Silybum marianum, ext.

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:

14 November 2018 - 12 December 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.
Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).

The Test Bottles
The number of test bottles was the follow:
- 10 bottles containing the test item and inoculum
- 10 bottles containing the reference item and inoculum (procedure control)
- 10 bottles containing only inoculum (inoculum control)
- 10 bottles containing the test item, reference item and inoculum (toxicity control)
The reference item and inoculum (procedure control) were also part of a concurrent study (Citoxlab study code: 18/163-322AN) performed in the same experimental period using the same batch of chemicals and solutions. Raw data sheets are clearly associate data with the appropriate study number. Copies of all raw data of positive and negative controls run in parallel were placed in the study file.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2°C according to guideline. The test flasks were placed into an incubator and kept at 20.1 - 23.8°C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 8.9 mg/L at the start of the test.
The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.4.
The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF THE TEST SOLUTIONS
The respective amount of Test Item was weighed in directly to reach the required test item concentration of 2.0 mg/L.
During the preparation of the test solutions they were mixed by mechanical stirring using ultrasonic bath for 10 minutes.
The chosen test item concentration was based on the measured chemical oxygen demand (COD): 3.04 ±0.055 mg test item and on the performed 14-d preliminary test.
The components were applied in the amounts/volumes in the test flasks:
1.) Test Item (flasks 1a and 1b)
Based on the measured chemical oxygen demand (COD) of 3.04 mg O2/mg test item, 6.8 mg of Test Item was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 2.0 mg/L test item, with a COD of about 6.08 mg O2/L).
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.24 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
* The concentration of the stock solution was: 360 mg/L.
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.40 litres of aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b)
The Test item (6.8 mg) and the reference item stock solution* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 2.0 mg/L test item (COD of 6.08 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).
* The concentration of the reference item stock solution was: 360 mg/L.
Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

COURSE OF THE TEST
Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.
Test item (flasks 1a and 1b) and procedure control (flasks 4a and 4b) test solutions were agitated weekly by magnetic stirrer about 2 hours before each oxygen measurement.

Measurement of Oxygen
The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.
Test item and procedure control test solutions were agitated weekly by magnetic stirrer about 2 hours before each oxygen measurement.

Measurement of Total Oxidised N, Nitrite and Nitrate
Because of possible nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the total oxidised nitrogen (nitrate and nitrite) concentrations were determined after each oxygen measurement.
The analysis was carried out with Lovibond MaxiDirect portable photometer using Nitrite LR tablet reagent, and Vario NitraX Reagent Set.

Measurement of Temperature
Temperature was measured continuously and registered on weekdays.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

22.2

Sampling time:

28 d

Key result

Parameter:

COD

Value:

3.04 g O2/g test mat.

Remarks on result:

other: ±0.055 mg O2/mg test item

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 70.8 % after 14 days, and to a mean of 80.0 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 36.4 % biodegradation was noted within 14 days and 44.0% biodegradation after 28 days of incubation.

Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

 

Treatment	Concentration [mg/L]	Flask No.	mg O2/L after n days of exposure
			0	7	14	21	28
Test item	2.0	1a	8.1	6.7	6.3	6.0	5.9
		1b	8.3	6.9	6.6	6.3	6.0
		mean	8.20	6.80	6.45	6.15	5.95
Reference item	3.6	2a	8.7	4.2	3.6	3.2	2.9
		2b	8.5	4.2	3.4	3.0	2.9
		mean	8.60	4.20	3.50	3.10	2.90
Inoculum control	-	3a	8.6	7.9	7.7	7.8	7.7
		3b	8.6	8.0	7.8	7.8	7.7
		mean	8.60	7.95	7.75	7.80	7.70
Toxicity control	Test item : 2.0 Reference item: 3.6	4a	8.3	3.5	2.8	2.4	1.7
		4b	8.3	3.6	2.5	2.2	1.5
		mean	8.30	3.55	2.65	2.30	1.60

 

Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration[mg/L]	Flask No.	mg O2/L after n days of exposure
			7	14	21	28
Test item	2.0	1a	0.75	0.95	1.30	1.30
		1b	0.75	0.85	1.20	1.40
Reference item	3.6	2a	3.85	4.25	4.70	4.90
		2b	3.65	4.25	4.70	4.70
Toxicity control	Test item : 2.0 Reference item: 3.6	4a	4.15	4.65	5.10	5.70
		4b	4.05	4.95	5.30	5.90

 

Total Oxidised Nitrogen at concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	mg total oxidised N/L after n days of exposure
			0	7	14	21	28
Test item	2.0	1a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		1b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Inoculum control	-	3a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		3b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Toxicity control	Test item : 2.0 Reference item: 3.6	4a	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
		4b	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

LOQ: 4.43 mg NO3/mL; LOQ: 0.03 mg NO2/mL;

(< LOQ: below the quantification level)

Equipment:

Lovibond Maxidirect Photometer using Nitrite LR tablet reagent and Vario NitraX Reagent Set.

BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration[mg/L]	Flask No.	BOD after n days of exposure
			7	14	21	28
Test item	2.0	1a	0.38	0.48	0.65	0.65
		1b	0.38	0.43	0.60	0.70
Reference item	3.6	2a	1.07	1.18	1.31	1.36
		2b	1.01	1.18	1.31	1.31
Toxicity control	Test item : 2.0 Reference item: 3.6	4a	0.74	0.83	0.91	1.02
		4b	0.72	0.88	0.95	1.05

 

BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration[mg/L]	Flask No.	BOD after n days of exposure
			7	14	21	28
Test item	2.0	1a	12.3	15.6	21.4	21.4
		1b	12.3	14.0	19.7	23.0
		mean	12.3	14.8	20.6	22.2
Reference item	3.6	2a	64.2	70.8	78.3	81.7
		2b	60.8	70.8	78.3	78.3
		mean	62.5	70.8	78.3	80.0
Toxicity control	Test item : 2.0 Reference item: 3.6	4a	31.5	35.3	38.7	43.3
		4b	30.7	37.6	40.2	44.8
		mean	31.1	36.4	39.5	44.0

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The Test Item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. 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.
The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.
Under the test conditions the percentage biodegradation of the Test Item reached a mean of 22.2 % after 28 days based on the COD of the test item.
According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD/COD. Therefore, the test item is considered not readily biodegradable.
The reference item Sodium benzoate was sufficiently degraded to a mean of 70.8 % after 14 days, and to a mean of 80.0 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.
In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 36.4 % biodegradation was noted within 14 days and 44.0% biodegradation after 28 days of incubation.
According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.0 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.
The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.
Based on the results of this study, the test item is considered not readily biodegradable.

Executive summary:

The Test Item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. 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.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the Test Item reached a mean of 22.2 % after 28 days based on the COD of the test item.

According to the test guidelines the pass level for ready biodegradability is removal of 60% ThOD/COD. Therefore, the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 70.8 % after 14 days, and to a mean of 80.0 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 36.4 % biodegradation was noted within 14 days and 44.0% biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.0 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.

Description of key information

Based on the results of this study, the test item is considered not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The Test Item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. 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.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of the Test Item reached a mean of 22.2 % after 28 days based on the COD of the test item.

According to the test guidelines the pass level for ready biodegradability is removal of 60% ThOD/COD. Therefore, the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 70.8 % after 14 days, and to a mean of 80.0 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 36.4 % biodegradation was noted within 14 days and 44.0% biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.0 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.