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

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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

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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.)

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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

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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

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Perc

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cp1cp2cp3-5

J6 Native Prairie

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J6N-NF

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J6S-F

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J6S Native Prairie

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The Maturity of the Nematode Community in J6N is Reflected in the Richness of the

Aboveground Plant Community

R2 = 0.8844

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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