 23.8%. The substance is therefore, not readily biodegradable under test conditions. In the toxicity test, more than 25% of biodegradation (based on ThOD) occurred in 14 days (38.5%) and the test item cannot be deemed to be inhibitory to microorganisms.

Description of key information

Key study: Test method OECD Guideline 301F. GLP study. The substance is not readily biodegradable under the test conditions.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Key study: The determination of the ready biodegradability of the substance in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4-D Method (GLP study). After correction, for oxygen uptake for interference by nitrification and by the blank inoculum control, the biodegradation of test item on 28th test day was 23.8%. The substance is therefore, not readily biodegradable under test conditions. In the toxicity test, more than 25% of biodegradation (based on ThOD) occurred in 14 days (38.9%). The test item is not inhibitory to the microorganisms.