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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:
weight of evidence
Study period:
August, 2013
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
not specified
Remarks:
Klimisch 4 study part of weight of evidence. GLP is not a requirement.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DNAN (CAS# 119-27-7) was purchased from AlfaAesar (Ward Hill, MA, USA).
- Purity: 98%

Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Methanogenic sludge, aerobic return activated sludge (RAS), and nitrifying sludge were used as inoculum. The methanogenic sludge was obtained from a full-scale anaerobic bioreactor treating brewery wastewater (Mahou, Guadalajara, Spain). RAS and the nitrifying inoculum were collected from local municipal wastewater treatment plants; Ina Road and Randolph Park Wastewater Reclamation Facilities (Tucson, AZ, USA), respectively. All sludge samples were stored at 4°C. The volatile suspended solid (VSS) content in the methanogenic, RAS, and nitrifying sludge was 7.92, 0.25, and 0.53% (wet wt), respectively. The aerobic inocula were centrifuged (20 min at 4000 rpm) and the supernatant was discarded before use in bioassays.
Initial conc.:
0 µmol/L
Based on:
test mat.
Initial conc.:
13 µmol/L
Based on:
test mat.
Initial conc.:
26 µmol/L
Based on:
test mat.
Initial conc.:
52 µmol/L
Based on:
test mat.
Initial conc.:
78 µmol/L
Based on:
test mat.
Initial conc.:
104 µmol/L
Based on:
test mat.
Initial conc.:
130 µmol/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CH4 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The basal mineral medium labeled “medium 1” contained (in mg/L): K2HPO4(250), CaCl2·2H2O (10), MgSO4·7H2O (100), MgCl2·6H2O (100), NH4Cl (280), NaHCO3 (4000), yeast extract (100). The basal medium termed “medium 2” contained (in mg/ L): NaH2PO4 (1500), Na2HPO4 (894), NH4Cl (164), NaHCO3 (899). All media were supplemented with 1 mL/ L of trace elementsolution.
- Test temperature: 30°C
- pH: 7.2
- pH adjusted: yes

TEST SYSTEM
Methanogenic toxicity assay
Assays were conducted in glass flasks (160 mL) with basal medium 1 (25 mL) supplemented with acetate (26 mM) and methanogenic sludge (1.5 g VSS/L). All flasks were sealed with butyl rubber stoppers and then flushed with N2/CO2(80:20, v/v) for 5 min to create anaerobic conditions. The flasks were pre-incubated overnight to ensure that the sludge was adapted to the assay conditions. The following day, DNAN (0–130 µM), MENA (0–500 µM), orDAAN (0–661 µM) were added from concentrated stock solutions. The methane content in the headspace of each flask was measured periodically until the production of methane became constant in the toxicant-free controls. The maximum specific methanogenic activity of the control was 0.16 g CH4-COD/ g VSS/d.

Nitrification inhibition assays
Assays were conducted in Erlenmeyer flasks (125 mL) containing basal medium 2 (50 mL) and nitrifying sludge (0.5 g VSS/L).The flasks were spiked with DNAN (0–520 µM) or MENA(0–480 µM) and then capped with cotton gauzes to facilitate gas exchange. Liquid samples were collected periodically for ammonium analysis. The nitrifying activity of the uninhibited control was 19.3 mg NH4+-N g/ VSS/d.

Microtox: Microtox®Model 500 analyzer (Strategic Diagnos-tics, Inc. SDIX, Newark, DE, USA) was used to measured changes in the bioluminescence produced by the marine bacterium Aliivibrio fischeri (lyophilized culture of A. fischeri NRRL-B-11177, AZUR Environmental, Carlsbad, CA, USA). DNAN (0–650 µM),MENA (0–1300 µM), DAAN (0–300 µM), and 2,4-dinitrophenol(0–650 µM) solutions were tested. Microbial inhibition in Microtox was measured at 25°C in triplicate experiments as previouslydescribed.

All experiments were conducted in duplicate. The bioassays using sludge were incubated at 30°C in an orbital shaker (115 rpm) in the dark. Flasks without toxicant were included in all the assays and served as uninhibited controls. The maximum specific O2 consumption, as well as the nitrifying and methanogenic activities were calculated from the slope of O2 consumption, ammonium concentration, and cumulative methane production; respectively. The activities were normalized with respect to the biomass concentration. The initial concentrations of toxicant causing 20, 50 and 80% reduction in activity compared to an uninhibited control were referred to as IC20, IC50 and IC80, respectively.
Key result
Parameter:
% degradation (CH4 evolution)
Value:
39
Sampling time:
100 h

  Methanogens Aerobics heterotrophs Nitrifers Microtox
  IC20 IC50 IC80 IC20 IC50 IC80 IC20 IC50 IC80 IC20 IC50 IC80

DNAN

(µ M)

16 41 70 158 NT NT 20 49 125 27 57 110
Validity criteria fulfilled:
not applicable
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The results indicate that DNAN causes strong acute cytotoxicity in methanogenic and nitrification microbial populations. Preliminary results suggest that microbial reductive transformation may reduce the inhibitory impact of DNAN.
Executive summary:

DNAN undergoes facile microbial reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN). The study investigated the inhibitory effect of DNAN, MENA, and DAAN toward various microbial targets in anaerobic (acetoclastic methanogens) and aerobic (heterotrophs and nitrifiers) sludge, and the bioluminescent bacterium, Aliivibrio fischeri, used in the Microtox assay. Aerobic heterotrophic and nitrifying batch experiments with DAAN could not be performed because the compound underwent extensive auto oxidation in these assays. DNAN severely inhibited methanogens, nitrifying bacteria, and A. fischeri (50% inhibitory concentrations (IC50) ranging 41–57 M), but was notably less inhibitory to aerobic heterotrophs (IC50> 390 M). Reduction of DNAN to MENA and DAAN lead to a marked decrease in methanogenic inhibition (i.e., DNAN > MENA ≈ DAAN). Reduction of all nitro groups in DNAN also resulted in partial detoxification in assays with A. fischeri. In contrast, reduction of a single nitro group did not alter the inhibitory impact of DNAN toward A. fischeri and nitrifying bacteria given the similar IC50 values determined for MENA and DNAN in these assays. These results indicate that reductive biotransformation could reduce the inhibitory potential of DNAN.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 2012
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
not specified
Remarks:
Klimisch 4 study part of weight of evidence. GLP is not a requirement.
Specific details on test material used for the study:
2,4-Dinitroanisole (DNAN; CAS # 119-27-7, 98% purity) was purchased from Alfa Aesar (Ward Hill, MA).
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Two different microbial mixed cultures were used in this investigation, anaerobic granular sludge (AGS), and aerobic return activated sludge (RAS). AGS was obtained from a full-scale upward-flow anaerobic sludge blanket reactor treating wastewater at a brewery (Mahou, Guadalajara, Spain).
- Method of cultivation: The sludge was washed and sieved to remove fine particles before use in the bioassays. RAS was obtained from a municipal wastewater treatment plant (Ina Road Wastewater Reclamation Facility, Tucson, AZ). The content of volatile suspended solids (VSS) in AGS and RAS was 7.92% and 0.19% of the wet weight, respectively. The sludge samples were stored in a refrigerator at 48°C.
- Pretreatment: none
- Concentration of sludge: Inoculated aerobic and microaerophilic bioassays were supplied with RAS (0.5 g VSS/L). Prior to addition, RAS was centrifuged (20 min at 2,880g) and the liquid medium was discarded. Anaerobic bioassays were inoculated with sieved AGS (1.5 g VSS/ L).
Duration of test (contact time):
ca. 750 h
Initial conc.:
40 µmol/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: The basal mineral medium was prepared using ultra pure water (NANOpure InfinityTM, Barnstead International, Dubuque, IA) and contained the following (in mg/ L): K2HPO4 (250), CaCl22H2O (10), MgSO47H2O (100), MgCl26H2O (100), NH4Cl (280), NaHCO3 (4,000), yeast extract (100), and trace element solution (1 mL/ L). The solution of trace elements contained (in mg/L): H3BO3 (50), FeCl24H2O (2,000), ZnCl2 (50), MnCl24H2O (50), (NH4)6Mo7O244H2O (50), AlCl36H2O (90), CoCl26H2O (2,000), NiCl26H2O (50),
- Test temperature: 30°C
- pH: 7.2
- pH adjusted: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The liquid volume in all bioassays was 100 mL. Aerobic biotransformation assays were conducted in glass Erlenmeyer flasks (250 mL) capped with cotton gauze and placed on an orbital shaker at 180 rpm. Microaerophilic biotransformation assays were performed in glass serum flasks (160mL) capped with cotton gauze and placed on an orbital shaker at 115 rpm. Due to the lower shaking intensity and the higher depth of the liquid medium, the shaking was notably less aggressive compared to the fully aerobic treatment. Anaerobic biotransformation assays were conducted in glass serum flasks (160 mL) sealed with butyl rubber stoppers and aluminum crimp caps. The culture medium and the headspace were flushed with N2/CO2 (80:20, v/v) for 5 min to create anaerobic conditions.
- Number of culture flasks/concentration: 2
- Measuring equipment: Samples were centrifuged immediately (10 min at 9,600g) and then 1596 Biotechnology and Bioengineering, Vol. 110, No. 6, June, 2013 analyzed by high-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD). Evaporation of the liquid in both the aerobic and microaerophilic conditions was monitored during the experimental period to adjust the final results according to the original liquid volume.

SAMPLING
- Sampling method: Liquid samples were collected periodically for analysis of DNAN and DNAN transformation products. Samples were centrifuged immediately (10 min at 9,600g) and then analyzed by high-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD). Anaerobic samples were spiked with 250 mg L1 ascorbic acid to prevent autoxidation of aromatic amines and other reduced metabolites. Based on sample stability tests performed, ascorbic acid did not chemically reduce DNAN and was therefore chosen as a suitable antioxidant for the anaerobic samples. Dissolved oxygen (DO) and oxidation-reduction potential (ORP) were monitored in experiments replicating the endogenous and cosubstrate conditions for aerobic, microaerophilic, and anaerobic conditions. These experiments were performed in duplicate.
- Sterility check if applicable: sterile meidum was used
- Sample storage before analysis: not specified
Key result
Parameter:
% degradation (test mat. analysis)
Value:
87
Sampling time:
700 h
Remarks on result:
other: using cosubstrate (acetate)
Details on results:
Aerobic Biotransformation
The DNAN biotransformation rate under fully aerobic conditions was very low. Under the best condition (with cosubstrate addition), almost 700 h was required to remove most (87%) of the added DNAN.

Microaerophilic Biotransformation
Under microaerophilic conditions, the biotransformation of DNAN was much faster in all treatments compared to the fully aerobic condition. In this case, the endogenous and cosubstrate treatments behaved similarly with 80% removal of DNAN in 35 and 52 h, respectively. In the treatments with live sludge, DNAN conversion was notably faster than with heat-killed sludge where only 8% of DNAN was transformed to other compounds during the whole experimental period of 150 h.

Anaerobic Biotransformation
Under anaerobic conditions, the DNAN biotransformation occurred considerably faster and a higher conversion was observed compared to the aerobic and microaerophilic conditions. In the treatment with H2 as cosubstrate, DNAN was nearly fully converted within 12 h. In the endogenous treatment, an almost complete conversion required only 33 h. The heat-killed sludge treatment also retained reducing capacity, converting 88% of the added DNAN by the end of the experimental period of 48 h.
Validity criteria fulfilled:
not applicable
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
DNAN was reduced by sludge to two main metabolites, MENA and DAAN, under all of the different conditions tested. The highest DNAN biotransformation rate was observed in anaerobic conditions enhanced by the addition of H2, as a cosubstrate. The nitro group in the ortho position was first reduced to an amine to yield MENA, then the para nitro group was reduced to an amine to produce DAAN. During the reductive biotransformation, coupling of DNAN intermediates occurred, yielding azo compounds. These products were further reduced to hydrazine dimers. In addition, a diversity of products was created by parallel pathways of N-methylation, and N-acetylation of primary amines, as well as O-demethylation and dehydroxylation of methoxy groups. These insights about the fate and biotransformation of DNAN will help in understanding environmental health risks from DNAN and provide clues for the bioremediation of DNAN contamination.
Executive summary:

DNAN biotransformation rates in sludge under aerobic, microaerophilic, and anaerobic conditions, detected biotransformationproducts, and elucidated their chemical structures were studied. The biotransformation of DNAN was most rapid under anaerobic conditions with H2 as a cosubstrate. The results showed that the ortho nitro group in DNAN is regioselectively reduced to yield 2-methoxy-5-nitroaniline (MENA), and then the para nitro group is reduced to give 2,4-diaminoanisole (DAAN). Both MENA and DAAN were identified as important metabolites in all redox conditions. Azo and hydrazine dimer derivatives formed from the coupling of DNAN reduction products in anaerobic conditions. Secondary pathways included acetylation and methylation of amine moieties, as well as the stepwise Odemethylation and dehydroxylation of methoxy groups. Seven unique metabolites were identified which enabled elucidation of biotransformation pathways. The results taken as a whole suggest that reductive biotransformation is an important fate of DNAN leading to the formation of aromatic amines as well as azo and hydrazine dimeric metabolites.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
February 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source
Justification for type of information:
Q(S)AR
Qualifier:
no guideline required
Principles of method if other than guideline:
Q(S)AR
GLP compliance:
no
Remarks:
GLP does not apply to QSARs for which there is no test article as such.
Oxygen conditions:
other: Q(S)AR
Inoculum or test system:
other: Q(S)AR
Duration of test (contact time):
0 s
Initial conc.:
0 other: Q(S)AR
Based on:
other: Q(S)AR
Parameter followed for biodegradation estimation:
other: Q(S)AR
Preliminary study:
Not required
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
2.364
Sampling time:
2.364 wk
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Value:
-0.032
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction
Details on results:
Criteria for the YES or NO prediction: If the Biowin3 (ultimate survey model) result is "weeks" or faster (i.e. "days", "days to weeks", or "weeks" AND the Biowin5 (MITI linear model) probability is >= 0.5, then the prediction is YES (readily biodegradable). If this condition is not satisfied, the prediction is NO (not readily biodegradable).
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Based on BIOWIN program results, BIOWIN3 and BIOWIN5 results demontrated that the substance is not readily biodegradable.
Executive summary:

If the Biowin3 (ultimate survey model) result is "weeks" or faster (i.e. "days", "days to weeks", or "weeks" AND the Biowin5 (MITI linear model) probability is >= 0.5, then the prediction is YES (readily biodegradable). If this condition is not satisfied, the prediction is NO (not readily biodegradable).

Based on BIOWIN program results, BIOWIN3 and BIOWIN5 results demontrated that the substance is not readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 2014
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
equivalent or similar to
Guideline:
other: OECD 209
GLP compliance:
not specified
Remarks:
Klimisch 4 study part of weight of evidence. GLP is not a requirement.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material media: The IMX-101 formulation containing 40–45% DNAN, 18–23%NTO, and 35–40% NQ was provided by Picatinny Arsenal.
- Analytical standards for DNAN were provided by BAE Holston.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The IMX-101 degrading enrichment culture was obtained by first selecting the fastest growing cultures that degraded IMX-101 when alternate carbon and nitrogen was present (phase I).Then the phase I culture that utilized IMX-101 as a sole source of nitrogen was selected and further enriched with IMX-101 providing nitrogen in the media (phase II). Four different feedstocks were used to enrich an IMX-101 degrading culture during phase I enrichment studies, including aerobic activated sludge.

Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge: Sart City wastewater treatment plan
- Laboratory culture: Aerobic cultures (i.e., activated sludge, soil and compost treatments) were grown in 250 mL Wheaton media bottles containing 75 mL of minimal media and capped with sterile foam stoppers to allow for oxygen exchange. Anaerobic cultures (i.e., anaerobic digestersludge) were inoculated into 60 mL amber VOA vials containing capped with Mininert®caps with 30 mL of a modified minimal media solution containing a five fold increase in TES compared toaerobic cultures. The VOA vials were sparged with compressed nitrogen gas (Airgas, Radnor Township, PA, 99% purity) to remove oxygen from the headspace. All cultures were incubated in the dark at 25°C on a shaker table at 150 rpm. During phase I enrichment studies microcosms were inoculated using 100 µL of each of the slurry feed stocks or 1 g of soil. Degradation of DNAN was observed over nine days by high pressure liquid chromatography tandem photo diode array (HPLC-PDA). At each sample point 2 mL aliquots were removed from the treatment and syringe filtered through a nylon 0.45 um filter (FisherBrand, Waltham, MA)into amber vials.
- Storage conditions: 4°C
- Storage length: until analysis
- Pretreatment: Soil microorganisms were enriched from a loamys and that had previously been exposed to IMX-101 for 110 days in the lab.
Duration of test (contact time):
ca. 9 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
15 mg/L
Based on:
test mat.
Initial conc.:
25 mg/L
Based on:
test mat.
Initial conc.:
50 mg/L
Based on:
test mat.
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Growth media used in aerobic studies contained 1800 mg/L K2HPO4, 600 mg/L KH2PO4, 200 mg/ L MgSO4,100 mg/ L NaCl, 100 mg/L KCl, and 40 mg/L CaCl. Vitaminsolution was added to provide 10 µg/L pyridoxine·HCl, 5 µg/L thiamine·HCl, 5 µg/L riboflavin, 5 µg/L calcium pantothenate, 5 µg/ L thioctic acid, 5 µg/ L p-aminobenzoic acid, 5 µg/ Lnico-tinic acid, 5 µg/L vitamin B12, 2 µg/ Lbiotin, and 2 µg/ L folic acid. Trace element solution (TES) was added to provide 2 mg/ Lnitrilotriacetic acid, 1 mg/ L MnSO4·H2O, 800 ug/ L FeCl3, 200 ug/ L CoCl2·6H2O, 200 ug/L ZnSO4·7H2O, 20 ug/L CuSO4·5H2O, 20 ug/L NiSO4·6H2O, 20 ug/L Na2MoO4·2H2O,20 ug/L Na2SeO4, 20 ug/L Na2WO4, and 8 ug/ L H3BO3. Chemicals were purchased from Fisher Scientific (Hampton, NH) excluding K2HPO4 (Sigma-Life science, St. Louis MO), glucose, nitrilotriacetic acid, Na2MoO4·2H2O, Na2SeO4, Na2WO4 (AcrosOrganics, Geel, Belgium) and vitamin B12 and NiSO4·6H2O (AlfaAesar, Ward Hill, MA). TES chemicals were purchased from (AcrosOrganics, Geel, Belgium) excluding thioctic acid and vitamin B12(MP Biomedical, Solon, OH).
- Additional substrate: none
- Test temperature: 25°C
- pH: 7-7.5
- pH adjusted: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 250 mL Wheaton media bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: sterile foam stopppers that allow for oxygen exchanges.
- Method used to create anaerobic conditions: vials were sparged with compressed notrogen gas.
- Measuring equipment: HPLC-PDA
Key result
Parameter:
% degradation (test mat. analysis)
Value:
80.5
Sampling time:
9 d
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
Using IMX-101 formulation containing 40–45% DNAN, 80.5% of DNAN has been biodegradated after 9 days, suggesting that the test compound is not readily biodegradable.
Executive summary:

In this study simultaneous aerobic degradation of the IMX-101 formulation constituents 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ) was observed and degradation products were examined. Degradation products over four days of incubation included: nitrourea, 1,2-dihydro-3H-1,2,4-triazol-3-one, and 2,4-dinitrophenol. The enrichment culture maximum specific growth rate of 0.12/h and half saturation constant of 288 mg/ L during degradation of IMX-101 as a sole nitrogen source suggest that enrichment culture growth kinetics may closely relate to those of other explosive and nitroaromatic compounds.

Using IMX-101 formulation containing 40–45% DNAN, 80.5% of DNAN has been biodegradated after 9 days, suggesting that the test compound is not readily biodegradable.

Description of key information

Four different independent sources of data including a QSAR (Epiwin) prediction have been considered for biodegradability. There is strong consistency in the results from these studies including the QSAR, leading to the conclusion that the combined weight is sufficient for expert judgement.

The QSAR predicts 2,4 –dinitroanisole is not readily biodegradable with a Klimisch score of 2.

The three experimental studies used different protocols and test conditions similar for inherent biodegradation, however, with a Klimisch score of 4 cannot considered to be relevant or reliable for this endpoint.

Based on the above, it is concluded that 2,4 –dinitroanisole is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information