1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of study: 29th August 2001; End of study: 22 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP

Qualifier:

according to guideline

Guideline:

other: Test Method Relating to New Chemical Substances (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, 1974; partially revised 1998)

GLP compliance:

yes

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Standard activated sludge
Source: Kurume Facility, Chemicals Evaluation and Research Institute, Japan
Date received: 19 July 2001
Culture: Approximately 1/3 of the total supernatant liquid was removed once a day, and an equivalent volume of 0.1% synthetic sewage was added and cultured
MLSS (mixed liquor suspended solids): 5300 mg/L

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Test conditions:
Temperature: 25±1°C (measured value: 25.0°C)
Duration: 28 days (BOD measurement)
Volume: 300 mL
Concentration: Test substance and aniline* (standard substance) 100 mg/L
Standard activated sludge :30 mg/L
*: Aniline: Kanto Kagaku, reagent grade, lot number 212G1294

Test structure and addition of test substance:
No. 1: Degradation activity confirmation system (aniline + standard activated sludge* + basic culture medium**)
Basic culture medium was placed in a culture bottle, 30.1 mg of aniline weighed in a glass cup was added and mixed, then standard activated sludge was added to the culture bottle.

No. 2: Basic sludge respiration system (standard activated sludge* + basic culture medium**)
Basic culture medium was placed in a culture bottle and standard activated sludge was added.

No. 3-No. 5: Test substance degradation system (test substance + standard activated sludge* + basic culture medium**)
Basic culture medium was placed in a culture bottle, 30.0 mg of test substance weighed in a glass cup was added and mixed, then standard activated sludge was added to the culture bottle.

No. 6:Stability in water system (test substance + Millipore water***)
300 mL of Millipore water was placed in a culture bottle, then 30.0 mg of test substance weighed in a glass cup was added.

*: MLSS (Mixed Liquor Suspended Solids) was measured with a portable sludge concentration meter before the start of the experiment, and the amount of standard activated sludge added was calculated based on that concentration as follows, from the amount of test solution and test sludge concentration.

Amount added (mL) = 30 (mg/L)/5300 (mg/L: MLSS) × 300 (mL) = 1.7

**: Basic culture medium was prepared by adding 6 mL each of solution A (Lot No. 303F9117; solutions A-C all made by Kanto Kagaku), solution B (Lot No. 303F9118), solution C (Lot No. 303F9119 and Solution D (0.0247 g of Kanto Kagaku reagent FeCl3•6H2O, Lot No. 211D2290, was filled up to 100 mL with distilled water), which are specified in JIS K 0102-1998 21 a), to Millipore water and mixing them to make 2 L. This was adjusted to pH 7 with 1N H3PO4. The amount basic culture medium added to each culture bottle was 300 mL minus the amount of standard activated sludge added (1.7 mL).

***: Deionized water purified by Milli-Q (Millipore), specific resistance 15.2 MΩ cm.

BOD measurement:
BOD was measured for 28 days using the apparatus below, and during the measurement period observation for abnormalities in the temperature of the constant temperature room of the apparatus, growth of activated sludge, the degree of dissolution of the test substance, and changes in the color of the test solution due to biodegradation was conducted.

Pretreatment of Test Solutions for Analysis after End of BOD Measurement
After measurement of BOD for 28 days, test solutions were pretreated according to the flow sheet in Figure 4, and pH, DOC and residual test substance concentration were measured. However, only pH was measured in culture bottle No. 1.

pH Measurement
In order to examine changes in pH during the 28-day period of BOD measurement, pH of the test solutions was measured

Dissolved Organic Carbon Concentration (DOC) Measurement
DOC was measured with the apparatus and under the conditions below.

1) Apparatus: Total organic carbon meter (TOC meter), Shimadzu, TOC-5000A

2) Measurement conditions:
Furnace temperature: TC 680°C
Air flow: 150 mL/min
Measurement sensitivity: × 5
Injection volume: 25 μL

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Details on results:

Results of Observation of Culture Bottle Contents after BOD Measurement
The liquid in the degradation activity confirmation system (No. 1) was light yellow, and those in the basic sludge respiration system (No. 2), the test substance degradation systems 1, 2 and 3 (Nos. 3, 4, 5) and in the stability in water system (No. 6) were colorless. More growth of sludge was observed in the degradation activity confirmation system than in the basic sludge respiration system, but growth of sludge was not observed in the test substance degradation system. The test substance was insoluble in water.


Results of pH Measurement
The pH of test solutions after the end of BOD measurement was all 7.2 in the test substance degradation system and was also 7.2 in the stability in water system.

Degradation Activity of Standard Activated Sludge
BOD degradation of aniline after 7 days was 53%, and the activity of the standard activated sludge was good.

Results of BOD Measurement
BOD after 28 days in the test substance degradation system against the theoretical oxygen demand (93.4 mg) was -1.3, -0.7 and -0.1 mg (after correction by the measured value in the basic sludge respiration system, in No. 3, 4 and 5), and was 0.3 mg in the stability in water system. Degradation from BOD after 28 days was respectively 0(-1), 0(-1) and 0%, with an average of 0%. Because degradation in test substance degradation systems 1 and 2 was negative, the calculated value is shown in parentheses.


Results of DOC Measurement
DOC after 28 days in the test substance degradation system against the theoretical oxygen demand (91.5 mg/L) was 0.0, 2.2 and 1.7 mg/L (after correction by the measured value in the basic sludge respiration system), and 0 mg/L in the stability in water system. DOC was low because the test substance did not dissolve in the test systems. Degradation was not calculated from DOC.

Results of Measurement of Residual Test Substance Concentration
The residual test substance concentration after 28 days in the test substance degradation system against the charged concentration (100.0 mg/L) was 97.1, 98.9 and 98.7 mg/L, and 101.4 mg/L in the stability in water system. Degradation from the residual test substance concentration after 28 days was 4, 2 and 3% respectively, with an average of 3%. There were no changes in HPLC retention time or peak shape during the experiment.

Measured value (after 28 days)

Measurement item	Test substance degradation system*			Stability in water system	Charged theoretical value
	1	2	3
BOD (mg)	-1.3	-0.7	-0.1	0.3	93.4
DOC (mg/L)	0.0	2.2	1.7	0.0	91.5
Test substance (mg/L)	97.1	98.9	98.7	101.4	100.0

*: Value after correction by the measured value of basic sludge respiration system

Degradation (after 28 days)

Degradation	Test substance degradation system*			Average
	1	2	3
Degradation from BOD (%)	0 (-1)**	0 (-1)**	0	0
Degradation from DOC (%)	Not calculated because test substance is insoluble in water
Degradation from residual test substance concentration (%)	4	2	3	3

**: Figures in parentheses show calculated degradation value when it was negative

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Average degradation of the test substance after 28 days from BOD was 0%, and average degradation from residual test substance concentration was 3%. The test substance was judged to have poor degradability.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 16 November 2012. Experimental Comletion date: 14 December 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of International Trade and Industry's Chemical Substances Control Law (Law Clause No 117, 1973)

Qualifier:

according to guideline

Guideline:

other: The methodology outlined in the MITI Gazette, 19 July 1974 under paragraph 7-1: "Method for Testing the Biodegradability of Chemical Substances by Micro-organisms"

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: A mixed population of active sludge micro-organisms were obtained from 10 different sites around the UK.

Details on inoculum:

- Source of inoculum/activated sludge:
a) Domestic sewage plants (x3)
- i) Liverpool; ii) Loughborough, Leicestershire; iii) Gloucester
b) Industrial sewage plant (x1)
- i) Derby
c) Freshwater samples (x3)
- i) Leeds and Liverpool Canal, Liverpool; ii) River Derwent, Belper, Derbyshire; iii) River Severn, Gloucester
d) Lake Water (x1)
- i) Allestree Lake, Derby
e) Sea Water Samples (x2)
- i) Huttoft, Eastern Coast; ii) Hightown, North Western Coast
- Sample types and volumes from each site: Sewage Plants: 1 L of return sludge at each sewage disposal plant; Freshwater, lake and sea: 1 L of surface water and 1 L of surface soil on the bank/beach which is in contact with the atmosphere.

The samples obtained from the sampling sites were mixed thoroughly and the mixture allowed to settle. The floating foreign matter was removed and the supernatant filtered through a coarse filter paper. The filtrate was then mixed with 2 L of synthetic sewage and transferred to a culture vessel. The pH of the culture mixture was measured using a WTW pH 340I pH and dissolved oxygen meter and adjusted to pH 7.0 ± 1.0 with sodium hydroxide or phosphoric acid and constantly aerated via a narrow bore glass pipette at a temperature of 25 ± 2 °C.

The culture was allowed to settle daily for approximately 30 min and approximately 1/3 of the volume of the supernatant removed. An equal volume of 0.1 % synthetic sewage was added and the aeration re-started again. Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionized water at a concentration of 0.1 % w/v. The pH of the synthetic sewage and culture was adjusted daily to within the range pH 7.0 ± 1.0 with sodium hydroxide or phosphoric acid.

The sludge was found to form a clear supernatant on settling and to have an active microflora including a variety of protozoa, including ciliates, invertebrates and a large population of motile bacteria. The sludge formed cloudy flocs when on aeration.

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

MINERAL MEDIUM
- Deionized water purified by reverse osmosis* was aerated for approximately 24 hours at 21 °C to give a dissolved oxygen concentration of 8.12 mg O2/L measured using a WTW pH 340I pH and dissolved oxygen meter. (*Elga Optima 15+ or Elga Purelab Option R-15 BP).
- To each L of aerated water, 3 mL of solutions 1- 4 was added:
Solution 1
K2HPO4: 21.75 g/L
KH2PO4: 8.50 g/L
Na2HPO4.12H2O: 44.60 g/L
NH4Cl : 1.70 g/L
Solution 2
MgSO4.7H2O: 22.50 g/L
Solution 3
CaCl2: 27.50 g/L
Solution 4
FeCl3.6H2O: 0.25 g/L
- pH: 7.0- 7.2

SEEDED DILUTION WATER
- Determination of the suspended solids level of the active sludge culture was carried out according to the method given in the Japanese Industrial Standard JISK 0102-1981. The suspended solids (ss) concentration of the culture was determined to be 1100 mg ss/L. Seeded dilution water was prepared by addition of 727.3 mL of the active sludge culture to a final volume of 800 mL of mineral medium to give a suspended solids level of 1000 mg ss/L and maintained on aeration at approximately 25 ºC for 4 days prior to use in the study in order to reduce the basal BOD of the seeded dilution water. On subsequent dilution in the preparation of the test series, suspended solids levels of 30 mg ss/L and 100 mg ss/L were obtained for the reference and test item preparations respectively.

TEST CONDITIONS
- Test temperature: 24- 25 °C
- pH: 6.6- 7.5
- Continuous darkness: yes; the test was conducted in diffuse light

TEST SYSTEM
- Number of culture flasks/concentration: The following test concentrations were prepared in 500 mL glass bottles:
1. Three replicate bottles containing inoculated mineral medium to act as the inoculum control for the test item vessels. These bottles contained 50 mL of seeded dilution water of suspended solids level of 1000 mg ss/L and 450 mL of mineral medium to give a final suspended solids level of 100 mg ss/L.
2. Three replicate bottles containing inoculated mineral medium to act as the inoculum control for the procedure control vessels. These bottles contained 15 mL of seeded dilution water of suspended solids level of 1000 mg ss/L and 485 mL of mineral medium to give a final suspended solids level of 30 mg ss/L.
3. Three replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control. These bottles were prepared by diluting a 50 mL aliquot of a 1000 mg/L stock solution of aniline with 435 mL of mineral medium and 15 mL of seeded dilution water of suspended solids level of 1000 mg ss/L to give a test concentration of 100 mg/L, and a final suspended solids concentration of 30 mg ss/L.
4. Eight replicate bottles containing inoculated mineral medium and the test item at a concentration of 30 mg/L. These bottles were prepared by dispersing an amount of test item (15 mg) with 450 mL of mineral medium and 50 mL of seeded dilution water of suspended solids level of 1000 mg ss/L to give a test concentration of 30 mg/L, and a final suspended solids concentration of 100 mg ss/L.
5. Four replicate bottles containing the test item in deionized water alone at a concentration of 30 mg/L. These bottles were prepared by dispersing an amount of test item (15 mg) with 500 mL of deionized water.
6. One bottle containing the test item in mineral medium alone at a concentration of 30 mg/L. This bottle was prepared by dispersing an amount of test item (15 mg) with 500 mL of mineral medium.

SAMPLING
On Day 0, one of each inoculated mineral medium at 30 and 100 mg ss/L, 1 procedure control, 1 test item in inoculated mineral medium, and 1 test item in deionized water vessel were sampled for compound specific analysis and/or pH determination. All remaining inoculum control, procedure control and test item vessels were placed in the CES Multi-Channel Aerobic Respirometer. On Day 28, one of each inoculated mineral medium at 30 and 100 mg ss/L, 1 procedure control, 3 test item in inoculated mineral medium, and 1 test item in deionized water vessel that were considered to have given the most consistent BOD values over the study period were selected for pH determinations and compound specific analysis. The remaining vessels which were not sampled were discarded and not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the study. The remaining test item vessels which were not sampled were stored deep frozen to allow for identification of degradation products if required.

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

65

Sampling time:

28 d

Remarks on result:

other: The test item attained 75 %, 83 % and 34 % degradation with a mean of 65 % degradation

Parameter:

% degradation (test mat. analysis)

Value:

82

Sampling time:

28 d

Remarks on result:

other: The results of the compound specific analyses showed that the test item attained 100 %, 95 % and 50 % degradation with a mean of 82 % degradation

Details on results:

The compound specific analyses conducted showed that whilst there was a significant decrease in measured concentrations in the test item plus inoculum vessels, a significant decrease was also observed in the test item in deionized water vessel thereby indicating that the test item was subject to hydrolysis over the 28-Day test period. Examination of chromatograms from the Day 28 analyses did not show the presence of any hydrolysis products, however as the method of analysis was specific to the parent test item and not any possible hydrolysis products this was to be expected.

BOD values showed that in the test item plus inoculum vessels, biodegradation of the test item was occurring and hence the higher losses of test item in the test item plus inoculum vessels when compared to the test item in deionized water vessel were probably the result of a combination of biodegradation and hydrolysis. As there was significant hydrolysis in the test item in deionized water vessel, the calculation of degradation rates based on the results of the compound specific analyses was skewed. Recalculation of the degradation rates based on loss of test item over the 28-Day period assuming a starting concentration of 30 mg/L gave degradation rates of 100%, 99% and 63% with a mean value of 87%.

Pre-study solubility work showed the test item had limited solubility in water. As hydrolysis and subsequent biodegradation is dependent on the test item first solubilising, the differing rates of biodegradation observed for the replicate test item plus inoculum vessels calculated from the BOD values were considered likely to be due to differing rates of solubilisation in the individual vessels. The relatively large variation in biodegradation rates between the individual test vessels was therefore considered not to have affected the integrity of the study as this was considered the result of physico-chemical parameters rather than biological parameters.

The following table summarizes the results from Days 7, 14, 21 and 28, calculated from oxygen consumption values and the compound specific test item analyses :

Identification		Degradation (%)
		Oxygen Consumption				Day 28
		Day 7	Day 14	Day 21	Day 28	Compound specific analyses
Test item (30 mg/L) plus inoculum	R1	12	17	53	75	100
	R2	40	36	86	83	95
	R3	0	0	1	34	50
	Mean	17	18	47	64	82

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The inherent biodegradability of the test material was assessed according to OECD guideline 302 C. The test item attained 75%, 83% and 34% degradation with a mean of 64% degradation calculated from oxygen consumption values after 28 days. The degradation rates from the compound specific analyses were 100%, 95% and 50% with a mean value of 82%. Therefore, the test item can be considered to be inherently biodegradable.

Description of key information

The inherent biodegradability of the test material was assessed according to OECD guideline 302 C. The test item attained 75%, 83% and 34% degradation with a mean of 64% degradation calculated from oxygen consumption values after 28 days. The degradation rates from the compound specific analyses were 100%, 93% and 49% with a mean value of 82%. Therefore, the test item can be considered to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

A study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous medium. The method followed are designed to be compatible with the OECD Guidelines for Testing of Chemicals (1981) No. 302C Inherent Biodegradability; Modified MITI (II) Test and in the MITI Gazette, 19 July 1974 under paragraph 7-1: Method for Testing the Biodegradability of Chemical Substances by Micro-organisms.

The test item was prepared as an aqueous dispersion at a concentration of 30 mg/L, inoculated with micro-organisms from a laboratory culture originating from 10 different sites throughout the UK and incubated in diffuse light between 24 and 25 °C for 28 days. The degradation of the test item was assessed by the measurements of oxygen consumption and compound specific analyses on Days 0 and 28. The test item attained a mean of 64% degradation calculated from oxygen consumption values after 28 days. The degradation rates from the compound specific analyses gave a mean value of 82%.

The following table summarizes the results from Days 7, 14, 21 and 28, calculated from oxygen consumption values and the compound specific test item analyses :

Identification		Degradation (%)
		Oxygen Consumption				Day 28
		Day 7	Day 14	Day 21	Day 28	Compound specific analyses
Test item (30 mg/L) plus inoculum	R1	12	17	53	75	100
	R2	40	36	86	83	95
	R3	0	0	1	34	50
	Mean	17	18	47	64	82

 The compound specific analyses conducted showed that whilst there was a significant decrease in measured concentrations in the test item plus inoculum vessels, a significant decrease was also observed in the test item in deionized water vessel thereby indicating that the test item was subject to hydrolysis over the 28-Day test period. Examination of chromatograms from the Day 28 analyses did not show the presence of any hydrolysis products, however as the method of analysis was specific to the parent test item and not any possible hydrolysis products this was to be expected.

BOD values showed that in the test item plus inoculum vessels, biodegradation of the test item was occurring and hence the higher losses of test item in the test item plus inoculum vessels when compared to the test item in deionized water vessel were probably the result of a combination of biodegradation and hydrolysis. As there was significant hydrolysis in the test item in deionized water vessel, the calculation of degradation rates based on the results of the compound specific analyses was skewed. Recalculation of the degradation rates based on loss of test item over the 28-Day period assuming a starting concentration of 30 mg/L gave degradation rates of 100%, 99% and 63% with a mean value of 87%.

Pre-study solubility work showed the test item had limited solubility in water. As hydrolysis and subsequent biodegradation is dependent on the test item first solubilising, the differing rates of biodegradation observed for the replicate test item plus inoculum vessels calculated from the BOD values were considered likely to be due to differing rates of solubilisation in the individual vessels. The relatively large variation in biodegradation rates between the individual test vessels was therefore considered not to have affected the integrity of the study as this was considered the result of physico-chemical parameters rather than biological parameters.

Under the conditions of this study, the test item can therefore be considered to be inherently biodegradable.