Tar acids, xylenol fraction

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The Little Miami wastewater treatment plant, Cincinnati, Ohio, USA
- Preparation of inoculum for exposure: Sludge was allowed to settle for about an hour, decanted, and t´hen aerated at room temperature for 24 hours
- Concentration of sludge: 30 mg/L municipal sewage sludge as dry matter

Duration of test (contact time):

20 - 40 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD medium
- Test temperature: 25 °C

TEST SYSTEM
- Culturing apparatus: Sapromat
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirred for an hour to ensure steady state of the endogenous respiration at the initiation of oxygen uptake measurements
- Measuring equipment: automated continuous oxygen measuring Voith Sapromat B-12 electrolytic respirometer (Voith-Morden, Milwaukee, WI)
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: carbon dioxide absorber (soda lime)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask
- Toxicity control: 1 flask
- Reference substance: 2 flasks

Reference substance:

aniline

Remarks:

100 mg/L

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

10 d

Remarks on result:

other: see comment in "Any other information on results" below

Parameter:

% degradation (O2 consumption)

Value:

80 - 95

Sampling time:

40 d

Details on results:

The oxygen uptake curves are not reported. However, the authors state that all test compounds revealed the lag phase, biodegradation phase and the plateau region within a period of 10 days, indicating that the 10-day window criteria is met.
first order biodegradation constant (hr-1): ln k = -6.247
maximum specific substrate uptake rate per unit biomass km = 14.4 / day (Aniline 16.1, Phenol 16.9)

Results with reference substance:

Degradation of reference substance aniline ≥ 60% within 28 days.

The specific oxygen uptake curve is not reported. According to the author "within a period of 10 days all the controls, test compounds and aniline revealed the lag phase, biodegradation phase and the plateau region". For the reference substance it is explicitly stated that "60% degradation of control substance aniline was achieved within a period of 28 days", further "within a period of 40 days all the compounds were degraded between 80% - 95%". Therefore, it can be concluded from this test that 60% biodegradation were reached within a period of 10 days and the test substance is readily biodegradable.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Executive summary:

In a test according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)), 80 to 95 % of 2,4-xylenol were degraded within 40 days. 2,4-xylenol is considered readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

94.5

Sampling time:

5 d

Details on results:

Initial degradation rate: (94.5%) 28.2 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,4-xylenol	94.5	28.2

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

2,4-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 94.5% after 5 days of incubation . 2,4-xylenol had degradation rates >15 mg COD/g/h which was the criteria for being readily biodegradable (Pitter 1976).

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The Little Miami wastewater treatment plant, Cincinnati, Ohio, USA
- Preparation of inoculum for exposure: Sludge was allowed to settle for about an hour, decanted, and t´hen aerated at room temperature for 24 hours
- Concentration of sludge: 30 mg/L municipal sewage sludge as dry matter

Duration of test (contact time):

20 - 40 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD medium
- Test temperature: 25 °C

TEST SYSTEM
- Culturing apparatus: Sapromat
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirred for an hour to ensure steady state of the endogenous respiration at the initiation of oxygen uptake measurements
- Measuring equipment: automated continuous oxygen measuring Voith Sapromat B-12 electrolytic respirometer (Voith-Morden, Milwaukee, WI)
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: carbon dioxide absorber (soda lime)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask
- Toxicity control: 1 flask
- Reference substance: 2 flasks

Reference substance:

aniline

Remarks:

100 mg/L

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

10 d

Remarks on result:

other: see comment in "Any other information on results" below

Parameter:

% degradation (O2 consumption)

Value:

80 - 95

Sampling time:

40 d

Details on results:

The oxygen uptake curves are not reported. However, the authors state that all test compounds revealed the lag phase, biodegradation phase and the plateau region within a period of 10 days, indicating that the 10-day window criteria is met.
first order biodegradation constant (hr-1): ln k = -5.87
maximum specific substrate uptake rate per unit biomass km = 18.5 / day (Aniline 16.1, Phenol 16.9)

Results with reference substance:

Degradation of reference substance aniline ≥ 60% within 28 days.

The specific oxygen uptake curve is not reported. According to the author "within a period of 10 days all the controls, test compounds and aniline revealed the lag phase, biodegradation phase and the plateau region". For the reference substance it is explicitly stated that "60% degradation of control substance aniline was achieved within a period of 28 days", further "within a period of 40 days all the compounds were degraded between 80% - 95%". Therefore, it can be concluded from this test that 60% biodegradation were reached within a period of 10 days and the test substance is readily biodegradable.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Executive summary:

In a test according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)), 80 to 95 % of p-cresol were degraded within 40 days. p-Cresol is considered readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

96

Sampling time:

5 d

Details on results:

Initial degradation rate: (96%) 55.0 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
p-cresol	96.0	55.0

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

p-cresol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 96% of p-cresol after 5 days of incubation. The degradation rates were >15 mg COD/g/h which was the criteria for being readily biodegradable.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The Little Miami wastewater treatment plant, Cincinnati, Ohio, USA
- Preparation of inoculum for exposure: Sludge was allowed to settle for about an hour, decanted, and t´hen aerated at room temperature for 24 hours
- Concentration of sludge: 30 mg/L municipal sewage sludge as dry matter

Duration of test (contact time):

20 - 40 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD medium
- Test temperature: 25 °C

TEST SYSTEM
- Culturing apparatus: Sapromat
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirred for an hour to ensure steady state of the endogenous respiration at the initiation of oxygen uptake measurements
- Measuring equipment: automated continuous oxygen measuring Voith Sapromat B-12 electrolytic respirometer (Voith-Morden, Milwaukee, WI)
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: carbon dioxide absorber (soda lime)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask
- Toxicity control: 1 flask
- Reference substance: 2 flasks

Reference substance:

aniline

Remarks:

100 mg/L

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

10 d

Remarks on result:

other: see comment in "Any other information on results" below

Parameter:

% degradation (O2 consumption)

Value:

80 - 95

Sampling time:

40 d

Details on results:

The oxygen uptake curves are not reported. However, the authors state that all test compounds revealed the lag phase, biodegradation phase and the plateau region within a period of 10 days, indicating that the 10-day window criteria is met.
first order biodegradation constant (hr-1): ln k = -5.77
maximum specific substrate uptake rate per unit biomass km = 17.3 / day (Aniline 16.1, Phenol 16.9)

Results with reference substance:

Degradation of reference substance aniline ≥ 60% within 28 days.

The specific oxygen uptake curve is not reported. According to the author "within a period of 10 days all the controls, test compounds and aniline revealed the lag phase, biodegradation phase and the plateau region". For the reference substance it is explicitly stated that "60% degradation of control substance aniline was achieved within a period of 28 days", further "within a period of 40 days all the compounds were degraded between 80% - 95%". Therefore, it can be concluded from this test that 60% biodegradation were reached within a period of 10 days and the test substance is readily biodegradable.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Executive summary:

In a test according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)), 80 to 95 % of m-cresol were degraded within 40 days. m-Cresol is considered readily biodegradable.

Reference 6

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

95.5

Sampling time:

5 d

Details on results:

Initial degradation rate: (95.5%) 55.0 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
m-cresol	95.5	55.0

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

m-cresol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 95.5% of m-cresol after 5 days of incubation. The degradation rates were >15 mg COD/g/h which was the criteria for being readily biodegradable.

Reference 7

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

95.5

Sampling time:

5 d

Details on results:

Initial degradation rate: (95.5%) 35.0 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,3-xylenol	95.5	35.0

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

2,3-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 95.5 % after 5 days of incubation . 2,3-xylenol had degradation rates >15 mg COD/g/h which was the criteria for being readily biodegradable (Pitter 1976).

Reference 8

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Secondary effluent from a predominantly domestic STP. Collected 15 June 2010 and filtered. Concentration of 1ml/L.

Duration of test (contact time):

>= 28 d

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

BOD determined

Details on study design:

Replicate standard BOD bottles were filled with the test concentration, prepared by addition of a sample stock solution to inoculated mineral medium. To check the intergrity of the test procedure a readily degradable reference chemical, sodium acetate, was tested and blank tests set up. Initial DO concentrations were measured and then incubated in the dark at ca 20 degrees C. Further measuresments of DO were made on bottles removed after 7, 14, 21 and 28 days. The theoretical OD was calculated based on information provided by the client and structures sourced from the Internet.

Reference substance:

other: Sodium acetate

Test performance:

The reference material was readily degradable at 71% degradation in < 10 days.

Parameter:

other: calculated BOD

Value:

39

Sampling time:

28 d

Details on results:

The xylenol mixture failed to meet the requirements for a pass in this test (>60% degradation relative to the COD value - a maximum of 39% was recorded). The ThOD, calculated based on information provided by the client and structures sourced from the internet, has a value of 2623 mg O2/g for HPV Xylenols.

Results with reference substance:

The reference material was readily degradable at 71% degradation in < 10 days.
The blank dissolved oxygen loss was 0.42mg/l (Guideline requires blank dissolved oxygen los of <1.5 mg/l).

	Incubation period (days)
	0	7		14		21			28
Incubator temperature (°C)	20.5	20.5		20.5		20.6			20.5
Average blank - DO (mg/l)	8.57	8.51		8.44		8.36			8.15
Replicate number	1	1	2	1	2	1	2	3	1	2	3
Test substance – DO (mg/l)	8.57	6.91	7.00	6.27	6.87	6.03	6.05	5.82	5.79	5.86	5.42
Test substance - BOD (mg O2/g)	-	658	621	893	646	959	951	1045	971	942	1123
Reference material – DO (mg/l)	8.57	5.65	5.82	5.63	5.64	5.39	5.49	5.46	5.14	5.26	5.09
Reference material – BOD (mg O2/g)	-	570	536	560	558	592	572	578	600	576	610

	Incubation period (days)
	7		14		21			28
Replicate number	1	2	1	2	1	2	3	1	2	3
Test substance (%)	25	24	34	25	37	36	40	37	36	43
Average degradation (%)	24		29		38			39
Reference material (%)	73	69	72	72	76	73	74	77	74	78
Average degradation (%)	71		72		74			76

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

HPV Xylenols failed to meet the requirements for a pass in this test with a maximum of 39% observed over 28 days. However because of the stringency of the test, this does not necessarily mean that the test substance is not degradable under environmental conditions. The results of the blank tests and reference material show that the tests were conducted correctly and the guidelines followed.

Executive summary:

HPV Xylenols failed to meet the requirements for a pass in this test (³60% degradation relative to the COD value - a maximum of 39% was recorded). However, because of the stringency of the test, this does not necessarily mean that the test substance is not biodegradable under environmental conditions, but indicates that more work would be necessary to establish biodegradability.

 

The guideline requires that the reference material degrades by at least 60% of its ThOD, and that the blank dissolved oxygen loss does not exceed 1.5mg/l after 28 days. Both of these criteria were satisfied for this test; a maximum value of 76% degradation of the sodium acetate was achieved, and the blank DO loss was 0.42mg/l.

 

The ThOD was calculated based on information provided by the client and structures sourced from the Internet.

Reference 9

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The Little Miami wastewater treatment plant, Cincinnati, Ohio, USA
- Preparation of inoculum for exposure: Sludge was allowed to settle for about an hour, decanted, and t´hen aerated at room temperature for 24 hours
- Concentration of sludge: 30 mg/L municipal sewage sludge as dry matter

Duration of test (contact time):

20 - 40 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: OECD medium
- Test temperature: 25 °C

TEST SYSTEM
- Culturing apparatus: Sapromat
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirred for an hour to ensure steady state of the endogenous respiration at the initiation of oxygen uptake measurements
- Measuring equipment: automated continuous oxygen measuring Voith Sapromat B-12 electrolytic respirometer (Voith-Morden, Milwaukee, WI)
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: carbon dioxide absorber (soda lime)

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask
- Toxicity control: 1 flask
- Reference substance: 2 flasks

Reference substance:

aniline

Remarks:

100 mg/L

Parameter:

% degradation (O2 consumption)

Value:

> 60

Sampling time:

10 d

Remarks on result:

other: see comment in "Any other information on results" below

Parameter:

% degradation (O2 consumption)

Value:

80 - 95

Sampling time:

40 d

Details on results:

The oxygen uptake curves are not reported. However, the authors state that all test compounds revealed the lag phase, biodegradation phase and the plateau region within a period of 10 days, indicating that the 10-day window criteria is met.
first order biodegradation constant (hr-1): ln k = -6.089
maximum specific substrate uptake rate per unit biomass km = 10.0 / day (Aniline 16.1, Phenol 16.9)

Results with reference substance:

Degradation of reference substance aniline ≥ 60% within 28 days.

The specific oxygen uptake curve is not reported. According to the author "within a period of 10 days all the controls, test compounds and aniline revealed the lag phase, biodegradation phase and the plateau region". For the reference substance it is explicitly stated that "60% degradation of control substance aniline was achieved within a period of 28 days", further "within a period of 40 days all the compounds were degraded between 80% - 95%". Therefore, it can be concluded from this test that 60% biodegradation were reached within a period of 10 days and the test substance is readily biodegradable.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Executive summary:

In a test according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)), 80 to 95 % of o-cresol were degraded within 40 days. o-cresol is considered readily biodegradable.

Reference 10

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

95

Sampling time:

5 d

Details on results:

Initial degradation rate: (95%) 54.0 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
o-cresol	95.0	54.0

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

o-cresol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 95% of o-cresol after 5 days of incubation. The degradation rates were >15 mg COD/g/h which was the criteria for being readily biodegradable.

Reference 11

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

89.3

Sampling time:

5 d

Details on results:

Initial degradation rate: (89.3%) 11.1 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,4-xylenol	89.3	11.1

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

3,5-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 89.3% after 5 days of incubation . 3,5-xylenol had degradation rates <15 mg COD/g/h which was the criteria for not being readily biodegradable (Pitter 1976).

Reference 12

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

94.3

Sampling time:

5 d

Details on results:

Initial degradation rate: (94.3%) 9.0 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,4-xylenol	94.3	9.0

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

2,6-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 94.3% after 5 days of incubation . 2,6-xylenol had degradation rates <15 mg COD/g/h which was the criteria for being readily biodegradable (Pitter 1976).

Reference 13

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

97.5

Sampling time:

5 d

Details on results:

Initial degradation rate: (97.5%) 13.4 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,4-xylenol	97.5	13.4

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

3,4-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 97.5% after 5 days of incubation. 3,4-xylenol had degradation rates < 15 mg COD/g/h which was the criteria for not being readily biodegradable (Pitter 1976).

Reference 14

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor deviations

Principles of method if other than guideline:

On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge taken from sewage treatment plant is cultivated in a 1000 mL volumetric cyclinder and aerated with pressure air. Every day 200 mL of the mixture is driven off so that the sludge age is 5 days. After driving off the 200 mL aeration is interrupted, and after sedimentation ca 600 mL of the liquid phase is driven off. The residue (200 mL of thickened activated sludge) is diluted to ca 800 mL and 600 mg/L starch or glucose, 600 mg/L peptone, 25 mL phosphate buffer pH 7.2 and the solution of the tested compound are added. Then the mixture is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200 mg/L COD.
- Concentration of sludge: 100 mg/L dry weight

Duration of test (contact time):

ca. 1 - ca. 20 d

Initial conc.:

200 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD removal

Details on study design:

TEST CONDITIONS
- Conduct: Test compound was sole source of carbon. To 1000 - 1500 mL of the biological medium such amount of the solution of the substance tested is added that the intial COD is 200 mg/L. Then an amount of the adapted activated sludge is dosed to the medium that the concentration of the dry matter is 100 mg/L. Simultaneouly a blank test is prepared to verify the activity of the inoculum. With volatile substances a test without the inoculum is carried out as well to differentiate the actual biological degradation and the losses due to volatilization. 50-80 ml of the mixture are taken, and after filtration through a medium porosity filter paper, the inital vlaues of COD or organic carbon of the liquid phase are determined. Initial levels in the beakers are marked and afterwards placed in a dark room at 20 +/- 3 degrees C on magnetic stirrers. The oxygenation capacity of this device was ca 11 mg oxygen h/L at 800 rev/min. At regular time intervals ca 50-80 ml of the sample are taken for analysis. The experiment was carried out until there is no decrease in COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If 90% of the initial COD is removed in 120h of incubation (degradation rate being more than 15 mg of COD g/h) then the test substance can be considered to be readily biodegradable.
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCI2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCI3; 6 H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10 g (NH4)2SO4 in 1 L distilled water], 20mL of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8 g K2HPO4 and 44.7 g Na2HPO4; 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000 mL with distilled water.

Parameter:

other: COD removal

Value:

94.5

Sampling time:

5 d

Details on results:

Initial degradation rate: (94.5%) 10.6 mg COD/g/h

Substance name	Amount removed (%)	Degradation rate mg/COD/g/h
2,4-xylenol	94.5	10.6

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable

Executive summary:

The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were the sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. Samples were taken at regular intervals and the COD measured until there was no more decrease in COD. After that time the total percentage of COD removed and the rate of degradation were evaluated.

2,5-xylenol is inherently degradable as shown by a test similar to OECD guideline 302 B with a removal of 94.5% after 5 days of incubation . 2,5-xylenol had degradation rates <15 mg COD/g/h which was the criteria for being readily biodegradable (Pitter 1976).

Reference 15

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: DIN 38412

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage treatment plant Hoechst AG, Frankfurt, Germany
- Concentration of sludge: 1.1 g/L

Duration of test (contact time):

10 d

Initial conc.:

50 - 400 mg/L

Based on:

DOC

Initial conc.:

200 - 1 000 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

DOC removal

Reference substance:

other: several other test substances

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

10 d

Remarks on result:

other: 2 day lag-phase; 90% degradation during 8 day log-phase

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Reference 16

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: DIN 38412

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage treatment plant Hoechst AG, Frankfurt, Germany
- Concentration of sludge: 1.1 g/L

Duration of test (contact time):

10 d

Initial conc.:

50 - 400 mg/L

Based on:

DOC

Initial conc.:

200 - 1 000 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

DOC removal

Reference substance:

other: several other test substances

Parameter:

% degradation (DOC removal)

Value:

96

Sampling time:

10 d

Remarks on result:

other: 2 day lag-phase; 90% degradation during 8 day log-phase

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Reference 17

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: DIN 38412

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage treatment plant Hoechst AG, Frankfurt, Germany
- Concentration of sludge: 1.1 g/L

Duration of test (contact time):

7 d

Initial conc.:

50 - 400 mg/L

Based on:

DOC

Remarks:

Exact concentration was not specified further.

Initial conc.:

200 - 1 000 mg/L

Based on:

COD

Remarks:

Exact concentration was not specified further.

Parameter followed for biodegradation estimation:

DOC removal

Reference substance:

other: several other test substances

Parameter:

% degradation (DOC removal)

Value:

100

Sampling time:

7 d

Remarks on result:

other: 3 day lag-phase; 95% degradation during 4 day log-phase

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Reference 18

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.5 (Degradation: Biochemical Oxygen Demand)

Principles of method if other than guideline:

Development of a BOD test for large volume experiments and quiescent conditions such as would be found in the environment (e.g. lake). System developed for determination of the rate, degree and extent of biodegradation of organic chemicals. Oxygen consumption, TOC, DOC and bacterial growth were determined during the test.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: St. Louis Metropolitan Sewer District manhole, Washington University campus, USA
- Preparation of inoculum for exposure: raw wastewater was settled and filtrated

Duration of test (contact time):

20 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: according to Standard Methods (for BOD testing) for the examination of Water and Wastewater (Am. Pub. Health Assn., New York 1965)
- Test temperature: 19.5 - 20.5 °C
- pH: not specified
- Inoculum: 2 mL/L settled, raw wastewater

TEST SYSTEM
- Culturing apparatus: BOD apparatus of Orford et al. 1953. Two autoclaved 9 L pyrex bottles with a connecting siphon. The one bottle was placed lower than the other in order to allow automatic flow of the medium from the higher bottle to the lower one at sampling time points. The lower bottle was kept full and closed in order to avoid air while the higher bottle was opened.
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: if re-aeration was necessary, filtered air was blown for 20-30 min directly into the lower bottle
- Measuring equipment: see details on analytical methods

SAMPLING
- Sampling frequency: on days 0, 1, 2, 3, 4, 5, 7, 9, 12, 16 and 20
- Sampling method: samples were taken from the lower bottle by opening the hose clamp of the siphon. The volume of the sample was replaced continuously through the siphon from the second bottle, which was covered but had access to the atmosphere. The first 50 mL were discarded in order to clear the sampling channel. Samples for dissolved oxygen: 125 mL in a reagent bottle; for COD and TOC analysis: 30 to 40 mL in a clean tube; for centrifugation: 40 mL (also for TOC analysis); nitrite determination: 50 mL in a nessler tube.

CONTROL AND BLANK SYSTEM
- Inoculum blank: included (no carbon source)
- Abiotic sterile control: not included
- Toxicity control: not included
- Reference substance: glucose and glutamic acid

Reference substance:

other: glucose-glutamic acid solution, 10 mg/L

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

5 d

Remarks on result:

other: BOD5 = 1.76 mg O2/mg test mat.

Parameter:

% degradation (O2 consumption)

Value:

86

Sampling time:

20 d

Remarks on result:

other: BOD20 = 2.16 mg O2/mg test mat.

Details on results:

TOC and COD data confirmed the data from the oxygen consumption.

Parameter:

BOD5

Value:

1.76 mg O2/g test mat.

Parameter:

BOD5

Value:

70 other: % of TOD

Results with reference substance:

The reference with glucose-glutamic acid solution (10 mg/L) showed immediate uptake of oxygen at the start of the test. A maximum uptake was observed the fifth day. After this timepoint no further O2 uptake was observed. The 5-day BOD was 1.49 mg O2/mg test mat. COD and TOC analysis verified the depletion of the substrate.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Reference 19

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 July 2004 to 25 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ASTM 1720-95 (sealed vessel test)

Qualifier:

according to guideline

Guideline:

ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3120 (Sealed Vessel Carbon Dioxide Production Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Collected from Wareham Wastewater Treatment Plan, Wareham, Massachusetts, US. Sludge was 2mm sieved, centrifuged in four 1L bottles for 10 minutes at 1500 rpm and the supernatant stored. Sludge was washed twice with tap water. Moisture content was determined to be 95. 44%.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 13.5 mL
- Composition of medium: Phosphate buffer: 10 mL/L, cclcium chloride: 1 mL/L, magnesium sulfate: 1 mL/L, ferric chloride, 1 mL/L.
- Solubilising agent (type and concentration if used): none
- Test temperature: 19.6 - 21.9°C
- pH: not stated
- pH adjusted: not stated
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration: 10 mg/L
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: no

TEST SYSTEM
- Culturing apparatus: 20 mL serum vials
- Number of culture flasks/concentration: 27
- Method used to create aerobic conditions:
- Method used to create anaerobic conditions:
- Measuring equipment: ThermoGlas Model 1200 Carbon Analyzer

- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: not used

SAMPLING
- Sampling frequency: Days 2, 4, 7, 10, 14, 21 and 28
- Sampling method: Acidification by injection of 100 µL of 4.9 M phosphoric acid through the septum, then shaking on orbital shaker at 200 rpm for at least 60 minutes prior to analysis.
- Sterility check if applicable: no
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: mineral media only
- Abiotic sterile control: not applicable
- Toxicity control: sodium benzoate

STATISTICAL METHODS: not applicable

Reference substance:

benzoic acid, sodium salt

Test performance:

The ultimate biodegradation of the reference substance, sodium benzoate, initially increased and peaked on day 10 at 94.9%. It then decreased until the end of the test, at 85.8% on day 28, due to continued CO2 evolution in the inoculum controls. Sodium benzoate is therefore considered to be readily biodegradable and thus confirmed the sensitivity of the test system.

The mean biodegradation value for ethyl phenols at the end of the test was 87.0% of the theoretical, indicating that biodegradation had occurred. The biodegradation value on day 7 was 73.9%. As the biodegradation reached >60% within the first 7 days, ethyl phenols are considered to be readily biodegradable.

Parameter:

% degradation (CO2 evolution)

Value:

26.9

Sampling time:

2 d

Parameter:

% degradation (CO2 evolution)

Value:

69.27

Sampling time:

4 d

Parameter:

% degradation (CO2 evolution)

Value:

73.9

Sampling time:

7 d

Parameter:

% degradation (CO2 evolution)

Value:

89.85

Sampling time:

10 d

Parameter:

% degradation (CO2 evolution)

Value:

89.07

Sampling time:

14 d

Parameter:

% degradation (CO2 evolution)

Value:

85.72

Sampling time:

21 d

Parameter:

% degradation (CO2 evolution)

Value:

86.98

Sampling time:

28 d

The ultimate biodegradation of the reference substance, sodium benzoate, initially increased and peaked on day 10 at 94.9%. It then decreased until the end of the test, at 85.8% on day 28, due to continued CO2 evolution in the inoculum controls. Sodium benzoate is therefore considered to be readily biodegradable and thus confirmed the sensitivity of the test system.

The mean biodegradation value for ethyl phenols at the end of the test was 87.0% of the theoretical, indicating that biodegradation had occurred. The biodegradation value on day 7 was 73.9%. As the biodegradation reached >60% within the first 7 days, ethyl phenols are considered to be readily biodegradable.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Ethyl phenols are considered to be readily biodegradable. The sensitivity of the test system was confirmed as the reference substance sodium benzoate is considered to be readily biodegradable.

Executive summary:

The ultimate biodegradation of the reference substance, sodium benzoate, initially increased and peaked on day 10 at 94.9%. It then decreased until the end of the test, at 85.8% on day 28, due to continued CO2 evolution in the inoculum controls. Sodium benzoate is therefore considered to be readily biodegradable and thus confirmed the sensitivity of the test system.

The mean biodegradation value for ethyl phenols at the end of the test was 87.0% of the theoretical, indicating that biodegradation had occurred. The biodegradation value on day 7 was 73.9%. As the biodegradation reached >60% within the first 7 days, ethyl phenols are considered to be readily biodegradable.

Reference 20

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 May - 17 Aug 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan), Biodegradation test

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Result with reference substance after 14 days is missing.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: SEK4693

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inoculum was provided by Chemicals Evaluation and Research Institute, Kurume Laboratory, Kurume, Japan.
- Method of cultivation: pH 7.0 ± 1.0 at 25.0 ± 2.0°C
- Concentration of sludge: 4616 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test temperature: 25.0 - 25.1°C
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)
- Measuring equipment: coulometer

SAMPLING
- Sampling frequency: continuous O2 meaurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

91

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Parameter:

% degradation (TOC removal)

Value:

98

Sampling time:

28 d

Details on results:

pH at the end of the test period was 7.05 - 7.17 in the inoculum and test substance.

Results with reference substance:

The biodegradation ratio according to oxygen consumption for aniline in the above used test system reached 53% degradation after 7 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Reference 21

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7 Jul - 21 Jul 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan)

Version / remarks:

July 13, 1974

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep in the dark and cool place

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): location: 10 sites (4 sewage plants, 3 rivers, 1 lake and 2 bays) around Japan, sampling depth: return sludge in sewage plants, surface water and surface soil of foreshore
- Laboratory culture: 5 L of the obtained sludge was mixed with 5 L of activated sludge which was in cubated for preveous 3 month. pH was adapted to 7.0 ± 1.0. The mixture was aerated. Thirty minutes after stopping of aeration, discard about one third of the whole volume of supernatant and add an e qual volume of a solution (pH adapted to 7.0 with NaOH) containing 50 g/L each of glucose, peptone and pottasium orthophosphate, to the settled material and re-commence aeration. This procedure was repeated once per day.
- Storage conditions: observed apperance and general condition of sludge, determined sediments, pH, temperature and dissolved oxygen and observed with a light microscope, if it is normal.
- Concentration of sludge: 5500 mg/L
- Water filtered: no

Duration of test (contact time):

14 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Composition of medium: recomended medium in the guideline
- Test temperature: 25 ± 1 °C
- pH adjusted: no
- Suspended solids concentration: 30 mg/L (?)

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: continuous O2 meaurement

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

>= 89 - <= 90

Sampling time:

14 d

Parameter:

% degradation (TOC removal)

Value:

>= 98 - <= 100

Sampling time:

14 d

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

14 d

Details on results:

pH at the end of the test period was 6.8 - 6.9 in the inoculum and test substance.

Results with reference substance:

The biodegrdation ration according to oxygen consumption for aniline in the above used test system reached 65% and 77% degradation after 7 days and 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Reference 22

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Jul - 22 Aug 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan), biodegradation test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

The percentage of the biodegradability after 14 days for aniline is missing.

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inoculum was provided by Chemicals Evaluation and Research Institute, Kurume Laboratory, Kurume, Japan.
- Method of cultivation: pH 7.0 ± 1.0 at 25.0 ± 2.0°C
- Concentration of sludge: 4771 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test temperature: 25 ± 1 °C
- pH adjusted: no
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: continuous O2 meaurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

5

Sampling time:

28 d

Parameter:

% degradation (TOC removal)

Value:

6

Sampling time:

28 d

Details on results:

To take into account in the volatility of the test substance, soda lime was washed by methanol. The test water and washed methanol were analyzed together.

pH at the end of the study period was 7.18 - 7.20 in the inoculum and test substance.

Results with reference substance:

The biodegradative ratio according to oxygen consumption for aniline in the above used test system reached 75% degradation after 7 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Reference 23

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Feb - 25 Mar 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan),

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

The percentage degradation of aniline after 14 days is missing.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)

SAMPLING
- Sampling frequency: continuous O2 meaurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

28 d

Parameter:

% degradation (TOC removal)

Value:

0

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

0

Sampling time:

28 d

Results with reference substance:

The biodegradation ratio according to oxygen consumption for aniline in the above used test system reached 74% degradation after 7 days.

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Reference 24

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Jun - 26 Jul 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan), biodegradatio test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

The percentage degradation of aniline after 14 days is missing.

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inoculum was provided by Chemicals Evaluation and Research Institute, Kurume Laboratory, Kurume, Japan.
- Method of cultivation: pH 7.0 ± 1.0 at 25.0 ± 2.0°C
- Concentration of sludge: 6663 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

TOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test temperature: 25 °C
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: continuous O2 meaurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

1

Sampling time:

28 d

Details on results:

Residual ratio according to TOC at the end of the study period was 64% and 66% in inoculum with test substance and water with test substance, respectively. Residual ratio according to the HPLC at the end of the study period was 64% both in inoculum with test substance and water with test substance. To be taken into acount in the volatility of the test substance, soda lime was washed with the methanol. Total residual ratio was 91% and 92% in inoculum with test substance and water with test substance, respectively.

pH at the end of the study period was 7.18, 7.24, and 7.23 in the test substance and inoculmn.

Results with reference substance:

The oxygen consumption for aniline in the above used test system reached 63% degradation after 7 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Reference 25

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Jul – 23 Aug 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Circular on Test Methods of New Chemical Substances (Japan), Biodegradation test

Version / remarks:

21 November, 2003

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): location: 10 sites (4 sewage plants, 3 rivers, 1 lake and 2 bays) around Japan, sampling depth: return sludge in sewage plants, surface water and surface soil of foreshore
- Laboratory culture: 5 L of the obtained sludge was mixed with 5 L of activated sludge which was incubated for preveous 3 month. pH was adapted to 7.0 ± 1.0. The mixture was aerated. Thirty minutes after stopping of aeration, discard about one third of the whole volume of supernatant and add an equal volume of a solution (pH adapted to 7.0 with NaOH) containing 50 g/L each of glucose, peptone and pottasium orthophosphate, to the settled material and re-commence aeration. This procedure was repeated once per day.
- Storage conditions: observed apperance and general condition of sludge, determined sediments, pH, temperature and dissolved oxygen and observed with a light microscope, if it is normal.
- Storage length: 18 - 24 h
- Concentration of sludge: 4300 mg/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation:

test mat. analysis

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: recomended medium in the guideline
- Test temperature: 25 ± 1 °C
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: closed oxgen consuption measuring machine with 300 mL culture bottle
- Number of culture flasks/concentration: 1 (water + test substance), 3 (inoculum + test sub.), 1 (inoculum + reference sub.), 1 (inoculum)
- Measuring equipment: Coulometer
- Details of trap for CO2 and volatile organics if used: soda lime

SAMPLING
- Sampling frequency: continuous O2 meaurement

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes

Reference substance:

aniline

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

2

Sampling time:

28 d

Parameter:

% degradation (test mat. analysis)

Value:

2

Sampling time:

28 d

Details on results:

pH at the end of the test period was 7.1, 7.3, and 7.1 in the inoculumn and test substance.

Results with reference substance:

The oxygen consumption for aniline in the above used test system reached 59% and 66% degradation after 7 days and 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Description of key information

Based on all available information (weight-of-evidence) and following an analogue read-across approach Tar acids, Xylenol fraction (CAS 84989-06-0) is described as to be inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Data on biodegradation in water are available for o-, m-, p-cresols, for 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-xylenols, mixed ethylphenols, trimethylphenols and for Tar acids, 3,5-xylenol fraction (Young et al. 1968, Pitter 1976, Wellens 1990, Desai et al. 1990, Gledhill 2004, Drake 2010, MITI 1981, 1994, 2001, 2005).

Monod kinetics of m- and p-cresol using an electrolytic respirometric test comparable to OECD guideline 301C and percentage biodegradation for the three cresol isomers and 2,4-xylenol were determined (Desai et al. 1990, OECD SIDS 2003). The results of this test are not comparable to those from standard tests on ready biodegradation. The test system and test conditions used by Desai et al. (1990) are nevertheless similar to those prescribed by the OECD 301 guidelines (e.g. high substance/inoculum ratio, non-adapted inoculum). Activated sludge from a wastewater treatment plant receiving predominantly domestic sewage was used as inoculum in a concentration of 30 mg/L. Within an incubation period of 40 days degradation of the cresol isomers and 2,4-xylenol (initial concentration 100 mg/L) was in the range of 80% to 95%. The specific oxygen uptake curves of the cresols are not reported in Desai et al. (1990). However, the authors state that all test compounds went through the lag phase, biodegradation phase and the plateau region within a period of 10 days. For the reference substance it is explicitly stated that "60% degradation of control substance aniline was achieved within a period of 28 days". Thus, it can be concluded from the test that this constituents of Tar acids, xylenol fraction are readily biodegradable. The first order degradation constants ln(k) [h-1] were determined to be -5.77 (m-cresol), -6.08 (o-cresol), -5.87 (p-cresol), and -6.25 (2,4-xylenol). From these values half-lives of 9.3 d resp. 10.3 d for m- and p-cresol can be calculated (OECD SIDS 2003). Further ready biodegradability tests according to OECD 301C are available for 2,4-, 2,5- and 2,6-xylenol, 4-ethylphenol and trimethylphenols (MITI 1981, 1994, 2001, 2005). Based on oxygen consumption the OECD 301C studies confirm ready biodegradability for 2,4-xylenol with a degradation rate of 91 % after 28 days (MITI 2001) and for 4-ethylenphenol with degradation rates of 89-90% after 14 days (MITI, 2001, 1994), while only negligible degradation was observed for the other investigated constituents 2,5- and 2-6-xylenol and trimethylphenols (MITI 1981, MITI 2001, MITI 2005).

Further, Pitter (1976) investigated the biological degradability of cresols in a study design similar to OECD 302B, however, an adapted inoculum was used. Within 5 days all 3 cresol isomers demonstrated 95% to 96% removal based upon COD with biodegradation rates ranging from 54 to 55 mg COD/g/h. In the same study also six xylenol isomers were tested. Results for 2,3-,2,4-, 2,5-, 2,6-, 3,4-, and 3,5-xylenol were in between 89.3% to 97.5% removal with degradation rates of 9.0 to 35 mg COD/g/h. The study design does not allow considering these substances as readily biodegradable, nevertheless, the potential for ultimate biodegradation is demonstrated.

In the study by Wellens (1990) the biodegradation of the three cresol isomers was examined according to OECD 302B guideline. An industrial activated sludge was used in this investigation. After 7 days 100% biodegradation was observed for o-cresol based on DOC removal. For p- and m-cresol 100% and 96% after 10 days were observed, respectively. As an industrial activated sludge was used, which must be considered as adapted, o-, m-, and p-cresol were considered as inherently biodegradable.

A further study on o-cresol is available by Young et al. (1968) conducted equivalent to EU guideline C.5. Under the conditions of this study, using non-adapted sewage sludge 70% and 86% were achieved after 5 and 20 days, respectively. Therefore, this constituent of Tar acids, xylenol fraction is considered readily biodegradable.

Mixed ethylphenols were used in a screening test by Gledhill (2004). The study was conducted under GLP and followed ASTM guideline 1720-95 and ISO 14593:1999. Domestic activated sludge was used as inoculum. 69%, 74%, 89%, and 87% biodegradation were observed at days 4, 7, 14, and 28, respectively. The sensitivity of the test system was confirmed as the reference substance sodium benzoate is considered to be readily biodegradable. Ethylphenols are considered to be readily biodegradable.

Tar acids, 3,5-xylenol fraction, a UVCB substance, is considered to be inherently biodegradable with 39% degradation after 28 days based on COD removal in a GLP study conducted according to OECD 301D (Drake 2010).

Although there is evidence that constituents like cresols, ethylphenols or some xylenols are readily biodegradable Tar acids, Xylenol fraction (CAS 84989-06-0) is considered as inherently biodegradable since rapid degradation cannot be shown for all constituents of the UVCB.

Results on biodegradation in water in the studies available are listed in the table below:

Source	Inoculum	method	Initial test substance concentration	p-cresol	m-cresol	o-cresol
Pitter, 1976	Activated sludge, adapted	Similar to OECD 302B	200 mg/L (COD)	96% (COD removal, 5d)	95.5% (COD removal, 5d)	95% (COD removal, 5d)
Desai et al., 1990	Activated sludge, non-adapted	Similar to OECD 301C	100 mg/L (test mat.)	>60% (10d), 80-95% (40d) (O2-consumption)	>60% (10d), 80-95% (40d) (O2-consumption)	>60% (10d), 80-95% (40d) (O2-consumption)
Young et al., 1968	sewage, domestic, non-adapted	Similar to EU Method C.5	10 mg/L (test mat.)	-	-	86% (20d) (O2-consumption)
Wellens, 1990	Activated sludge, industrial	OECD 302B	50 – 400 mg/L (DOC)	100% (DOC removal, 10d)	96% (DOC removal, 10d)	100% (DOC removal, 7d)

				2,3-xylenol	2,4-xylenol	2,5-xylenol
Pitter, 1976	Activated sludge, adapted	Similar to OECD 302B	200 mg/L (COD)	95.5% (COD removal, 5d)	94.5% (COD removal, 5d)	94.5% (COD removal, 5d)
Desai et al., 1990	Activated sludge, non-adapted	Similar to OECD 301C	100 mg/L (test mat.)	-	>60% (10d), 80-95% (40d) (O2-consumption)	-
MITI 2001, MITI 2005	mixed inoculum	OECD 301C	100 mg/L /test mat.)	-	91% (28d) (O2 -consumption)	0% (28d) (O2 -consumption)

				2,6-xylenol	3,4-xylenol	3,5-xylenol
Pitter, 1976	Activated sludge, adapted	Similar to OECD 302B	200 mg/L (COD)	94.3% (COD removal, 5d)	97.5% (COD removal, 5d)	89.3% (COD removal, 5d)
MITI, 2001	mixed inoculum	OECD 301C	100 mg/L (test mat.)	1% (28d) (O2 -onsumption)	-	-

				Mixed ethylphenols	Tar acids, 3,5-xylenol fraction	4 -ethylphenol
Drake, 2010	Activated sludge, non-adapted	OECD 301D	-	-	39% (calculated BOD, 28d)	-
Gledhill, 2004	Activated sludge, non-adapted	OPPTS 835.3120 / ISO 14593 / ASTM 1720-95	10 mg/L (COD)	86.98% (CO2-evolution, 28d)	-	-
MITI 1994	mixed inoculum	OECD 301C				89 -90% (14d) (O2 -consumption)

				trimethylphenol	2,3,6 -trimethylphenol	-
MITI 1981 MITI 2001	mixed inoculum	OECD 301C	100 mg/L (test mat.)	7% (28d) (O2 -consumption)	1% (28d) (O2 -consumption)	-