earthworms as eco-engineers

Earthworms as Eco-engineers in the Restoration of Oil and Brine-

Impacted Soils Following RemediationNitya AlahariKerry Sublette

Eleanor JenningsCenter for Applied Biogeosciences

University of TulsaGreg ThomaDuane Wolf

University of ArkansasKathleen Duncan

University of OklahomaTim Todd

Kansas State UniversityMac A. Callaham, Jr.USDA-Forest Service

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 organisms– Loss of vegetation

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

Project Summary• Objective: to determine the efficacy of the introduction of non-

indigenous earthworms to accelerate the restoration of remediated oil and brine spill sites

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

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

• Within 60 days:– earthworms indigenous to the area were detected in significant

numbers both outside and inside of the enclosures – very few E. fetida could be found.

• Thus all enclosures were accessible to indigenous earthworms and in reality there were only four treatments: hay + fertilizer (HF), hay only (H), fertilizer only (F), and no treatment (N).

• The greatest earthworm activity was assumed to occur in those enclosures where there was food (surface hay)

Metrics Used to Evaluate Treatments

• Soil nutrients• Microbial biomass as PLFA

– PLFA = phospholipid fatty acid analysis• N-cycling bacteria• Nematode numbers and community structure• 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

Project Timeline• Ripped and tilled sites• Installed earthworm enclosures and added

amendments (fertilizer and/or hay)• 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

Filling enclosure with homogenized soil at hydrocarbon site

Filling earthworm enclosure with homogenized soil

Hay

Earthworm enclosures installed and amendments added at hydrocarbon site

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

• Most worm observations at soil moisture concentrations of 22-26%

0

2

4

6

8

10

12

16-18 18-20 20-22 22-24 24-26 26-28 28-30 30-32

Percent Moisture

No.

of O

bser

vatio

ns o

f W

orm

s

Brine SiteHydrocarbon site

TreatmentBrine Site

August 2005 October 2005 June 2006

HF 26.0 ± 2.1 23.3 ± 2.8 29.4 ± 3.3

H 25.7 ± 3.3 24.2 ± 3.4 29.2 ± 2.9

F 26.1 ± 1.6 22.5 ± 3.0 25.9 ± 4.0

N 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 ± 6.4

H 21.7 ± 4.5 18.2 ± 4.6 23.2 ± 8.4

F 20.7 ± 4.3 19.3 ± 4.8 28.4 ± 13.3

N 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

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 11.7 ± 8.1 a 0.28 ± 0.53 a 1.51 ± 0.31 a

H 1.74 ± 0.69 a 14.6 ± 9.0 b 25.7 ± 19.6 ab 0.11 ± 0.03 a 1.69 ± 0.38 a

F 1.50 ± 0.55 a 6.5 ± 2.5 a 15.7 ± 17.6 ab 0.10 ± 0.008 a 1.51 ± 0.25 a

N 1.85 ± 1.04 a 12.5 ± 8.1 ab 34.5 ± 24.4 b 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 a

F 2.85 ± 0.67 a 2.52 ± 1.08 a 22.5 ± 10.7 a 0.11 ± 0.006 a 1.61 ± 0.30 a

N 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 a

N 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 a

N 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

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 5,686

F 7,126 ± 3,099 a 11,033 ± 4089 a 6,700

N 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

Evaluation of Metrics

• Of all the metrics used to evaluate treatments, only one was capable of distinguishing between treatments –nematode numbers and community structure– Trophic groups– Colonizer-persister classes

Free-living NematodesBrine Site

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Free

-livi

ng N

emat

odes

(#/1

00 g

) August 2005 October 2005 June 2005

Maturity IndexBrine Site

HF H F N

Treatment

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Mat

urity

Inde

x

August 2005 October 2005 June 2006

Brine SiteAugust 2005

HF H F N

Treatment

0

100

200

300

400

500

600

700

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Brine SiteOctober 2005

HF H F N

Treatment

0

20

40

60

80

100

120

140

160

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

Free-living NematodesHydrocarbon Site

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Free

-livi

ng N

emat

odes

(#/1

00 g

) August 2005 October 2005 June 2006

Maturity IndexHydrocarbon Site

HF H F N

Treatment

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Mat

urity

Inde

x

August 2005 October 2005 June 2006

Hydrocarbon SiteAugust 2005

HF H F N

Treatment

0

200

400

600

800

1000

1200

1400

1600

1800

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

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Hydrocarbon SiteJune 2006

HF H F N

Treatment

0

100

200

300

400

500

600

700

800

Nem

atod

es (#

/100

g)

Total Herbivores Total Microbivores Total Fungivores Total O/P

Preliminary Conclusions

• Indigenous earthworms quickly re-populate remediated oil and brine spill sites when hay and moisture are made available

• Nematode numbers and community structure – indicated more intense nutrient cycling in treatments that

included hay– sensitive metrics for assessing soil ecosystem restoration in

these sites• The availability of hay on the soil surface likely

correlates with earthworm activity