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Nematodes as Ecological Indicators inMonitoring Soil Ecosystem RecoveryFollowing Bioremediation of aTerrestrial Crude Oil Spill
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Nematodes as Ecological Indicators in Monitoring Soil Ecosystem Recovery
Following Bioremediation of a Terrestrial Crude Oil Spill
Kerry Sublette, Eleanor M. Jennings, Jennifer Busch-Harris, and Chintan Mehta
Center for Applied Biogeosciences, University of Tulsa, Tulsa, OKKathleen Duncan
University of Oklahoma, Norman, OKJosh Brokaw
Oklahoma State University, Stillwater, OK Tim Todd
Kansas State University, Manhattan, KS Greg Thoma
University of Arkansas, Fayetteville, AR
Oklahoma Reported Oil Spills1993-2002
Average Size of Crude Oil Spills by Year
0
10
20
30
40
50
60
70
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
Year
Ave
rage
Spi
ll Vo
lum
e (B
bl)
*
* Does not include one 80,000 bbl spill
Produced Water Salinity Varies Geographically (USGS)
Oklahoma Brine Spills 1993-2002
How Much Crude Oil and Brine Were Spilled in Oklahoma from 1993 Through 2002?
• 620,000 bbl of crude oil based on reported spills
• 1,029,000 bbl of saltwater– This is equivalent to
four Exxon-Valdez-size saltwater spills in Oklahoma during this ten-year period!
– 28,000 tons of salt spilled (1993 – 2002)
Remediation of Crude Oil Spills
• Fertilizer– Provide N and P for hydrocarbon degrading
bacteria• Organic matter
– Increases O2 infiltration– Decay products help rebuild soil structure– Substrate for soil fauna
• Tilling– Aeration– Mixing– Distribute oil in the soil to create more oil-water
interface
Restoration of Remediated Oil Spills
• Both the original spill and the remediation process disrupt soilecology– Disruptions in N and P cycling– Reduced diversity of soil microbes and invertebrates– Loss of vegetation
• All levels of ecosystem affected– Producers– Consumers– Decomposers
• Is restoration of the soil ecosystem the real definition of “clean” for a high value site?– Left to nature restoration is a lengthy process
Research Objectives
• Determine the effect of a fertilizer amendment on subsequent recovery of the soil ecosystem impacted by crude oil and
• Identify sensitive ecological indicators useful in monitoring the recovery of soil ecosystems impacted by crude oil – Lowest possible cost– Readily available analysis– Easy to understand and implement
J6N
TPH 33,500 mg/kg
J6S
TPH 4,800 mg/kg
January 1999
Tilled Hay Tilled Hay and Fertilizer
Tilled Hay
Tilled Hay and Fertilizer
J6S
J6N
J6 – Prairie
(No Treatment)
J6N -Control (Tilled Hay)
J6S – Control (Tilled Hay)
J6N-NF & J6N-F
J6S-NF & J6S-F
J6-C
May 1999
1999
2000
2001
2002
2003
Remediation Phase
Restoration Phase
J6 Timeline
Last tilling event
0
5000
10000
15000
20000
25000
30000
35000
40000
May-99
Jun-99 Apr-00 Oct-00 Apr-01 Jun-01 Oct-01 Mar-02
Sampling Time
TPH
(mg/
kg)
J6N-FJ6N-NF Regulatory limit
generally 10,000 mg/kg
Monitoring Ecological Indicators
• Soil chemistry – nutrients– NH4
+ and NO3-
– Total N– Total C– N mineralization– Plant available P
• Soil microbiology– PLFA– DNA– Nematodes
• Re-vegetation patterns
Mineralization rates in J6N in 2002 (mg/kg-d)
Sampling Date J6N-F J6N-NF
March 2002 0.58 ± 0.11 -0.06 ± 0.25June 2002 0.72 ± 0.35 0.38 ± .38
October 2002 0.36 ± 0.25 -0.08 ± .22
Nutrient Analysis Reveals Disruption in N Cycling
Phospholipid Fatty Acids (PLFA)
• Viable biomass (concentration of PLFA)• Community structure (relative proportions of
structural groups and specific biomarkers)• Metabolic status and environmental stress
(changes in membrane permeability through structural changes in PLFA)
8 10 12 14 16 18 20 22 24 26 28
Sample Moisture (% by wt.)
0
10000
20000
30000
40000
50000
60000
70000
[PLF
A],
pmol
e/g
r = 0.45, p = 0.0003
[PLFA] Strongly Influenced by Soil Moisture
Comparison of Total PLFA Concentrations in Test Plots During Remediation and Restoration
Plot Remediation PhaseMean ± std. dev.*
Restoration Phase2002
Mean ± std. dev.
Restoration Phase 2003
Mean ± std. dev.
J6N-F 32340 ± 17587 ab 13194 ± 5467 ab 30603 ± 9400 ab
J6N-NF 28454 ± 12410 ab 11578 ± 5831 a 22583 ± 12731 a
J6C 47941 ± 28362 ab 17446 ± 6800 abc 37559 ± 17280 ab
J6S-F 33370 ± 19571 ab 19598 ± 2738 abc 37583 ± 20289 ab
J6S-NF 22842 ± 13229 a 11108 ± 5340 a 38925 ± 23430 ab
J6N-NP 66069 ± 46787 b 22022 ± 2146 c 44517 ± 22654 b
J6S-NP Samples not collected at all time points
21964 ± 2146 bc 36781 ± 18247 ab
*Repeated Measures ANOVA
Relative Amounts of PLFA Structural Groups in Impacted Plots
PlotTerBrSats MidBrSats BrMonos
Remediation Phase (N=7)
(% of Total PLFA)*± std. dev.
(% of Total PLFA)± std. dev.
(% of Total PLFA)± std. dev.
J6N-F 16.8 ± 2.1 ab 10.0 ± 4.2 b 3.3 ± 1.4 aJ6N-NF 14.8 ± 2.5 a 5.5 ± 2.7 a 3.9 ± 2.2 aJ6S-F 19.1 ± 3.1 bc 11.7 ± 5.6 b 4.0 ± 2.4 aJ6S-NF 16.3 ± 2.1 ab 10.4 ± 4.6 b 4.7 ± 2.5 aJ6C 20.5 ± 1.8 c 12.0 ± 3.1 b 3.9 ± 1.5 aRestoration Phase (N=6)
J6N-F 21.5 ± 2.4 ab 13.1 ± 3.1 b 4.5 ± 0.17 aJ6N-NF 19.0 ± 3.6 a 9.8 ± 1.5 a 4.6 ± 0.51 aJ6S-F 23.6 ± 3.7 bc 16.5 ± 3.9 c 4.3 ± 0.48 aJ6S-NF 19.4 ± 3.9 a 14.9 ± 2.2 cb 4.9 ± 0.87 aJ6C 25.7 ± 3.9 c 16.9 ± 4.1 c 4.3 ± 0.26 a
J6N-NP: uncontaminated prairie
3/99 4/00 4/01 6/01 10/01 3/02 6/02 9/02 4/03 6/03
Uncontaminated sites:Little obvious change seasonally or yearly
DGGE Analysis4-yeartimeline
19992003
J6, 2 yr. after contamination and treatment
N-NF N-F NP C S-NF S-F SPUncontaminated sites:Higher diversityMore equal proportions
Contaminated sites:Lower diversityCertain sequences dominant
Most contaminated leastN-NF, N-F, S-NF, S-F
treated
Shifts in Dominant Groups After Hydrocarbon Impact
Slow-growing organisms adapted to low nutrient conditions like:
AcidobacteriaActinobacteria
Native Prairie Soils
Fast-growing, nutritionally versatile organisms like:
γ-Proteobacteria
Oil-impacted Soils
Evaluation of Ecological Indicators
• Biomarker analyses (PLFA and DNA) could distinguish between the fertilized and non-fertilized sections of J6N (higher initial TPH concentration); harder for J6S– Lowest possible cost: PLFA or DNA analysis costs
> $300/sample– Readily available analysis: Limited number of
commercial labs and universities– Easy to understand and implement: Requires
extensive training
0
50
100
150
200
250
H F M O H F M O H F M O H F M O
J6N-F J6N-NF J6-C J6N-P
num
ber/
100
g of
soi
l
A pr-01 Jun-01 Oct-01 M ar-02 Jun-02 Oct-02 A pr-03 Jun-03
H : H erbivo res F : F ungivo resM : M icro bivo res O: Omnivo res
(698) (313.5)
Nematode Trophic Groups – J6N
0
50
100
150
200
250
H F M O H F M O H F M O H F M O
J6S-F J6S-NF J6-C J6S-P
num
ber/
100
g of
soi
l
Apr-01 Jun-01 Oct-01 Mar-02 Jun-02 Oct-02 Apr-03 Jun-03
H: Herbivores F: FungivoresM: Microbivores O: Omnivores
(330)
Nematode Trophic Groups-J6S
Nematode Maturity Indices in Test Plots During Remediation and Restoration
Plot 2001Mean ± std. dev.*
2002Mean ± std. dev.
2003Mean ± std. dev.
J6N-F 1.41 ± 0.51 ab 2.05 ± 0.08 b 1.76 ± 0.42 a
J6N-NF 1.02 ± 0.02 a 1.47 ± 0.36 a 1.24 ± 0.21 a
J6C 2.18 ± 0.07 b 2.09 ± 0.11 b 2.39 ± 0.24 b
J6S-F 1.88 ± 0.26 ab 2.20 ± 0.26 b 2.31 ± 0.28 b
J6S-NF 1.91 ± 0.65 ab 2.07 ± 0.19 b 2.44 ± 0.18 b
J6N-NP 2.27 ± 0.06 b 2.29 ± 0.16 b 2.34 ± 0.05 b
J6S-NP 2.20 ± 0.06 b 2.38 ± 0.22 b 2.42 ± 0.08 b
*Repeated Measures ANOVA
J6N-F
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
J6 Native Prairie
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
J6N-NF
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2
J6C
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
J6S-F
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
J6S-NF
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
J6S Native Prairie
0
20
40
60
80
100
Apr01
Jun 01
Oct01
Mar02
Jun02
Oct02
Apr03
Jun03
Oct03
Perc
ent o
f Tot
al
cp1cp2cp3-5
The Maturity of the Nematode Community in J6N is Reflected in the Richness of the
Aboveground Plant Community
R2 = 0.8844
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
%cp3-5
Num
ber o
f Pla
nt S
peci
es
July 2003
Conclusions
• What are the consequences of not using fertilizer when bioremediatinghydrocarbons in soil?– Disruption of N cycling in the soil– Slower recovery of soil ecosystem
• What is the best ecological indicator of soil ecosystem recovery?– Nematode numbers and community
structure (trophic and colonizer-persister)
Conclusions• Advantages of nematode
numbers and community structure as ecological indicators:– Provide integrative picture
of the restorative capacity of an impacted soil ecosystem
– Reflect aboveground attributes of plant communities
– Obtained at low cost– Results easy to interpret– Allows consideration of land
use in regulatory decisions since direct comparisons to target ecosystems can be made
Future Work• Generating database of attributes of nematode
community structure in oil and brine impacted sites– Different stage of re-vegetation– Different climate zones
• Correlating with:– Contaminant concentrations
• Hydrocarbons• Brine components (Na+, Cl-, B, SAR, EC)
– Nutrient levels (NH4+, NO3
-, plant available P, %C, %N)– Soil texture– Vegetation
• Above-ground biomass• Plant species diversity
Acknowledgements
• This work was funded by the U.S. Department of Energy
• The authors thank The Nature Conservancy for access to the site