monitoring recovery of a crude oil-contaminated saltmarsh following in-situ remediation treatments

Monitoring recovery of a crude oil-contaminated saltmarsh following in-situremediation treatments

K. Lee’, A.D. Venosa2, M.T. Suida#, COW.Greer4, G.Wohlgeschaffenl, C. Cobanli], G.H. Tremblayi, J. Gauthierl &K.G. Doe6lFisherim and Oceans Canada2U.S. Environmental Protection Agency3University of Cincinnati, USA4National Research Council Canada‘Fisheries and Oceans Canada, Mont-JoIi, QC, Canada6Environment Canada

Abstract

Wetlands are among the most sensitive of habitats to oil spills. To determine thesignificance of nutrient enrichment in enhancing wetland restoration in thepresence and absence of plants, an intentional spill was conducted on a saltmarsh in Atlantic Canada. The study evaluated 6 experimental treatments:unoiled, unoiled with nutrients (nitrate and phosphate fertilizer), naturalattenuation, nutrient addition with intact plants, nutrient addition with plants cutback, and nutient addition with intact plants and tilling to enrich oxygenpenetration. Remediation success was quantified by determining the rates of oilloss, plant recovery and reduction in sediment and interstitial water toxicity.Based on the experimental results, on an operational scale, natural attenuation isthe recommended clean-up strategy for the ecotype under study. Significant plantrecovery was observed after 20 weeks and approximately 90°/0 of the resolved n-alkanes and 70°A of the parent and alkyl-substituted polyaromatic hydrocarbons(PAH) were biodegraded.

© 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.Web: www.witpress.com Email [email protected] from: Coastal Environment, CA Brebbia (Editor).ISBN 1-85312-921-6

128 C“oastalEnvirontnen[

1 Introduction

Saltmarshes provide nursery support for coastal fisheries, a habitat for wildlife[1] and a protective mechanism against shoreline erosion [2, 3]. Unfortunately,they are among the most vulnerable habitats to oil spills due to the potentialpersistence of residual oil stranded within wetland sediments and the sensitivitytheir flora to physical disruptions resulting from traditional oil spill clean-upoperations [4, 5, 6]. To address this concern, bioremediation andphytoremediation, based on the stimulation of indigenous oil-degrading bacteriaby nutrient enrichment and the inherent ability of plants to accumulate or degradeenvironmental contaminants [7], have been identified as potential cleanupstrategies.

The present study was conducted on a saltmarsh dominated by the wetlandplant species, Spartina altern#70ra (Conrod’s Beach, Nova Scotia, 44°42’ N; 63°11’ W). The objective was to determine the rates of natural recovery and thesignificance of nutrient enrichment in enhancing wetland restoration following acrude oil spill, in the presence and absence of wetland plants.

2 Methods

2.1 Experimental design

The experiment consisted of 3 blocks of 6 plots each. Plots were 3 x 3 m. Thetreatments which were replicated in each block were (1) unoiled control withintact plants, (2) unoiled control with intact plants + nutrients, (3) naturalattenuation, (4) oil + nutrients, (5) oil + nutrients + plants cut back to suppressplant activity, (6) oil + nutrients + disking (tilling) to enrich oxygen penetration.MESA Iight crude oil ‘weathered’ by forced air evaporation to 13.8% loss byweight was homogeneously applied to the surface of selected experimental plotsto a final concentration of 35 #kg (1.8 L/m*). Experimental nutrientamendments added to each plot consisted of 1,280 g N and 550 g P in the form ofcommercial fertilizer — pnlled NH4N03 and Ca(HzPOJzHzO — with re-applications when the interstitial-N concentrations fell below 5 mglL.

2.2 Chemical analysis

Residual oil in the sediments was determined by solvent extraction withdichloromethane and subsequent gas chromatography/mass spectrometry(Venosa et al., 2002). To quantify the extent of biodegradation, all resolvedhydrocarbons were normalized to the conserved biomarker Cso-17a(H), 21 (3(H)-hopane in the MESA oil [8],

2.3 Biological analysis

At Week O, 9 and 20 sampling point intervals, 3 samples of vegetation (each 625cm2) were removed from each experimental plot for subsequent biomass (dry-


Coastai Environment 129

weight after 72 hours at 105°C) measurements, For comparative purposes thedata was normalized to the Day O value for each treabnent to account forbetween-plot variability.

Potential microbial hydrocarbon degradation rates of representative alkaneand PAH components within sediment samples were determined by quantifyingthe respiration rate of the added radioisotopes, 14C-hexadecane and 14C-phenanthrene [9, 10, 11]. These were considered representative n-alkane andPAH class components of the test oil,

Denitritication is a primary process that regulates the nitrogen cycle inwetland sediments. Microbial denitrification activity was monitored on eachsampling occasion by placing a gas chamber on each plot and taking airspacesamples over 30 minutes, which were subsequently analyzed for nitrous oxide.

Nucleic acid-based molecular techniques can identify bacterial species by theunique sequence of molecular codes in their genes. One of most useful methodsfor determining the diversity of bacterial communities is denaturing gradient gelelectrophoresis (DGGE) which provides a ‘community fingerprint’ [12], Anapproximation of the number and identity of possible bacterial species was madeby comparison to reference databases containing the 16S rDNA sequences ofcurrently known organisms [13].

In the Microtox” Solid Phase Test [14, 15, 16] the bacterium, Vibriojsheri, wasexposed to test sediments. A significant decrease in bioluminescence relative towater-only controls was indicative of sediment toxicity. Toxicity levels werecalculated as the concentration of sample that would result in a 500/0reduction inluminescence (’effective concentration,’ EC50). To account for interference ffomdifferences in sample grain size distribution, turbidity, and to a lesser extent, color ofthe sample dilutions, sample test results were compared with results of unoiledsediments from the immediate study area.

The Amphipod Test measured the effects of sediment samples on survival ofsediment-dwelling Eohaustorius estuarius [17]. The mean percent survival andthe mean weight of animals in each treatment were compared with mean percentsurvival and mean weight of amphipods in reference control sediments todetermine if the treatments caused a significant decrease in organism survival orgrowth. The results were reported as percent mortality.

3 Results

3,1 Oil analysis

Detailed analysis of chemical data (hopane-normalization of specific targetcompounds, the ratio of phenanthrene to C4-phenanthrene, etc.) provided clearevidence of biodegradation for the alkane and PAH fractions within theintegrated samples (3 cm thick sods) that extended to the depth of oil penetration[ 18]. After the first 20 weeks, approximately 90% of the resolved n-alkanes weredegraded within the experimental plots. The corresponding reduction of parentand alkyl-substituted PAHs was only about 700A. Statistical analysis indicated


130 Coastal E}qvlronnlent

that significant treatment differences (p<O,O1) were only observed for the alkanefraction,

3,2 Recovery of vegetation

By Week 9, growth (biomass) of the predominant plant species, S. alternzj70ra,wassignificantly suppressed by oiling to 55-73’% of the Day O levels (Figure 1). Thecontrol and control + nutients showed an increase from seasonal growth, withnutrient addition ampli&ing this increase. Cutting of plants suppressed recovery.Stem and leaf biomass was destroyed by the tilling. By Week 20, the oil and oil +nutrients plots recovered to within 13 and 11‘Yo of the Day O values, although thedifferences were not statistically significant. The following spring, there was someevidence of changes in species composition within the treated plots, Anotheropportunistic plant species that was more tolerant to the altered conditionsin~reaaed its pffcentage of cover,

280

260

240

220

200

180

180

140

120

100

80

60

40

20

00920 0920 0920 0920 0920 0920

Control Control Oil Oil Oil OilNutrients Nutrients Nutrients Nutrients

cut Disking

Figure 1: Total plant biomass collected from plots at Weeks O, 9 and 20.

3.3 ,Microbial oil degradation potential

Time-series changes (Week 4, 7, 9, 12, 16, 20 and 62) in the turnover time of theradiotracers were calculated with the actual concentrations of residual


Coastal Envi.oumenf 131

hexadecane and phenanthrene in each sample determined by GC/MS to accountfor dilution by the unlabeled fraction of the specific substrates under study.

The results of the added 14C-labeled hexadecane studies clearIy illustrated thestimulation of indigenous organisms with the potential to degrade alkanes withinthe first 10 weeks after the application of oil (Figure 2). A stimulator effect onpotential hexadecane degradation rates by the addition of nutrients to unoiledsediments was also observed, However, within the oiled sediments, remedialtreatments based on nutrient additions did not appear to cause a stimulatoreffect that could be adequately resolved by measurement of hexadecane

900G

%’ 800n; 400

.; 300

g 2(30

oE 100

?0


cut Disking

Figure 2: Hexadecane turnover time (kl SE) at Week 4,7,9, 12, 16,20 and 62.

respiration rates, Natural attenuation appeared to be relatively effective, Theseradiotracer studies agree with the chemistry that showed that 87°A of the target n-alkanes were removed from the test sediments within 20 weeks, Similarobservations were made for the biodegradation of PAHs (represented by the 3-ringed phenanthrene) with the exception that nutrient amendments to the unoiledcontrol sediments had no stimulator effect (Figure 3).

3.4 Denitri!ication activity

The seasonal average of denitrification activity plotted against treatment showedthat in all cases where nutrients were applied, there was a net positivedenitrification potential (Figure 4), Natural attenuation and the unoiled controlshowed a net negative denitrification potential. These results indicate thatnurnent application resulted in increased denitrification activity in the sediments.


132 Coastal Environment

G> 2000c 10051- 0

. T I


cut Disking

Figure 3: Phenanthrene turnover time (il SE) at Week 4,7,9, 12, 16,20 and 62,

800 --- --–— – ————.

-----

~

———

,

7-

,- 1

~ :~ ()!- ———.——. _—u.- 1

—- — Oil3* -200- ;i, Nutrients

Disking

5Oil ——

* -400 ‘Nutrients Oil#-

z NutrientsControl

n. .. Nutrientsbut

-600 -- Control ~—

Figure 4: Average denitrification activity June to November 2000 (*1 SE).


Coastal Environment 133

3.5 Changes in microbial community structure

Total microbial community DNA extracts were obtained from the sediments ofexperimental Block I one day after oiling, 4 weeks after oiling and 16 weeks afteroiling. Representative examples from the Unoiled control, Oiled (naturalattenuation) and Oil + nutrients are illustrated (Figure 5). They clearly

Id 4 wk 16wk

N03- Nat. Unoiled N03- Nat. Unoiled NO;Attn. NH4+ Attn. NH4+

Figure 5: DGGE, two GC clamps (a and b), arrows indicating fiagrnents thatappeared or increased in intensity,

demonstrated differences in the microbial community composition. In addition todistinct differences in fkagment presence with treatment and time, certainfragments of 16S rDNA, amplified fkom the sediment extracts increased ordecreased in intensity.


134 Coastal Environtnen[

3.6 Microtox Solid Phase Test

Based on Environment Canada’s ocean-dumping guidelines [19] which sets theregulatory threshold for toxicity at 1,000 mg/L, all unoiled sediment sampleswere deemed non toxic. EC50 data normalized to the controls (Figure 6) showed

140

0Control Control Oil Oil Oil Oil

Nutrients Nutrients Nutrients Nutrientscut Disking

Figure 6: Microtox toxicity, Week 0,2,4,7,9, 12, 16,20 and 62 (M SD)

an initial detrimental response in sediments treated with nutrients or the test oil.Subsequent recovery by natural attenuation was observed. By week 9 alltreatments were deemed non-toxic.

3.7 Amphipod survival test

Mortality was high in all of the oiled treatments, but began to decrease by Week12 largely as the result of natural processes (Figure 7). Addition of nutrientsaccompanied by disking appeared to cause the most rapid rates of detoxification(recovery) within the oiled plots, However, the results of chemical analyses(GC/MS) indicated that this observation could also be attributed to the physicalremoval of oil (enhanced dispersion w-ith tides) mediated by disking operations.By Week 62, the difference from the Oil treatment was highly significant: 32%

vs. .5°/0 mortality.


10090

80

g 70

30

20

100

Coastal Envit-onmet7t 135

I


cut Disking

Figure 7: Amphipod survival toxicity test, Weeks 0,2,7, 12,20 and 62 (*1 SD)

4 Discussion

Results of chemical analysis, bioassessments and bioassay tests suggested thatnatural attenuation was the primary process that reduced residual oilconcentrations and toxic effects. The biotest results showed that the remediationstrategies under evaluation (bioremediation and phytoremediation by nutrientamendment and physical aeration of sediments by disking) were not effective onan operational scale, in spite of the significant changes observed in microbialresponse, It was also evident in the results of some biotests (amphipod survivaltest, lMicrotox test) that possible detrimental effects may be linked to the additionof fertilizers.

Based on the evidence of oil bioremediation (90V0 of resolved n-alkanes and70?40 of parent and alkyl-substituted PAH within 20 weeks) and recovery ofecosystem structure and function in the oiled, but untreated plots, this studyprovides support for the consideration of natural attenuation (natural recovery) asan option for operational oil spill response in north-temperate salt marshenvironments.


136 Coastal En~ironment

5 References

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[2]

[3]

[4]

[5]

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[7]

[8]

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Mitsch, W.J. & Gosselink, J.G. (eds). Wetlands, John Wiley & Sons:Toronto, 2000.Broome, S.W., Seneca, E.D. & Woodhouse, W.W, Jr. Tidal salt marshrestoration. Aquatic Botany, 32, pp. 1-22, 1988.Kennish, M.J. Pollution in estuarine and marine environments (Chapter 1).Pollution Impacts of Marine Biotic Communities, CRC Press: Boca Raton, p.1, 1997.Teal, J,M., Farnngton, J.W., Bums, K.A., Stegeman, J.J., Tnpp, B.W.,Woodin, B, & Phinney, C. The West Falmouth oil spill aller 20 years: fate offuel oil compounds and effects on animals. Marine Pollution Bulletin, 24,pp. 607-614, 1992,

Owens, E.H., Sergy, G.A., McGuire, BE. & Humphrey, B, The 1970 Arrowoil spill: what remains on the shoreline 22 years later? Proc, of the 16[hArctic and Marine Oil Spill Program (MOP) Technical Seminar,Environment Canada: Ottawa, pp. 1149-1168, 1993.Lee, K, & Levy, E,M. Bioremediation: waxy crude oils stranded on low-energy shorelines. Proc. of the 1991 Oil Spill Conf, American PetroleumInstitute: Washington, pp. 541-547, 1991,McIntyre, T. & Lewis, G.M. The advancement of phytoremediation as aninnovative environmental technology for stabilization, remediation, orrestoration of contaminated sites in Canada a discussion paper. Journal ofSoil Contamination, 6(3), pp. 227-241, 1997,

Prince, R.C., Elmendorf, D.L,, Lute, J.R., Hsu, C. S., Haith, C,E,, Senius, J.D.,Dechert, G.J,, Douglas G.S, & Butler, E.L. 17a(H), 21 ~(H) -hopane as aconserved internal marker for estimating the biodegradation of crude oil.Environmental Science and Technolo~, 28, pp. 142-145, 1994.Lee, K. & Levy, E.M. Enhancement of the natural biodegradation ofcondensate and crude oil on beaches of Atlantic Canada. Proc, of 1989 OilSpill Conference, American Petroleum Institute: Washington, pp. 479-486,1989.

[10] Caparello, D.M, & LaRock, PA, A radioisotope assay for the quantificationof hydrocarbon potential in environmental samples. Microbial Ecology, 2,pp. 28-42, 1975.

[1 l] Walker, J.D. & Colwell, R.R. Measuring potential activity of hydrocarbondegrading bacteria. Applied Environmental Microbiology, 31, pp. 189-197,1976.

[12] Muyzer, G., De Waal, E.C, & Uitterlinden, A,G, Profiling of complexmicrobial populations by denaturing gradient gel electrophoresis analysis ofpolymerase chain reaction-amplified genes coding for 16S Rma, AppliedEnvironmental Microbiology, 59, pp. 695-700, 1993.

[13] Maidak, B,L,, Cole, JR., Lilbum, T.G., Parker, C,T. Jr., Saxman, P.R.,Stredwick, J. M., Garrity, G.M., Li, B., Olsen, G.J., Pramanik, S,, Schmidt,T.M. & Tiedje, J.M. The RDP (Ribosomal Database Project) continues.Nucleic Acids Research, 28, pp. 173-174,2000.


Coastal Environment 137

[14] AZUR Environmental, MicrotoxOmni for Windows 95/98, ver. 1.18, CD-ROM, AZUR Environmental: Carlsbad, 1999.

[15] Lee, K., Siron, R. & Tremblay, G.H. Effectiveness of bioremediation inreducing toxicity in oiled intertidal sediments. Microbial Processes forBioremediation, eds. Hinchee et al., Battelle Press: Columbus, pp. 117-127,1995.

[16] Microbics Corporation. Detailed solid-phase test protocol. Microtox Manual -A Toxicip Testing Handbook Vol. II Detailed Protocols, MicrobicsCorporation: Carlsbad, pp. 153-178, 1992.

[17] Environment Canada, Biological Test Method: Acute Tesl for SedimentToxicity Using Marine or Estuarine Amphipods Report EPS l/RM/26,Environment Canada: Ottawa, 83 p,, 1992.

[18] Venosa, A.D., Suidan, M. T., Lee, K,, Cobanli, S.E., Garcia-Blanco, S. &Haines, J.R. Bioremediation of oil-contaminated coastal freshwater andsaltwater wetlands. (in the same volume of this publication), 2002.

[19] Tay, K.L,, Doe, K. G., MacDonald, A.J, & Lee, K. Monitoring oj the BlackPoint Ocean Disposal Site Saint John Harbour, New Brunswick 1992-1994,Environment Canada: Atlantic Region, Internal Report ISBN O-662-25655-5,Cat. no. En40-214/9E, 133 pp, 1997.


monitoring recovery of a crude oil-contaminated saltmarsh following in-situ remediation treatments

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