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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 November 22, 2021 to January 04, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection : 23 and 24 may 2019 Statement : 30 Jan 2020

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

The inoculum was a fresh sample of activated sludge and was collected from the aeration tank of a sewage treatment plant receiving predominantly domestic sewage.

Laboratory preparation: A filtration through a fine sieve (about 1 mm) was performed in order to remove coarse particles. After removal of any coarse particles, the sludge was washed by decantation in a mineral medium until sludge was considered free from excess substrate or inhibitor.

Inoculum pre-conditioning: Pre-conditioning consisted in aerating sludge (in mineral medium) for 5-7 days before the test and at the test temperature. The optimum volume of inoculum for the test was 0.05 to 5 mL per litre of medium depending on the concentration of the effluent.

The sludge was not be pre-adapted to the test item.

Sample site : Water treatment plant "STEP de Lille", 33290 Blanquefort (France) Tank 2

Date of sampling : November 17, 2021

Suspended solid : 3.5 g/L

Activated sludge preparation :

1: Filtration through a 1 mm sieve

2: Three successive decantations into mineral medium

3: Activated sludge aeration during 5 days

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test duration : 28 days
Light intensity :Darkness
Temperature : 21.7 to 21.9°C, (constant temperature ± 1°C), see Appendix 1
Incubation conditions : Test flasks incubated in environmental simulation chamber under continuous stirring

A special preparation for the test item was used because the test item was poorly soluble in water. Due to its properties, administration of the test item was accomplished with the help of silica gel. For this purpose, approximately 0.03 g of test item was added to 10 g of silica gel present in a 50 mL flask. The flask was closed with a screw top with aluminium foil and the content was mixed vigorously. Then 0.2 g of silica gel dosed with test item was added to the respective test bottles in order to obtain a nominal concentration of 2 mg test item/L, and finally the bottles were filled with the inoculated mineral medium and closed. The test was carried out without adjustment of the pH.

The test medium was the mineral medium, a weakly saline aqueous medium. The test item was tested at 2 mg/L in mineral medium.

The temperature was within the range of 20°C and 24°C, and was constant within ± 1°C. Tests flasks were incubated in the dark and were agitated periodically during the incubation (about three times a week).

Experimental plan :

Inoculum control : 12 flasks : Inoculum only
Inoculum control silica gel : 12 flasks : Inoculum and silica gel (0.2 g/flask)
Test item : 12 flasks Test item and inoculum
Reference item : 12 flasks : Sodium benzoate and inoculum
Toxicity control : 6 flasks : Test item, sodium benzoate and inoculum


The analysis of the dissolved Oxygen at zero-time was immediately measured at the start of the experimentation with a probe Oxygen (WTW) introduced directly into the bottle. This probe was provided with an integrated stirrer.
Duplicate bottles, at least, were removed from each series for dissolved Oxygen analysis at time intervals (at least weekly) over the 28 days incubation. In order to characterize the 10 days-window, additional measures were performed.

In the calculation of percentage of degradation, the mean values of the duplicate measurement of the parameter in both test vessels and inoculum blank were used.

First, we calculated the BOD after each time period by subtracting the Oxygen depletion (mg O2/L) of the inoculum control from that exhibited by the test item. Divided this corrected depletion by the concentration (mg/L) of the test item, to obtain the specific BOD as mg Oxygen per mg test item. Then, we calculated the percentage of biodegradation by dividing BOD by the specific ThOD.

Biological Oxygen Demand: BOD = (mgO2 uptake by test item - average mg O2 uptake by controls ) / mg test item in vessel
Biodegradation percentage: = (BOD / ThOD ) x 100

Reference substance:

benzoic acid, sodium salt

Preliminary study:

no

Parameter:

% degradation (O2 consumption)

Value:

44.6

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

62.7

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

65.1

Sampling time:

28 d

Details on results:

The pass level for readily biodegradability, 60% removal of ThOD, has been reached in the 10 day window: the degradation for the 10 Day window beginning was on Day 7 (44.6%), and the end of the 10 day window was on Day 14, the mean degradation was 62.7%.


Moreover, The degradation rate of the test item reached 65.1% (60.4% to 69.9%), following the ThODNH4, after 28 days of incubation



Therefore, the test item can be considered as readily biodegradable, according to the OECD guideline 301D for testing of chemicals.

Results with reference substance:

Sodium benzoate was biodegraded to 79.9% of its ThOD after 14 days of incubation and 81.1% (82.9% and 79.3%) at Day 28

The pass level for readily biodegradability, 60% removal of ThOD, has been reached in the 10 day window.

 

Moreover, The degradation rate of the test item reached 65.1% (60.4% to 69.9%), following the ThOD_NH4, after 28 days of incubation

Therefore, the test item can be considered as readily biodegradable, according to the OECD guideline 301D for testing of chemicals.

 

control

	Day 0		Day 2		Day 7		Day 10		Day 14		Day 21		Day 28
Bottle 1-2	9.2	9.2	9.1	9.1	8.6	9.0	8.8	8.7	8.6	8.6	8.2	8.2	7.9	7.9
Mean	9.20		9.10		8.80		8.75		8.60		8.20		7.90
O2 consumption	/		0.10		0.40		0.45		0.60		1.00		1.30

 

Sodium benzoate

	Day 0		Day 2		Day 7		Day 10		Day 14		Day 21		Day 28
Bottle 1-2	9.3	9.3	5.0	4.7	4.1	3.8	2.7	2.5	2.0	2.1	1.5	1.5	1.1		1.4
Mean	9.30		/		/		/		/				/
O2 consumption			4.20	4.50	4.80	5.10	6.15	6.35	6.70	6.60	6.80	6.80	6.90	6.60
BOD (mgO2/mg)			0.84	0.90	0.96	1.02	1.23	1.27	1.34	1.32	1.36	1.36	1.38	1.32
% degradation			50.5	54.1	57.7	61.3	73.9	76.3	80.5	79.3	81.7	81.7	82.9	79.3
Mean % degradation	/		52.33		59.5		75.1		79.9		81.7		81.1
ThOD	1.665		mg O2/ mg sodium benzoate

 

Silica gel Control

	Day 0		Day 2		Day 7		Day 10		Day 14		Day 21		Day 28
Bottle 1-2	9.3	9.3	9.2	9.2	8.9	8.8	8.7	8.7	8.6	8.6	8.0	8.2	7.9	7.9
Mean	9.30		9.20		8.85		8.70		8.60		8.10		7.90
O2 consumption	/		0.10		0.45		0.60		0.70		1.20		1.40

 

TEST ITEM

	Day 0		Day 2		Day 7		Day 10		Day 14		Day 21		Day 28
Bottle 1-2	9.3	9.3	8.9	9.0	6.1	6.0	5.3	5.4	4.7	4.6	4.5	4.1	4.1	3.5
Mean	9.30		/		/		/		/				/
O2 consumption			0.30	0.20	2.75	2.85	3.40	3.30	3.90	4.00	3.60	4.00	3.80	4.40
BOD (mgO2/mg)			0.15	0.10	1.38	1.43	1.70	1.65	1.95	2.00	1.80	2.00	1.90	2.20
% degradation			4.8	3.2	43.8	45.4	54.0	52.4	61.9	63.5	57.2	63.5	60.4	69.9
Mean % degradation	/		4.0		44.6		53.2		62.7		60.4		65.1
ThOD	3.148		mg O2/ mg sodium benzoate

 

Toxicity control

	Day 0		Day 2		Day 7		Day 10		Day 14		Day 21		Day 28
Bottle 1-2	9.3	/	5.0	/	3.7	/	3.2	/	2.8	/	0.9	/	0.5	/
Mean	9.30		4.00		1.00		3.20		2.80		0.90		0.50
O2 consumption			5.20		7.90		5.50		5.80		7.20		7.40
BOD (mgO2/mg)			0.74		1.13		0.79		0.83		1.03		1.06
% degradation			35.6		54.0		37.6		39.7		49.2		50.6

 

 

 

	Values for   OECD 301D	Validity criteria for the test
Oxygen depletion in the Control after 28 days	< 1.5 mg/L	OK   (Control: 1.30 mg/L)  (Silica gel Control 1.40 mg/L)
Dissolved Oxygen in the bottles (test item) during the study	≥ 0.5 mg/L	OK (3.5 to 4.1 mg/L)
Variation between test item replicates at the end of the test	< 20%	OK (Cv =3.7%)
Biodegradadability of the reference item within 14 days	≥ 60%	OK (79.9% Day 14)
Biodegradadability of the toxicity control at 14 days	≥ 25% (ThOD)	OK (39.7%)(with ThODNH4)

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

All the validity criteria were successful.


The pass level for readily biodegradability, 60% removal of ThOD, has been reached in the 10 day window: the degradation for the 10 Day window beginning was on Day 7 (44.6%), and the end of the 10 day window was on Day 14, the mean degradation was 62.7%.

Moreover, The degradation rate of the test item reached 65.1% (60.4% to 69.9%), following the ThODNH4, after 28 days of incubation

Therefore, the test item can be considered as readily biodegradable, according to the OECD guideline 301D for testing of chemicals.

Executive summary:

The readily biodegradability of the tested substance was assessed according to the OECD guideline for testing of Chemicals No 301 D " Closed bottle test " over a period of 28 days with GLP statement.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.
The test item loading rate was 2 mg/L (corresponding to an oxygen demand of about 3.148 mg O2/ mg test item)  (ThODNH4).

Sodium benzoate was biodegraded to 79.9% of its ThOD after 14 days of incubation and 81.1% (82.9% and 79.3%) at Day 28.

The toxicity control containing inoculum, test item (2 mg/L) and sodium benzoate (5 mg/L) was not considered to be inhibitory to the activity of the inoculum. There was no inhibition of the test item to benzoate. The percentage of biodegradation was 39.7% (withThOD_NH4) on Day 14 (validity criterion ≥ 25%).

The pass level for readily biodegradability, 60% removal of ThOD, has been reached in the 10 day window: the degradation for the 10 Day window beginning was on Day 7 (44.6%), and the end of the 10 day window was on Day 14, the mean degradation was 62.7%. The time window criterion should however not be applied according to section 4.1.2.9.5 of CLP Commission regulation (EU) No 286/2011 of 10 March 2011 because test item is a UVCB.The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should therefore not be used as a pass/fail criterion.

The degradation rate of the test item reached 65.1% (60.4% to 69.9%), following the ThODNH4, after 28 days of incubation

Therefore, the test item can be considered as readily biodegradable, according to the OECD guideline 301D for testing of chemicals.

Description of key information

The substance is readily biodegradable, at day 28, the test item reached 65.1% of degradation.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

One key study is available to assess the biodegradation in water of the test substance.

The readily biodegradability of the tested substance was assessed according to the OECD guideline for testing of Chemicals No 301 D " Closed bottle test " over a period of 28 days with GLP statement.

The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Degradation rate of test item was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.
The test item loading rate was 2 mg/L (corresponding to an oxygen demand of about 3.148 mg O2/ mg test item)  (ThODNH4).

Sodium benzoate was biodegraded to 79.9% of its ThOD after 14 days of incubation and 81.1% (82.9% and 79.3%) at Day 28.

The toxicity control containing inoculum, test item (2 mg/L) and sodium benzoate (5 mg/L) was not considered to be inhibitory to the activity of the inoculum. There was no inhibition of the test item to benzoate. The percentage of biodegradation was 39.7% (withThOD_NH4) on Day 14 (validity criterion ≥ 25%).

The pass level for readily biodegradability, 60% removal of ThOD, has been reached in the 10 day window: the degradation for the 10 Day window beginning was on Day 7 (44.6%), and the end of the 10 day window was on Day 14, the mean degradation was 62.7%. The time window criterion should however not be applied according to section 4.1.2.9.5 of CLP Commission regulation (EU) No 286/2011 of 10 March 2011 because test item is a UVCB.The biodegradation of the test substance is therefore an addition of different biodegradation curves and the time window should therefore not be used as a pass/fail criterion.

The degradation rate of the test item reached 65.1% (60.4% to 69.9%), following the ThODNH4, after 28 days of incubation

Therefore, the test item can be considered as readily biodegradable, according to the OECD guideline 301D for testing of chemicals.