7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

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:

09 January - 08 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

Paragraph (m)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch No. of test material: 06-262-PP-01
- Appearance: Off-white powder
- Purity: 95.71%
- Storage condition of test material: Approximately +4°C in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Storage conditions: Continuous aeration in the laboratory at 21°C
- Storage length: Used on the day of collection
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
- Pretreatment: The test material was dispersed directly in the culture medium. An amount of test material (42.0 mg) was dispersed in approximately 400 mL of culture medium and subjected to ultrasonication for approximately 15 minutes prior to dispersal in inoculated culture medium. The volume was adjusted to 3 L to give a final concentration of 14.0 mg/L, equivalent to 10 mg carbon/L.
- Initial cell/biomass concentration: The suspended solids concentration was 2.8 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: mg carbon/L

Initial conc.:

14 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The culture medium used in the study was that recommended in the OECD Guidelines.
- Test temperature: approximately 21°C
- pH: Day 28 pH 7.4 - 7.5
- pH adjusted: no
- Aeration: Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 mL of culture medium and 32.1 mL of inoculum and aerated overnight. The culture vessels were sealed and CO2-free air was bubbled through the solution at a rate of approximately 40 mL/minute and stirred continuously by a magnetic stirrer.
- Suspended solids concentration: 30 mg/L suspended solids
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels each containing 3 L of solution
- Number of culture flasks/concentration: 2 replicates per concentration
- Test performed in open system: No, the culture vessels were sealed
- Details of trap for CO2: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
- Sample storage before analysis: The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Day 12 were stored at approximately -20°C prior to analysis. The samples taken on Day 18 were also stored at approximately -20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that degradation of the test material had met the 10-day window validation criterion and therefore additional analysis was considered to be unnecessary.
- Other: On Day 28, 1 mL of concentrated HCl was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated culture medium only prepared in duplicate.
- Toxicity control: A toxicity control was prepared containing the test material and sodium benzoate at a final concentration of 14.0 mg/L test material plus 17.1 mg/L sodium benzoate, equivalent to a total of 20 mg carbon/L (one vessel only).
- Other: The standard material (sodium benzoate) , in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.

Reference substance:

benzoic acid, sodium salt

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 28.92 mg/L.
The IC content of the test material suspension in the mineral medium at the start of the test was below 5% of the total carbon (TC) content.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20%.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

86

Sampling time:

28 d

Details on results:

The test material attained 86% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. These increases were considered to be due to CO2 present in solution being driven off by the addition of HCl on Day 28 and resulted in an increase in the percentage degradation value for the test material from 86% on Day 28 to 91% on Day 29.

The toxicity control attained 76% degradation after 14 days and 95% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The increase in inorganic carbon in the first absorber vessel on Day 29 resulted in an increase in the percentage degradation value for the toxicity control from 95% on Day 28 to 106% on Day 29. Degradation values in excess of 100% were considered to be due to sampling/ analytical variation.

Observations made throughout the test period showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. On Days 0 and 6 of the test period the contents of the test material and toxicity control vessels were observed as light brown dispersions with a few particles of undissolved test material visible on the surface and dispersed throughout. No undissolved standard material was visible in the toxicity control vessel. Further observations made on Days 13, 20 and 27 of the test showed the test material and toxicity control vessels to contain light brown slightly cloudy dispersions with fine particles of undissolved test material visible on the surface and dispersed throughout. No undissolved standard material was visible in the toxicity control vessel.

Results with reference substance:

Sodium benzoate attained 63% degradation after 14 days and 88% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The increase in IC in the first absorber vessels on Day 29 resulted in a decrease in the percentage degradation value for the standard material from 88% on Day 28 to 85% on Day 29. The decrease in degradation obtained on Day 29 was considered to be due to the increases in IC within the replicate control vessels being greater than those within the replicate standard material vessels.

IC analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for DOC gave percentage degradation values of 100% for both replicates. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from IC analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation and hence CO2 evolution occurring.

Table 1: Percentage biodegradation values

Day	% Degradation Sodium benzoate	% Degradation Test material	% Degradation Test material plus sodium benzoate toxicity control
0	0	0	0
1	20	3	21
2	43	6	38
3	41	15	44
6	42	35	66
8	54	39	69
10	61	51	82
12	63	61	79
14	63	60	76
16	66	60	87
20	81	71	98
22	84	75	89
24	84	73	91
27	82	73	93
28	88	86	95
29*	85	91	106

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

 

Table 2: Total and inorganic carbon values in the culture vessels on Day 0

Test vessel	Total carbon* (mg/L)	Inorganic carbon* (mg/L)	IC content (% of TC)
Sodium benzoate 10 mg C/L R1	8.77	-0.88	0
Sodium benzoate 10 mg C/L R2	10.41	0.27	3
Test material 10 mg C/L R1	10.24**	0.20	2
Test material 10 mg C/L R2	10.40**	0.32	3
Test material plus sodium benzoate toxicity control 20 mg C/L	20.59**	0.52	3

R1-R2: Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentration being less than control values

** Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test material and sodium benzoate where applicable.

 

Table 3: Dissolved organic carbon (DOC) values in the culture vessels on Days 0 and 28

Test vessel	DOC* concentration
	Day 0		Day 28
	mg C/L	% of nominal carbon content	mg C/L	% of initial carbon concentration	% degradation
Sodium benzoate 10 mg C/L R1	9.66	97	<control	0	100
Sodium benzoate 10 mg C/L R2	10.15	102	<control	0	100

R1-R2: Replicates 1 and 2

* Corrected for control values.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria given in the OECD Test Guidelines were satisfied.

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 86% degradation after 28 days and satisfied the 10-Day window validation criterion. The test material, Formononetin can therefore be considered to be readily biodegradable.

Description of key information

The test material attained 86% degradation after 28 days and satisfied the 10-Day window validation criterion. The test material, Formononetin can therefore be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable