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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Analysis of radio labelled test substance and comparison with standards
- Short description of test conditions: isolation fo contaminated site adapted strains of various microorganisms and cultivation under pH 6+8 at 27°C.
- Parameters analysed / observed: Radio cpm of HPLC and TLC spearated fractions of metabolised medium.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
NTO*: Commissariat a© l'Energie Atomique (Centre du Ripault, Monts, France)
Urazole: Aldrich
5-Amino-1,2,4-triazol-3-one (ATO) and
1,2,4-triazol-3-one (TO): Self preparation

- Expiration date of the lot/batch: not reported (self preparation)
- Purity test date: not available

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not reported
- Specific activity: not reported
- Locations of the label: 14C
- Expiration date of radiochemical substance: not reported (self preparation)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not applicable

- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: not reported
- Final preparation of a solid: not applicable
Radiolabelling:
yes
Remarks:
14C
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment: freshwater
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): not reported
- Storage conditions: 27° C, unsealed
- Storage length: not reported
- Temperature (°C) at time of collection: not reported
- pH at time of collection: not reported
- Electrical conductivity: not reported
- Redox potential (mv) initial/final: not reported
- Oxygen concentration (mg/l) initial/final: not reported
- Hardness (CaCO3): not reported
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): initial bio mass not reported.
- Water filtered: no
Details on inoculum:
- Source of inoculum wastewater (e.g. location, contamination history) contaminated site, not further spcecified
- Storage conditions: not reported
- Storage length: not reported
- Temperature (°C) at time of collection: not reported
- pH at time of collection: 3
- Electrical conductivity: not reported
- Redox potential (mv) initial/final: not reported
- Oxygen concentration (mg/l) initial/final: not reported
- Dissolved organic carbon (%): not reported
- Biomass (e.g. in mg microbial C/100 mg, CFU:
- Biomass concentration (mg/L) used in test: 7 g/ 25 mL
- Chemical oxygen Demand (COD)- Water filtered: yes/no- Source of inoculum wastewater: NTO contaminated site
- Storage conditions: not reported
- Storage length: not reported
- Temperature (°C) at time of collection: not reported
- pH at time of collection: 3
- Electrical conductivity: not reported
- Redox potential (mv) initial/final: not reported
- Oxygen concentration (mg/l) initial/final: not reported
- Dissolved organic carbon (%): not reported
- Biomass (e.g. in mg microbial C/100 mg, CFU: not reported
- Biomass concentration (mg/L) used in test: 7 g/ L
- Chemical oxygen Demand (COD)- Water filtered: no
Duration of test (contact time):
ca. 14 d
Initial conc.:
12 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 7 g in 25 ml (Erlenmeyer flasks)
- Composition of medium: Per L
0.5 g KH2PO4,
1 g K2HPO4,
30 g D-glucose,
10 g corn steep,
0.5 g MgSO4,
2 g NaNO3,
0.5 g KCl,
0.02 g FeSO4
placed at 27³C on a rotary shaker (200 rpm).

- Additional substrate: Glucose
- Solubilising agent (type and concentration if used): none
- Test temperature: 27
- pH: 6+8
- pH adjusted: yes
- CEC (meq/100 g):
- Aeration of dilution water: not reported
- Suspended solids concentration: not reported
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: not reported
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: not reported
- Method used to create aerobic conditions: rotary shaker
- Method used to create anaerobic conditions: not applicable
- Method used to control oxygen conditions: not reported
- Measuring equipment: not reported

- Test performed in closed vessels due to significant volatility of test substance: not applicable
- Test performed in open system: not applicable
- Details of trap for CO2 and volatile organics if used: not reported
- Other:

SAMPLING
- Sampling frequency: 24 h and 14 d
- Sampling method used per analysis type: not specified
- Sterility check if applicable: not applicable
- Sample storage before analysis: not reported
- Other:

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank: not applicable
- Abiotic sterile control: not applicable
- Toxicity control: not applicable
- Other:

STATISTICAL METHODS:
Not reported
Reference substance:
not required
Compartment:
abiotic control measured at end of test
% Recovery:
100
St. dev.:
1
Remarks on result:
not determinable
Key result
% Degr.:
ca. 100
Parameter:
radiochem. meas.
Sampling time:
16 d
Key result
Compartment:
entire system
DT50:
ca. 24 h
Type:
(pseudo-)first order (= half-life)
Temp.:
27 °C
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), degradation in solution, 27 °C
maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): no

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
100% degredation of NTO into ATO
Final degradation products
40% CO2
Rest Urea and Hydroxyurea
- Range of maximum concentrations in % of the applied amount at end of study period:
on the - the and -th day of incubation, respectively. At the end of the study period, the corresponding concentrations were - and -- % of the applied amount, respectively.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

STERILE TREATMENTS (if used)
- Transformation of the parent compound:
- Formation of transformation products:
- Formation of extractable and non-extractable residues:
- Volatilization:

RESULTS OF SUPPLEMENTARY EXPERIMENT (if any):
Validity criteria fulfilled:
yes
Conclusions:
The substance NTO has been assessed for its degradation potential in a scientific study which was well described.
The breakdown products have been analysed and compared by and with different radio labelled merthods and standard substances
IT was found that NTO can in 24Hrs be metabolised by microorganisms into the first metabolite ATO, which in a further is cleaved and as ultimate degradation product CO2, Urea and hydroxyurea has been identified.

Thus, it is concluded that NTO has been assessed as ultimately biodegradable by a method which is a scientific but non-standard method. The documentqation is appropriate to conclude on the validity of the test results.
Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010-2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: HPLC-UV coupled identification of degradation products
- Short description of test conditions: Transformation by microorganisms collected on contaminated land site under aerobic and anaerobic lab scale conditions.
- Parameters analysed / observed: Breakdown of DNAN into its metabolites 2-amino-4-nitroanisole and 2-hydroxylamino-4-nitroanisole.
GLP compliance:
not specified
Radiolabelling:
no
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
natural soil
Details on inoculum:
- Temperature (°C) at time of collection: not reported
- pH at time of collection: not reported
- Oxygen concentration (mg/l) initial/final: not reported
- Dissolved organic carbon (%): not reported
- Source of activated sludge (e.g. location, contamination history): not aplicable
- Laboratory culture: yes
- Method of cultivation: glucose/succrose supplemented LB and MS medium
- Storage conditions: not reported
- Storage length: not reported
- Preparation for exposure: not reported
- Pretreatment: not reported
- Biomass concentration (mg/L) used in test: not specified
Duration of test (contact time):
ca. 35 d
Parameter followed for biodegradation estimation:
test mat. analysis
other: Metabolite analysis: 2-A-4-NAN (2-amino-4-nitroanisole) and HA-4-NAN (2-hydroxylamino-4-nitroanisole)
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment:
- Composition of medium:
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:
- pH:
- pH adjusted: yes/no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: no
- Other:

TEST SYSTEM
- Culturing apparatus: erlenmayer flasks
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: Rotary shaker
- Method used to create anaerobic conditions: n.a.
- Method used to control oxygen conditions: nots specified
- Measuring equipment: not specified

- Test performed in closed vessels due to significant volatility of test substance: n.a.
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: n.a.
- Other:

SAMPLING
- Sampling frequency:
- Sampling method used per analysis type:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance:
not required
Remarks:
analysis of degradation products, not looking for toxiticy
Test performance:
Soil microbial community readily transforms DNAN in aerobic microcosms supplemented with a nitrogen (NH4Cl) and carbon sources (glucose and succinate).
DNAN disappeared at the rate of 1.7 ± 0.2 nmol h-1 g-1 and the initial 4 μmols were completely removed in 8 days. We observed disappearance of 88% of DNAN in only 28 h after fresh culture medium was supplied to actively transforming microcosms (data not shown); this is most likely because microbial populations adapted tothe prevailing conditions and/or they increased in number.

In soil microcosms supplemented with the carbon sources alone, DNAN removal began slowly during at least the first 8 days and was completed after 34 days. DNAN only slowly disappeared in unamended microcosms with the loss of 0.58 μmols after 35 days and 0.65 μmol after 5 months of aerobic incubation (data not shown). This suggests that DNAN may persist under natural conditions at least in soils with no history of contamination with DNAN.

No significant loss of DNAN occurred in the sterile controls.
Key result
% Degr.:
ca. 100
Parameter:
test mat. analysis
Sampling time:
35 d
Key result
Compartment:
biologically active treatment at end of test
DT50:
ca. 20 d
Type:
(pseudo-)first order (= half-life)
Other kinetic parameters:
first order rate constant
Transformation products:
yes
Validity criteria fulfilled:
not applicable
Conclusions:
The parental compound DNAN has been shown to be pricipally be convertable into the degradation products 2-A-4-NAN, 2-hydroxylamino-4-nitroanisole (2-HA-4-NAN)
by indigenous microorganisms collected from a contaminated land site. These degradation products are assumed to be be further biodegradable.

Description of key information

NTO is demonstrated to be ultimately biodegradable by different microbial species, belonging to various bacteria and fungi. The substance can totally transformed to urea and a polar compound assumed to be urea/ hydroxyurea.

 

The first step of the microbial metabolisation is the nitroreduction of NTO within 24 hours to ATO. This is followed by a two week lasting period where the triazolone ring of ATO is cleaved to urea and probably hydroxyurea as second step. This reaction consists on the hydrolysis of the pseudo `guanido' group of ATO.

 

The metabolising strains are adapted to the substance and are collected from waste water contaminated with NTO. From this source source two strains could be isolated:

- Penicillium chrysogenum CNCM. I-2081 and

- Bacillus licheniformis CNCM. I-1915

Both strains were able to biodegrade the substance NTO to ATO and further to urea and hydroxyurea.

 

Besides, a screening of microorganisms from various microbial collections including eight bacteria and 22 fungi, three strains were also able to reduce NTO to ATO. Identified strains are:

- Beauveria bassiana ATCC 7159,

- Rhizopus arrhizus ATCC 11145,

- Cylindrocarpon radicicola ATCC 11011

However, none of these strains have been demonstrated to further degrade ATO.

In another paper it was also demonstrated, the substance DNAN can be degraded by infidigenous microbial communities. DNAN and TNO share the same nitro-group structure what has been shown before to be degraded, followed by the cleavage of the also comparable nitrozole core.

Key value for chemical safety assessment

Half-life in freshwater:
6 d
at the temperature of:
27 °C

Additional information