2,4-xylenol

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:

supporting study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The measurement of oxygen consumption is one of the oldest means of assessing biodegradability, and respirometry is one technique for measuring oxygen consumption. E l e c t r o l y t i c respirometry i s the most commonly employed method for biodégradation studies. Generally i t i s used for biochemical oxygen demand (BOD) determination

The oxygen uptake by a stirred solution, or suspension, of the test substance in a mineral medium, inoculated with specially grown, unadapted micro-organisms, is measured automatically over a period (20-40 d) in a darkened, enclosed respirometer at 25 + 1°C. Evolved carbon dioxide is absorbed by soda lime. Biodegradation is expressed as the percentage oxygen uptake. The percentage primary biodegradation is also calculated from supplemental specific chemical analysis made at the beginning and end of incubation.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Remarks:

activated sludge sample from wastewater treatment plant receiving predominantly domestic sewage

Details on inoculum:

The microbial inoculum was an activated sludge sample from The Little Miami wastewater treatment plant in Cincinnati, Ohio, which receives predominantly domestic sewage. The sludge was allowed to settle for about an hour, decanted, and then aerated at room temperature for 24 h. The dry weight of sludge was determined by drying samples, in duplicates, of 1 ml, 2 ml and 3 ml at 105°C overnight. A concentration of 30 mg/1 of sludge as dry matter was used in the experiment. The total volume of the synthetic medium in the 500 ml capacity reactor vessels was brought up to a final volume of 250 ml.

Duration of test (contact time):

ca. 20 - ca. 40 d

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Electrolytic respirometry i s the most commonly employed method for biodégradation studies. Generally i t i s used for biochemical oxygen demand (BOD) determination. The data collection is automatic with electrolytic respirometry, so manual collection and analysis of samples i s not required to follow the course of substrate degradation. Since the respirometer can be automated, the length of the experiment is less important and thus unacclimated biomass can be used in the experiments.

Electrolytic respirometry studies were conducted using an automated continuous oxygen measuring Voith Sapromat B-12 electrolytic respirometer. This system consists of a temperature-controlled water bath which contains the measuring units, a recorder for digital indication, a plotter for direct presentation of the oxygen uptake curves of substrates, and a cooling unit for conditioning and continuous recirculation of water bath contents. The system used had 12 measuring units each connected to the recorder. Each unit, as shown in Figure 1, consisted of a reaction vessel A, with a carbon dioxide absorber (soda lime) mounted in a stopper, an oxygen generator B, and a pressure indicator C. Interconnected by hoses, the vessels form a sealed measuring system so that barometric pressure fluctuations do not adversely affect the results. The magnetic stirrer in the sample to be analyzed provides vigorous agitation, thus ensuring effective gas exchange. Microorganism activity in the sample creates a pressure reduction that is recorded by the pressure indicator which controls both the electrolytic oxygen generation and plotting of the measured values. The CO2 generated is absorbed by soda lime.

The N 2/O 2 ratio in the gas phase above the sample is maintained constant throughout the experiment by an on/off feedback control loop. As oxygen is depleted, pressure reduction is created in the sample and, as a result, the level of the sulphuric acid in the pressure indicator rises and comes in contact with a platinum electrode. This completes the circuit and triggers the generation of oxygen by electrolysis. The electrolytic cell provides the required amount of oxygen to the reaction vessel by electrolytic dissociation of a CUSO4 -H2 SO4 solution thus alleviating the negative pressure. As the level of the electrolyte in the pressure indicator decreases, the contact with the electrode is broken. This switches off the electrolytic cell. The amount of oxygen supplied to the sample is recorded directly in milligrams per liter by the recorder. The recorder is connected to a microcomputer which records data from the measuring units every 15 minutes.

The concentration of the test substances was 100 mg/1 of medium. Aniline was used as the biodegradable reference substance at a concentration of 100 mg/1. The stock solutions were made in distilled water for aniline and phenol with concentration of 5 g/1. Other substances were added directly to the reaction vessels using microliter syringes.

The typical experimental system consisted of duplicate flasks for the reference substance aniline and the test substances and a single flask for toxicity control (test substance + aniline at 100 mg/1) and an inoculum control. The contents of the reaction vessels were stirred for an hour to ensure a steady state of the endogenous respiration at the initiation of oxygen uptake measurements. Then the test substance and aniline were added. The reaction vessels were incubated at 25°C in the temperature-controlled water bath and stirred continuously throughout the run. The microbiota of the activated sludge used as inoculum were not preacclimated to the test substances. The incubation period of the experimental run was between 20 to 40 days.

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

> 80

Remarks on result:

other: . Within a period of 40 days the test substances were degraded between 80%-95%

Remarks:

The incubation period of the experimental run was between 20 to 40 days.

Details on results:

Oxygen uptake curves for the test substances, reference substance aniline, the toxicity control (aniline + test substance) and the endogenous control systems were generated over a period of 20 to 40 days. Within a period of 10 days all the controls, test substances and aniline revealed the lag phase, biodegradation phase and the plateau region. Figures 2 illustrate the representative oxygen uptake curves generated for two of the test substances, phenol. These data reveal that 2,4-dimethyl phenol, were biodegradable at the concentration level of 100 mg/1 under the experimental conditions. According to OECD, the results of the degradation experiment were valid because 60% degradation of control substance aniline was achieved within a period of 28 days. Within a period of 40 days all the test substances were degraded between 80%-95%.

Results with reference substance:

The results of the degradation experiment were valid because 60% degradation of control substance aniline was achieved within a period of 28 days.

The difference between the total oxygen uptake of the toxicity control (test substance + aniline), and of the corresponding test substance, was approximately equal to the total oxygen uptake of the control substance aniline.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance. The concentration of the test substances was 100 mg/L of medium. Aniline was used as the reference substance at a concentration of 100 mg/L. The unadapated activated sludge sample was collected from The Little Miami wastewater treatment plant, which receives predominantly domestic sewage. A concentration of 30 mg/L of sludge as dry matter was used in the experiment. The experimental system consisted of duplicate flasks for the reference substance aniline and the test substances and a single flask for toxicity control (test substance + aniline at 100 mg/L) and an inoculum control. The contents of the reaction vessels were stirred for an hour to ensure a steady state of the endogenous respiration at the initiation of oxygen uptake measurements. Then the test substance and aniline were added. The reaction vessels were incubated at 25°C in the temperature-controlled water bath and stirred continuously throughout the run. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values automatically by electrolytic respirometer. The incubation period of the experimental run was between 20 to 40 days. The estimation of Monod kinetic parameters were obtained for all the compounds by a graphical method. Within a period of 10 days all the controls, test substances and aniline revealed the lag phase, biodegradation phase and the plateau region. The test substance was degraded in excess of 80% within a period of 40 days. The test substance was not found to be inhibitory towards the biodegradation of aniline in the sludge. The results of the degradation experiment were valid because 60% degradation of control substance aniline was achieved within a period of 28 days. Under the study conditions, the test substance can be considered to be inherently biodegradable (Desai, 1990).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 may 2001 to 17 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Biodegradation study of chemical substances by microorganisms in Kanpogyo, No.5, Yakuhatsu, No.615, 49 Kikyoku, No.392

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Activated sludge

Details on inoculum:

Concentration of activated sludge: 30 mg/L
Active sludge: Standard active sludge obtained from Chemical Substances Safety Center of Chemical Examination and Research Institute, Japan on May 17, 2001 and incubated under control at 25.0±2.0°C, dissolving oxygen of 5 mg/L or above and pH 7.0+-1.0 (Active sludge No.130517).
Suspended solid concentration measurement result of active sludge (From May 25, 2001 to May 28, 2001): 4616 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

other: residual test substance amount measurement

Details on study design:

Study system
Coulometer: Type OM-2001A, made by Ohkura Electric Co., Ltd.
Data sampler: DS-3, made by Asahi Techneion Co., Ltd.

Test conditions
Test temperature: 25.0-25.1 °C
Test solution volume: 300 mL
Test water: Self-made water by ultrapure water maker, made by Japan Millipore
Environmental factors considered to affect reliability of the study result: None

Types and frequency of observation, measurement, examination and analysis:
Observation of temperature, test substance dissolving status, test solution color and proliferative state of the sludge: Observed once per day (except total 8 days of weekends).
Oxygen consumption measurement: 28-day continuous measurement automatically by coulometer.
pH measurement of the test solution: Measured immediately after taking out from coulometer after completion of the study.
Residual test substance amount measurement: Analyzed by LC after pre-treatment of the test solution at completion of the study.
Total organic carbon (TOC) amount measurement: Analyzed by TOC analyzer after pre-treatment of the test solution at completion of the study

Reference substance:

aniline

Remarks:

Lot No.: PM805L

Key result

Parameter:

% degradation (O2 consumption)

Value:

91

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Remarks:

(average biodegradation by direct quantificaton was 100%)

Key result

Parameter:

% degradation (TOC removal)

Value:

98

Sampling time:

28 d

Remarks on result:

other: average degradation

Details on results:

Confirmation of test conditions: Biodegradation of aniline calculated by oxygen consumption was 53 % after 7 days of the start of the exposure, therefore, this study is valid.

Residual rate and degradation degree:
As shown in the Table 1, average biodegradation of test substance calculated by oxygen consumption was 91 % after 28 days of the start of the exposure. Average biodegradation by direct quantification was 100 %.


Residual total organic carbon (TOC) amount :

As shown in Table 2, average total organic carbon residual rate in (Test Substance Degradation) area and (Test Substance-Water) area are 1 % and 82 % respectively. Average biodegradation was 98 %.
Residual rate of K-1227C by direct quantification for (Test Substance-Water) area was 78 % and total organic carbon residual rate was 82 %. The biodegradation was low. Because the test substance was considered to volatilize during the exposure period, washing solution of soda lime of (Test Substance-Water) area irradiated in methanol was analyzed by LC. As a result, residual amount of K-1227C was 5.3 mg and mass balance added residual amount in the test solution at the completion of the study was good result of 96 %.


Observation and measurement performed during and after exposure study: Observation results of once per day (except total of 8 days of weekends) performed during the exposure study period are shown in Table 3.
Observation and measurement results at the completion of the study of the test solutions are shown in Table 4.

Table 1:

	No.	Test area	Biodegradation
			(%)			Ave.
Result by oxygen consumption	3	TSD*	98			91
	4	TSD*	84
	5	TSD*	91
	No.	Test area	Residual rate		Biodegradation
			(%)	Ave.	(%)	Ave.
Result by direct quantification	3	TSD*	0	0	100	100
	4	TSD*	0		100
	5	TSD*	0		100
	6	TS-W**	78		-

*TSD: (Test Substance Degradation) area, **TS-W: (Test Substance-Water) area Note) No.: Shows coulometer detector No.

Table 2:

No.	Test area	TOC amount^*2		Residual rate		Biodegradation
		(mg)	Ave.	(%)	Ave.	(%)	Ave.
3	TSD*	0.2	0.3	1	1	99	98
4	TSD*	0.3		1		98
5	TSD*	0.5		2		97
6	TS-W**	19.3		82		-

*TSD: (Test Substance Degradation) area, **TS-W: (Test Substance-Water) area

 Note) No.: Shows coulometer detector No.

*2: The values of (Test Substance Degradation) and (Test Substance-Water) areas are shown after subtracted the values of (Basic Respiratory) area and Ultrapure Water Blank used for the study respectively.

Table 3:

Temperature of the constant temperature water bathof the coulometer	25.0-25.1°C throughout the period.
Dissolving state of the test substance	(Test Substance Degradation) area: Good throughout the period. (Test Substance-Water) area:Good throughout the period.
Color of the test solution	(Aniline Degradation) area:        0-3 days after; Slightly white turbid.        4,5 days after; Not observed (weekend).        6-28 days after: White turbid to light yellow. (Test Substance Degradation) area:        0-3 days after; Slightly white turbid.        4,5 days after; Not observed (weekend).        6-28 days after: White turbid. (Test Substance-Water) area:        0-28 days after: Colorless.
Proliferative state of the sludge	(Aniline Degradation) area:        0-3 days after; No proliferation.        4,5 days after; Not observed (weekend).        6 days after: Proliferation was observed. (Test Substance Degradation) area:        0-3 days after; No proliferation.        4,5 days after; Not observed (weekend).        6 days after: Proliferation was observed.

Substance confirmation of the test substance and stability confirmation under storage conditions:

Substance confirmation: The test substance identity was confirmed comparing spectra and test substance spectra

Stability confirmation: IR analysis was performed on the start dates of the exposure study, compared them and confirmed that the test substance is stable throughout the study period.

Validity criteria fulfilled:

yes

Remarks:

Biodegradation of aniline calculated by oxygen consumption was 53 % after 7 days of the start of the exposure, therefore, this study is valid.

Interpretation of results:

readily biodegradable

Conclusions:

Under the study conditions, the test substance was determined to be readily biodegradable with 91% biodegradation after 28 days.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, according to the biodegradation study of chemical substances by microorganisms in Kanpogyo, No.5, Yakuhatsu, No.615, 49 Kikyoku, No.392 ( method similar to MITI (I) (OECD 301C), in compliance with GLP. The stability of test substance was performed by IR analysis on the start and completion dates the study. The test substance at a concentration of 100 mg/L was exposed to active sludge treatment micro-organisms incubated under control conditions (i.e., 25.0±2.0°C, dissolving oxygen of 5 mg/L or above and pH 7.0±1.0) for 28 d. Aniline was used as the reference substance. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values automatically by coulometer for 28 d. TheSuspended solid concentration of active sludge was measured to be 4616 mg/L.The residual test substance and Total organic carbon (TOC) amount was analysed by LC and TOC analyser respectively after pre-treatment of the test solution at completion of the study. The temperature, test substance dissolving status, test solution color and proliferative state of the sludgewere recorded daily during experimental period(except total 8 days of weekends).Test pH was measuredimmediately after taking out from coulometer after completion of the study. The test substance was confirmed to be stable throughout the study period. The average biodegradation of test substance calculated by oxygen consumption was 91 % after 28 d. The average biodegradation of test substance by average residual rate and average total organic carbon residue was 100 and 98% respectively. Biodegradation of aniline calculated by oxygen consumption was 53 % after 7 days of the start of the exposure, therefore, the study was considered to be valid. Under the study conditions, the test substance was considered to be readily biodegradable with 91% degradation after 28 days (KTSC, 2001).

Description of key information

Based on the available studies, the test substance can be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Study 1: A study was conducted to determine the ready biodegradability of the test substance, according to the biodegradation study of chemical substances by microorganisms in Kanpogyo, No.5, Yakuhatsu, No.615, 49 Kikyoku, No.392 ( method similar to MITI (I) (OECD 301C), in compliance with GLP. The stability of test substance was performed by IR analysis on the start and completion dates the study. The test substance at a concentration of 100 mg/L was exposed to active sludge treatment micro-organisms incubated under control conditions (i.e., 25.0±2.0°C, dissolving oxygen of 5 mg/L or above and pH 7.0±1.0) for 28 d. Aniline was used as the reference substance. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values automatically by coulometer for 28 d. TheSuspended solid concentration of active sludge was measured to be 4616 mg/L.The residual test substance and Total organic carbon (TOC) amount was analysed by LC and TOC analyser respectively after pre-treatment of the test solution at completion of the study. The temperature, test substance dissolving status, test solution color and proliferative state of the sludgewere recorded daily during experimental period(except total 8 days of weekends).Test pH was measuredimmediately after taking out from coulometer after completion of the study. The test substance was confirmed to be stable throughout the study period. The average biodegradation of test substance calculated by oxygen consumption was 91 % after 28 d. The average biodegradation of test substance by average residual rate and average total organic carbon residue was 100 and 98% respectively. Biodegradation of aniline calculated by oxygen consumption was 53 % after 7 days of the start of the exposure, therefore, the study was considered to be valid. Under the study conditions, the test substance was considered to be readily biodegradable with 91% degradation after 28 days (KTSC, 2001).

Study 2: A study was conducted to determine the ready biodegradability of the test substance. The concentration of the test substances was 100 mg/L of medium. Aniline was used as the reference substance at a concentration of 100 mg/L. The unadapated activated sludge sample was collected from The Little Miami wastewater treatment plant, which receives predominantly domestic sewage. A concentration of 30 mg/L of sludge as dry matter was used in the experiment. The experimental system consisted of duplicate flasks for the reference substance aniline and the test substances and a single flask for toxicity control (test substance + aniline at 100 mg/L) and an inoculum control. The contents of the reaction vessels were stirred for an hour to ensure a steady state of the endogenous respiration at the initiation of oxygen uptake measurements. Then the test substance and aniline were added. The reaction vessels were incubated at 25°C in the temperature-controlled water bath and stirred continuously throughout the run. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values automatically by electrolytic respirometer. The incubation period of the experimental run was between 20 to 40 days. The estimation of Monod kinetic parameters were obtained for all the compounds by a graphical method. Within a period of 10 days all the controls, test substances and aniline revealed the lag phase, biodegradation phase and the plateau region. The test substance was degraded in excess of 80% within a period of 40 days. The test substance was not found to be inhibitory towards the biodegradation of aniline in the sludge. The results of the degradation experiment were valid because 60% degradation of control substance aniline was achieved within a period of 28 days. Under the study conditions, the test substance can be considered to be inherently biodegradable (Desai, 1990).

Based on the available studies, the test substance can be considered to be readily biodegradable.