A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June 1998 - 07 August 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

anaerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

A sample of secondary effluent was obtained on the day of the test from Thorndon sewage treatment works, a trickling-filter plant that treats predominantly domestic waste.
It was maintained under aerobic conditions in the laboratory until required, and vacuum-filtered through a Whatman's GFC filter paper immediately before use. The filtrate was used as the source of inoculum for the test (1 drop/litre test medium).

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Details on study design:

Four groups of four BOD bottles were filled with Mineral Salts Medium, inoculum and test substance and/or sodium benzoate according to the schedule shown below, care being taken to avoid the introduction of air bubbles during preparation and transfer of media to bottles

Group Additions to MSM
1 Inoculum
2 Inoculum + sodium benzoate (2 mg/l)
3 Inoculum + test substance (30 mg/l)
4 Inoculum + test substance (30 mg/l)
+ sodium benzoate (2 mg/l)

Sodium benzoate was added as an aqueous stock solution (1 g/l).

A stock solution of the test material was prepared in acetone (207.5 mg/25 ml; 8.312 rng/rnl) and aliquots (1 rnl) were added to BOD bottles (nominal capacity 277 rnl). The acetone was then evaporated in a gentle stream of nitrogen whilst the bottle was continuously rotated to leave the required weight of test substance deposited onto the wall of the BOD bottle. Test concentrations quoted in this report refer to the test substance as received; no allowance has been made for a purity of less than 100%.

The concentrations of dissolved oxygen (DO) and the temperatures of the contents of duplicate vessels from each group were measured, using a YSI dissolved oxygen meter fitted with a self-stirring DO/temperature bottle probe, at the start of the test and after incubation in darkness for five days at approximately 20°C.

The pH of each control, test and reference mixture was measured after oxygen and temperature measurements.

MANOMETRIC RESPIROMETRY TEST
Inoculum
A sample of activated sludge was obtained from Oakley Sewage Treatment Works, a sewage plant treating predominantly domestic waste. In the laboratory, the sampie was maintained under aerobic conditions until required.

The concentration of suspended solids in a homogenised sample was determined before the start of the test. Aliquots (25 ml) of the activated sludge were filtered through dried and preweighed Whatman's GFC filter papers which were then dried again at 105˚C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then determined and the volume required to give a solids level of 100 mg/I in the test mixture was calculated.

The correct operation of the electrolytic cells, the cornpurer control system and the data recording system was confirmed before the start of the test.

Seven electrolytic respirometry flasks (nominal capacity 550 rnl) were prepared and an appropriate volume of mineral salts medium or ultrapure water was added to each. An electrolytic celI containing the required volume of a copper sulphate solution (1M) acidified with sulphuric acid (1%), and an empty CO, absorber trap were then fitted to each and the flasks were placed in a temperature-controlled water bath to equilibrate overnight. Each flask was placed on an electrically operated magnetic stirrer which was set to give a vortex depth of at least 2 cm in each test mixture.

On the day of the test, a stock solution of ANOX BF (206.3 mg / 25ml: 8.252 mg/ml) was prepared in acetone and aliquots (2 ml) were added to a second set of three, clean flasks that had been previously rinsed with acetone and allowed to dry The acetone was then evaporated in a gentle stream of nitrogen whilst the flask was rotated to leave the required weight of test substance deposited on the wall of the flask. Pre-equilibrated equilibrated medium was then transferred to the flasks containing the test substance. The reference substance was added as an aqueous stock solution (2.75 g/I).
Test and control mixtures were prepared according to the following schedule. In each case the final volume was 550 ml.

Flask number Contents
1 and 2 Control - Inoculated MSMS
3 Reference - Inoculated MSMS + sodium benzoate (100 mg/l)
4 and 5 Test - Inoculated MSMS + test substance (30 mg/l)
6 Abiotic control - Ultrapure water + test substance (30 mg/l)
7 Ultrapure water alone

The CO, absorber (KOH, 1.5 ml of a 10 M solution) was added to the trap in each flask and the flasks were sealed, returned to the water bath and covered with black polythene to minimise contact with light. The electrolytic cells were then connected to a computer-controlled current generating and , data recording system and the test was initiated.

The data recording system was programmed to record the amount of oxygen generated in units of 0.4 mgO2.

The manometric electrolytic cells fitted to each flask comprised two concentrically arranged connected chambers; the inner chamber, which was connected to the headspace above the test mixture, contained a platinum anode and the outer which was vented to the atmosphere, contained a copper cathode. A platinum level sensor fitted in the outer (atmospheric) chamber monitored the level of the electrolyte.

As biodegradation progressed, oxygen in the medium and the headspace above the test mixture was consumed, and the CO2 produced was absorbed. The absorption of CO2 caused a reduction in the pressure of the gas in the headspace above the test mixture which in turn changed the level of the electrolyte and caused a current (20.1 mA) to flow between the anode and cathode for periods of one minute, under the control of the level sensor.

The flow of current generated oxygen at a rate of 0.1 mg/minute at the anode and this flow was maintained until a sufficient quantity had been produced to return the electrolyte to its original level and so replace the oxygen consumed during degradation. The flow of current was automatically terminated by the level sensor when a sufficient amount of oxygen had been produced.

The record of the cumulative oxygen demand by each cell made each hour was printed at the end of the study.

The correct operation of the magnetic stirrers was verified: and the temperature of the water bath measured, on each day of the test.

Reference substance:

other: Sodium benzoate

Preliminary study:

Oxygen consumption in bottles containing ANOX BF alone at 30 mg/1 was negligible (<1% of its
ThOD) which indicates that the test substance was not degradable under the conditions of this
preliminary test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

36.2

Sampling time:

28 d

Details on results:

Experimental values (test substance):
8.7 % degradation after 7 d
18.3 % degradation after 14 d
30.5 % degradation after 21 d
36.2 % degradation after 28 d

Key result

Parameter:

ThOD

Value:

2.78 other: mgO2/mg

Results with reference substance:

Experimental values (reference substance):
71.6 % degradation after 7 d
78.1 % degradation after 14 d
86.2 % degradation after 21 d
87.9 % degradation after 28 d


Calculated ThOD 1.67 mgO2/mg

MICROBIAL INHABITION ASSAY

Sodium benzoate was degraded to 42% of its ThOD after five days of incubation which indicates that the inoculum was viable and exerting normal biodegradative activity. In the presence of test substance at 30 mg/l, degradation of benzoate was unaffected at 5 1% of its ThOD which indicates that the test substance was not inhibitory to the microbial inoculum.

Oxygen consumption in bottles containing inoculated mineral salts medium alone at five days (0.2 mgO2/l) was acceptable for this assay system.

 

 

MANOMETRIC RESPIROMETRY TEST
Cumulative consumption of oxygen in the controls (60 4 and 61.2 mg/O2) was considered to be acceptable for cultures containing activated sludge at 100 mg solids/l. The degradation of sodium benzoate was rapid and achieved 63% of its ThOD after 4 days and 88% after 28 days. These results confirm that the inoculum was viable and that the validity criterion for the degradation of the reference substance (60% in 14 days) had been achieved. Oxygen consumption by the mixture of ANOX BF in ultrapure water (abiotic control) was equivalent to 3% of its ThOD after 28 days of incubation which indicated that it was not subject to a significant level of abiotic degradation. Mean oxygen consumption by biotic mixtures containing ANOX BF was equivalent to 11% of the ThOD of the mixture (45.87 mg/O2) after eight days and 36% by the end of the test on Day 28. Degradation was slow but progressive throughout; a degradation plateau was not attained in the test.
The pH of the biotic mixtures ranged from 7.4 to 7.6 at the end of the test; the pH of the abiotic mixture containing ANOX BF was 8.4. Temperatures of the water bath ranged from 20.6 to 23.1 "C.

 

Microbial inhibition assay – BOD (mgO2/mg) and percentage degradation

Test group	Mean oxygen conc. (mgO2/l)		BOD (mgO2/mg)	% degradation BOD:ThOD
	Day 0	Day 5
Inoculated mineral salts	8.8	8.6	(0.2)	-
Sodium benzoate (2mg/l)	8.7	7.1	0.70	42
ANOX BF (30 mg/l)	8.3	8.0	< 0.1	0
Sodium benzoate (2 mg/l) + ANOX BF (30 mg/l)	8.4	6.4	0.85	51

The values in brackets gives the oxygen consumption in mgO2/l of the control group

 

Manometric respirometry test – cumulative consumption of oxygen and percentage degradation

Day	Cell 1	Cell 2	Mean Cells 1 & 2	Cell 3	Corrected Cell 3 (mgO2)	Cell 3 - % degradation	Cell 4	Corrected 4 (mgO2)	Cell 4 - % degradation	Cell 5	Corrected Cell 5 (mgO2)	Cell - 5 % degradation	Mean % degradation Cells 4 and 5	Cell 6	Cell 7	Cell 6 – Cell 7 (mgO2)	Cell 6 - % degradation
0	0	0	0	0	0.0	0.0	0	0.0	0.0	0	0.0	0.0	0.0	0	0	0	0.0
1	116	112	114	376	26.2	28.5	100	-1.4	-3.1	108	-0.6	-1.3	-2.2	12	8	0.4	0.9
2	188	184	186	632	44.6	48.8	168	-1.8	-3.9	192	0.6	1.3	-1.3	32	24	0.8	1.7
3	240	236	238	772	53.4	58.1	236	-0.2	-0.4	248	1.0	2.2	0.9	48	40	0.8	1.7
4	280	276	278	856	57.8	62.9	280	0.2	0.4	288	1.0	2.2	1.3	48	40	0.8	1.7
5	308	304	306	912	60.6	65.9	312	0.6	1.3	316	1.0	2.2	1.7	52	44	0.8	1.7
6	324	324	324	960	63.6	69.2	352	2.8	6.1	340	1.6	3.5	4.8	52	44	0.8	1.7
7	352	348	350	1008	65.8	71.6	404	5.4	11.8	376	2.6	5.7	8.7	52	44	0.8	1.7
8	388	388	388	1056	66.8	72.7	452	6.4	14.0	428	4.0	8.7	11.3	52	44	0.8	1.7
9	420	420	420	1096	67.6	73.6	496	7.6	16.6	468	4.8	10.5	13.5	68	60	0.8	1.7
10	440	440	440	1128	68.8	74.9	524	84	18.3	492	5.2	11.3	14.8	72	60	1.2	2.6
11	444	444	444	1152	70.8	77.0	536	9.2	20.1	500	5.6	12.2	16.1	72	60	1.2	2.6
12	448	448	448	1164	71.6	77.9	544	9.6	20.9	504	5.6	12.2	16.6	72	60	1.2	2.6
13	460	460	460	1196	73.6	80.1	560	10.0	21.8	520	6.0	13.1	17.4	72	60	1.2	2.6
14	480	484	482	1228	74.6	81.2	588	10.6	23.1	544	6.2	13.5	18.3	72	60	1.2	2.6
15	508	512	510	1268	75.8	82.5	628	11.8	25.7	580	7.0	15.3	20.5	72	60	1.2	2.6
16	524	532	528	1288	76.0	82.7	656	12.8	27.9	600	7.2	15.7	21.8	72	60	1.2	2.6
17	528	536	532	1304	77.2	84.0	672	14.0	30.5	616	8.4	18.3	24.4	72	60	1.2	2.6
18	556	564	560	1332	77.2	84.0	708	14.8	32.3	652	9.2	20.1	26.2	84	72	1.2	2.6
19	556	564	560	1336	77.6	84.4	708	14.8	32.3	652	9.2	20.1	26.2	84	72	1.2	2.6
20	556	564	560	1340	78.0	84.9	716	15.6	34.0	656	9.6	20.9	27.5	84	72	1.2	2.6
21	564	572	568	1360	79.2	86.2	736	16.8	36.6	680	11.2	24.4	30.5	84	72	1.2	2.6
22	568	572	570	1364	79.4	86.4	470	17.0	37.1	680	11.0	24.0	30.5	84	72	1.2	2.6
23	568	572	570	1368	79.8	86.8	744	17.4	37.9	688	11.8	25.7	31.8	84	72	1.2	2.6
24	568	576	572	1376	80.4	87.5	752	18.0	39.2	696	12.4	27.0	33.1	84	72	1.2	2.6
25	576	584	580	1388	80.8	87.9	764	18.4	40.1	708	12.8	27.9	34.0	84	72	1.2	2.6
26	584	592	588	1396	80.8	87.9	772	18.4	40.1	724	13.6	29.6	34.9	84	72	1.2	2.6
27	600	608	604	1412	80.8	87.9	792	18.8	41.0	744	14.0	30.5	35.8	84	72	1.2	2.6
28	604	612	608	1416	80.8	87.9	796	18.8	41.0	752	14.4	31.4	36.2	84	72	1.2	2.6

 

Cells 1 and 2 = Inoculated Mineral salts medium alone (Control)

Cell 3 = Inoculated mineral salts medium plus sodium benzoate (100 mg/l)

Cells 4 and 5 = Inoculated mineral salts medium plus test substance (30 mg/l)

Cell 6 = test substance (30 mg/l) in UllP water (abiotic mixture)

Cell 7 = UllP water alone (abiotic control)

ThOD of test material = 2.78 mgO2/mg

ThOD of test mixture = 45.87 mgO2

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The five-day inhibition assay was conducted before the main test to determine whether ANOX BF, at a concentration of 30 mg/l, inhibited the normal degradative activity of the microbial inoculum on the reference substance, sodium benzoate. In this preliminary test. the test substance was neither inhibition, at this concentration nor was it readily degradable.

In the Manometric Respirometry test, ANOX BF was degraded to 11% of its ThOD after eight days and 36% by the end of the test (Day 28). Degradation was slow but progressive throughout; a degradation plateau was not attained in the test. The level of abiotic degradation was insignificant (equivalent to 3% of the ThOD after 28 days).

Substances are considered to show evidence of inherent. primary biodegradation in this type of test if a significant level of degradation (i.e. >30%) has been achieved after 28 days. According to this definition. ANOX BF can be considered to show evidence of primary. inherent biodegradation.

The result obtained for the rate of degradation of sodium benzoate (63% of its ThOD after 4 days and 88% after 28 days) fulfils the validity criteria for this test.

Executive summary:

The inherent biodegradability of ANOX BF was assessed by a Manometric Respirometry method based on OECD Procedure 302C, Inherent Biodegradability, Modified MITI test (II) and OECD Procedure 301 F. Ready Biodegradability, Manometric Respirometry test.

 

A five-day microbial inhibition test was performed under the conditions of the Closed Bottle test (EC Procedure C.4-E1 OECD Procedure 301D). This showed that ANOX BF at a nominal concentration of 30 mg/l did not inhibit degradation of the reference substance sodium benzoate. In this preliminary test. ANOX BF showed no evidence of biodegradation.

 

In the Manometric Respirometry test, ANOX BF was added to two flasks containing mineral salts medium inoculated with activated sludge (100 mg solids/litre) and to one flask containing ultrapure water alone (abiotic control) to give a nominal concentration of 30 mg/l. Controls comprised two flasks containing inoculated mineral salts medium alone. one containing inoculated mineral salts medium plus the reference substance sodium benzoate (30 mg/l) and one containing ultrapure water alone. The contents of the flasks were vigorously stirred to give a vortex depth of > 2 cm.

 

Each flask was fitted with a trap containing potassium hydroxide (1.5 ml, 10M), to absorb any carbon dioxide (CO2) produced during degradation, and a manometric electrolytic cell containing copper sulphate (1M) which automatically replaced the oxygen consumed during degradation. The operation of the electrolytic cell and recording of oxygen demand was controlled by a computer. Degradation of the test or reference substance was expressed as the cumulative amount of oxygen consumed as a percentage of the respective Theoretical Oxygen Demand (ThOD).

 

Sodium benzoate was degraded by 63% of the ThOD of the mixture (91.9 mggO2) after four days and 88% after 28 days. Cumulative oxygen consumption in the controls after 28 days (60.4 and 61 -2 mgO,) was considered to be acceptable for an inoculum level of 100 mg solids/l. These results confirm that the inoculum was viable and that the test was valid.

 

Mean oxygen consumption by biotic mixtures containing ANOX BF was equivalent to 11% of the ThOD of the mixture (45.87 mgO2) after eight days and 36% by the end of the test on Day 28. Degradation was slow but progressive throughout; a degradation plateau was not attained in the test. The level of abiotic degradation was insignificant (equivalent to 3% of the ThOD after 28 days).

 

Substances are considered to show evidence of inherent, primary biodegradation in this type of test if a significant levels of degradation (i.e. >30%) has been achieved after 28 days. ANOX BF can therefore be considered to show evidence of primary, inherent biodegradation.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1992 - July 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: 92/69/EWG, C.4-D

GLP compliance:

no

Remarks:

Study conducted prior to the mandatory requirement to conduct studies to the GLP directive

Oxygen conditions:

aerobic

Inoculum or test system:

not specified

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Ready biodegradability
Biodegradability has been assessed by means of manometric respirometry.
A measured volume of inoculated medium containing a known amount of test substance is stirred in a closed flask. The consumption of oxygen is determined from the change in volume of air within the apparatus.
Evoled CO2, is absorbed in soda lime. The amount of oxygen taken up by the substance (corrected for blank) is expressed as a percentage of the theoretical oxygen demand calculated from the formula of the compound (2.905 mgO2/mg test substance).
A reference substance (Na-benzoate) is run in parallel for checking the inoculum.

Chemical oxygen demand
A predetermined amount of the test substance, which contains not more than 30 mg ThOD (Theoretical Oxygen Demand), is oxidized by a boiling mixture of 25 rnl potassium dichromate 0.25 N and concentrated sulfuric acid. Silver sulfate is added to the mixture as a catalyst for a more complete oxidation of organic substances such as straight chain aliphatic compounds. After two hours boiling, the mixture is cooled to room temperature and diluted with water: the excess of dichromate is titrated with ferrous ammonium sulfate 0.25 M.

Reference substance:

other: Sodium benzoate

Key result

Parameter:

% degradation (CO2 evolution)

Value:

34.5

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
0 % degradation after 3 d
0 % degradation after 5 d
.4 % degradation after 9 d
2.3 % degradation after 12 d
6.9 % degradation after 14 d
13.8 % degradation after 16 d
23.7 % degradation after 19 d
32 % degradation after 23 d
33.2 % degradation after 26 d
34.5 % degradation after 28 d

Key result

Parameter:

COD

Value:

2.77 other: mg O2/mg test mat

Remarks on result:

other: Mean

Results with reference substance:

Points of degradation plot (reference substance):
65.2 % degradation after 3 d
75.4 % degradation after 5 d
89.7 % degradation after 9 d
89.8 % degradation after 12 d
90 % degradation after 14 d
92.7 % degradation after 16 d
92.9 % degradation after 19 d
96.5 % degradation after 23 d
96.6 % degradation after 26 d
96.8 % degradation after 28 d

Time (Days)	Biodegradation (%)		Time (Days)	Biodegradation (%)
	ANOX BF*	Reference		ANOX BF*	Reference
0	0	0	16	13.8	92.7
3	0	65.2	19	23.7	92.9
5	0	75.4	23	32.0	96.5
9	0.4	89.7	26	33.2	96.6
12	2.3	89.8	28	34.5	96.8
14	6.9	90.0

 

*mean values of two determination

 

Test number	1	2	3
COD (mgO2/mg substance)	2.82	2.76	2.74

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable

Conclusions:

The biodegradation of the test substance ANOX BF measured by manometric respirometry is 34.5 % after 28 days of incubation;  the COD of the substance is 2.77 mgO2/mg substance.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2001 - 26 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

ACTIVATED SLUDGE
1) Mixed liquor suspended solid (MLSS) : 4500 mg/L
2) Source : Chemical Evaluation and Research Institute, Japan
3) Date of receipt : July 19, 2001

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Remarks:

measured with HPLC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The test substance was exposed to the activated sludge in a closed-system oxygen consumption measuring apparatus. The biochemical oxygen demand (BOD) was measured over a 28 day period, After this period, the concentrations of the dissolved organic carbon (DOC) and the residual test substance in the test bottles were measured. The biodegradability of the test substance was evaluated from these results.

ACTIVATED SLUDGE
1) Mixed liquor suspended solid (MLSS) : 4500 mg/L
2) Source : Chemical Evaluation and Research Institute ,Japan
3) Date of receipt : July 19, 2001

EXPOSURE CONDITIONS_
1) Temperature : 25 +/- 1˚C
2) Exposure period : 28 days
3) Test volume : 300 mL
4) Concentration : test substance (bottles 3-6) : 100 mg/L
aniline* (bottle 1) : 100 mg/L
activated sludge (bottles 1-5) : 30 mg/L
* Reference substance : KANTO CHEMICAL Co., INC. Lot No.212G1294

Test bottle contents :
Bottle 1 : Aniline + activated sludge + basal medium 29 µL (30 mg) of aniline was added to the basal medium*, then activated sludge was added.
Bottle 2 : Activated sludge + basal medium
Activated sludge was added to the basal medium**.
Bottles 3-5 : Test substance + activated sludge + basal medium
30 mg of the test substance was added to the basal medium, then activated sludge was added.
Bottle 6 : Test substance + purified water
30 mg of the test substance was added to 300 mL of purified water***.
** The total volume of the basal medium and the sludge was held fixed at 300 mL.
*** Grade A4, Japanese Industrial Standards KO557

BOD MEASUREMENT
The BOD was measured for 28 days
1)Apparatus : closed system oxygen consumption measuring apparatus Ohkura Electric Co., Model OM-2001 (M.S.I. ID NO.D).

pH MEASUREMENT
After the exposure period, 20 mL of the test solution in each test bottle was transferred into a 20-mL glass beaker for pH measurement. After pH measurement, the test solution was returned to each bottle for measurement of residual. test substance concentration.
1)Apparatus : pH meter, ORION Research, Model 720

DOC MEASUREMEN2
The concentration of the DOC was measured as follows.
1 ) Apparatus : TOC analyzer, Shimaclzu Co., model TOC-5000A
2 Conditions : Furnace temperature : 680˚C (TC)
Air flow rate : 250 mL/min.
Sensitivity: x5
Injection volume : 50 ILL
3) Calibration curve
The following standard solutions were injected into the TOC analyzer
The calibration curve was prepared by the data processor of the analyzer.
Standard solutions
TC (total carbon) 20 and 50 mg C/L aqueous solutions of potassium biphthalate.
IC(inorganic carbon) 0 mg C/1 purified water (purified by Milli-Q) and 10 mg C/1 aqueous solution of sodium hydrogen carbonate and sodium carbonate.
4) Measurement of DOC in the bottles
Ten millilitre of the test solution in each bottle was transferred into a 10-mL centrifuge tube and centrifuged at 3000 rpm for 10 minutes. Five millilitre of supernatant was used for the DOC measurement. The remaining supernatant and the precipitate were returned to each bottle for the measurement of the residual test substance concentration.

Reference substance:

aniline

Key result

Parameter:

other:

Value:

>= 11 - <= 48

Sampling time:

28 d

Remarks on result:

other: Degradability based on residual test substance

Details on results:

OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD
The solution in the bottles 1 and 5 were cloudy, in the bottles 2, 3, 4 and 6 were colorless. Growth of the sludge was observed in bottle I, in contrast with the control bottle (bottle 2). No growth was observed in bottles 3, 4 and 5.

pH MEASUREMKNT
After 28 days of exposure, the pi1 was determined to be 6.9, 7.1, 6.9 and 8.0 for bottles 3, 4, 5 and 6, respectively.

ACTIVITY OF SLUDGE
The degradability of aniline, based on the BOD measurements was 62 % after 7 days. The activity of the sludge was thus shown to be satisfactory.

DEGRADABILITY BASED ON BOD
BODk1 in bottles 3, 4, and 5 (as corrected with the value in bottle 2) were 6.7, -1.0 and 3.9 mg, and BOD in bottle 6 was 0.0 mg, respectively.
(*I Maximum theoretical value = 88.9 mg)
The degree of degradability based on the BOD measurements were 8, O(ca1culated value = -1) and 4 % for bottles 3, 4, and 5, respectively.

DEGRADABILITY BASED ON DOC
DOC*> in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 18.0, 3.9, and 23.0 mg/L, respectively. DOC in bottle 6 was 2.0 mg/L.
(*2 Maximum theoretical value = 78.9 mg/L)

Degradability was not calculated because the test substance was insoluble in water.

DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE CONCENTRATION
The test substance*' was detected at concentrations of 67.2, 91.8, 53.5 are 103.6 mg/L in bottles 3, 4, 5 and 6, respectively.

(*3 Initial concentration = 100 mg/l)
The degree of degradability based on the residual test substance concentration was calculated to be 35, 11 and 48 for bottles 3, 4 and 5, respectively.
A new peak was detected on the HPLC chromatograms for bottles 3-5 with retention time of about 2.2 min.

Key result

Parameter:

BOD5

Value:

>= 0 - <= 8 other: mg/L

SUMMARY OF DOC MEASUREMENT

Degradability based on BOD

 

Bottle No.	Test substance	ThOD mg	Day 7		Day 14		Day 21		Day 28
			BOD mg	% degradability	BOD mg	% degradability	BOD mg	% degradability	BOD mg	% degradability
1	Aniline	90.2	59.7	62	70.5	71	71.1	71	71.1	71
2	-	-	3.6	-	6.3	-	6.8	-	6.7	-
3	ANOX BF	88.9	3.4	0	6.3	0	7.0	0	13.4	8
4	ANOX BF	88.9	2.1	0 (-2)	5.2	0 (-1)	5.8	0 (-1)	5.7	0 (-1)
5	ANOX BF	88.9	2.1	0 (-2)	4.7	0 (-2)	5.0	0 (-2)	10.6	4
6	ANOX BF	88.9	0.0	-	0.0	-	0.0	-	0.0	-

Where % degradability was calculated to be negative, this value is shown in parentheses

 

pH measurement

Bottle No.	pH
	Day 0	Day 28
1	-	7.7
2	7.0	7.1
3	-	6.9
4	-	7.1
5	-	6.9
6	7.9	8.0

 

Results of DOC measurement

Bottle No.	Organic carbon	DOC mg/L
2	-	1.1
3	78.9	19.1
4	78.9	5.0
5	78.9	24.1
6	78.9	2.0

 

Degradability based on residual test substance

Bottle No.	Concentration mg/L	Degradability %
2	< 1	-
3	67.2	35
4	91.8	11
5	53.5	48
6	103.6	-

 

RESULT OF DOC MEASUREMENT

	Bottle 2		Bottle 3		Bottle 4		Bottle 5		Bottle 6		Calibration curve
	TC		TC		TC		TC		TC		TC standard area
	Area	mg/L	Area	mg/L	Area	mg/L	Area	mg/L	Area	mg/L	20 mg/L	50 mg/L
Measure 1	878	1.279	13457	19.610	3618	5.271	16743	24.390	1400	2.040	13860	34435
Measure 2	893	1.301	13159	19.170	3585	5.223	16689	24.310	1378	2.008	13880	34437
Measure 3	892	1.300	13423	19.560	3586	5.225	16905	24.630	1401	2.041	13968	34610
Mean	888	1.293	13346	19.447	3596	5.240	16779	24.443	1393	2.030	13909	34494

 

	Bottle 2		Bottle 3		Bottle 4		Bottle 5		Bottle 6		Calibration curve
	IC		IC		IC		IC		IC		IC standard area
	Area	mg/L	Area	mg/L	Area	mg/L	Area	mg/L	Area	mg/L	0 mg/L	10 mg/L
Measure 1	111	0.157	260	0.368	175	0.248	227	0.321	0	0	0	7034
Measure 2	145	0.205	262	0.371	186	0.263	242	0.343	0	0	0	7072
Measure 3	126	0.178	275	0.389	205	0.290	229	0.324	0	0	0	7082
Mean	127	0.180	266	0.376	189	0.267	233	0.329	0	0	0	7063

 

	TC (mg/L)	IC (mg/L)	TOC (mg/L)
Bottle 2	1.293	0.180	1.113
Bottle 3	19.447	0.376	19.071
Bottle 4	5.240	0.267	4.973
Bottle 5	24.443	0.329	24.114
Bottle 6	2.030	0.000	2.030

 

TOC-5000A Conditions

TC Furnace Temp. : 680°C

TC Catalyst: 25L

Springe Size: Normal

 

Measurement Mode: TOC

 

Air flow r a t e: 150 ml/min

 

No of Injects: 3

 

Range: x 5

 

Inj vol: 50µL

 

 

CALCULATION OF RECOVERY AND DETECTION LIMIT

	Peak area mAb.sec (A)	Concentration in solution mg/L		Recovered mass mg (D)	Added mass mg (E)	Recovery & (F)	Detection limit mg/L (G)
		For HPLC analysis (B)	In bottle (C)
Recovery test 1	4413292	1433.4	95.6	28.7	30.0	96	-
Recovery test 2	4529207	1471.0	98.1	29.4	30.0	98	-
Blank	<2000	<0.7	<0.1	-	0.0	-	1
						Average recovery = 97%

 

Standard solution

Concentration (H) : 1500 mg/L

Peak area (I) : 4618494 mAbs. sec

Volume of solution for HPLC analysis (J) : 0.020 L

Volume of test solution for HPLC analysis (K) : 0.300 L

Volume of test solution in bottle (L) : 0.300 L

 

Equation: B = H x A ÷ I C = H x A ÷ I x J ÷ K      D = C x L                      F = D ÷ E x 100 G: raise decimal fractions to units

 

Calculation of the detection limit

Retention time (min)	1500 mg/L std.		Blank
	Peak area mAb. sec	Rate of peak area (%)	Peak area mAb. sec
2.5	275638	6.0	-
5.7	116385	2.5	-
6.3	129733	2.8	-
7.0	2325708	50.4	< 1000
7.9	1771030	38.3	-
Total	4618494	100.0	< 2000

 

The minimum detectable peak area of the test substance was calculated by the following equation based on the minimum detectable peak area of 1000 mAbs-sec for the largest peak (retention time 7.0 min. ) in HPLC chromatogram.

 

Minimum detectable peak area (mAbs. sec) 4618494 x (1000 (mAbs-sec) ÷ 2325708 (dbs. sec) = 1986 (mAbs-sec) (< 2000 (mAbs.sec))

 

RESIDUAL TEST SUBSTANCE CONCENTRATION IN TEST BOTTLE

Bottle No.	Peak area mAb. sec (A)	Concentration in solution (mg/L)
		For HPLC analysis (B)	In bottle (C)
2	< 2000	< 0.7	< 1
3	3146970	977.9	67.2
4	4299421	1336.1	91.8
5	2506017	778.8	53.5
6	4849234	1506.9	103.6

 

Standard solution

Concentration (D) : 1500 mg/L

Peak area (E) : 4826956 mAbs. sec

Volume of solution for HPLC analysis (F) : 0.020 L

Volume of test solution for HPLC analysis (G) : 0.300 L

Average recovery (H) : 97%

 

Equation: B = D x A ÷ E             C = D x A ÷ E x F ÷ G ÷ H x 100

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

From the degradability results based on the BOD and the residual test substance concentration, it is concluded that the test substance was not readily biodegradable under the conditions of this test.
As a new peak was detected on the HPLC chromatograms for bottles 3-5, it is suggested that the test substance has been transformed during the exposure period.

Executive summary:

The test substance was exposed to the activated sludge in a closed-system oxygen consumption measuring apparatus. The biochemical oxygen demand (BOD) was measured over a 28 day period, After this period, the concentrations of the dissolved organic carbon (DOC) and the residual test substance in the test bottles were measured. The biodegradability of the test substance was evaluated from these results. Study was conducted in accordance with "Ready biodegradability, Modified MITI test" in OECD Guidelines for Testing of Chemicals No. 301C.

Results of the study are summarised as follows:

Measured values (day 28)

	Bottle No.
	3*	4*	5*	6	Theoretical value
BOD (mg)	6.7	-1.0	3.9	0.0	88.9
DOC (mg/L)	18.0	3.9	23.0	2.0	78.9
Test substance (mg/L)	67.2	91.8	53.5	103.6	100.0

* value corrected with BOD or DOC value of Bottle 2.

Degradabilities (%)

	Bottle No.
	3	4	5	Average
BOD	8	0 (-1)**	4	4
DOC	NA***	NA***	NA***	-
Test substance	35	11	48	31

** where % degradability was calculated to be negative, this value is shown in parentheses.

*** degradability was not calculated because the test substance was insoluble in water.

From the degradability results based on the BOD and the residual test substance concentration, it is concluded that the test substance was not readily biodegradable under the conditions of this test. As a new peak was detected on the HPLC chromatograms for bottles 3-5, it is suggested that the test substance has been transformed during the exposure

Description of key information

The biodegradation of the test substance ANOX BF measured by manometric respirometry is 34.5 % after 28 days of incubation;  the COD of the substance is 2.77 mgO2/mg substance. A peak derived from transformed product was detected at the retention time of approximately 1.2 minutes under the conditions of ESI negative mode on the HPLC chromatograms of bottles 3, 4 and 5. The molecular ion peak of m/z 277 (M-H) was detected on the MS Spectra. From these results, the structure of the transformed product was estimated to be 3-[3,5-Bis(tert-butyl)-4-hydroxyphenyl]propionic acid, MW 278.39.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater