2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.

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Paper from: Coastal Environment, CA Brebbia (Editor).
ISBN 1-85312-921-6

Bioremediation of oil-contaminated coastal

freshwater and saltwater wetlands

A. D. Venosa], M. T. Suidan2, K. Lee3, S. E. Cobanli3,

S. Garcia-Blanco2 and J. R. Hainesl
1US. Environmental Protection Agency, Cincinnati, Ohio, U.S.A.
2Universip of Cincinnati, Cincinnati, Ohio, U.S.A.
3Fisheries and Oceans-Canada, Hal@x, Nova Scotia, Canada.

Abstract

Two field studies involving intentional releases of crude oil onto a freshwater
wetland and a salt marsh were conducted in Canada in the summers of 1999and
2000, respectively. The objective of both studies was to determine the role of
nutrients in enhancing wetland restoration in the presence and absence of wetland
plants, The experiments involved several replications of the following oiled
treatments: (1) natural attenuation, (2) ammonium nitrate addition with intact
plants, (3) ammonium nitrate addition with plants cut back to suppress plant
activity, and (4) sodium nitrate addition to separate the effects of ammonium-N
from nitrate-N. A fertilized, unoiled treatment was also included. For the salt
marsh study, tilling was added as another treatment. Time series data from both
studies were analyzed by GC/MS to monitor oil degradation. Results from both
field experiments indicate that significant alkane and PAH biodegradation
occurred (more so in the salt marsh). Biodegradation rates were not enhanced by
any of the amendments in the freshwater wetland experiment, but substantial
restoration of the wetland ecosystem was accelerated in the amended treatments.
Significant treatment effects were observed in the salt marsh study in regards to
alkane but not PAH degradation, and levels of restoration similar to those
observed in the freshwater wetland were not evident in the salt marsh ecosystem.

1 Introduction

The sudden discharge of organic chemical pollution into a wetland environment

can cause substantial damage to these sensitive ecosystems, including wildlife
morbidity and mortality, a lowering of ecological diversity, disruptions to food
chain interactions, erosion, and unsightly harm to the landscape. Scientists and
 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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140 Coastal Envirot?ment

spill responders have been trying to find ways to accelerate cleanup of such
environments to prevent further damage or future reoccurrences. Restoration is
ofien difficult due to the fragility of the marsh substrate. In addition to the stress
caused by exposure of wetland vegetation to chemical pollution, traditional spill
cleanup activities may do more damage to the highly sensitive marsh than the
contamination itself. For example, foot and mechanical traffic on the wetland
during cleanup can trample vegetation and push the hydrocarbons deeper into the
anaerobic sediments where they may persist for years. A restoration methodology
that both initiates and accelerates biotic restoration and enhances contaminant
removal is needed. Bioremediation is one technology that offers promise in
Bahan dan metode
converting the toxic compounds to nontoxic products with minimum disruption to
2,1 Situs.
the local environment. This paperlahan
Lokasi discusses
basahresults
air tawarfrom field di
terletak research
pantai conducted
selatan St Lawrence
on a freshwater wetlandSungai and dekat
a salt desa
marsh, both in Canada,
Ste, Croix de Lotibiniere to (46
further our71 "45'W),
37 'N,
understanding of the controlling
kira-kirafactors
20 km that would
barat dayahinder our ability
Kota Quebec. to restore
Spesies the utama
tanaman
contaminated ecosystem. yang menempati situs tersebut adalah Scirpus pungens. Pasang
surut di Ste. Croix dianggap sebagai campuran, terutama
The research was designedsemi-diurnal pasangthe
to: (1) determine dengan kisaran of
effectiveness sekitar 4 m.addition
fertilizer Situs rawa garam
terletak rates
to accelerate the biodegradation di Pantai Conrod,
of residual oildekat mulut Petpeswick
in intentionally Inlet (44 42 'N; 63
contaminated
11' W) di Pantai Timur Nova Scotia.
marsh ecosystems; and (2) determine the extent of recovery of the stressedJenis tanaman lahan basah
yang dominan di daerah ini adalah Spartina alternij70ra
ecosystems with and without addition of inorganic fertilizers

2 Materials and Methods

2,1 Sites.

The freshwater wetland site was located on the south shore of the St Lawrence
River near the village of Ste, Croix de Lotibiniere (4637 N, 71 45W),
approximately 20 km southwest of Quebec City. The predominant plant species
occupying the site was Scirpus pungens. The tide at Ste. Croix is considered to be
a mixed, mainly semi-diurnal tide with a range of about 4 m. The salt marsh site
was located at Conrods Beach, near the mouth of Petpeswick Inlet (4442 N;
6311 W) on the Eastern Shore of Nova Scotia. The predominant wetland plant
species in the area was Spartina alternij70ra

2,2 Test Oil

Mesa light crude oil from the PetroCanada refinery in Montreal with an API
gravity of 29,7 and flash point of 4C was used in both studies. The oil was
artificially weathered by forcing air through it with a compressor for 130 hours to
evaporate the light fraction, The volume loss was approximately 13.80A. The oil
was applied to the t%eshwater marsh plots at the rate of 15 g/kg of sediment and to
the salt marsh plots at a rate of 35 g/kg,
 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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ISBN 1-85312-921-6

Coastal Envirownent 141

2.3 Experimental Design

2.3.1 Freshwater Wetland Study

The experimental area consisted of 20 plots each measuring 5 m x 4 m. A 0.5 m
perimeter buffer zone around each plot served as a no-sampling zone (except for
nutrient samples). Five treatments included a no oil control and four oiled
treatments consisting of a natural attenuation control with no amendments
(Treatment A), a plot receiving ammonium nitrate and orthophosphate nutrients
but with the wetland plants continually cut back to ground surface to suppress
photosynthetic activity and growth (Treatment B), a plot receiving the same
nutrients as Treatment B but with the plants left intact (Treatment C), and a plot
similar to Treatment C but with only nitrate (no ammonium) serving as the
nitrogen source (Treatment D). The no-oil control (Treatment E) also received the
same nutrients as Treatments B and C. The fertilizers used for Treatments B, C,
and E were prilled NH4N03 and Ca(H2POJ2H20. The fertilizer for Treatment D
was a powdered form of NaNO~ and the same phosphate source.

Four replications of the five treatments were set out in blocks (Blocks I-IV), The
null hypothesis tested was that addition of inorganic fertilizers does not enhance
natural rates of hydrocarbon disappearance nor the restoration of the stressed
freshwater wetland ecosystem beyond the natural rate.

2,3.2 Salt Marsh Study

The experimental area consisted of 18 3m x 3 m plots, The six treatments included
an untreated, unoiled plot (A), an unoiled plot with nutrients added (B), a natural
attenuation plot (oil, no amendments) (C), a plot receiving nutrients (D), a plot
receiving nutrients but the plants cut back to suppress growth (E), and a plot
receiving nutrients and gently disked daily to introduce oxygen to the subsurface
(F). Each of the 6 treatments was replicated three times in blocks, and the null
hypothesis was the same as in the freshwater study.

2.4 Nutrient and Oil Application

Nutrient amendments to all plots were applied manually using a whirlybird

spreader during a falling low tide prior to oiling. In the freshwater study,
following nutrient application but before oil was applied, the top 2-3 cm surface
sediment in all plots was manually raked using cast iron rakes to facilitate
penetration of the oil into the sediment. In subsequent fertilizer applications,
nutrients were manually spread over the plots as evenly as possible without
raking. The oil was applied uniformly using a pump/spray apparatus, The
sediment was then raked again to incorporate the nutrients and the oil into the top
2-3 cm of soil, The same method of oil application was used in the salt marsh
study without the raking.
 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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ISBN 1-85312-921-6

In the freshwater study, the amount of nutrients initially added and at subsequent
reapplication periods was 1,000 g-N and 300 g-P per plot. Nutrients were added at
weekly intervals to maintain relatively high interstitial pore water nitrogen
concentrations. Oil was applied at the rate of 12 L/plot or 0.6 L/m2, In the salt
marsh study, N and P were added to each plot at the rate of 1280 and 550 g/plot,
respectively. Reapplication was performed when the interstitial N concentration
fell below 5 mg/L, Oil was applied at the rate of 8 L/plot or 1.8 L/mz,

2.5 Sampling Design

2.5.1 Freshwater Wetland Study

At each sampling event, 9-cm deep core samples 7 cm in diameter were collected
from random subsampling zones using a tulip bulb planter, The 6 subsamples
from each plot were combined into 2 composites per plot, One was processed, the
other archived. Samples for oil analyses (gas chromatography/mass spectrometry
or GC/MS) were frozen and shipped in dry ice to the laboratory, For oil chemistry,
samples were frozen (-18 to -20C) until extraction.

The first sampling event (week O) took place at low tide the day after the initial
application of oil and nutrients (June 11, 1999). Subsequent sampling events
occurred at weeks 1, 2, 4, 6, 8, 12, 16, 21, 49, and 65 in 1999 and 2000. No
fertilizer applications were made during the second year.

For the weekly measurements of nutrients and sediment pH, six samples were
collected using the tulip bulb planter from different locations within the perimeter
buffer zone of each plot and composite into one sample. In addition, random
samples were collected daily from each of the five treatments within one single
block to closely monitor nutrient concentrations. The blocks were rotated on a
daily basis.

2.5.2 Salt Marsh Study

The sampling approach was similar to the freshwater study. Eight sampling events
occurred during the summer of 2000 (weeks O, 2, 4, 7, 9, 12, 16, and 20) and one
additional sampling event took place in the summer of 2001 (week 62), Sod
samples measuring -25 cm x 25 cm were collected by cutting the roots about 3 cm
below the surface with sharp knives having serrated cutting edges,

2.6 Analytical

Marsh sediment samples (both studies) were extracted in dichloromethane (DCM)

using a Soxhlet apparatus. The detailed procedure has been described elsewhere
(Garcia-Blanco et al. [1]). Changes in the concentration and composition of
remaining crude oil in the sediments were determined by GC/MS with a Hewlett-
Packard 5890 series II gas chromatography coupled with a Hewlett-Packard 5971A
mass selective detector (MSD) and a Hewlett-Packard 7673 autosampler. The
detailed method can be found elsewhere (Garcia-Blanco et al., [2]). All analytes
 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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Paper from: Coastal Environment, CA Brebbia (Editor).
ISBN 1-85312-921-6

were normalized to the conservative biomarker C30-17a-(H), 21 ~(H) -hopane,

naturally present in the tested oil (Prince et al., [3]) for quantification of
biodegradative losses.

3 Results

3.1 Physical Loss of Oil

When conducting field trials of bioremediation, it is important to be able to

distinguish between loss due to washout and loss due to biodegradation. By
quantifying the concentration of the biologically refractory biomarker hopane, one
can easily make that distinction. Since hopane is resistant to biological attack, any
loss measured is due to physical processes. Figure 1 summarizes the physical loss
of oil from the plots in both studies. The upper panel shows the hopane
persistence in the freshwater marsh study, while the lower panel presents the salt
marsh data. Error bars are quite wide, signifying that variability of oil
concentration was high despite the uniformity with which it was applied.
Nonetheless, the data from both studies indicate that hopane persisted for the
duration of the experimental periods. One additional graph is noted in the lower
panel, It shows the loss of hopane in the tilled plots. Note that hopane disappeared
more readily from those plots than the untilled plots. Tilling caused extensive
damage to the root structure of the plants, so less retention of oil (hopane)
occurred as a result compared to the untilled plots.

3,2 Biodegradation

Figures 2 and 3 summarize the biodegradation of hopane-normalized alkanes and

PAHs, respectively. The upper panel in each figure shows the results for the
freshwater wetland, the lower panel for the salt marsh. The biodegradation
behavior is extremely different in the two studies. Only about 30-33% removal of
alkanes and PAHs occurred in the freshwater wetland study regardless of
treatment, whereas over 93% alkane and 70% PAH removal occurred in the salt
marsh. The main reason for the reduced biodegradation effectiveness in the St.
Lawrence study was due to the raking of the oil into the subsurface, causing
penetration of oil several cm below the surface, In a highly saturated wetland
environment, anaerobic conditions predominate only a few mm below the surface.
Under such an environment, biodegradation of hydrocarbons is extremely slow.
Excess nutrients will not accelerate biodegradation if oxygen is limiting.

Figure 4 is a plot of the phenanthrene to C4-phenanthrene ratio in both studies to

show that losses of PAHs were caused by biodegradation as opposed to physical
mechanisms. It is well known that, as the number of alkyl groups increases on the
parent ring structure, biodegradation effectiveness declines. Thus, the
phenanthrene to C4-phenanthrene ratio should decline as biodegradation ensues.
This is evident for all treatments in both panels in Figure 4, more so in the lower
panel (salt marsh). Thus, biodegradation indeed occurred in both studies, since
physical loss was somewhat limited (as evidenced in Figure 1).
 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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ISBN 1-85312-921-6

144 Coastal Emvrotment

1.6
Freshwater Marsh Site
1.4
1.2
1.0 [1 T
0.8
~TTT1
0

j.

T1

T
0.6
1 1
0.4 :111 1 1
0.2
0,0
14
12
10
8
6
4
2
0
0 5 10 15 20 25

time, weeks

Figure 1. Persistence of hopane averaged over all plots.

 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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Paper from: Coastal Environment, CA Brebbia (Editor).
ISBN 1-85312-921-6

Coastal Environment 14.5

Freshwater Marah

NA

v NH, Cut

+ NH, Intact
1-
N03 Intact

? 100 t

1 I 1 I 1 1 !
0
Salt Marah

NA

v Nuts

+ Nuts Cut

Nuts Tilled

0
o 20 40 60 80 100 120 140 160

time, days

Figure 2. Biodegradation of hopane-nomalized total alkanes.

 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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ISBN 1-85312-921-6

146 Coastal En~ivotvnent

140
Freshwater Marsh
120
Pm
100
T NH, Cut
80
NH, Intact
60
NO, Intact
40

20
1 I I I 1 1 1

14; Salt Marsh

120
NA
100 *
v Nuts

80
Nuts Cut

60
Nuts Tllkd

40

20

0
o 20 40 60 80 100 120 140 160

time, days

Figure 3. Biodegradation of hopane-normalized total PAHs.

 2002 WIT Press, Ashurst Lodge, Southampton, SO40 7AA, UK. All rights reserved.
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ISBN 1-85312-921-6

Coastal Environment 147

Freshwater Marsh
1.00
NA
; 0.80 ~ NH,.CUT

; +
. NH, Intaci
z 0.60
&
* NO,-intact

$ 0.40
z
&
0.20

1 1 1 ! I 1 t
0.00
1.00 Salt Marsh

0
,= NA
~ 0.80
v Nuts
L
E 0.60 Nuts, Cut
*
Nuts, Tilled
* 0.40
x
n
0.20 v

I 1 I 1 I I , I
0.00
o 20 40 60 80 100 120 140 160

time, days

Figure 4, Phenanthrene-C4 -Phenanthrene Ratio, both studies.

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148 Coastal Environment

Statistically, however, as determined both by non-linear regression analysis and

analysis of variance, no treatment differences were evident in the St. Lawrence
River study for either alkanes or PAHs (p > 0.05), In the salt marsh study,
however, significant treatment differences existed among treatments (p << 0.01)
for alkanes (all treatments receiving nutrients compared to the natural attenuation
control) but not PAHs (p> 0.05).

4 Discussion

The two studies described in this paper demonstrated that phytoremediation might
be a useful oil cleanup countermeasure if penetration has not taken place below
the subsurface. They also confirmed that aerobic biodegradation is a primary
hydrocarbon removal mechanism in wetlands. Addition of inorganic nutients in
the form of ammonium nitrate and orthophosphate can accelerate hydrocarbon
disappearance when the oil is present mostly on the surface. The hypothesis that
plants may transfer oxygen into the rhizosphere to stimulate bioremediation of
penetrated hydrocarbons was not supported from these studies. It is highly likely
that the oxygen transported into the root zone by plant tissue is used mostly by the
plants themselves and is insufficient to support accelerated hydrocarbon oxidation
by microbes in the rhizosphere.

References

[1] Garcia-Blanco, S., Motelab, M., Venosa, AD., Suidan, M. T., Lee, K,, King,
D,W, Restoration of the oil-contaminated Saint Lawrence River Shoreline:
Bioremediation and Phytoremediation. Proceedings of 200] International Oil
Spill Conference. American Petroleum Institute, Washington DC, PP303-308,
2001..
[2] Garcia-Blanco, S., Suidan, M. T., Venosa, A. D,, Huang, T., Cacho-Rivero, J.
Microcosm study of effect of different nutrient addition on bioremediation of fuel
oil #2 in soil from Nova Scotia coastal marshes. Proceedings of 2001
International Oil Spill Conference. American Petroleum Institute, Washington
DC, pp309-3 14,2001,
[3] Prince, R. C., Elmendorf, D, L,, Lute, J, R,, Hsu, C. S., Haith, C. E., Senius, J.
D,, Dechert, G. J., Douglas, G. S, and Butler, E, L, 17a(H), 21 b(H) -Hopane as a
conserved internal marker for estimating the biodegradation of crude oil,
Environmental Science and Technology, 28, 142-145, 1994.
[4] Hambrick, G, A, R, D. Delaune, and W. H. Patrick. Effect of estuarine
sediment pH and oxidation-reduction potential on microbial hydrocarbon
degradation Appl, Environ. Microbiology, 40,365-369, 1980.