Methenamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: public available literature (non GLP)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)

Deviations:

not applicable

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
1) pH 7.8

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, adapted

Details on inoculum:

The study included several independent activated sludge samples collected at aeration chambers of biological wastewater treatment stations of different chemical plant. As an additional reference in yeast integration experiments, the activated sludge from a typical minicipal household wastewater treatment station was also used. Theactivated sludge was applied in experiments within two weeks of collection. The sludge was stored at 4°C and upon experiment, it was reactivated by placing 50-100 ml suspension in a 250 ml flask ans by aerobically cultivationg in a rotary shaker for 48 h at room temperature. Integration of the activated sludge with H. polymorpha was performed by adding the appropriate number of yeast cells to 20-50 mL of the sludge suspension and by cultivating the mixture under aerobic conditions for 24 h at 25°C.

Duration of test (contact time):

48 h

Initial conc.:

2 300 mg/L

Based on:

other: bound and anbound formaldehyd (mainly methenamine) in wastewater experiments

Initial conc.:

1 600 mg/L

Based on:

other: pure methenaine

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

1) Measurement of methenamin in wastewater
2) Measurement of pure methenamine

Reference substance:

not specified

Preliminary study:

no data

Test performance:

A good test performance was documented.

Parameter:

% degradation (test mat. analysis)

Value:

9

Sampling time:

48 h

Remarks on result:

other: Wastewater, pH 8.1

Parameter:

% degradation (test mat. analysis)

Value:

74

Sampling time:

48 h

Remarks on result:

other: Wastewater, pH 4.5

Parameter:

% degradation (test mat. analysis)

Value:

44

Sampling time:

48 h

Remarks on result:

other: pure substance, pH 5.3

Details on results:

1) Wastewater:
Very poor degradation with H. polymorpha at high ph values of 8.1. At lower pH (4.5) a degradation of 74% was observed.
2) Pure methenamine:
In a model system at pH 5.3, H. polymorpha dens culture (at about 10(exp 7) cells/mL) was able to biodegrade methenamineat initial concentrations up to 1600 mg/L to about 44%.

Results with reference substance:

no reference substance

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

Methenamine is biodegradable in wastewater at low pH conditions.

Executive summary:

Methenamine biodegrandation was measured in wastewater containing high amounts of metheamine, formaldehyde and methanol and also in a model system using pure methenamine. The measurement was done at different pH values, by determining the methenamin concentrations colorimentrically after 48 h incubation.

Results:

- Wastewater: Very poor degradation with H. polymorpha at high ph values of 8.1. At lower pH (4.5) a degradation of 74% was observed.

- Pure methenamine: In a model system at pH 5.3, H. polymorpha dens culture (at about 10(exp 7) cells/mL) was able to biodegrade methenamineat initial concentrations up to 1600 mg/L to about 44%.

Thus, methenamine can be regarded biodegradable in wastewater at low pH conditions.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-08-31 to 2009-09-28

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: sewage, household treating

Details on inoculum:

Specification
Effluent from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage.
Source
The effluent was taken from the effluent channel of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf. Date of collection: 25. Aug. 2009, batch no: 25082009.
Pre-Treatment
The effluent was kept aerobic during transport and storage. Before usage, the effluent was left to settle for one hour. The effluent was aerated for six days at room temperature in or-der to lower the oxygen consumption of the blanks. 0.5 mL/L test medium were used.

Duration of test (contact time):

28 d

Initial conc.:

1.93 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Start of the Test
The test series were prepared in aspirator bottles as follows:
•Control
The aspirator bottle was filled up with one third of medium, then, the necessary amount of inoculum was added. The bottle was then filled to the necessary volume and thoroughly mixed.
•Test Item, Positive Control and Toxicity Control
One third of the medium was filled in the aspirator bottle. The respective amount of the appropriate stock solution(s) and inoculum was added. The bottle was filled up with medium to the necessary volume and thoroughly mixed.
The test series were prepared in aspirator bottles as follows:
The test series solutions were filled in the test vessels through the stopcocks of the aspira-tor bottles using a tube in order to avoid bubbles. The test vessels were completely filled with the test solution. The oxygen concentration of each test series was measured imme-diately in one test vessel. To disable aeration, the other vessels were closed immediately with a glass stopper.
Sampling
For each test series, seven samplings were made (on days 0, 3, 9, 15, 21, 23 and 28). For each sampling, the oxygen content of two vessels of each test series was measured (one vessel on day 0).
For the correction of the oxygen uptake caused by nitrification, the concentrations of nitrite and nitrate were determined in each test vessel after oxygen measurement.
O2-Determination
Determinations of dissolved O2 were performed using an oxygen electrode.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

NA

Test performance:

The test is considered to be valid.

Key result

Parameter:

% degradation (O2 consumption)

Value:

35

Sampling time:

28 d

Details on results:

For the determination of the biological results, the data were evaluated without considera-tion of nitrification.
• The test item Methenamine (Hexamethylene tetramine crystalline) can be considered as „not readily biodegradable“.
• The degree of biodegradation was 35.3 % after 28 days (without consideration of ni-trification).
• The 10-day-window was day 8 – day 18. At the end of the 10-day-window, the pass level was missed.

Results with reference substance:

The positive control reached the pass level already on day 3.

no remarks

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The following data were determined for the test item Methenamine (Hexamethylenetetramine crystalline):
10-day-window: day 8 - 18
degradation at the end of 10-day-window: 22%
degradation at the end of the test: 35%

Executive summary:

This study was performed in order to evaluate aerobic elimination and degradation potential of Methenamine (Hexamethylenetetramine crystalline) in a test for ready biodegradability, using a test item concentration of 1.9 mg/L (corresponding to an theoretical oxygen consumption of 6.2 mg/L).

The test item was tested using a concentration of 1.93 mg Methenamine/L (corresponding to a theoretical oxygen consumption of 6.15 mg/L) in test medium following OECD 301D and EU-Guideline C.4-E.

Sodium benzoate was chosen as positive control. Degradation of the positive control was 68% after three days.

Effluent from a sewage treating plant was used as inoculum (conc. 0.5 mL effluent /L). The Biodegradation was determined for 28 days.

The O2-uptake caused by nitrification was not considered for the determination of the deg-radation, as no increase in nitrate/nitrite concentration was observed.

The following data were determined for the test item Methenamine:

10-day-window: day 8 - 18

degradation at the end of 10-day-window: 22%

degradation at the end of the test: 35%

Although some degradation was noted, Methenamine is not readily biodegradable in this OECD 301D/EU C.4-E test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: public available literature (non GLP)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)

Deviations:

not specified

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

no details given.

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

In a ring-test performed according to the EEC Respirometry Test with 21 participating laboratories the biodegradion of methenamine was determined.

Reference substance:

aniline

Preliminary study:

no preliminary study.

Test performance:

Positive control was valid. Test performance in the laboratories was good.

Key result

Parameter:

% degradation (DOC removal)

Value:

39

St. dev.:

33

Sampling time:

28 d

Details on results:

In a ring-test performed according to the EEC Respirometry Test, the results varied considerably. In 5 out of 21 laboratories degradation achieved > 70 % related to DOC. The mean DOC-elimination was 39 % and the standard deviation 33 %.

Results with reference substance:

Aniline, used as the control substance , behaved as expected on over 97% occasions.

Validity criteria fulfilled:

yes

Interpretation of results:

other: different results in the participating laboratories

Conclusions:

In a ring-test performed according to the EEC Respirometry Test, the results varied considerably. In 5 out of 21 laboratories degradation achieved > 70 % related to DOC. The mean DOC-elimination was 39 % and the standard deviation 33 %.

Executive summary:

In a ring-test performed according to the EEC Respirometry Test, the results varied considerably. In 5 out of 21 laboratories degradation achieved > 70 % related to DOC. The mean DOC-elimination was 39 % and the standard deviation 33 %.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: public available literature (non GLP, pH not indicated)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

ISO 7827 (Evaluation in an Aqueous Medium of the "Ultimate" Aerobic Biodegradability of Organic Compounds - Method by Analysis of Dissolved Organic Carbon (DOC))

Deviations:

no

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: swage effluent and activated sludge

Details on inoculum:

inocula concentrations were either 0.5 mL effluent/L or 30 mg activated sludge/L

Duration of test (contact time):

28 d

Initial conc.:

>= 10 - <= 50 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Different Methods were compared:
The dissolved organic carbon (DOC) die-away test as described in the ISO method was used; initial DOC concentrations were 10-50 mg/L and inocula concentrations were either 0.5 mL effluent/L or 30 mg activated sludge solids/L. Both the modified MITI and EEC versions of the respirometric were employed, using 100 mg test substance/L and 30 mg suspended solids/L as inoculum. Test were carried out at either 20 or 25°C.

Reference substance:

not specified

Preliminary study:

no preliminary study

Test performance:

no data

Parameter:

other: ThOD

Value:

47

Sampling time:

28 d

Remarks on result:

other: MITI-test

Key result

Parameter:

% degradation (DOC removal)

Value:

54 - 97

Sampling time:

28 d

Remarks on result:

other: MITI-test

Key result

Parameter:

% degradation (DOC removal)

Value:

79 - 108

Sampling time:

28 d

Remarks on result:

other: DOC removal test

Key result

Parameter:

other: ThOD

Value:

28 - 83

Sampling time:

28 d

Remarks on result:

other: EEC test

Details on results:

An MITI (I) test was conducted where biodegradation after 28 days was approximately 47 % as determined by ThOD. DOC-elimination attained 54 % and 97 % in the two parallel vessels. No information about the pH during the study is submitted. Since hydrolysis of methenamine is highly pH-dependent it remains unclear up to which level hydrolysis might have contributed to the degradation observed in the study. The important deviation of DOC-elimination in the two assays is not useful to assess the biodegradation potential of the substance.
In a DOC Die Away Test a DOC removal of 79-108 % was observed in several assays if 30 mg/l activated sludge was used. With effluent as inoculum, lower biodegradation levels were observed. Since no pH was documented, the degree of hydrolysis contributing to degradation remains unknown.
Biodegradation levels of 28-83 % related to ThOD and of 95-98 % related to DOC were achieved in another EEC Respirometry Test. The pH value was not documented.

Results with reference substance:

not indicated.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

Degradation of methenamine was observed in 3 different test. It is not clear if degradation was due toabiotic or biotic mechanisms, however a complete elimination of methenamine was noted.

Executive summary:

Three different screening test were conducted:

An MITI (I) test was conducted where biodegradation after 28 days was approximately 47 % as determined by ThOD. DOC-elimination attained 54 % and 97 % in the two parallel vessels. No information about the pH during the study is submitted. Since hydrolysis of methenamine is highly pH-dependent it remains unclear up to which level hydrolysis might have contributed to the degradation observed in the study. The important deviation of DOC-elimination in the two assays is not useful to assess the biodegradation potential of the substance.

In a DOC Die Away Test a DOC removal of 79-108 % was observed in several assays if 30 mg/l activated sludge was used. With effluent as inoculum, lower biodegradation levels were observed. Since no pH was documented, the degree of hydrolysis contributing to degradation remains unknown.

Biodegradation levels of 28-83 % related to ThOD and of 95-98 % related to DOC were achieved in another EEC Respirometry Test. The pH value was not documented.

Description of key information

The degradation of methenamine is strongly dependent on the pH where abiotic and biotic pathways are possible. With decreasing pH abiotic degradation (mainly hydrolysis) increases. In a new state of the art study (Muckle, 2009) Methenamine was shown to be not ready biodegradable at neutral pH. The following data were determined for the test item: 10-day-window: day 8 - 18 degradation at the end of 10-day-window: 22% degradation at the end of the test: 35%. However, other studies clearly indicate a good biodegradation on the chosen test conditions. Taken all together, hydrolysis seems to be the major degradation pathway formethenaminein the environment. At acidic pH-levels, methenamine is quickly degraded hydrolytically. At neutral and basic pH, the rate of hydrolysis decreases, and degradation is supported by microbial activity. Consequently, methenamine can be regarded as degradable but not as “ready biodegradable”.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

Key studies:

In a new state of the art study with Methenamine (Muckle, 2009), the test item was tested using a concentration of 1.93 mg/L (corresponding to a theoretical oxygen consumption of 6.15 mg/L) in test medium following OECD 301D and EU-Guideline C.4-E. Sodium benzoate was chosen as positive control. Degradation of the positive control was 68% after three days. Effluent from a sewage treating plant was used as inoculum (conc. 0.5 mL effluent /L). Biodegradation was measured for 28 days. The O₂-uptake caused by nitrification was not considered for the determination of the degradation, as no increase in nitrate/nitrite concentration was observed.

The following data were determined for the test item Methenamine:

10-day-window: day 8 - 18

degradation at the end of 10-day-window: 22%

degradation at the end of the test: 35%

Therefore Methenamine, is not readily biodegradable following OECD 301D/EU C.4-E (neutral pH conditions).

In a ring-test performed according to the EEC Respirometry Test, the results varied considerably. In 5 out of 21 laboratories degradation achieved > 70 % related to DOC. The mean DOC-elimination was 39 % and the standard deviation 33 % (Painter and King 1985).

Biodegradation levels of 28-83 % related to ThOD and of 95-98 % related to DOC were achieved by Painter and King (1986) in another EEC Respirometry Test. The pH value was not documented.

In a DOC Die Away Test a DOC removal of 79-108 % was observed in several assays if 30 mg/l activated sludge was used. With effluent as inoculum, lower biodegradation levels were observed (Painter and King 1986). Since no pH was documented, the degree of hydrolysis contributing to degradation remains unknown, nevertheless this study clearly indicates a good degradation of methenamine.

Methenamine biodegrandation was also measured in wastewater containing high amounts of metheamine, formaldehyde and methanol and also in a model system using pure methenamine (Kaszycki, 2002). The measurement was done at different pH values, by determining the methenamin concentrations colorimentrically after 48 h incubation.

Results:

- Wastewater: Very poor degradation with H. polymorpha at high ph values of 8.1. At lower pH (4.5) a degradation of 74% was observed.

- Pure methenamine: In a model system at pH 5.3, H. polymorpha dens culture (at about 10(exp 7) cells/mL) was able to biodegrade methenamineat initial concentrations up to 1600 mg/L to about 44%.

Thus, methenamine can be regarded biodegradable in wastewater at low pH conditions.

Supporting studies:

Painter and King (1986) conducted a MITI (I) test where biodegradation after 28 days was approximately 47 % as determined by ThOD. DOC-elimination attained 54 % and 97 % in the two parallel vessels. No information about the pH during the study is given. Since hydrolysis of methenamine is highly pH-dependent it remains unclear up to which level hydrolysis might have contributed to the degradation observed in the study. The deviation of DOC-elimination in the two assays does not allow a valid assessment of the biodegradation potential of the substance.

Van Ginkel and Stroo (1992) performed a Closed Bottle Test, into which 2 mg of activated sludge were introduced (related to dry weight) instead of 5 ml effluent/l. This does not correspond to the low inoculum concentration usually applied in the test. The result of the test was 70 % biodegradation after 28 days.

Bodik et al. (1991) examined the elimination of methenamine in a continuous and semicontinuous pilot plant. Analysis was carried out by monitoring the test substance, no buffered medium was used. The inoculum was adapted to methenamine for 100 days. Then the effects of the age of the sludge on pH and methenamine-elimination were determined. Due to nitrification the pH value reached the acid range. With increasing age of the sludge the pH value decreased and methenamine elimination increased: at a sludge age of 10 and 50 d methenamine-elimination attained 18 % and 53 % at pH-values of 6.6 and 5.2, respectively.

Drtil et al. (1991) performed a biodegradation test of methenamine in acid medium by using a biofilm reactor. The elimination level correlated with the pH: 15 % at pH 6.4 and 44 % at pH 4.2 using a sludge retention time of 35 d.

Gomolka and Gomolka (1984) concluded that methenamine present in aerated municipal sewage is highly resistant to biodegradation. The process should be preceded by an extended adaption of the microorganisms.

Swope and Kenna (1950) obtained a BOD of approximately 2 % of ThOD after 5 days at pH = 7. The performance of the test was insufficiently documented.

In a study by Choules and Adams (1984), efforts were made to biolocically degrade methenamine. Initially, sewage organisms were grown aerobically in tanks containing 150 ppm methenamine as the only carbon and nitrogen source. Media flow was 1 L/hr. The biologically floc appeared active for a time but died out and disappeared within 7 days. Attempts to isolated methenamine degrading organisms from the influent and effluent line failed. Upward flow columns were prepared, filled with gravel, and inoculated with various sewage organisms. Methenamine media concentrations (150 to 600 ppm) stop and go flows, various intensities of bubbling and the presence of formaldehyde degrading organisms were tested. Samples were taken from the columns at various times, and pure cultures isolated and culture selection methods applied. At various times during these procedures the methenamine concentration would drop 15 to 25 % below the initial media concentration, but results were not consistent. Although many efforts were made to optimize conditions, only a small degradation of methenamine was obtained. It is considered possible that the test organisms had incomplete ability to attack the methenamine molecule and only degraded naturally occurring partially-hydrolyzed molecules of methenamine.

In conclusion, methenamine can be regarded as degradable in the environment, mainly due to hydrolysis and supported by biodegradation.