Climbazole

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-31 to 2019-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Version / remarks:

January 2001

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge
- Source of activated sludge: Sewage plant at 31137 Hildesheim, Germany. It is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste
- Pretreatment: The activated sludge was washed once with tap water and maintained in an aerobic condition by aeration at room temperature until use
- Biomass concentration used in test: 3.9 g/L mean dry weight (during acclimatisation); 2.7 g/L mean dry weight (during test item application)

Domestic sewage
- Source of domestic sewage: Sewage plant at 31137 Hildesheim, Germany. It is well suited as it receives predominantly municipal sewage and hardly industrial chemical waste. Collected from the primary sedimentation tank of the treatment plant
- Dissolved organic carbon: 75.9 -136 mg/L
- Storage conditions: at 4 °C to reduce microbal activity
- Pretreatment: At sampling the sewage was filtered to remove coarse particles. DOC adjustment of the domestic sewage with synthetic sewage was not necessary Tap water or ultrapure water for domestic and synthetic sewage was not necessary

Duration of test (contact time):

38 d

Initial conc.:

2 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

STUDY DESIGN
- System: Gas Flow through system, test item and organic medium were continuously pumped through the system.
- Test vessels: Activated sludge unit consisting of an aeration vessel (approx. 500 mL, activated sludge volume 372 mL) and a separator (approx. 250 mL)
- Replicates: 1
- Aeration: Aeration chamber with activated sludge was continuously purged with air to maintain the sludge in aerobic conditions
- Settling tank: activated sludge was pumped back into the aeration chamber by an airlift pump

SET UP
- Volume of activated sludge in the aeration chamber: 372 mL
- Sludge adhering to the walls were re-suspended daily, tubes and tubings were cleaned to prevent growth of biofilm

INCUBATION
- Temperature: 21.0 – 21.3 °C
- pH: 7.2 - 8.0
- Aeration: continuously
- Airflow: adjusted to an oxygen level at least 2 mg/L
- Sludge retention time: 10 d; 1/10 of the volume (37.2 mL) of the activated sludge in the aeration vessel was removed as surplus sludge once per day directly from the aeration vessel by a syringe.
- Mean hydraulic retention time: 6 h,
- Influent flow organic medium: 62 µL/h
- Influent flow test item solution: 124 µL/h
- Wastage sludge rate: ca. 37.2 mL/d

TEST ITEM APPLICATION
- Test item was applied to the system after the removal of DOC was sufficient (> 80 %)
- Test item concentraion in the influent: 2 µg/L
- Test item was continiously applied by adding directly to the activated sludge to avoid biodegradation before it reached the aeration vessel. The elimination, sorption and primary degradation of the test item was determined by LC-MS/MS.

TEST DURATION
- Duration: 54 days
- Acclimatisation period: 16 days
- Test item application period: 38 days
- Plateau phase: 22 days

MEASUREMENTS
- Influent volumes: at least weekdays
- DOC of the effluents: At least 2 x per week
- DOC of the organic media (influent): Every new batch
- Temperature, pH-value, oxygen concentration in the aeration vessel (activated sludge): At least weekly, depended on the stability of the system, measured in the surplus sludge
- Suspended solids(activated sludge): At least 2 x per week, obligatory in parallel to activated sludge analysis

TEST ITEM SAMPLING PROCEDURE
- Approx. 100 mL of the effluent were sampled directly from the outlet tube of the settling tank. Therefore, the tube was transferred in a glass vessel. Analysis of activated sludge was done from the surplus sludge
- Influent: Every new batch during adaption and plateau phase
- Influent Sewage: at least every new batch during the adaption and plateau phase
- Effluent: at least 3x per week in the adaption phase and daily in the plateau phase
- Activated sludge: at least 3x per week in the adaption phase and daily in the plateau phase

Key result

% Degr.:

46.6

St. dev.:

6.7

Parameter:

test mat. analysis

Sampling time:

22 d

Remarks on result:

other:

Remarks:

mean degradation rate of the plateau phase

Transformation products:

not measured

Details on results:

TEST CONDITIONS
- Temperature in the aeration vessel: 21.0 – 21.6 °C
- pH: was mostly in the range of 7.5 ± 0.5 and was only once slightly above 8.0
- Oxygen Concentration in the activates sludge: >= 5.42 mg/L, mostly even > 7.00 mg/L
- Suspended Solids: 1.8 – 4.6 g/L
- Biodegradation of organic medium (DOC): 75-90%, The slight decrease was probably due to variations in the quality and composition of the organic medium. Nevertheless, the unit worked stable and efficiently throughout the study

RESULTS
- Plateau phase: after 17 days of test item application, the plateau phase was started. The plateau phase lasted about three weeks and in total 22 samplings of the effluent and sludge were done. Influent (each new stock solution), effluent and sludge samples were analysed.
- Elimination of the test item: s. table 1
- Load on the activated sludge: s. table 2
- Mean elimination rate of the test item in the effluent: 46.6 % of the influent concentration during the plateau phase (95 % CI: 43.1 – 50.2).
- Mean test item concentration in the sludge: 1.4% during the plateau phase of total influent load (95 % CI: 1.2-1.6).

Tab. 1: Elimination of the test item

Study Phase	Study Day	Day of Test Item Appl.	Conc. Influent (Organic medium)	Conc. Influent (Stock solution)	Conc. Effluent	Elimination
			ng/L	ng/L	ng/L	%
Acclimatization	1	-15	221	-	-	-
	2	-14	206		77	64.9
	5	-11	259		120	41.7
	7	-9	282		130	49.6
	9	-7	223		116	48.2
	12	-4	128		112	12.7
	15	-1	159		81	37.0
Adaptation	19	3		1600	770	56.2
	21	5	161		1059	39.8
	23	7			1118	36.5
	26	10		2000	1454	17.4
	28	12	204		1205	31.5
	30	14			1484	32.7
Plateau Phase	33	17	204	2000	1281	41.9
	34	18			1005	54.4
	35	19	153		971	55.9
	36	20			1157	46.3
	37	21			1268	41.1
	38	22			1265	41.2
	39	23			1215	43.6
	40	24			1392	35.4
	41	25	123		1398	35.1
	42	26			1237	41.7
	43	27			960	54.8
	44	28			946	55.4
	45	29			968	54.4
	46	30			1208	43.1
	47	31			1052	50.4
	48	32	163		1286	39.4
	49	33			1230	43.2
	50	34			1117	48.4
	51	35			1224	43.4
	52	36			971	55.1
	53	37			1073	50.4
	54	38			1047	51.6

Tab. 2: Load of Activated Sludge with the test item

Study Phase	Study Day	Day of Test Item Appl.	Influent Load absolute	Conc.	Sludge absolute	% of Load
			ng/L	ng/g DW	ng	%
Acclimatization	1	-15	657	-	-	-
	2	-14	986	42	91	27.6
	5	-11	1677	57	132	10.2
	7	-9	2447	50	100	4.9
	9	-7	3199	41	72	2.5
	12	-4	4053	46	85	2.2
	15	-1	4625	52	85	1.9
Adaptation	19	3	14911	1077	1836	14.9
	21	5	20100	1129	1738	9.9
	23	7	25334	1105	1679	7.4
	26	10	33196	1228	1977	6.5
	28	12	39627	1043	1637	4.5
	30	14	46188	627	875	2.0
Plateau Phase	33	17	56028	711	1049	2.0
	34	18	59308	96	142	0.3
	35	19	62589	721	992	1.7
	36	20	65793	754	1085	1.7
	37	21	68997	940	1189	1.8
	38	22	72201	750	994	1.4
	39	23	75406	1074	988	1.4
	40	24	78610	619	749	1.0
	41	25	81814	640	806	1.0
	42	26	84972	764	764	0.9
	43	27	88131	743	1151	1.4
	44	28	91289	717	1191	1.4
	45	29	94447	1067	1494	1.6
	46	30	97606	1369	1841	1.9
	47	31	100764	1476	1897	1.9
	48	32	103923	1496	1802	1.8
	49	33	107141	1152	1595	1.5
	50	34	110360	1049	1201	1.1
	51	35	113579	988	1023	0.9
	52	36	116798	1065	958	0.8
	53	37	120017	1064	1053	0.9
	54	38	123236	1091	1156	1.0
			Mean at Plateau Phase			1.4
			SD			0.4
			95 % CI			1.2 – 1.6

Validity criteria fulfilled:

yes

Conclusions:

Based on the analysis of the test item in the effluent, the mean elimination rate at the plateau phase in relation to the total influent concentration was calculated. The elimination of the test item in the effluent was 46.6 % in a continuously operating activated sludge unit. The mean load of the test item in the sludge during the plateau phase was 1.4 %.
Based on the outcome of this study it can be concluded that the main elimination process of the test item in a sewage treatment plant is biodegradation.

Description of key information

The elimination of the test item in the effluent was 46.6 % in a continuously operating activated sludge unit (OECD 303 A). The mean load of the test item in the sludge during the plateau phase was 1.4 %. Based on these results it can be concluded that the main elimination process of the test item in a sewage treatment plant is biodegradation.

Additional information

The elimination and primary degradation of the test item in an activated sludge unit was determined according to OECD guideline 303A and GLP. The activated sludge unit was started with dosing the organic medium. After a short acclimatization period of the activated sludge (DOC elimination > 80 %), the application of the test item was started. Samples of the influent, effluent and sludge were taken at regular intervals and LC-MS/MS analysis was carried out to determine the effluent concentration and fraction adsorbed on the activated sludge. During acclimatization, the activated sludge unit was inoculated with activated sludge. The DOC elimination of the organic medium reached a degradation rate > 80 % already after 1 day. The dosage of the test item was started after an acclimatization period of 16 days. The nominal concentration of the test item stock solution was 1 mg/L. The stock solution was dosed at a constant rate of 124 µl/h into the activated sludge unit, resulting in an influent concentration of 2 µg test item/L.

Based on the analysis of the test item in the effluent, the mean elimination rate at the plateau phase in relation to the total influent concentration was calculated. The elimination of thet test item was 46.6 % of the influent concentration in a continuously operating activated sludge unit. The mean load of the test item in the sludge during the plateau phase was 1.4 % of total influent load.

Based on these data it can be concluded that the main elimination process of the test item in a sewage treatment plant is biodegradation.