Xylene

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, acceptable without restriction

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

Non-adapted activate sludge was obtained from the sewage plant at Hildesheim. It is well suited as it receives predominantly municipal sewage and hardly any chemical waste. The sludge was washed in chlorine free tap water and resuspended in mineral medium for 2 hours before homogenisation with a blender. The supernatant was decanted after sedimentation and maintained for 6 days with continual CO2-free aeration.
The soil was Standard soil from LUFA at Speyer. It is well suited and certified field fresh soil for biodegradation studies. The soil was mixed according to OECD 301 with mineral salts and shaken overnight. The supernatant after sedimentation was maintained with CO2-free aeration until test start when it was used as inoculum.
Colony forming units in test vessel: Approximately 10 (7) to 10 (8) CFU/L.
Inoculum in test: 10 mL/L activated sludge, 7 mL/L soil

Duration of test (contact time):

28 d

Initial conc.:

16 mg/L

Based on:

test mat.

Initial conc.:

50.4 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Test vessel: 250 mL test solution in 500 mL brown glass bottle fitted with a rubber sleeve with soda lime to absorb evolved CO2. Bottles were closed with OxiTop measuring heads.
- Test medium: OECD 301F mineral salts medium
- Temperature: 21.3-22.1°C
- pH: 7.78 at start and 7.69 to 7.72 at end in test vessel, 7.66 to 7.78 at start and 7.65 to 7.98 at end in control vessels
- Conditions: Continual stirring in an incubator in the dark
- Observations: Oxygen consumption was measured every 112 minutes. The difference between the test and inoculum control replicates was used to determine the oxygen depletion, with biodegradation calculated as the relation of the BOD to the ThOD. Results were calculated using Sigma Plot SPSS software.
- Replicates: 3 per test treatment
- Functional control: 1 replicate
- Inoculum control: 2 replicate of test medium without test or reference item
- Toxicity control: 1 replicate of test medium with test and reference item

Reference substance:

benzoic acid, sodium salt

Remarks:

45 mg/L test concentration, 75.2 mg/L ThOD in vessel

Preliminary study:

Not applicable

Test performance:

The toxicity control reached 84% biodegradation after 14 days and 89% after 28 days showing that the test item did not exhibit inhibitory effects on the micro-organisms.

Key result

Parameter:

% degradation (O2 consumption)

Value:

98

Sampling time:

28 d

Details on results:

A mean of 98% biodegradation was achieved after 28 days. 10% biodegradation was reached in 2 days, with 60% biodegradation reached within 5 days, thereby satisfying the 10 day window criterion.

Results with reference substance:

The functional control reached >60% biodegradation within 2 days with a maximum of 93% biodegradation on day 27 which met the validity criteria.

Biodegradation [%] of the test item, m-xylene, in comparison to the
Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item	1	84	100	100	100
	2	81	100	100	100
	3	75	89	94	95
	mv	80	96	98	98
Functional Control		79	88	91	91
Toxicity Control 16mg/L Test Item + 45 mg/L Reference Item		69	84	87	89

                            mv = mean value

Validity criteria fulfilled:

yes

Remarks:

Functional control: 60% in 2 days. Inoculum control: 39.5 mg O2/L on day 28. Toxicity control: 25% within 1 day. pH: 7.65-7.98 on day 28. Difference in replicates: 5% on day 28.

Interpretation of results:

readily biodegradable

Conclusions:

The substance was found to be readily biodegradable reaching 60% biodegradation after 5 days and 98% biodegradation after 28 days.

Executive summary:

Biodegradation testing was carried out following OECD 301F guidelines using a mixed inoculum of non-adapted sludge and soil micro-organisms. m-xylene was found to be readily biodegradable, with 60% biodegradation after 5 days and after 28 days the mean biodegradation was found to be 98%.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, acceptable without restriction

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

Non-adapted activate sludge was obtained from the sewage plant at Hildesheim. It is well suited as it receives predominantly municipal sewage and hardly any chemical waste. The sludge was washed in chlorine free tap water and resuspended in mineral medium for 2 hours before homogenisation with a blender. The supernatant was decanted after sedimentation and maintained for 6 days with continual CO2-free aeration.
The soil was Standard soil from LUFA at Speyer. It is well suited and certified field fresh soil for biodegradation studies. The soil was mixed according to OECD 301 with mineral salts and shaken overnight. The supernatant after sedimentation was maintained with CO2-free aeration until test start when it was used as inoculum.
Colony forming units in test vessel: Approximately 10 (7) to 10 (8) CFU/L.
Inoculum in test: 10 mL/L activated sludge, 7 mL/L soil

Duration of test (contact time):

28 d

Initial conc.:

16 mg/L

Based on:

test mat.

Initial conc.:

50.9 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Test vessel: 250 mL test solution in 500 mL brown glass bottle fitted with a rubber sleeve with soda lime to absorb evolved CO2. Bottles were closed with OxiTop measuring heads.
- Test medium: OECD 301F mineral salts medium
- Temperature: 21.3-22.1°C
- pH: 7.79 at start and 7.71 to 7.76 at end in test vessel, 7.66 to 7.79 at start and 7.65 to 7.98 at end in control vessels
- Conditions: Continual stirring in an incubator in the dark
- Observations: Oxygen consumption was measured every 112 minutes. The difference between the test and inoculum control replicates was used to determine the oxygen depletion, with biodegradation calculated as the relation of the BOD to the ThOD. Results were calculated using Sigma Plot SPSS software.
- Replicates: 3 per test treatment
- Functional control: 1 replicate
- Inoculum control: 2 replicate of test medium without test or reference item
- Toxicity control: 1 replicate of test medium with test and reference item

Reference substance:

benzoic acid, sodium salt

Remarks:

45 mg/L test concentration, 75.2 mg/L ThOD in vessel.

Preliminary study:

Not applicable

Test performance:

The pass level for ready biodegradation in the functional control (> 60 % degradation) was reached within 2 days.

Key result

Parameter:

% degradation (O2 consumption)

Value:

94

Sampling time:

28 d

Details on results:

Biodegradation was calculated based on the ThOD of 3.14 mg O2/mg test item.
The biodegradation of the test item in comparison to the readily degradable functional control and the toxicity control reached the 10 % level (beginning of biodegradation) within 4 days. The course of the degradation was rapid and the 1st and 3rd test item replicates reached the pass level of 60 % within 7 days and the 2nd test item replicate on day 8. The mean biodegradation on day 28 was 94 %. After a test period of 28 days, the test item is classified as readily biodegradable in the 10-d-window and within the 28 day period of the study.

Results with reference substance:

In the toxicity control, the biodegradation achieved 79 % after 14 days. After 28 days the biodegradation came to 86 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Biodegradation [%] of the test item, m-xylene, in comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item	1	61	89	95	94
	2	58	83	90	88
	3	69	97	100	100
	mv	63	90	95	94
Functional Control		79	88	91	91
Toxicity Control		63	79	83	86

                                    mv = mean value

Validity criteria fulfilled:

yes

Remarks:

Functional control: 60% in 2 days. Inoculum control: 39.5 mg O2/L on day 28. Toxicity control: 25% within 1 day. pH: 7.65-7.98 on day 28. Difference in replicates: 12% on day 28

Interpretation of results:

readily biodegradable

Conclusions:

The substance was found to be readily biodegradable after 28 days.

Executive summary:

Biodegradation testing was carried out following OECD 301F guidelines using a mixed inoculum of non-adapted sludge and soil micro-organisms. o-xylene was found to be readily biodegradable, with 60% biodegradation after 8 days and after 28 days the mean biodegradation was found to be 94%.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, acceptable without restriction

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

Non-adapted activate sludge was obtained from the sewage plant at Hildesheim. It is well suited as it receives predominantly municipal sewage and hardly any chemical waste. The sludge was washed in chlorine free tap water and resuspended in mineral medium for 2 hours before homogenisation with a blender. The supernatant was decanted after sedimentation and maintained for 6 days with continual CO2-free aeration.
The soil was Standard soil from LUFA at Speyer. It is well suited and certified field fresh soil for biodegradation studies. The soil was mixed according to OECD 301 with mineral salts and shaken overnight. The supernatant after sedimentation was maintained with CO2-free aeration until test start when it was used as inoculum.
Colony forming units in test vessel: Approximately 10 (7) to 10 (8) CFU/L.
Inoculum in test: 10 mL/L activated sludge, 7 mL/L soil

Duration of test (contact time):

28 d

Initial conc.:

16.2 mg/L

Based on:

test mat.

Initial conc.:

51 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Test vessel: 250 mL test solution in 500 mL brown glass bottle fitted with a rubber sleeve with soda lime to absorb evolved CO2. Bottles were closed with OxiTop measuring heads.
- Test medium: OECD 301F mineral salts medium
- Temperature: 21.3-22.1°C
- pH: 7.78 at start and 7.71 to 7.73 at end in test vessel, 7.66 to 7.78 at start and 7.65 to 7.96 at end in control vessels
- Conditions: Continual stirring in an incubator in the dark
- Observations: Oxygen consumption was measured every 112 minutes. The difference between the test and inoculum control replicates was used to determine the oxygen depletion, with biodegradation calculated as the relation of the BOD to the ThOD. Results were calculated using Sigma Plot SPSS software.
- Replicates: 3 per test treatment
- Functional control: 1 replicate
- Inoculum control: 2 replicate of test medium without test or reference item
- Toxicity control: 1 replicate of test medium with test and reference item

Reference substance:

benzoic acid, sodium salt

Remarks:

45 mg/L test concentration, 75.2 mg/L ThOD in vessel

Preliminary study:

Not applicable

Test performance:

The toxicity control reached 82% biodegradation after 14 days and 87% after 28 days showing that the test item did not exhibit inhibitory effects on the micro-organisms.

Key result

Parameter:

% degradation (O2 consumption)

Value:

90

Sampling time:

28 d

Details on results:

A mean of 90% biodegradation was achieved after 28 days. 10% biodegradation was reached in 4 days, with 60% biodegradation reached within 7 days, thereby satisfying the 10 day window criterion.

Results with reference substance:

The functional control reached >60% biodegradation within 2 days with a maximum of 93% biodegradation on day 27 which met the validity criteria.

Biodegradation [%] of the test item, p-xylene, in comparison to the Functional Control and Toxicity Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item	1	64	83	84	83
	2	66	91	95	97
	3	61	86	90	91
	mv	64	87	90	90
Functional Control		79	88	91	91
Toxicity Control		66	82	86	87

                                     Mv = mean value

Validity criteria fulfilled:

yes

Remarks:

Functional control: 60% in 2 days. Inoculum control: 39.5 mg O2/L on day 28. Toxicity control: 25% within 1 day. pH: 7.65-7.98 on day 28. Difference in replicates: 14% on day 28

Interpretation of results:

readily biodegradable

Conclusions:

The substance was found to be readily biodegradable reaching 60% biodegradation after 7 days and 90% biodegradation after 28 days.

Executive summary:

Biodegradation testing was carried out following OECD 301F guidelines using a mixed inoculum of non-adapted sludge and soil micro-organisms. p-xylene was found to be readily biodegradable, with 60% biodegradation after 7 days and after 28 days the mean biodegradation was found to be 90%.

Description of key information

Based on read across from the xylene isomers and ethylbenzene, the Reaction Mass of Ethylbenzene and Xylene is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

There are no biodegradation studies available for the Reaction Mass of Ethylbenzene and Xylene. It is difficult to assess the persistence of the substance using standard test methods, as the tests measure the properties of the whole substance but do not provide information on the individual constituents. Instead, it is more appropriate to consider whether the representative constituents of the substance are persistent. Therefore, data has been read across from the xylene isomers and ethylbenzene as these are the major constituents of the substance.

 

Ethylbenzene and the xylene isomers are structurally similar substances, consisting of a benzene ring with one and two methyl functional groups attached respectively; the different xylene isomers having the two methyl functional groups attached at different locations. The presence and position of the second methyl functional group on the benzene ring is not expected to change the biodegradation properties of the substance significantly. The similarities in structure, water solubility and behaviour in the aquatic environment indicate that the biodegradation of the components of this substance are likely to be similar. All of the components are relatively soluble in water, with limited potential to partition to soils or sediment. The water solubility (Yalkowsky and He 2003) and log partition coefficient (Hansch et al. 1995) of ethylbenzene, m-, o- and p-xylene are 177 mg/L and 3.15, 146 mg/L and 3.2, 170.5 mg/L and 3.12, and 156 mg/L and 3.15 respectively.

 

Data is available for all three xylene isomers from GLP-compliant guideline studies (Dr Noack 2015). The studies followed OECD guideline 301F (ready biodegradability: manometric respirometry test) using a mixture of sewage and soil micro-organisms. m-xylene reached 60% biodegradation after 5 days and 98% biodegradation after 28 days. o-xylene reached 60% biodegradation after 8 days and 94% biodegradation after 28 days. p-xylene reached 60% biodegradation after 7 days and 90% biodegradation after 28 days. The studies show that the xylene isomers are readily biodegradable, meeting the 10 day window criterion. In addition to the key studies, additional data on the biodegradability of the xylene isomers and ethylbenzene are available from a number of sources.

 

The EU RAR (2007) concludes that ethylbenzene is readily biodegradable. The Risk Assessment Report is peer-reviewed by the Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE), now renamed Scientific Committee on Health and Environmental Risks (SCHER) which gives its opinion to the European Commission on the quality of the risk assessment. This Draft Risk Assessment Report has undergone a discussion in the Competent Group of Member State experts with the aim of reaching consensus by interpreting the underlying scientific information.

 

Exxon Biomedical Sciences (1995) conducted an OECD 301F test with p-xylene, showing 68% biodegradation in 10 days and 87.8% biodegradation in 28 days. This GLP compliant guideline study found p-xylene to be readily biodegradable. Although greater than 20% variability was observed between replicates at the start and end of the 10 day window, all three replicates had reached >60% biodegradation by day 11, so this was not considered to have invalidated the results of the test.

 

Exxon Biomedical Sciences (1996) conducted a GLP-compliant study with o-xylene following OECD guideline 301F. The study was conducted with three replicates, each containing approximately 36 mg/L of test item. The biodegradation reached approximately 70% by day 28, as an average of the three replicates (47.41, 79.5 and 82.1 %), and the positive control material degraded by approximately 94% by day 28. However, only one replicate met the 10 day window criterion, with one other replicate reaching 10% degradation by day 11 but not achieving 60% degradation until day 25 and the final replicate not reaching 60% biodegradation by day 28. As the difference in the extremes of the replicates at the end of the test is greater than 20%, the validity criteria for the OECD guideline 301F were not met, however the study does show that o-xylene underwent significant degradation over the course of the test.

 

Bridie et al. (1979) report the results of a 5 day BOD/ThOD test. This study predates the implementation of GLP and the OECD guidelines for biodegradation screening tests. The study authors report that they followed guidelines in operation at the time, although the level of detail in the publication is limited, in particular the concentration of the test substance is not reported. The study followed an APHA 219 method (1971) for determination of the biological oxygen demand (BOD) and ASTM D1251 -67 method (1974) for determination of the chemical oxygen demand (COD). The study is used in the draft SIDS for xylenes and demonstrates that the xylene isomers can be biodegraded by non-adapted sewage sludge, with a 5 day BOD/ThOD of 80, 52 and 44% for m-, o- and p-xylene respectively.

 

MITI (2001) reports that 100% biodegradation (based on BOD and GCMS) was observed in an OECD 301C study with m-xylene. In a second study with p-xylene 38% biodegradation based on BOD and 92% biodegradation based on GCMS analysis was reported. An earlier study with ethylbenzene, CITI (1992), the precursor to MITI, reported 81-126% biodegradation based on BOD over two weeks in an OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II)). We have been unable to obtain copies of these reports so have not been able to assess their reliability or confirm that the validity criteria were met. However, as the results have been taken from regulatory review articles, they are considered to be suitable for use to support this endpoint.

 

BIOWIN indicates that ethylbenzene and o-, m- and p-xylene biodegrade fast (linear and non-linear model predictions), with primary biodegradation timeframes of days-weeks and ultimate biodegradation timeframes of weeks. The MITI linear and non-linear models predict that o-, m- and p-xylene will biodegrade fast and the overall ready biodegradability predictions are “yes”. However, the MITI linear and non-linear models predict that ethylbenzene will not biodegrade fast and the overall ready biodegradability prediction is “no”. The BioHCwin results estimate that the half-lives of o-, m- and p-xylene would be 4.44 days and the half-life of ethylbenzene would be 5 days. A screening assessment for persistence using the combined results of some of the Biowin models (ECHA (2012) Guidance on information requirements and chemical safety assessment Chapter R11: PBT assessment) indicates that the components in this substance would not meet the screening criteria for persistence. The use of QSAR estimates to predict the biodegradability of the substance is considered valid as ethylbenzene and the xylene isomers are included in the training and validation sets of the BIOWIN and BioHCwin models.

 

Overall, based on the available data for its major constituents (o-, m-, p-xylene and ethylbenzene) it has been concluded that the substance is readily biodegradable.