4-[bis(phenylsubstituted)substituted]phthalic acid

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:

Experimental starting date: October 20, 2015: Experimental completion date: November 17, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

impurity

Test material form:

solid: particulate/powder

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes and inland sea; return sludge from sewage plants). Activated sludge, which was prepared and controlled in the test laboratory, was used in this study. The activated sludge, which was cultivated for 18 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method: glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0 ± 1.0.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation 3

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Performance of biodegradability test:
Preparations for test:
a) Decision of additive amount of activated sludge
Additive amount of activated sludge into the test vessel was 2.72 ml on the basis of the concentration of suspended solid in the activated sludge which was determined by the following methods;
Method: In accordance with Japanese Industrial Standards (JIS) K 0102-2013 Section 14.1
Date: October 19, 2015
Result: 3310mg/L
b) Preparation of basal culture medium
The basal culture medium (2L) was prepared at the same proportion as the following method; purified water was added to each 3 ml aliquot of solutions A, B, C and D, which are described in JIS K 0102-2013 Section 21, in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.
c) Validity of activated sludge
Aniline was used as a reference item in order to confirm that the sludge was sufficiently active.

Preparation of test solutions:
a) Addition of test item of aniline
1) Test solution (water + test item) (n=1, Vessel No. 1)
In one test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to 300 ml of purified water, so that the concentration of the test item reached 100 mg/l. After stirring the test solution for 18 hours, the pH was the test solution was measured.
2) Test solution (sludge + test item) (n=3, Vessel Nos. 2, 3 and 4)
In each test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to the basal culture medium [the volume subtracting the volume (2.72 ml) of activated sludge from 300 ml], so that the concentration of the test item reached 100 mg/l. After stirring the test solution for 18 hours, the pH of the test solution was measured and adjusted to 7.0.
3) Test solution (sludge + aniline) (n=1, Vessel No. 6)
In one test vessel, the basal culture medium [the volume subtracting the volume (2.72 ml) of activated sludge from 300ml] was added and stirred for 18 hours. Then, 29.6 µl (30 mg) of aniline was taken out by micro syringe and added to the basal culture medium so that the concentration of aniline reached 100 mg/L.
4) Test solution (control blank) (n=1, Vessel No. 5)
In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (2.72 ml) of activated sludge from 300ml]. After stirring the test solution for 18 hours, the pH of the test solution was measured.
b) Inoculation of activated sludge
The activated sludge was added to each test vessel described in 20, 3) and 4), so that the concentration of the suspended solid reached 30 mg/l.

Instruments and conditions of incubation:
Instruments for incubation:
Closed system oxygen consumption measuring apparatus - Temperature controlled back with measuring unit
Vessel – Glass
Absorbant for carbon dioxide – Soda lime No.1
Conditions of incubation:
Temperature – 25 ± 1 ºC
Duration – 28 days
Stirring method – each test solution was stirred by a magnetic stirrer

Observation and measurement:
Observation of test solution: During the incubation period, the appearance of the test solution was observed once a day.
Measurement of biochemical oxygen demand (BOD): During the incubation period, BOD of the test solutions was measured continuously by a closed system oxygen consumption measuring apparatus. The incubation temperature was measured and recorded once a day.

Reference substance

Reference substance:

aniline

Results and discussion

Test performance:

See validity of test conditions.

% Degradation

% Degradation 1

Parameter:

other: % degradation by BOD

Value:

65

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

75

Sampling time:

28 d

% Degradation 3

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

For the reference substance, 91% biodegradation was calculated by BOD after 28 days.

Any other information on results incl. tables

Analytical results of the test solutions after 28 days were as follows:

		Water + test item	Sludge + test item			Theoretical amount
		Vessel No.1	Vessel No.2	Vessel No.3	Vessel No.4
BOD*	Mg	1.1	29.6	18.5	32.3	41.4
Residual amount and percentage residue of DOC	mgC	14.6	3.7	3.4	3.7	15.1
	%	96	24	22	25	-
Residual amount** and percentage residue of test item (by HPLC)	Mg	0	0	0	0	30.0
	%	0	0	0	0	-
Produced amount and percentage production of ammonia nitrogen (by HPLC)	mgN	0	0.6	1.0	0.6	1.8
	%	0	34	54	35	-
Produced amount and percentage production of nitrite nitrogen (by HPLC)	mgN	0	0	0	0	1.8
	%	0	0	0	0	-
Produced amount and percentage production of nitrate nitrogen (by HPLC)	mgN	0	0	0	0	1.8
	%	0	0	0	0	-
Detection of converted product (by LC-MS)	-	Detected ***				-

* The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item)

** Calculated from the total peak area on the chromatograms

*** Determination of each converted product could not be performed because the structural estimation about the most of them was difficult.

Percentage biodegradation:

The percentage decrease of the test item, the ratio of decreased amount to the theoretical amount was calculated instead of the percentage biodegradation by the chemical analysis because the percentage residue of the test item in the test solution (water + test item) (0%) was less than 90%.

		Sludge + test item
		Vessel No.2	Vessel No.3	Vessel No.4	Average
Percentage biodegradation by BOD	%	72	45	78	65
Percentage biodegradation by DOC	%	75	77	74	75
Percentage decrease of test item (by HPLC)	%	100	100	100	100

 

Results of qualitative analysis of converted products:

Ten peaks of the converted products in total were found on HPLC chromatograms in the test solution (water + test item) and (sludge + test item). These converted products were treated as A to J in elution order. From the qualitative analysis by LC-MS [with photo-diode-array (PDA) detector], seven peaks of the converted products were detected in total on PDA chromatograms and total ion current (TIC) chromatograms of positive and negative ion in the test solution (water + test item). In the test solution (sludge + test item), two peaks of the converted products were detected in total on PDA chromatograms and TIC chromatograms of positive and negative ion.

From the results of the mass spectral analysis of the converted products (see attached background material), two of them were presumed to be benzenesulfonic acid and 4-hydroxyphthalic acid, and the structures of the others could not be presumed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Difference between the max value and the min value of the % biodegradation by BOD did not meet validity criteria but not considered to effect validity of test results.

Interpretation of results:

other: see conclusion

Conclusions:

Under the test conditions pf this study, most of the test item was biodegraded and plural converted products were produced. The converted products are inferred to be eventually degraded by microorganisms.

Executive summary:

Summary

This study was aimed at evaluating the biodegradability of CIM-43 by microorganisms

 

Test method

OECD guidelines for testing of chemical, No. 301C

 

Conditions of incubation

Concentration of test item: 100 mg/L

Concentration of activated sludge: 30 mg/L ( as concentration of suspended solid)

Volume of test solution: 300 ml

Incubation temperature: 25 ± 1 ºC

Incubation duration: 28 days (under dark conditions)

 

Measurement and analysis for calculation of percentage biodegradation or decrease

a) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

b) Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

c) Determination of test item by high-performance liquid chromatography (HPLC)

Results

		Sludge + test item
		Vessel No.2	Vessel No.3	Vessel No.4	Average
Percentage biodegradation by BOD	%	72	45	78	65
Percentage biodegradation by DOC	%	75	77	74	75
Percentage decrease of test item (by HPLC)*	%	100	100	100	100

* The percentage decrease of the test item, the ratio of decreased amount to the theoretical amount, was calculated instead of the percentage biodegradation by the chemical analysis because the percentage residue of the test item in the test solution (water + test item) (0%) was less than 90%.

Conclusion

Under the test conditions of this study, most of the test item was biodegraded and plural converted products were produced. The converted products are inferred to be eventually degraded by microorganisms.