4,4'-benzylidenedianiline

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From the 17th of December 2009 to the 15th of January 2010

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Version / remarks:

July 17th 1992 version

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: DATIPH
Physical form: Solid
Solubility in water: < 1 g/L
Colour: yellow brown
CAS no.: 603-40-7
Chemical name: 4,4’-Benzylidenedianiline
Molecular formula: C19H18N2
Theoretical carbon content: 83.2%
Test substance storage: At room temperature protected from light
Stability: Stable under storage conditions
Expiry date: 31 October 2010

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant, not adapted; 0.2 g/l dry matter in the final mixture.

Duration of test (contact time):

28 d

Initial conc.:

ca. 60.1 mg/L

Based on:

TOC

Remarks:

60.1 mg/L of 4,4'-Benzylidenedianiline represents a theoretical concentration of 50.0 mg of organic carbon/L

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The activated sludge was used after sampling from the treatment plant without adaptation. However, the sludge will be pre-conditioned for 3 days (aerated but not fed). Prior to the test the sludge was washed twice with tap water. The test material was directly added to give a final DOC concentration of about 50 mg/l. The activated sludge was applied to give a dry substance content of 0.2 g/l. The dry substance content of the activated sludge is gravimetrically determined in regular intervals after drying of the sludge at 105°C.

For each test series the following number of test flasks was set up:
Test suspension (T), 2 replicates: containing activated sludge + test medium + test substance
Inoculum blank (B), 2 replicates: containing activated sludge + test medium
Procedure control (R), 2 replicates : containing activated sludge + test medium + diethyleneglycol as ready biodegradable reference compound

The test vessels were stirred (100 r.p.m.) and aerated with synthetic CO2-free air for a maximum test period of 28 days. The air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH. The pH-value was checked at the beginning and was adjusted to pH 7.4 +/- 0.2 with HCI. At the end of the test the pH was recorded.
The elimination of the test material was followed by DOC determinations at regular intervals. First samples were analyzed before the addition of the sludge and 3 h after starting the test. The trapped CO2 in the gas-absorption bottles was determined as inorganic carbon (IC).

Reference substance:

diethylene glycol

Parameter:

% degradation (inorg. C analysis)

Value:

< 20

Sampling time:

28 d

Details on results:

Based on the data of the individual DOC determinations (Table 2, Figure 1) DATIPH reached an elimination of 66% after 28 days calculated with respect to the applied TOC.
The main elimination process occurred during the first day, when 90% of the initial DOC was removed. Maximum elimination was attained after 1 day of incubation. Biodegradation was observed without an adaptation period (lag phase).
It has to be mentioned that after an initial DOC elimination due to adsorption, the DOC values increased again during the first 27 days, most probably due to a slow dissolution process.
The positive control, diethyleneglycol, showed 98% biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70% within 14 days of incubation.
No biodegradation of DATIPH based on CO2 evolution and calculated as % ThCO2 was observed after 28 days of incubation underpinning that the observed DOC decrease was due to elimination not degradation. The mineralization of the procedure control with diethyleneglycol reached 84% based on ThCO2 (Table 3).
Based on the mineralization data DATIPH can neither be termed as “inherently, ultimately biodegradable” nor as “inherently, primarily biodegradable” under the conditions of the OECD guideline no. 302, since less than 20% degradation was attained after 28 days of contact time. However, a major part of DATIPH will be eliminated in sewage treatment plants.

Table 1: DOC concentration of test suspension, inoculum blank and procedure control and calculation of degradation data.

	Inoculum blank (B) *	Procedure control with diethylene glycol (R)			Test suspension with test material (T) *
Time (days)	DOC (mg/l)	DOC (mg/l)	DOC net. (mg/l)	Degradation (%)	DOC (mg/l)	DOC net. (mg/l)	Degradation (%)
0	0.8	50.7	50.0		n.d.
0.125	1.3	51.2	49.8	3	n.d.	50.0	0
1	1.2	49.3	48.1	6	6.4	5.2	90
4	1.7	28.9	27.1	47	9.8	8.0	84
7	3.1	3.6	0.5	99	13.1	9.9	80
11	2.0	2.9	0.9	98	13.2	11.3	78
14	2.0	2.9	0.8	98	13.9	11.9	76
18	2.3	2.7	0.5	99	15.8	13.5	73
21	2.1	2.3	0.2	100	16.1	14.0	72
25	2.0	2.7	0.7	99	17.1	15.0	70
27	2.1	2.5	0.4	99	19.6	17.4	65
28	1.6	2.0	0.4	99	18.6	17.0	66

The DOC elimination is calculated with respect to the applied theoretical TOC concentration of 50.0 mg C/L.

Since the test substance was insoluble, the calculation of biodegradation was based on the theoretically applied TOC values calculated from the added amount of the test substance and the theoretical carbon content.

Table 2: IC concentrations, calculated from the concentrations in the gas adsorption bottles of test suspension, inoculum blank and procedure control and corresponding degradation data.

Time (days)	Test suspension IC (mg/l)	Procedure control IC (mg/l)	Inoculum blank IC (mg/l)	Biodegradation Test suspension (% ThCO2)	Biodegradation Procedure control (% ThCO2)
0	nd	nd	nd	-	-
7	20.9	59.7	24.9	-8	71
14	27.2	71.8	31.8	-9	82
21	31.9	84.8	41.4	-19	89
28	39.7	92.3	51.5	-24	84

Validity criteria fulfilled:

yes

Remarks:

The test was considered valid, since the degradation of the reference compound reached more than 70% within 14 days of incubation.

Interpretation of results:

not inherently biodegradable

Conclusions:

Based on the mineralization data DATIPH can neither be termed as “inherently, ultimately biodegradable” nor as “inherently, primarily biodegradable” under the conditions of the OECD guideline no. 302, since less than 20% degradation was attained after 28 days of contact time.

Executive summary:

The biodegradability of DATIPH exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions.

Based on the data of the individual DOC determinations DATIPH reached a biodegradation of 66% after 28 days, calculated with respect to the applied TOC.

The main elimination process occurred during the first day, when 90% of the initial DOC was removed. Maximum elimination was attained after 1 day of incubation. No significant adaptation period (lag phase) was observed.

It has to be mentioned that after an initial DOC elimination due to adsorption, the DOC values increased again during the first 27 days, most probably due to a slow dissolution process.

The positive control, diethyleneglycol, showed 98% biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70% within 14 days of incubation.

No biodegradation of DATIPH based on CO₂evolution and calculated as%ThCO₂was observed after 28 days of incubation underpinning that the observed DOC decrease was due to elimination not degradation. The mineralization of the procedure control with diethyleneglycol reached 84% based on ThCO₂.

Based on the mineralization data DATIPH can neither be termed as “inherently, ultimately biodegradable” nor as “inherently, primarily biodegradable” under the conditions of the OECD guideline no. 302. since less than 20% degradation was attained after 28 days of contact time. However, a major part of DATIPH will be eliminated in sewage treatment plants.

Description of key information

The biodegradability of 4,4'-Benzylidenedianliline exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions.

Based on the mineralization data, 4,4'-Benzylidenedianiline can neither be termed as “inherently, ultimately biodegradable” nor as “inherently, primarily biodegradable” under the conditions of the OECD guideline no. 302. since less than 20% degradation was attained after 28 days of contact time. However, a major part of DATIPH will be eliminated in sewage treatment plants.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed