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Ecotoxicological assessment of anaerobic bioremediation of sludge contaminated by the
explosive 2,4,6-trinitrotoluene (TNT)
Veronica Ribé
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BIOREX Biological Remediation of Explosives
Project Objectives- Develop a method for microbial degradation of
explosives - Remediate sludge and soil contaminated with
explosives- Characterization of the degradation process
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Project Partners- Mälardalen University- Nammo Demil Division- FOI (Swedish Defense Research Agency)- Cesium Innovation Company- Eurenco Bofors- Bofors Test Center- KCEM (Competence Centre for Energetic Material)
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PhD Research ProjectAims:- Chemical characterization of the degradation
process- Ecotoxicological testing during the degradation
to identify toxic intermediates in the process
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Why microbiological degradation of explosives?
- Contamination of soils during the manufacture, employment and disposal of explosives worldwide
- Most explosive substances are environmentally very stabile compounds- Many explosives, such as TNT, are very toxic to living organisms- Desirable to develop a cheap, non-toxic degradation method
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Project IdeaTNT sludge
BioslurryOrganic material
WaterCO2
Bacterial cultureSubstrate
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Current Experimental Set-up
TNT-contaminated sludge mixed with an adapted bacterial culture in a sand slurry
Left picture: Jianjun Hao, UC David Plant Pathology Department, University of California
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Current Experimental Set-up- 16-day degradation of TNT in bioslurry batch
experiments (5 & 10% TNT)- TNT-adapted bacterial culture from a
denitrifying activated sludge- Static, reducing conditions- Monitoring and adjustment of pH and nutrient
levels
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Chemical characterisation of the degradation of TNT
- Frequent sampling during the degradation process
- Chemical structural analysis of the extracted samples
- Characterisation of the step-wise degradation of TNT by the bacterial culture
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Chemical analysisGas chromatography:- How much of the
explosive is degraded?
- What metabolites are formed in the process?
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Ecotoxicological testing of metabolites in the degradation process
- Frequent sampling during the degradation process
- Toxicity testing of the samples
- Ecotoxicological characterisation of the degradation process
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Ecotoxicity testsConsumers:
Heterocypris incongruens
(Ostracodtoxkit)
Decomposers:Vibrio fischeri (Microtox)
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Project Outcomes- Identifying the metabolites of the degradation
process- Determining the combined toxicity of the
metabolites during the degradation
- Optimizing the bacterial degradation of explosives
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Results
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Chemical characterization- Monoaminodinitrotoluene
s (4a-2,6-dnt and 2a-4,6-dnt) and dinitrotoluenes(2,6-dnt and 2,4-dnt) foundin µg/g levels
- Trace levels of nitrotoluenes, dinitrobenzenes and dinitrotoluenes (2,5-dnt, 2,3-dnt and 3,4-dnt) were detected
- Concentrations of trinitrobenzene could not be quantified
- The presence of diaminonitrotoluene (2,6-dia4-nt) could not be verified
- Degradation of TNT was low
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Chemical characterizationChromatogram from the analysis of a 5%TNT bioslurry
sample taken on day 16 of the degradation
Minutes8 9 10 11 12 13 14 15 16 17 18 19 20 21
mVo
lts
-200
-100
0
100
200
300
400
500
600
700
800
900
mVo
lts
-200
-100
0
100
200
300
400
500
600
700
800
900
2,6-
dnt
8,00
1
(2,5
-dnt
)
2,4-
dnt
9,24
12,
3-dn
t 9,
408
9,
954
10
,228
10
,323
(3,4
-dnt
)
11
,676
11
,786
12
,366
12
,459
12
,764
tnt
12,9
88
13
,296
tnb
13,
451
13
,527
13
,629
13
,747
13
,796
13
,949
14
,049
14
,106
2,4-
dna
14,
259
14
,484
15
,491
16
,054
17
,047
(2,4
dia-
6-nt
)
4a-2
,6dn
t 18
,699
18
,977
2,6d
ia-4
nt 1
9,26
4
19,4
89
2a-4
,6dn
t 19
,774
20
,095
20
,540
20
,782
20
,877
20
,944
24
,898
Detector 11003std4.met2007-04-04 17-29-24.dat
Nam eRetention T im e
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Ecotoxicological characterizationHeterocypris incongruens Toxicity
(Ostracodtoxkit)- Mortality of control organisms exceeded the
validity limit in all tests
- 100% mortality of all test organisms in slurry samples
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Ecotoxicological characterizationVibrio Fischeri toxicity (Microtox)
Toxicity of slurry leachates to Vibrio Fischeri
0
20
40
60
80
100
120
0 5 10 15 20
Day of degradation
Toxi
city
effe
ct %
5% TNT slurry10% TNT slurry
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Conclusions- The bacterial culture did not degrade TNT efficiently
under the experimental conditions
- The results of the ostracod toxicity test were invalid due to high mortality of the control organisms
- Microtox was used successfully to assess the toxicity of slurry leachates
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Future research- Optimized TNT
degradation by environmentally adapted microorganisms in a bioslurry reactor
- Extended ecotoxicity testing using aquatic organisms and enzymatic assays
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Acknowledgements
FOI Swedish Defence Research Agency
Nammo Demil Division
