Using CSIA for Biodegradation Assessment: Potential, Practicalities and Pitfalls
B. Sherwood LollarUniversity of Toronto
S. Mancini, M. Elsner, P. Morrill, S. Hirschorn,N. VanStone, M. Chartrand, G. Lacrampe-Couloume,E.A. Edwards, B. SleepG.F. Slater
CSIA as Field Diagnostic Tool
Environmental Forensics:(Philp)
Biodegradation& Abiotic Remediation (BSL)
EPA 600/R-08/148
Restoration Technology Transfer
Fundamental PrinciplesStandard Methods & QA/QCDecision Matrices
Outline
• FAQ: Common Pitfalls/Misconceptions• Source Differentiation• What is Fractionation?• Verification of MNA and/or Enhanced
Remediation using CSIA– Fingerprint of biodegradation?
• Where to be Careful• CSIA as Early Warning System &
Diagnostic Tool – Case study
FAQ Sheet
• Sample collection: adaptation of standard 40 mL VOA vial
• Turnaround: approximately 4 weeks• Cost: less than cost of one additional
monitoring well - can reduce uncertainty & risk, and drive decision making
• QA/QC: more than 50 year history of standardization and cross-calibration
• Tracer: but naturally occurring
Commercial CSIA (currently ~ a dozen labs worldwide)
• C most widely available (H, Cl)
• Petroleum hydrocarbons (including both aromatics and alkanes)
• Chlorinated ethene and ethanes
• Chlorinated aromatics
• MTBE and fuel oxygenates
• PAHs, PCBs, pesticides
Compound Specific Isotope AnalysisCompound Specific Isotope Analysis
Natural abundance of two stable isotopes
of carbon: 12C and 13C
CSIA measures R or isotope ratio
(13C/12C) of individual contaminant
x 100013C in ‰ =(13C/ 12C sample – 13C/ 12Cstandard)
13C/ 12C standard
Source differentiation of TCE
-35
-33
-31
-29
-27
-25
ACP PPG DOW ICI
Source/Manufacturer
13C
(‰
) Slater et al. (1998)van Warmerdam et al. (1995)
DATA FROMJendrzejewski et al. (2001)
MI
Principles of Fractionation
to - Before degradation
t1 - Post degradation
Preferential degradationof T12CE
T12CE
Remaining TCE progressivelyisotopically enriched in 13Ci.e. less negative 13C value
T12CE T12CE
T12CE
T12CET12CE
T12CE
T12CE
T12CE
T12CE
T13CET13CE
T13CE
T13CE
T13CE
T13CE T12CE
k12C > k13C
Sherwood Lollar et al. (1999)
-35
-30
-25
-20
-15
-10
0 0.2 0.4 0.6 0.8 1
Fraction of TCE remaining
13C
(in
‰)
Sherwood Lollar et al. (1999)Org. Geochem. 30:813-820
Biodegradation of TCE
Increasing Biodegradation
R = RR = Roo f f (α – 1)(α – 1)
0
5
10
15
20
25
0 250 500 750 1000 1250 1500
Hours
Ch
lori
nat
ed e
then
e (i
n u
mol
es)
cisDCE
TCE
VC
Ethene
Slater et al. (2001)ES&T 35:901-907
-80
-70
-60
-50
-40
-30
-20
-10
0
10
20
0 250 500 750 1000 1250 1500
Hours
13C
Ch
lori
nat
ed e
then
e
cisDCETCE VC
Ethene
Slater et al. (2001)ES&T 35:901-907
Fractionation of Daughter Products
• Breakdown Products – initially more negative 13C values than the compounds from which they form
• Products subsequently show isotopic enrichment trend (less negative values) as they themselves undergo biodegradation
• Combining parent and daughter product CSIA is valuable (a recurring theme …)
CSIA: Verification of Degradation
• Chlorinated ethenes (Hunkeler et al., 1999; Sherwood Lollar et al., 1999; Bloom et al., 2000; Slater et al., 2001; Slater et al., 2002; Song et al., 2002; Vieth et al., 2003, Hunkeler et al., 2004; VanStone et al., 2004; 2005; Chartrand et al., 2005; Morrill et al., 2005; Lee at el., 2007; Liang et al, 2007)
• Chlorinated ethanes (Hunkeler & Aravena 2000; Hirschorn et al. 2004; Hirschorn et al., 2007; VanStone et al., 2007; Elsner et al., 2007)
• Aromatics (Meckenstock et al., 1999; Ahad et al., 2000; Hunkeler et al., 2000, 2001; Ward et al., 2001; Morasch et al., 2001, 2003; Mancini et al. 2002, 2003; Griebler et al., 2003; Steinbach et al., 2003)
• MTBE (Hunkeler et al., 2001; Gray et al., 2002; Kolhatkar et al., 2003; Elsner et al., 2005, Kuder et al., 2005; Zwank et al., 2005; Elsner et al., 2007; McKelvie et al., 2007)
Biotic and Abiotic Degradation
• Chlorinated ethenes (Hunkeler et al., 1999; Sherwood Lollar et al., 1999; Bloom et al., 2000; Slater et al., 2001; Slater et al., 2002; Song et al., 2002; Vieth et al., 2003, Hunkeler et al., 2004; VanStone et al., 2004; 2005; Chartrand et al., 2005; Morrill et al., 2005; Lee at el., 2007; Liang et al, 2007; Elsner et al. 2010)
• Chlorinated ethanes (Hunkeler & Aravena 2000; Hirschorn et al. 2004; Hirschorn et al., 2007; VanStone et al., 2007; Elsner et al., 2007)
• Aromatics (Meckenstock et al., 1999; Ahad et al., 2000; Hunkeler et al., 2000, 2001; Ward et al., 2001; Morasch et al., 2001, 2003; Mancini et al. 2002, 2003; Griebler et al., 2003; Steinbach et al., 2003)
• MTBE (Hunkeler et al., 2001; Gray et al., 2002; Kolhatkar et al., 2003; Elsner et al., 2005, Kuder et al., 2005; Zwank et al., 2005; Elsner et al., 2007; McKelvie et al., 2007)
CSIA as Restoration Tool
• Isotopic enrichment in 13C in remaining contaminant (less negative 13C values) a dramatic indicator of biodegradation
• Extent of fractionation predictable and reproducible – Quantification (rates) possible in many cases
• CSIA can distinguish mass loss due to the strong fractionation in degradation (biotic and abiotic)
• versus small- or non-fractionating processes such as volatilization, diffusion, dissolution, sorption , etc.
Non-conservative vs. Conservative
Degradatio
n
% contaminant remaining
Non-degradative
100 75 50 25
Ch
an
ge in
13C
/12C
0
Non-conservative vs. Conservative
Degradatio
n
% contaminant remaining
Non-degradative
100 75 50 25
Ch
an
ge in
13C
/12C
0
Fractionation isabout breaking Bonds
100 255075
Degradatio
n
0
Non-degradative
Non-conservative vs. Conservative
% contaminant remaining
Ch
an
ge in
13C
/12C
100 255075
Degradatio
n
0
Non-degradative
Non-conservative vs. Conservative
% contaminant remaining
Ch
an
ge in
13C
/12C
100 255075
Degradatio
n
0
Non-degradative
Non-conservative vs. Conservative
% contaminant remaining
Ch
an
ge in
13C
/12C
Where to be careful
• Processes that drive towards low fraction remaining (air sparging)
• High Kow; high TOC (sorption)• Vadose zone (volatilization)• Hydrogen isotope effects can be larger• Fractionation is a function of different
microbial pathways (e.g. aerobic versus anaerobic)
• Be an informed customer
Case Study I: CSIA as early warning system for bioremediation: Kelly AFB
P. Morrill, G. Lacrampe-Couloume, G. Slater, E. Edwards, B. Sleep, B. Sherwood Lollar, M.
McMaster and D. Major
JCH (2005) 76:279-293
Early Warning System
• Stable carbon isotopes have potential to provide significant added value in early stages of biodegradation
• Monitoring 13C values of PCE and TCE may provide evidence of degradation prior to breakdown products such as VC and ethene rising above detection limits for VOC
Morrill et al. (2005)
Case Study II: CSIA to trouble-shoot potential
cisDCE stall
M. Chartrand, P. Morrill, G. Lacrampe-Couloume, and
B. Sherwood Lollar
ES&T (2005) 39:4848-4856
CSIA at Fractured Rock Site
0
6
18
24
30
36
40
12
Dep
th B
elow
Gro
und
Sur
face
(m
)
0 m 120 mground water flow direction
5B/T 1B 2B 3B 4B
fill
silt & peat
clay
till
fracturedbedrock
SUPPORT PILES
Manufacturing Building
A A`
Disposal well
6B
DNAPL0
6
18
24
30
36
40
12
Dep
th B
elow
Gro
und
Sur
face
(m
)
0 m 120 mground water flow direction
5B/T 1B 2B 3B 4B
fill
silt & peat
clay
till
fracturedbedrock
SUPPORT PILES
Manufacturing Building
A A`
Disposal well
6B
DNAPL
A`
N
Approximate ground water flow direction under undisturbed conditions
Manufacturing Building
Disposal Well
Well 7B
Well 5B/T
Well 2B
Well 1B
Well 3B
Excavation Activities
Ground water flow direction in pilot treatment area during isotope study (June - November 2002)
0 20m
Approximate Scale
Estimated TCE Plume
N N
Approximate ground water flow direction under undisturbed conditions
Manufacturing Building
Disposal Well
Well 7B
Well 5B/T
Well 6B
Well 2B
Pilot Treatment Area
Well 1B
Well 3B
Well 4B
Excavation Activities
Ground water flow direction in pilot treatment area during isotope study (June - November 2002)
0 20m
Approximate Scale
Estimated TCE Plume
A
Chartrand et al. (2005) ES&T 39:4848-4856
Fluctuation in VOC
0
10
20
30
40
50
60
70
80
2/2
8/0
1
4/1
9/0
1
6/8
/01
7/2
8/0
1
9/1
6/0
1
11
/5/0
1
12
/25
/01
2/1
3/0
2
4/4
/02
5/2
4/0
2
7/1
3/0
2
9/1
/02
10
/21
/02
12
/10
/02
Sampling Date
Co
nce
ntr
atio
n (
mg
L
-1)
Biostimulation
BioaugmentationBegin Isotope
Study
Nov-02Sep-02Jul-02Jun-02Mar-02Mar-01 May-01 Jul-01 Sep-01 Nov-01 Jan-02 Apr-02Feb-01
Sampling Date
VC
TCE
cisDCE
ETH
Chartrand et al. (2005)
CSIA as Diagnostic Tool
• Initial apparent successful production of VC and ethene
• Confused and potentially compromised by fluctuations in hydrogeologic gradients
• Periodic spikes in cisDCE & VC due to – Incomplete reductive dechlorination?– Dissolution (rebound) from NAPL phase?– Mixing of groundwater?
Chartrand et al. (2005)
Fluctuation in VOC
0
10
20
30
40
50
60
70
80
2/2
8/0
1
4/1
9/0
1
6/8
/01
7/2
8/0
1
9/1
6/0
1
11
/5/0
1
12
/25
/01
2/1
3/0
2
4/4
/02
5/2
4/0
2
7/1
3/0
2
9/1
/02
10
/21
/02
12
/10
/02
Sampling Date
Co
nce
ntr
atio
n (
mg
L
-1)
Biostimulation
BioaugmentationBegin Isotope
Study
Nov-02Sep-02Jul-02Jun-02Mar-02Mar-01 May-01 Jul-01 Sep-01 Nov-01 Jan-02 Apr-02Feb-01
Sampling Date
VC
TCE
cisDCE
ETH
VC
cisDCE
Chartrand et al. (2005)
Continued 13C enrichment despite VOC fluctuations
-30
-25
-20
-15
-10
Jun-02 Jul-02 Sep-02 Nov-02
Sampling Date
δ13C
(‰
)
well 2B
well 4B
well 3B
well 5B
Continuing Biodegradationof cisDCE
Cha Chartrand et al. (2005)
Fluctuation in VOC
0
10
20
30
40
50
60
70
80
2/2
8/0
1
4/1
9/0
1
6/8
/01
7/2
8/0
1
9/1
6/0
1
11
/5/0
1
12
/25
/01
2/1
3/0
2
4/4
/02
5/2
4/0
2
7/1
3/0
2
9/1
/02
10
/21
/02
12
/10
/02
Sampling Date
Co
nce
ntr
atio
n (
mg
L
-1)
Biostimulation
BioaugmentationBegin Isotope
Study
Nov-02Sep-02Jul-02Jun-02Mar-02Mar-01 May-01 Jul-01 Sep-01 Nov-01 Jan-02 Apr-02Feb-01
Sampling Date
VC
TCE
cisDCE
ETH
VC
cisDCE
Chartrand et al. (2005)
Continuing Net Biodegradation
-50
-40
-30
-20
-10
Jun-02 Jul-02 Sep-02 Nov-02
Sampling Date
δ13C
(‰
)
well 2B
well 5B
well 3B
well 2B
well 4B
VC
Ethene
Chartrand et al. (2005)
CSIA as Restoration Tool
• Verification of remediation – direct evidence for transformation
• Sensitive tracer – early warning system• Cost effectiveness - diagnostic for trouble-
shooting and optimization (Chartrand et al., 2005; Morrill et al 2009)
• Quantification of remedial effectiveness (Morrill et al., 2005; Hirschorn et al. 2007)
• Resolution of Abiotic versus Biotic degradation for chlorinated solvents (VanStone et al., 2008; Elsner et al. 2008; 2010)
More information?