4,4-bis(methoxymethyl)biphenyl

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 December 2008 and 14 January 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2008. Date of signature: 14/02/09.

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-bis(methoxymethyl)-1,1'-biphenyl

EC Number:

700-008-0

Cas Number:

3753-18-2

Molecular formula:

C16 H18 O2

IUPAC Name:

4,4'-bis(methoxymethyl)-1,1'-biphenyl

Constituent 2

Reference substance name:

4,4-bis(methoxymethyl)-biphenyl

IUPAC Name:

4,4-bis(methoxymethyl)-biphenyl

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on
15 December 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture: not applicable

- Method of cultivation: not applicable

- Storage conditions: The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and

- Storage length: used on 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. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.4 g/l prior to use.
* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

- Pretreatment: as above

- Concentration of sludge: not stated

- Initial cell/biomass concentration: as above

- Water filtered: yes

- Type and size of filter used, if any: not applicable

Duration of test (contact time):

29 d

Initial test substance concentration

Initial conc.:

12.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The culture medium used in this study was that recommended in the OECD Guidelines.
Solution a
KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 22.50 g/l
Solution d FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water* was added the following volumes of solutions
a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

- Additional substrate: none
- Solubilising agent (type and concentration if used): none used
- Test temperature: 21°C
- pH: 7.4
- pH adjusted: no
- CEC (meq/100 g): not stated in report
- Aeration of dilution water: not applicable
- Suspended solids concentration: 3.4 g/l
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 26.5 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
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.


- Method used to create aerobic conditions: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.

- Method used to create anaerobic conditions: not applicable

- Measuring equipment: 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.

- Test performed in closed vessels due to significant volatility of test substance: no

- Test performed in open system: no

- Details of trap for CO2 and volatile organics if used: 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:
CO2 analysis: Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessel was sampled on Days 0 and 29. The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.

DOC analysis: Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon content in the test media. The samples were centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.


- Sampling method:
CO2 analysis: On Day 28, 1 ml of concentrated hydrochloric acid 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.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

DOC analysis: On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 68°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.


- Sterility check if applicable: not described in report
- Sample storage before analysis: samples were analysed immediately.


CONTROL AND BLANK SYSTEM
- Inoculum blank: none
- Abiotic sterile control: none
- Toxicity control: For the purposes of the test, a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (37.8 mg) was dispersed in approximately 400 ml of culture medium and subjected to high shear mixing at approximately 7500 rpm for 15 minutes prior to dispersal in inoculated culture medium. An aliquot (51.4 ml) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 12.6 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

- Other:

STATISTICAL METHODS: None

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Preliminary study:

Not applicable

Test performance:

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. The contents of the test material vessels were light brown dispersions with tiny particles of test material on the surface and the contents of the toxicity control vessel was a light brown dispersion with tiny particles of test material on the surface with no undissolved standard material visible.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: The test material attained 0% degradation so cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Details on results:

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
The toxicity control attained 28% degradation after 14 days and 45% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 69% degradation after 14 days and 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 69% degradation after 14 days and 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 83% and 86% respectively for Replicates R1 and R2. The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

Any other information on results incl. tables

Inorganic Carbon Values on Each Analysis Occasion

Day	Control (mg IC)				Sodium Benzoate (mg IC)				Test Material (mg IC)				Test Material plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	0.70	0.58	0.58	0.70	0.70	0.47	0.47	0.47	0.47	0.47	0.35	1.52	0.47	0.47
2	5.57	-	5.22	-	13.34	-	13.92	-	1.63	-	4.52	-	7.66	-
6	21.11	-	16.26	-	33.79	-	34.37	-	12.11	-	14.19	-	29.18	-
8	17.89	-	19.38	-	39.33	-	39.22	-	17.66	-	17.54	-	28.55	-
10	20.75	-	20.97	-	43.20	-	42.52	-	22.00	-	22.12	-	33.17	-
14	26.29	-	25.95	-	45.90	-	47.94	-	24.37	-	25.39	-	43.18	-
21	27.60	-	26.59	-	46.19	-	50.14	-	25.58	-	26.48	-	45.40	-
28	31.14	-	31.69	-	53.87	-	53.65	-	31.81	-	32.48	-	62.72	-
29	29.62	1.51	29.84	1.51	54.44	1.63	55.89	2.55	29.39	1.28	30.17	1.51	56.78	1.51

R₁– R₂= Replicates 1 and 2

Abs= CO₂ absorber vessels

Percentage Biodegradation Values

Day	% Degradation Sodium Benzoate	% Degradation Test Material	% Degradation Test Material plus Sodium Benzoate Toxicity Control
0	0	0	0
2	27	0	4
6	51	0	17
8	69	0	17
10	73	4	21
14	69	0	28
21	70	0	31
28	74	2	52
29*	87	0	45

*Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 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/lR1	9.42	-0.48	0
Sodium Benzoate 10 mg C/l R2	8.67	-0.57	0
Test Material 10 mg C/l R1	10.42**	0.35	3
Test Material 10 mg C/l R2	9.97**	0.08	1
Test Material plus Sodium Benzoate Toxicity Control 20 mg C/l	20.16**	0.11	1

 

R₁– R₂= Replicates 1 and 2

*Corrected for control values. Negative values are due to measured concentrations 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

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	9.91	99	1.72	17	83
Sodium Benzoate 10 mg C/l R2	9.24	92	1.32	14	86

R₁– R₂= Replicates 1 and 2

*Corrected for control values

pH Values of the Test Preparations on Day 28

Test Vessel	pH
ControlR1	7.8
Control R2	7.8
Sodium Benzoate 10 mg C/l R1	7.9
Sodium Benzoate 10 mg C/l R2	7.8
Test Material 10 mg C/l R1	7.8
Test Material 10 mg C/l R2	7.9
Test Material plus Sodium Benzoate Toxicity Control 20 mg C/l	7.8

R₁– R₂= Replicates 1 and 2

Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Standard Material	R1	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
	R2	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible	Light brown dispersion, no undissolved standard material visible
Test Material	R1	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface
	R2	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface	Light brown dispersion, tiny particles of test material on surface
Toxicity Control		Light brown dispersion, tiny particles of test material on surface. No undissolved standard material visible	Light brown dispersion, tiny particles of test material on surface. No undissolved standard material visible	Light brown dispersion, tiny particles of test material on surface. No undissolved standard material visible	Light brown dispersion, tiny particles of test material on surface. No undissolved standard material visible	Light brown dispersion, tiny particles of test material on surface. No undissolved standard material visible

 

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Executive summary:

Introduction.A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008, and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Methods.The test material, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.The test material attained 0% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.