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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-06-15 to 1999-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
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))
GLP compliance:
yes
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): Activated sludge from Iaboratory wastewater plants treating municipal sewage.
- Pretreatment: no adapation phase
- Concentration of sludge: 30 mg/L dw
Duration of test (contact time):
28 d
Initial conc.:
78 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- pH adjusted: yes

SAMPLING
- Sampling frequency: On day 0, 1, 3, 5, 7, 10, 14, 17, 21, 24, 27 and 28 of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Other: inhibition control, adsorption control


Reference substance:
aniline
Parameter:
% degradation (DOC removal)
Value:
< 10
Sampling time:
28 d
Results with reference substance:
Degradation degree of the reference substance after 14 days (% DOC): 92

DOC decrease [%]:

Day

RS

IH

PC

AC

TS1

TS2

TS mv

0

0

0

0

0

0

0

0

1

-2

3

-5

-3

1

-6

-3

3

38

12

-3

-1

1

-5

-2

5

96

48

0

2

1

0

1

7

98

49

-5

 

1

-3

-1

10

96

48

-6

 

-1

-3

-2

14

95

46

-7

 

-3

-5

-4

17

96

49

-10

 

1

-7

-3

21

100

49

-8

 

-1

-3

-2

24

96

43

-17

 

-3

-16

-10

27

99

46

-15

 

-8

-25

-17

28

92

43

-20

 

-13

-18

-16

mv = mean value, RS = reference substance, IH = inhibition control, PC = abiotic control,

AC = adsorption control, TS = test substance

Duration of the adaption phase (days): -

Duration of the degradation phase (days): -

Degradation of the test substance at the end of the 10 -day window (% DOC): -

Degradation degree of the test substance at the end of the test (% DOC): <10

Degradation of the reference substance after 14 days (%DOC): 90 -100

Degradation degree in the inhibition control after 14 days (% DOC): 40 -50

Physico-chemical (abiotic) elimination of the test substance (% DOC):<10 at the end of the test

Elimination of the test substance by adsorption (% DOC): <10 after 5 days

DOC values [mg/L]:

Day

BC1

a        b

BC2

a        b

RS

a       b

IH

a        b

PC

a        b

AC

a       b

TS1

a        b

TS2

a        b

0

0.8

0.6

0.5

0.6

 21.0

 21.1

 41.3

 41.7

 20.6

 20.7

 21.1

21.1

21.2

21.2

 20.8

 20.8

1

1.7

1.7

0.8

1.0

 22.3

 22.2

 41.0

 41.1

 21.8

 21.5

 22.6

 22.2

 21.6

 21.7

 22.7

 22.9

3

0.7

0.8

0.9

0.9

 13.5

 13.7

 36.6

 36.6

 21.2

 21.5

 21.4

 21.6

 21.2

 21.2

 22.0

 22.1

5

0.6

0.7

0.6

0.7

 1.4

 1.5

 21.8

 21.8

 20.7

 20.7

 20.8

 20.8

 21.0

 21.2

 20.7

 21.2

7

0.6

0.8

1.1

1.0

 1.2

 1.5

 21.5

 22.0

 21.8

 21.8

 

 

 21.2

 21.3

 21.6

 21.7

10

1.0

1.2

0.7

0.7

 1.8

 1.7

 21.9

 22.2

 21.9

 22.0

 

 

 21.6

 21.7

 21.8

 21.7

14

1.0

1.1

0.8

0.8

 1.8

 1.9

 22.9

 22.9

 22.1

 22.3

 

 

 22.2

 22.0

 22.3

 22.1

17

1.2

1.4

0.8

0.8

 1.7

 1.9

 21.7

 22.1

 22.5

 22.8

 

 

 21.3

 21.5

 22.7

 22.6

21

1.3

1.4

1.9

1.9

 1.8

 1.8

 22.9

 22.5

 22.3

 22.5

 

 

 22.7

 22.4

 22.5

 22.6

24

1.4

1.2

1.0

1.0

 1.8

 2.2

 24.5

 22.3

 23.7

 24.9

 

 

 22.2

 22.6

 24.3

 24.6

27

0.7

0.8

0.8

0.8

 1.1

 1.1

 22.8

 23.7

 23.6

 23.9

 

 

 23.0

 23.1

 26.5

 25.5

28

1.0

0.8

1.5

1.5

 2.7

 2.7

 24.7

 24.6

 24.6

 25.1

 

 

 24.5

 24.0

 24.5

 25.1

a/b = DOC single value, mv = mean value, RS = reference substance, IH = inhibition control, PC = abiotic control,

AC = adsorption control, TS = test substance

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The substance N-methylmorpholine oxide was not readily biodegradable in an experimental study according to OECD guideline 301A.
Executive summary:

The biodegradation of N-methylmorpholine oxide was studied according to OECD guideline 301A with unadapted sludge from a predominantly industrial WWTP. The test substance was applied at 78 mg/L (initial concentration).

Biodegradation was assessed on days 0, 3, 5, 7, 10, 14, 17, 21, 24, 27 and 28.

Biodegradation was below 10% based on DOC removal after the incubation period of 28 days. Therefore, the test substance is not readily biodegradable.

The study is valid and satisfies the guideline criteria for a ready biodegradability test according to OECD guideline 301A.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
after 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Study performed according to OECD guideline 302B (modified Zahn-Wellens test). The test was well performed and the results can be considered reliable (Klimisch 2, because no information on GLP and not all information on materials and methods is reported in a detailed way).
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly industrial, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Lenzing AG's WWTP, at that time receiving pulp and paper mill waste waters as well as waste waters of a viscose fibre production plant and a small portion of domestic waste water.
- The plant was a 2-stage biological system with high load in the first stage (sludge age 2-3 days) and low load in the second stage (sludge age > 14 days).
- Mixtures of equal portions of the sludges from the first and second stages were used.
- Preparation for inoculation: supplemented with urea and phosphate to a ration of COD:N:P of about 100:5:1 because the sludge did not contain N or P.
- Concentration of sludge: 2g/L - 4.5g/L suspended solids
- Initial cell/biomass concentration: no data
Duration of test (contact time):
28 d
Initial conc.:
ca. 30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: COD
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
only in the additional test with 600 mg/L glucose as additional substrate
Details on study design:
TEST CONDITIONS
- Composition of medium: nutrient cocktail as in guideline OECD 302B
- Additional substrate: in a separate test the effect was studied of adding 600 mg/L glucose as additional substrate
- Test temperature: room temperature
- pH: no data
- Aeration only if oxygen concentration < 2 mg/L
- Suspended solids concentration: not reported
- Continuous darkness: yes
- Continuous stirring

BIODEGRADATION MEASUREMENTS
- Biodegradation was monitored in the liquid phase via COD (in some cases also DOC) analysis. The samples were filtered to separate the sludge from the liquid. In the separate test to study the effect of adding glucose as additional substrate test material analysis was performed using HPLC-UV absorption.

CONTROL AND BLANK SYSTEM
- No data
- No toxicity was observed during the tests
Parameter:
% degradation (test mat. analysis)
Value:
> 90
Sampling time:
21 d
Details on results:
Substance inherently biodegradable according to this test using adapted sludge from a WWTP with both short and long living sludge organisms.
> 90% degradation (based on test material analysis) was observed within 21 days. When glucose was added (600 mg/L) as additional substrate, NMMO degradation was speeded up and 100% degradation was observed after ca. 14 days already. To ensure not to determine glucose degradation instead, degradation of NMMO was followed by substrate specific analysis. The results of NMM and morpholine analyses were not clearly shown but ultimate biodegradation is reported in the publication.
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
This study was performed according to the Zahn-Wellens method (OECD guideline 302B) using adapted sludge from a WWTP containing both long- and short-living organisms. > 90% of the test substance NMMO was degraded after 21 days (based on test material analysis). Degradation was enhanced (speeded up) by the addition of glucose as additional substrate. The results of NMM and morpholine analyses were not clearly shown but the publication reports ultimate biodegradation. The test substance NMMO can be considered inherently biodegradable.
Executive summary:

The biodegradation of N-methylmorpholine oxide was studied according to the Zahn-Wellens method (OECD guideline 302B) to assess inherent biodegradability of the test substance with adapted sludge from a predominantly industrial WWTP. The test substance was applied at 30 mg/L.The sludge was allowed to adapt for 2 -3 weeks. Glucose was added in an additional experiment to stimulate microbial activity. The detection limit of the test substance by HPLC was at 1 mg/L.

Biodegradation was above 90% based on COD/DOC within 21 days. Adaptation of the sludge and a sludge age af >= 15 days are considered critial in the study for degradation. Addition of glucose increased biodegradation to 100% after 14 days. Therefore, the test substance is inherently biodegradable using adapted sludge of appropriate sludge age.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Study performed according to OECD guideline 302B (modified Zahn-Wellens test). The test was well performed and the results can be considered reliable (Klimisch 2, because no information on GLP and not all information on materials and methods is reported in a detailed way).
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly industrial, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Lenzing AG's WWTP, at that time receiving pulp and paper mill waste waters and a small portion of domestic waste water
- The sludge age in the plant was only 2-3 days
- Preparation for inoculation: supplemented with urea and phosphate to a ration of COD:N:P of about 100:5:1 because the sludge did not contain N or P.
- Concentration of sludge: 2g/L - 4.5g/L suspended solids
- Initial cell/biomass concentration: no data
Duration of test (contact time):
28 d
Initial conc.:
ca. 30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: COD
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: nutrient cocktail as in guideline OECD 302B
- Additional substrate: none
- Test temperature: room temperature
- pH: no data
- Aeration only if oxygen concentration < 2 mg/L
- Suspended solids concentration: not reported
- Continuous darkness: yes
- Continuous stirring

BIODEGRADATION MEASUREMENTS
- Biodegradation was monitored in the liquid phase via COD (in some cases also DOC) analysis. The samples were filtered to separate the sludge from the liquid.

CONTROL AND BLANK SYSTEM
- No data
- No toxicity was observed during the tests
Parameter:
other: COD
Value:
< 20
Sampling time:
28 d
Details on results:
Substance not readily or inherently biodegradable according to this test using unadapted sludge from a WWTP with high loading and low sludge age (2-3 days, no slowly reproducing organisms present).
Interpretation of results:
not inherently biodegradable
Conclusions:
This study was performed according to the Zahn-Wellens method (OECD guideline 302B) using unadapted sludge from a WWTP with high loading and low sludge age (2-3 days, no slowly reproducing organisms present). Less than 20% of the initial COD was degraded after 28 days and therefore the substance was considered not inherently biodegradable.
Executive summary:

The biodegradation of N-methylmorpholine oxide was studied according to the Zahn-Wellens method (OECD guideline 302B) to assess inherent biodegradability of the test substance with unadapted sludge from a predominantly industrial WWTP. The test substance was applied at 30 mg/L.

Biodegradation was below 20% based on COD after the incubation period of 28 days. Therefore, the test substance is not inherently biodegradable.

Description of key information

Available data include inherent biodegradation tests (Zahn-Wellens test, OECD302B) and a ready biodegradability test according to the DOC Die-Away test (OECD301A). The latter study is considered reliable (Klimisch 1) and is used as key study for this endpoint. All information together justifies the conclusion that the substance is ultimately and inherently biodegradable, not fulfilling specific criteria.
The results of the BASF study (OECD 301A) indicates that NNMO is not readily biodegradable. The results of the Zahn-Wellens tests (OECD302B) with unadapted and adapted sludge reported by Meister and Wechsler (1998) indicate that NMMO is inherently biodegradable, not fulfilling specific criteria, but that adaptation of the sludge is required for efficient breakdown. Additional experiments indicated that the substance is ultimately biodegradable and no persistent breakdown products are formed. This study is considered reliable (Klimisch 2) and is used as key study for this endpoint.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

The reliable key study determined the ready biodegradability of NMMO in a DOC Die-away test according to OECD 301A guidelines and in the presence of activated sewage sludge with 30 mg/L suspended solids. The test period was 28 days; after 28 days on average < 10% biodegradation was observed. The results show that the substance is not readily biodegradable.

Significant biodegradation however was observed in an OECD 302B test (BASF AG 2001) (ultimate biodegradability of 90 -100%) concluding that the substance is inherently biodegradable.

Meister and Wechsler (1998) thoroughly investigated the biodegradation of NMMO, NMM and morpholine using a variety of experiments, including guideline studies (OECD302B, Zahn-Wellens test for inherent biodegradation), lab-scale and pilot-scale experiments. This study was considered reliable (Klimisch 2). A first Zahn-Wellens test with unadapted sludge from a WWTP with high load and only short-living sludge organisms (sludge age 2-3 days) indicated < 20% degradation (based on COD) in 28 days. However, a second Zahn-Wellens test with adapted sludge from a WWTP with both short- and long-living sludge organisms (sludge age >= 14 days) indicated > 90% degradation (based on test material analysis) within 21 days. Based on this study NMMO can be considered inherently biodegradable without fulfilling specific criteria. The other experiments (lab-scale, pilot-scale) indicated that NMMO is ultimately biodegradable, and adaptation of the sludge speeds up the degradation. For successful adaptation a high sludge age is recommended, indicating that slowly-reproducing (long-living) sludge organisms are more efficient degraders of the substance. The degradation takes place in several steps. First, NMMO is reduced to NMM. The next step (rate-limiting) is the demethylation of NMMO to morpholine. This step is followed by much faster ring cleavage and further degradation.

Additionally, these authors investigated degradation of NMMO under anaerobic conditions. Hereto two yeast species (Saccharomyces carlbergensis and Saccharomyces cerevisiae) were exposed to the substance during 14 days. Mainly Saccharomyces carlbergensis significantly degraded NMMO to NMM (complete degradation to NMM), indicating that the substance is also biodegradable under anaerobic conditions. However, no further breakdown of NMM occurred in these experiments and no further experimentation was undertaken.