Of Earthworms and Nematodes In Restoration of E&P Sites

Kerry Sublette, Eleanor JenningsCenter for Applied Biogeosicences

University of Tulsa

Kathleen DuncanUniversity of Oklahoma

Greg Thoma, Duane WolfUniversity of Arkansas

Josh BrokawWashington State University

Mac A. CallahanUSDA Forest Service

Tim ToddKansas State University

Project Objective:Accelerated Restoration of Oil- and

Brine-impacted Sites• Both the original spill and the remediation process

disrupt soil ecology– Disruptions in N and P cycling– Reduced diversity of soil microbes and

invertebrates– Loss of vegetation

• All levels of ecosystem affected– Producers– Consumers– Decomposers

Project Summary• This project began as an effort to determine the

efficacy of the introduction of non-indigenous earthworms to remediated oil and brine spill sites in order to accelerate the restoration of these sites in terms of soil quality and health.

• Original treatments to be investigated included all combinations of the following plus no treatment:– inoculation with E. fetida– fertilizer addition – hay cover

• Treatments were applied in enclosures designed to retain E. fetida and exclude other macrofauna.

Project Summary• Within 60 days earthworms indigenous to the area

were detected in significant numbers both outside and inside of the enclosures and very few E. fetidacould be found.

• This observation led to the recognition that all enclosures were accessible to indigenous earthworms and in reality there were only four treatments: – hay + fertilizer (HF)– hay only (H) – fertilizer only (F) – no treatment (N)

• We were left with the assumption that earthworm activity correlates with the availability of food (surface hay)

Project Timeline

• Ripped and tilled sites• Installed earthworm enclosures and added

amendments (fertilizer and/or hay)• After 30 days inoculated with Eisenia fetida

– 5 worms per enclosure per worm treatment• Initial conditions:

– 350-600 mg/kg Na+ in brine site– 9,500-16,000 mg/kg total extractable hydrocarbons in

hydrocarbon site• Sampling:

– August 2005– October 2005– June 2006– September 2006

Metrics Used to Evaluate Treatments

• Soil nutrients• Microbial biomass as PLFA

– PLFA = phospholipid fatty acid analysis• N-cycling bacteria• Nematode numbers and community structure• Plant biomass• No acceptable metric for earthworm numbers was

identified– Earthworms were observed but were not confined to the

enclosures; therefore, direct counts were unreliable– Earthworm castings were damaged when the layer of damp

decomposing hay was removed to expose the surface in HF and H treatments

Block 1 Block 2 Block 3 Block 4

Original Experimental Design – Brine Site

(Similar layout at Hydrocarbon Site)

Worms + Hay + Fertilizer

Worms + Hay

Worms + Fertilizer

Hay + Fertilizer

Worms

Hay

Fertilizer

No Treatment

Filling enclosure with homogenized soil at hydrocarbon site

Filling earthworm enclosure with homogenized soil

Hay

Earthworm enclosures installed and amendments added at hydrocarbon site

Pantyhose cover added after 30 days

Entire site covered with hay for moisture and temperature control

Earthworm Observations

• At the first sampling, earthworms were found in the top 15 cm of most enclosures– A. trapezoides– Diplocardia sp.– Very few E. fetida

• Average worms/enclosure: 1.74 with surface hay and 0.63 without surface hay-supports positive correlation of worms with surface hay

TreatmentBrine Site

August 2005 October 2005 June 2006

HF 26.0 ± 2.1 23.3 ± 2.8 29.4 ± 3.3H 25.7 ± 3.3 24.2 ± 3.4 29.2 ± 2.9F 26.1 ± 1.6 22.5 ± 3.0 25.9 ± 4.0N 25.7 ± 1.6 21.9 ± 2.5 29.6 ± 3.3

NativePrairie

13.7 ± 1.5(n=4)

TreatmentHydrocarbon

Site

August 2005 October 2005 June 2006

HF 22.0 ± 3.8 18.9 ± 3.9 23.1 ± 6.4H 21.7 ± 4.5 18.2 ± 4.6 23.2 ± 8.4F 20.7 ± 4.3 19.3 ± 4.8 28.4 ± 13.3N 18.2 ± 4.9 19.4 ± 4.1 21.3 ± 6.5

NativePrairie

13.5 ± 1.7(n=4)

Soil Moisture

Mean ± std. dev. with n=8 or 9 unless otherwise indicated

Earthworms and Soil Health

• Earthworm castings – contain higher concentrations of SOM and bioavailable

nutrients than the surrounding bulk soil– exhibit greater microbial activity and higher rates of

respiration than bulk soil – lead to the formation of stable soil aggregates which

increase the permeability of the soil to air and water• Earthworm burrows create pathways for root growth, water

movement, and nutrient transport • Earthworm-related effects stimulate the uptake of nutrients by

plants which results in increased growth rates of plants and greater levels of biomass

Sample Date

TreatmentBrine Site

NO3-Nmg/kg

NH4-Nmg/kg

P(mg/kg)

%N %C

August 2005

HF 1.65 ± 0.74 a 7.7 ± 4.8 a 25.7 ± 19.6 ab 0.28 ± 0.53 a 1.51 ± 0.31 a

H 1.74 ± 0.69 a 14.6 ± 9.0 b 11.7 ± 8.1 a 0.11 ± 0.03 a 1.69 ± 0.38 aF 1.50 ± 0.55 a 6.5 ± 2.5 a 34.5 ± 24.4 b 0.10 ± 0.008 a 1.51 ± 0.25 aN 1.85 ± 1.04 a 12.5 ± 8.1 ab 15.7 ± 17.6 ab 0.38 ± 0.72 a 1.45 ± 0.24 a

October 2005

HF 3.42 ± 1.37 a 2.78 ± 1.00 a 24.0 ± 12.5 a 0.12 ± 0.02 a 1.67 ± 0.25 a

H 4.21 ± 3.29 a 2.05 ± 0.97 a 6.4 ± 1.7 b 0.12 ± 0.008 a 1.57 ± 0.28 aF 2.85 ± 0.67 a 2.52 ± 1.08 a 22.5 ± 10.7 a 0.11 ± 0.006 a 1.61 ± 0.30 aN 4.95 ± 3.74 a 1.99 ± 1.14 a 6.8 ± 1.3 b 0.12 ± 0.007 a 1.54 ± 0.23 a

Brine Site Nutrients1,2

1 All values are mean ± std. dev. with n=8 or 92 Different letters following values for a given sample data indicate significant differences at p<0.05

Sample Date

TreatmentHydrocarbon

Site

NO3-N(mg/kg)

NH4-N(mg/kg)

P(mg/kg)

%N %C

August 2005

HF 12.1 ± 6.2 a 3.25 ± 0.57 a 89.2 ± 51.9 a 0.20 ± 0.01 a 3.63 ± 0.30 a

H 10.1 ± 6.0 a 3.20 ± 0.82 a 27.3 ± 7.1 b 0.19 ± 0.01 a 3.66 ± 0.30 a

F 18.7 ± 24.4 a 3.00 ± 0.33 a 69.2 ± 30.2 a 0.18 ± 0.008 a 3.51 ± 0.30 aN 8.7 ± 7.2 a 2.72 ± 0.41 a 27.7 ± 11.5 b 0.19 ± 0.009 a 3.53 ± 0.25 a

October 2005

HF 6.6 ± 0.9 a 1.29 ± 0.24 a 66.4 ± 18.3 a 0.20 ± 0.01 a 3.68 ± 0.22 a

H 7.5 ± 4.4 a 1.41 ± 0.15 ab 22.9 ± 7.2 b 0.19 ± 0.01 a 3.62 ± 0.34 a

F 8.0 ± 3.9 a 1.62 ± 0.40 b 55.0 ± 13.7 a 0.20 ± 0.01 a 3.76 ± 0.33 aN 5.4 ± 0.8 a 1.50 ± 0.34 ab 25.5 ± 9.5 b 0.19 ± 0.02 a 3.69 ± 0.20 a

Hydrocarbon Site Nutrients1,2

1All values are mean ± std. dev. with n=8 or 92Different letters following values for a given sample data indicate significant differences at p<0.05

Earthworms and Soil Health

• Earthworm castings – contain higher concentrations of SOM and bioavailable

nutrients than the surrounding bulk soil– exhibit greater microbial activity and higher rates of

respiration than bulk soil – lead to the formation of stable soil aggregates which

increase the permeability of the soil to air and water• Earthworm burrows create pathways for root growth, water

movement, and nutrient transport • Earthworm-related effects stimulate the uptake of nutrients by

plants which results in increased growth rates of plants and greater levels of biomass

Soil Viable Biomass in Terms of Concentration of Phospholipids (pmoles/g)1

TreatmentBrine Site

August 20052 October 20052 June 20063

HF 8,315 ± 3,464 a 11,497 ± 3936 a 7,168

H 9,917 ± 4,863 a 12,843 ± 3790 a 5,686F 7,126 ± 3,099 a 11,033 ± 4089 a 6,700N 6,893 ± 1,483 a 11,038 ± 3111 a 7,827

NativePrairie

10,333

TreatmentHydrocarbon

Site

August 20052 October 20052 June 20063

HF 7,623 ± 1,356 a 9,301 ± 3082 a 10,270

H 8,489 ± 2,975 a 8,146 ± 1970 a 6,974

F 6,872 ± 2,339 a 7,577 ± 2751 a 6,929

N 7,270 ± 1,944 a 8,248 ± 3870 a 8,874

NativePrairie

23,718

2mean ±std.dev with n=8 or 9

3Eight-fold composite

1Concentration of phospholipids proportional to concentration of viable microbial biomass

Ecological Indicators - Nematodes

• Nematodes are the numerous multicellular animals on earth; most are microscopic round worms

• In soil environments they are classified as mesofauna – confined to and move about in thin films of moisture coating soil particles

• Nematodes may be classified as to their mode of feeding based on morphology (particularly mouth structure):– Microbivores feed on bacteria– Fungivores feed on fungi– Herbivores feed on plant material

(including roots)– Omnivores feed on all of the above– Predators feed on other nematodes

• Nematode analyses obtained at reasonable cost through university ag extension services

Earthworms and Soil Health

• Earthworm castings – contain higher concentrations of SOM and bioavailable

nutrients than the surrounding bulk soil– exhibit greater microbial activity and higher rates of

respiration than bulk soil – lead to the formation of stable soil aggregates which

increase the permeability of the soil to air and water• Earthworm burrows create pathways for root growth, water

movement, and nutrient transport • Earthworm-related effects stimulate the uptake of nutrients by

plants which results in increased growth rates of plants and greater levels of biomass

Brine SiteAugust 2005

HF H F N

Treatment

0

100

200

300

400

500

600

700

800

900

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Brine SiteOctober 2005

HF H F N

Treatment

0

100

200

300

400

500

600

700

800

900

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Brine SiteJune 2006

HF H F N

Treatment

0

100

200

300

400

500

600

700

800

900

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Lots of microbivoreswith hay All numbers

low; community structure shifting from microbivores to fungivores?

Fungivoresdominate

Very few herbivores

Hydrocarbon SiteAugust 2005

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Hydrocarbon SiteOctober 2005

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Hydrocarbon SiteJune 2006

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Lots of microbivoreswith hay

Herbivores increasing; microbivoresstill dominant with hay

Preliminary Conclusions• If the native soil surrounding a remediated oil or brine spill contains

earthworms – build it and they will come!– Organic matter and moisture– Look for burrows and castings

• Nutrient cycling stimulated by surface organic matter and presumably earthworm activity

• Nematode numbers and community structure are sensitive metrics of soil ecosystem status in these types of remediated spills

Understanding the status of a soil ecosystem can be really tricky when you look too close

We get a better picture when we step back and take an integrative approach

Acknowledgement

This work was funded by

the Integrated Petroleum Environmental Consortium (IPEC)