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 Marine Pollution Bulletin 64 (2012) 2681–2691

Contents lists available at SciVerse ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Effects of an experimental in situ diesel oil spill on the benthic community

of unvegetated tidal flats in a subtropical estuary (Paranaguá Bay, Brazil)
Aline Gonzalez Egres ⇑, César C. Martins, Verônica Maria de Oliveira, Paulo da Cunha Lana
Centro de Estudos do Mar, Universidade Federal do Paraná, Caixa Postal 61, 83255-976 Pontal do Sul, Pontal do Paraná, PR, Brazil

a r t i c l e i n f o a b s t r a c t

Keywords: The effects of diesel oil on benthic associations from unvegetated tidal flats in a subtropical estuary were
Diesel experimentally evaluated using a Multivariate Before and After/Control and Impact Model (M-BACI).
Benthic macrofauna Impacted treatments were contrasted with controls in 14 successive periods before and after the oil spill.
In situ experiment An acute effect was recorded just after the impact, but the recovery to pre-disturbance population levels
Recolonization
was extremely fast. The increase in the total density of the benthic community after the disturbance was
M-BACI
the result of an increase in the densities of Heleobia australis, oligochaetes, and ostracods, observed in
both impacted and control treatments, as a reflection of background variability and not the presence
of the contaminant. The experimental spill had little influence on the biological descriptors of the benthic
associations, which were resilient or tolerant to oil disturbance at the temporal (147 days) and spatial
(cm2) scales used in the experiment.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Oil spills are among the main sources of organic contamination
in marine environments and may cause negative effects on the bio-
ta in its various levels of organization (ITOPF, 1997; Clark, 1997;
Fleeger et al., 2003; NRC, 2003; Ruiz et al., 2005). Under normal
conditions, much of the oil is removed by physical forces such as
tidal movements, evaporation, and dispersion; however, an impor-
tant fraction can be introduced into the deep anaerobic layers of
the sediment, where it may persist for years (Readman et al.,
2002). Unlike exposed rocky or sandy beaches, estuarine habitats
are relatively protected from strong winds and currents and tend
to gather fine grained sediments, which tend to accumulate and
bind toxicants (Sanz-Lázaro and Marín, 2009), as it often happens
in tidal flats, salt marshes and mangroves.
Estuaries are habitats to diversified benthic populations that play
an important role as transfers of matter and energy between trophic
levels. Benthic organisms live in close association with the substrate,
which tends to accumulate and retain organic and inorganic con-
taminants, particularly when poorly oxygenated (Hyland et al.,
2005). It is difficult for these organisms to avoid exposure to adverse
conditions because of their relatively sedentary nature. Thus, ben-
thic animals have been frequently used on assessments of the inten-
sity and extent of damage caused by oil spills, mainly because they
reflect with greater precision and speed the changes in physical
and chemical parameters (Dauer, 1993; Poulton et al., 1998; Muniz
⇑ Corresponding author. Tel./fax: +55 41 3511862.
E-mail address: egres.aline@gmail.com (A.G. Egres).
et al., 2005; Gomez Gesteira and Dauvin, 2005; Borja and Dauer,
2008; Ocon et al., 2008; Dauvin et al., 2010).
Pollutants tend to affect the structure of benthic associations by
changing their abundance and composition. Species that are tolerant
and opportunistic become relatively more numerous after oil spills,
while those considered sensitive become increasingly rare or disap-
pear (Carman et al., 2000; Belan, 2003). Surviving or recolonizing
species can form population patches after acute impacts in affected
areas, benefiting from the exclusion of those previously dominant.
Thus, the composition of benthic communities can be changed tem-
porarily or permanently in comparison with pre-disturbance
conditions (Glasby and Underwood, 1996).
Reliable biological indicators of environmental quality need to
demonstrate logical connections between their responses and the
variables of interest and the idea that a taxonomic group or species
is representative of particular environmental conditions should be
initially treated as a hypothesis to be tested (Goodsell et al., 2009).
Toxicological approaches in laboratories or in situ have been widely
used for environmental risk assessment, both presenting specific
advantages and limitations (Carman et al., 2000; Bhattacharyya
et al., 2003; Schratzberger et al., 2003; Chung et al., 2004; Lu and
Wu, 2006). However, laboratory bioassays conducted under strictly
controlled conditions might not reflect the so-called natural vari-
ability (Morales-Caselles et al., 2008a). This creates uncertainty
as to the validity of result extrapolations when compared to field
studies (Sibley et al., 1999).
In fact, animal associations do not exhibit simple or linear re-
sponses to disturbance in their natural habitat. They are more
likely to resist or persist until reaching a threshold of disturbance

0025-326X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.marpolbul.2012.10.007
2682 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691
(Hyland et al., 2005). In this context, the simulation of small- or
meso-scale in situ manipulative experiments is often the best
way to assess impacts because they best reflect the ecological real-
ity responding to cumulative and synergistic effects (Glasby and
Underwood, 1996).
These experimental approaches are very simple from a conceptual
and methodological point of view and do not require sophisticated
equipment. Additionally, they are reliable tools because they use dif-
ferent lines of evidence and analyze the whole community through
key species (Sanz-Lázaro and Marín, 2009), which putatively support
ecosystem ecological integrity (structure and productivity).
The subtropical estuarine complex of Paranaguá Bay in south-
ern Brazil is one of the largest and best preserved in the southern
hemisphere (Lana et al., 2001). Its economic importance is related
to fishing activities, urban and tourist areas and industries, associ-
ated with fertilizer plants, fuel terminal and the main South Amer-
ican grain shipping port, being the third most important in loading
and unloading operations (Santos et al., 2009; Martins et al., 2010).
While not characterized by a large volume of oil operations, the
port hosts the Transportation Terminal of Paranaguá (TEPAR)
among its retro-port zone components, which operates refining,
storing, and transporting oil and derivatives. Intense ship traffic
in the bay, together with smaller fishing and leisure boats, must
be added as potential impact vectors. The most common fuel in
most of these vessels is the marine diesel oil, which regardless of
its tendency to persist less time due to its high evaporation rate
(FINGAS, 2001), has acute toxic effects on benthic plants and ani-
mals (Lytle and Peckarsky, 2001).
Paranaguá Bay has a wide variety of intertidal environments
such as mangroves, marshes, and unvegetated tidal flats, which
are most exposed to oil impacts. Local unvegetated tidal flats are
resting and feeding areas for migratory birds and fishing areas
for local fish-folk communities (Ryo et al., 2011). These systems,
usually dominant in confined low-energy areas along the south-
eastern and southern Brazilian coasts, are indeed potential targets
of accidental oil spills (Noernberg and Lana, 2002).
In this context, this work aims: (i) to evaluate, through a field
experiment, the acute or immediate effects of diesel oil on the
benthic macrofauna from unvegetated subtropical tidal flats ex-
pressed by the mortality rates and changes in vital signs of the
numerically dominant species; (ii) to assess the short-term effects
(weeks to months) of diesel oil expressed by variations in total mac-
rofaunal density, species numbers, and densities of the numerically
dominant macrobenthic species; (iii) to assess recovery speed to be-
fore-impact population levels, e.g. local resilience. We hypothesize
that macrofaunal recovery in intertidal areas of a subtropical estuary
after a small-scale oil spill will be fast and mostly determined by the
mobility strategies of individual species.
2. Materials and methods
2.1. Study area
The Paranaguá Bay (25°300 S; 48°250 W), located in the coastal
plain of Paraná State, is an estuarine system bordered by man-
groves, marshes, and extensive tidal plains. Unvegetated tidal flats
are the most common traits of the bay. They may be covered by
banks of macro-algae or diatoms films, which are the main primary
producers (Siqueira et al., 2006). The numerical dominance of poly-
chaetes and oligochaetes followed by crustaceans and mollusks are
recurrent (Faraco and Lana, 2004).
The in situ experiment was performed in unvegetated tidal flats
along the Cotinga Channel (Fig. 1), a sub-estuary from Paranaguá
Bay, with about 15 km in extension, which receives the freshwater
input from the Maciel, Correias, Almeidas, Guaraguaçú, and Itiberê
rivers (Lana et al., 2001).
2.2. Experimental design
An in situ experimental M-BACI approach was carried out be-
tween November 18, 2009 and April 13, 2010, with the simulation
of a single acute impact (January 29, 2010) using pre-established
temporal scales presented in Table 1. The Multivariate Before and
After/Control and Impact (M-BACI) model is a sampling strategy
appropriate for analyses over planned impacts (Keough and
Mapstone, 1997; Underwood, 2000; Downes et al., 2004).
Three impacted treatments were contrasted with three controls
over 14 successive sampling times, seven before and seven after
the spill (Table 1). Spatial and temporal sampling were replicated
to allow for a proper interpretation of the interactions between
treatments (impacted and control) and sampling times (before
and after) making the inferences more trustworthy (Underwood,
2000).
The contaminant used in the experiment was the marine diesel
oil donated by TRANSPETRO S.A. This fuel is destined to small, mid
size ships, and to auxiliary engines in large carriers. The experi-
mental spill was performed in three unvegetated tidal flats located
between the mouths of the Maciel and Guaraguaçu rivers: Area 1
(25°330 03100 S and 48°250 07400 W); Area 2 (25°320 70600 S and
48°250 82600 W); and Area 3 (25°320 55200 S and 48°270 14300 W) (Fig. 1).
Impacted treatments with diesel oil spill and the corresponding
control treatments were established in each tidal area, 40 m apart
and positioned at similar tidal levels, parallel to the coastline.
Twelve squares of 1 m2 arranged in rows and with centralized
sampling areas of 0.35  0.35 m were defined for each treatment;
walking circulation areas were also pre-established to avoid tram-
pling during sampling (Fig. 1).
The experimental spill was done during the low tide when the
unvegetated tidal flats were emersed, thus optimizing the time for
the oil to percolate into the sediment. Around 2500 ml of diesel were
dumped in each centralized sampling area. Immediately after the
discharge, the oil was contained by wooden square artifacts properly
allocated to prevent the dispersion of the product and cross-contam-
ination of the control treatments. The amount of oil tested was de-
fined based on pilot experiments to identify threshold effects on
the benthic fauna from increasing oil volumes. In lesser oil volumes,
no massive mortality rates or loss of vital signs, as indicated by active
locomotion in the case of polychaetes and crustaceans and ciliary
activity in the mantle of gastropods or bivalves, were observed.
2.3. Faunal sampling and processing
Three sampling units were randomly taken from each treatment
(impacted and control), in the three unvegetated tidal flats on each
of 14 sampling days (7 before and 7 after the oil spill), using a corer
of 10 cm in diameter and 8 cm in length. The samples were sorted in
the laboratory through 500 lm mesh sieves. The retained material
was fixed in 4% formaldehyde for 48 h and subsequently preserved
in 70% alcohol. The benthic macrofauna was counted and identified
to the lowest taxonomic level (usually to species).
The samples collected on the first day after the impact were ta-
ken to the laboratory and analyzed prior to fixation to assess the
immediate (acute) responses of organisms to oil. Organisms were
classified as alive and dead and quantified with the aid of a stereo-
scopic microscope. Alive organisms usually showed active mobility
and responses to mechanical or light stimuli.
2.4. Sampling of environmental variables
Sediment samples were collected in each treatment for particle
size analysis on the first and last day of the experiment (November
18, 2009 and April 13, 2010, respectively). Additional sediment
samples were collected in each treatment to determine the
 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691
Fig. 1. Map of Paranaguá Bay, showing the three unvegetated tidal flats (d) and an schematic display of the sampling design with impacted ( ) and control treaments ( ).
aliphatic hydrocarbon concentrations and organic matter content
one day after and in the last sampling date after the experimental
oil spill (January 30, 2010).
The top 2 cm of undisturbed surface sediment was placed into
pre-cleaned aluminum foil and plastic bags for hydrocarbons and
organic matter analyses, respectively, and stored at 20 °C. The
sediments were oven dried (40 °C), carefully homogenized with a
mortar and stored in clean glass bottles at room temperature until
organic compounds analysis.
2.5. Analyses of aliphatic hydrocarbons
The analytical procedure described by Martins et al. (2007) was
adopted for sample preparation. Approximately 5 g of sediment
was Soxhlet extracted over 8 h using 80 mL of a mixture of (1:1)
dichloromethane (DCM) and n-hexane. A mixture of surrogates
was added prior to sample extraction (hexadecene and eicosene).
The DCM/n-hexane extract was purified using the column chroma-
tography with 5% deactivated alumina (1.8 g) and silica (3.2 g). Elu-
tion was performed with 10 mL of n-hexane. Around 1 ll of the
aliphatic hydrocarbons concentrated extract fractions were deter-
mined by gas chromatography.
The analytical procedure for the determination of aliphatic
hydrocarbons followed Martins et al. (2012). The analyses were
performed with an Agilent GC model 7890A equipped with a flame
2683
ionization detector and an Agilent DB5 capillary fused silica col-
umn coated with 5% diphenyl/dimethylsiloxane (30 m, 0.25 mm
ID and 0.25 lm film thickness). Hydrogen was used as the carrier
gas. The oven temperature for aliphatic hydrocarbons were pro-
grammed from 40 °C, holding for 2 min, 40–60 °C at 20 °C min1,
then to 250 °C at 5 °C min1, and finally to 320 °C at 6 °C min1,
where this temperature was held for 20 min. Compounds were
individually identified by matching retention times with results
from standard mixtures of (C10–C40) n-alkanes within the range
of 0.25–10.0 lg L1.
2.6. Analyses of organic matter, grain size and water salinity
Organic matter content was measured by weight difference
after the sediment was burned at 550 °C for 1 h (Dean, 1974). For
sediment texture analyses, the samples were screened through
sieves ranging between 1.5 and 4.0 phi (phi = log 2 in diameter
(in mm)) and the silt–clay fraction was processed by pipetting (Su-
guio, 1973). These analyses were performed in the Geological
Oceanography Laboratory (LOGEO) from the Marine Studies Center
(CEM/UFPR).
Water salinity was measured using a portable refractometer.
Surface sediment temperature was measured using a digital ther-
mometer in Celsius degrees. Temperature and salinity measure-
ments were taken on all samplings days.
2684 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691
Table 1
Schedule of sampling, adopting the M-BACI sampling design.
Before Day-73 Day-45
18/11/09 16/12/09
Impact Experimental spill
29/01/10
After Day-1 Day-2
30/01/10 31/01/10
Fig. 2. M-BACI sampling design used in this study; NB: number of replicates before oil spill; NA: number of replicates after the spill.
2.7. Data processing
The significance of differences in total macrofaunal density (N),
number of species (S), and the density of the seven numerically
dominant species between treatments were tested by ANOVAs, fol-
lowing the routines described by Underwood (2000) and Downes
et al. (2004) for the M-BACI sampling strategy. The experimental
model included four factors with the following structure (Fig. 2):
period (two levels, before and after impact, fixed and orthogonal),
sampling time (seven levels, sampling dates before and after im-
pact, fixed and nested within each period), treatment (two levels,
control and impact, fixed and orthogonal), and location (three lev-
els, random and within nested in treatment). The statistical signif-
icance of ANOVA’s was built in the R 2.12.0 free software.
A non-metric multidimensional scaling (n-MDS), derived from
similarity matrices using the Bray-Curtis index, was used to
visualize the variation trends of the benthic associations between
treatments and periods. Average values of three treatment repli-
cates were considered for the n-MDS analysis. The significance of
differences between treatments was tested by a permutational mul-
tivariate analysis of variance (Anderson, 2001) using the PERMANO-
VA software, version 1.6 (Anderson, 2005).
3. Results
3.1. Environmental parameters
Sediment temperature and water salinity remained relatively
uniform in the three sampled areas during the studied period.
Day-32 Day-15 Day-3 Day-2 Day-1
29/12/09 15/01/10 26/01/10 27/01/10 28/01/10
Day-3 Day-15 Day-32 Day-45 Day-73
01/02/10 12/02/10 01/03/10 15/03/10 13/04/10
The sediment temperature ranged from 24.0 to 31.0 °C, whereas
the water salinity ranged between 19.0 and 35.0 PSU.
Local sediments are composed of very fine sand with low organ-
ic matter content. No relevant sediment texture variation was ob-
served between the first and last day of the experiment, except in
the impact treatment in the first (73) and last (+73) sampling day
in area 1 (Table 2).
3.2. Aliphatic hydrocarbons
The identification of oil contamination in impacted and control
areas, before and after spill experiment was based on the evalua-
tion of total aliphatic hydrocarbons, the presence of unresolved
complex mixture (UCM) and the predominance of odd or even n-
alkanes expressed by Carbon Preferred Index (CPI).
Estuarine or intertidal sediments may be considered uncontam-
inated by oil if the concentration of total aliphatic hydrocarbons is
below 10 lg.g1 (Maioli et al., 2011). Higher concentrations of total
aliphatic hydrocarbons (10.7–30.9 lg g1) were recorded in the
sediments of impacted treatments, one day after the diesel oil spill
(Table 3; Fig. 3). Pre-impact concentrations were reestablished
after 73 days, with the exception of the impacted treatment from
the area 2, which still contained significant concentrations of total
aliphatic hydrocarbons, up to 13.7 lg g1 (Table 3; Fig. 3).
The unresolved complex mixture (UCM) is considered to be a
mixture of many structurally complex isomers and homologues
of branched and cyclic hydrocarbons that cannot be resolved by
capillary GC columns and therefore appear as a hump in the GC
chromatogram (Bouloubassi and Saliot, 1993). This feature is nor-
mally associated with degraded or weathered petroleum residues
 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691 2685

Table 2
Sedimentological variables and percentages of organic matter in the first (73) and last (73) days of the experiment.

(%) Area 1 Area 2 Area 3

Control Impact Control Impact Control Impact
(73) (+73) (73) (+73) (73) (+73) (73) (+73) (73) (+73) (-73) (+73)
Coarse sand 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0
Mean sand 0.2 0.2 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0
Fine sand very 28.9 35.7 26.2 8.3 6.2 6.3 6.2 14.2 1.1 0.8 14.2 20.3
Fine sand 57.6 55.0 54.8 82.2 75.7 82.1 84.8 73.4 78.7 87.6 77.3 74.9
Silt 9.5 7.1 6.9 3.1 12.9 7.6 5.8 4.3 7.6 8.8 3.8 2.9
Clay 3.6 1.8 11.8 6.2 4.5 3.5 2.6 7.9 12.2 2.6 4.5 1.8
Organic matter 3.6 2.6 2.9 4.2 4.7 2.1 3.4 2.7 2.9 1.2 4.6 2.1

Table 3 between 5.6 and 6.4, indicating the predominance of recent higher
Concentrations of aliphatic hydrocarbons (AHs) and unresolved complex mixture plants n-alkanes. However, one day after the experimental oil spill,
(UCM), in lg g1, and Carbon Preferred Index (CPI) for the sediments analyzed.
the CPI values decreased to 2.4, 2.6 and 4.9 in the area 2, 3, and 1,
Day Control Impact respectively, which indicates some oil-genic influence (Table 3).
AHs UCM CPI AHs UCM CPI
Area 1 3.3. Macroinvertebrates
(73) 1.88 ND 6.3 3.98 ND 5.6
(+1) 1.93 ND 6.1 10.7 7.34 4.9
(+73) 1.38 ND 6.2 1.73 ND 5.9
A total of 21,870 individuals were recorded, belonging to 90
benthic taxa or morphotypes, including polychaetes (91.7% of spe-
Area 2
(73) 2.77 ND 5.8 2.31 ND 6.4
cies), crustaceans (6.03%), molluscs (1.73%), and insects (0.59%).
(+1) 2.55 ND 5.8 30.9 20.9 2.4 The dominant taxa were two unidentified morphotypes of oligo-
(+73) 2.46 ND 6.0 13.7 10.6 5.6 chaetes (25.1%), one morphotype of ostracod (21.2%), the gastro-
Area 3 pods Heleobia australis (10.6%) and Bulla striata (5.1%), the
(73) 1.42 ND 5.9 2.67 ND 5.9 polychaetes Glycinde multidens (4.1%), Mediomastus sp. (3.7%),
(+1) 1.84 ND 5.8 16.4 10.7 2.6 and Heteromastus sp. (3.2%), which contributed to 73.3% of the total
(+73) 0.73 ND 6.3 0.74 ND 5.5
density of organisms.
ND – Not detectable.

3.4. Effects of the experimental spill

(Readman et al., 2002). The levels of UCM (7.34–20.9 lg g1) con-
firmed the presence of hydrocarbons in the sediments one day
after the acute impact and their persistence in area 2 (Table 3).
The carbon preference index (CPI25-35), a measure of biologically
synthesized n-alkanes, indicates the relative contributions from
natural (biogenic/terrestrial; CPI > 1, typically between 4 and 7)
compared to anthropogenic (petroleum pollution; CPI  1) sources
(Aboul-Kassim and Simoneit, 1996; Didyk et al., 2000). In the sam-
ples from control area and before the impact, the CPI values ranged
Considering the experiment as a whole, no significant statistical
differences were detected in total macrofaunal density, number of
species, and density of numerically dominant taxa between the im-
pacted and control treatments before and after the experimental
spill (TrPe, p < 0.05, Table 4). However, there were significant dif-
ferences in total macrofaunal density, number of species and den-
sity of Glycinde multidens and ostracods between treatments from
time to time within each period (TrTe(Pe), p < 0.05, Table 4).
Oligochaete densities varied significantly between sites (i.e. tidal

Fig. 3. Concentrations of total aliphatics (lg g1) in the impacted treatments (I) and controls (C) in the three study areas: A1 (Maciel); A2 (Rasa Island); A3 (Guaraguaçu).
Sampling: (73), first day; (+1), a day after the impact; and (+73) last day of the experiment.
2686 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691

Table 4 Heteromastus sp. did not show significant variations in any of the
Summary of the ANOVAs for the M-BACI model for total macrofaunal density (N), interactions (Table 4 and Fig. 4I). There was a marked decrease in
number of species (S) and densities of numerically dominant species. Source of
variation: treatment = T; period = Pe; time = T; location = Lo. Significant interaction
the density of oligochaetes shortly after the impact and a recovery
(p < 0.05) indicated with. trend with a gradual increase from day 2, mainly in area 1 (Fig. 4F).
The similarity analysis indicated relative similarity between the
Source of variation df Sq F P
benthic associations in the impacted and control treatments, ex-
N cept on the first day after the spill. More than between treatments,
TrPe 1 29143,25 9,734763 0,035525
TrTe(Pe) 12 4481,411 3,48322 0,00098
major differences were observed in the distribution of replicates or
Lo(Tr)Pe 4 2993,73 2,326906 0,069616 sampling units, before and after the impact, indicating marked
Lo(Tr)Te(Pe) 48 1286,571 1,28812 0,123258 temporal variability during the whole experiment (Fig. 5). The PER-
S MANOVA indicated significant differences in the interactions at the
TrPe 1 114,6825 2,496545 0,189247 smallest spatial and temporal scales (Table 5) indicating that the
TrTe(Pe) 12 48,80423 4,780823 4,32E-05 benthic associations varied before and after the impact, and be-
Lo(Tr)Pe 4 45,93651 4,499903 0,003618
tween periods in the different sites.
Lo(Tr)Te(Pe) 48 10,20833 1,431553 0,05066
Bulla striata
TrPe 1 18,89286 0,532193 0,506109
TrTe(Pe) 12 36,6627 0,328619 0,980103 4. Discussion
Lo(Tr)Pe 4 35,5 0,318197 0,864413
Lo(Tr)Te(Pe) 48 111,5661 2,45822 1,28E05 Our working hypothesis was not refuted, and faunal recovery
Glycinde multidens was extremely fast, despite massive mortality just after the exper-
TrPe 1 35,06349 7,33887 0,053591 imental spill. The in situ experimental approach allowed for a con-
TrTe(Pe) 12 13,11905 2,125134 0,032665 sistent monitoring of the relationship between the presence of
Lo(Tr)Pe 4 4,777778 0,773945 0,547574
Lo(Tr)Te(Pe) 48 6,17328 1,409118 0,058649
diesel oil and variation in total macrofaunal density, number of
species, and densities of numerically dominant species. The M-
Heleobia australis
TrPe 1 1325,73 4,315606 0,106335
BACI manipulative model (Keough and Mapstone, 1997; Under-
TrTe(Pe) 12 185,4563 0,876424 0,575495 wood, 2000; Downes et al., 2004) indicated that the observed alter-
Lo(Tr)Pe 4 307,1944 1,45173 0,231624 ations were more related to the natural temporal variability in the
Lo(Tr)Te(Pe) 48 211,6058 1,972796 0,000838 benthic community structure than to the oil impact itself.
Oligochaeta Timescales to be used in planned impacts should be based on
TrPe 1 38,11111 0,052423 0,830129 the scales in which the local communities naturally vary. Other-
TrTe(Pe) 12 743,5807 2,743372 0,006522
wise, variations in macrobenthic associations might eventually
Lo(Tr)Pe 4 726,9921 2,682169 0,042513
Lo(Tr)Te(Pe) 48 271,0463 1,346655 0,086954 mask the adequate recognition of variability caused by the pres-
ence of oil (Spellerberg, 2005). Hence, the timescale of 147 days
Ostracoda
TrPe 1 8285,813 19,27058 0,011784 adopted in this experiment clearly incorporated processes that
TrTe(Pe) 12 665,4153 3,720993 0,000541 are associated with the temporal dynamics of benthic associations.
Lo(Tr)Pe 4 429,9722 2,404398 0,062513 The increase in the total macrobenthic density after the distur-
Lo(Tr)Te(Pe) 48 178,8274 1,419488 0,054828 bance was the result of a clear increase in the densities of Heleobia
Heteromastus sp. australis, oligochaetes, and ostracods, observed in both impacted
TrPe 1 0,142857 0,026686 0,878157 and control treatments, as a clear reflection of natural variability
TrTe(Pe) 12 7,205026 1,674197 0,102936
Lo(Tr)Pe 4 5,353175 1,243891 0,305077
and not the presence of the contaminant.
Lo(Tr)Te(Pe) 48 4,303571 0,985909 0,507231 The experimental oil spill had an immediate and acute effect of
Mediomastus sp.
the oil spill on the benthic macrofauna, evidenced by the high mor-
TrPe 1 3,813492 0,23577 0,652675 tality of organisms, one day after the contamination. However, the
TrTe(Pe) 12 13,60053 1,069538 0,405706 recovery to pre-impact population levels was extremely fast, and
Lo(Tr)Pe 4 16,1746 1,271961 0,294061 recolonization was already completed after day 2, as shown by
Lo(Tr)Te(Pe) 48 12,71627 1,544337 0,02341
the elevated total macrofaunal density in comparison with pre-dis-
turbance levels. This response pattern was defined by Glasby and
Underwood (1996) as a ‘‘pulse’’ disturbance, i.e. a short-term ef-
flats), within each treatment, and before and after the oil spill fect, with a sudden drop in density followed by a rapid recovery
(Lo(Tr)Pe < 0.05, Table 4 and Fig. 4F). in the absence of new disturbances.
The effects of oil on the densities of Bulla striata, Heleobia Very fast recovery of benthic associations after small-scale dis-
australis, and Mediomastus sp. were expressed in the interaction turbances has been previously reported (Faraco and Lana, 2003;
of the smaller spatial and temporal scales analyzed, with variations Negrello Filho et al., 2006) and variously associated to the migra-
over time from one site to another (Lo(Tr)Te(Pe), p < 0.05, Table 4 tion of juveniles and adults from adjacent populations (Negrello
and Fig. 4C, E, and H). Filho et al., 2006), larval recruitment (Carman et al., 2000), or
Despite the absence of overall differences in benthic variables high tolerance to toxic compounds by the recolonizing species
before and after impact considering the experiment as a whole, (Schratzberger et al., 2003). In our study, the main recolonization
acute exposure to oil-contaminated sediment caused high mortal- vector was the active migration of adults from the surrounding
ity rates of the benthic macrofauna. The few remaining alive organ- sediments since their density began to increase on the second
isms on the first day after the impact were not active and capitellid day after the impact and few juveniles were recorded. This accel-
polychaetes presented signs of hemolysis (personal observation). erated pattern of recolonization of intertidal macrobenthic spe-
However, a quick recovery to pre-impact population levels was ob- cies in a subtropical estuary is probably associated with the
served in the subsequent samplings (Fig. 4A–I). Although the poly- evolutionary history of adaptation to natural stressors such as
chaete Glycinde multidens was sensitive and displayed high desiccation, high temperatures during tidal exposure, and a corre-
mortality rates just after the impact, its pre-impact density was spondingly reduction in feeding and respiration times (Bolam
recovered after day 15 (Fig. 4D). The populations of the polychaete et al., 2004).
 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691 2687

Although the simulated diesel oil spill has not caused signifi-
cant changes in the overall structure of the benthic associations
at a 147-day time scale, variations in the response patterns of
individual species were rather evident. The polychaete Glycinde
multidens proved initially sensitive to oil, with a marked drop in
population densities just after the experimental spill, but soon
recovered to pre-impact levels. This initial sensitivity of this vagile
predator species to oil has already been observed near the study
site (Faraco and Lana, 2003). Peso-Aguiar et al. (2000) also re-
ported that G. multidens was absent in a contaminated area at
chronically impacted sites near an oil refinery in the Todos os San-
tos Bay (Bahia) suggesting their low tolerance. On the other hand,
Venturini and Tommasi (2004) observed that Glycinde multidens
was numerically dominant in sublitoral sediments with high con-
centrations of PAHs also in Todos os Santos Bay. Venturini et al.
(2008) also recorded high abundances of Glycinde multidens in
sites moderately contaminated by total aliphatics hydrocarbons
(>23 lg g1) and presence of UCM. Despite the inconsistency of
these trends, populations of a same species are known to respond
differently to the same contaminants in distinct areas possibly be-
cause of different histories of exposure to contamination (Carman
et al., 2000).

A
250

Control
Total density of macrobenthos

Impact
200
0.095 m−2
150
100
50
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

B
25
Number of species 0.095 m−2
20
15
10
5
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

C
50
Density of B. striata 0.095 m−2
40
30
20
10
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day
Fig. 4. Temporal variability of average values of (A) total macrofaunal density (B) species number and average densities of (C) Bulla striata, (D) Glycinde multidens, (E) Heleobia
australis, (F) Oligochaeta (G) Ostracoda (H) Mediomastus sp. and (I) Heteromastus sp. before (73, 45, 32, 15, 3, 2, 1) and after impact (1, 2, 3, 15, 32, 45, 73) in the
treatments (impact and control).
2688 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691

12
Density of G. multidens 0.095 m−2
10
8
6
4
2
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

E
Density of H. australis 0.095 m−2
60
40
20
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

F
100
Density of Oligochaeta 0.095 m−2
80
60
40
20
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day
Fig. 4. (continued)

The two morphotypes of oligochaetes proved tolerant or resil-
ient to the impact as shown by their high population densities after
day 2 in the impacted treatments. Similar results were reported by
Gomez Gesteira and Dauvin (2000) and Ocon et al. (2008) who re-
corded high densities of oligochaetes in oil impacted sites.
Field and laboratory studies have suggested that ostracods are
sensitive to oil as expressed by high mortality rates in contami-
nated sediments (Ruiz et al., 2005). Similar responses were re-
corded by Carman et al. (2000) in an experimental microcosm
with initial mortality followed by a later change in the trophic
structure. In our experimental analysis, live ostracods were not
found in the impacted treatments shortly after the spill, but high
densities were recorded after day 2. The recolonization patterns
varied greatly and inconsistently between the treatments for the
remaining analyzed taxa making it difficult to clearly define the
processes that effectively controlled their distribution.
Descriptive or experimental analyses of the effects of oil spills
on intertidal benthic macrofauna have produced contradictory re-
sults. Although some studies report decreases in density and rich-
ness of taxa, others do not record significant effects or even report
increases in densities of organisms. Chung et al. (2004), using
experimental mesocosms, observed that the density of deposit-
feeders animals (both superficial or burrowers) decreased soon
after an oil spill. Some populations recovered pre-disturbance den-
sities only 42 days after the exposure. Lu and Wu (2006) recorded
fast recolonization in oil contaminated sediments, but the domi-
 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691 2689

80
Density of Ostracoda 0.095 m−2
60
40
20
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

H
25
Density of Mediomastus sp.

20
0.095 m−2
15
10
5
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day

I
10
Density of Heteromastus sp.

8
0.095 m−2
6
4
2
0

−73 −45 −32 −15 −3 −2 −1 1 2 3 15 32 45 73

Day
Fig. 4. (continued)

nant species were very distinct from the control sediments for the
first five months. Lu (2005) observed a strong negative correlation
between the abundance, species numbers, and diversity of macro-
benthos and the total concentration of hydrocarbons in sediments
in Singapore. Andersen et al. (2008) observed that the abundance
and richness of the intertidal macrobenthos from impacted and
control sites were very similar just one month after an oil spill in
Gladstone (Australia). Ocon et al. (2008) recorded high densities
of tolerant organisms in oil contaminated sites in the Río de la Plata
estuary (Uruguay), while the sensitive species were usually absent.
The severity of oil impacts on the benthic macrofauna depends
on many factors, most notably (1) the oil amount; (2) composition;
(3) form (fresh or emulsified); (4) occurrence (i.e. solution, suspen-
sion, dispersion, or adsorption to the particulate matter); (5) expo-
sure time; (6) involvement of juvenile and adult forms; (7) season;
(8) natural environmental stress associated with fluctuations in
temperature, salinity, and other variables; and (9) type of affected
environment (Kennish, 1997). The hydrocarbon analyses indicated
that all impacted sediments were effectively contaminated by the
diesel oil spillage. However, a trend of fast decrease in the total
concentration of aliphatic hydrocarbons was observed during the
experimental period.
Significant changes in hydrocarbon composition occur due to
biological (biodegradation and bioturbation) and physical pro-
2690 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691

Fig. 5. MDS of mean densities (0.095 m2) of benthic macrofauna in impact (s) and control (r) treatments, before (white) and after (black) the experimental impact.

spatial (cm2) and temporal scales (147 days). The initial recoloni-
Table 5
PERMANOVA based on Bray-Curtis dissimilarities, without data transformation. p- zation and subsequent succession of the benthic community was
Values were obtained from 9999 permutations. not delayed in the oil contaminated sediments. There were no sig-
nificant changes in overall abundance and number of species ex-
Source g MQ F p(MC)
cept just after the oil spill. The observed temporal variability was
PexTr 1 2016.4709 0.7012 0.6237
not related to changes in the density and composition of tolerant
PexLo(tr) 4 2875.7472 4.0654 10.0001
Te(Pe)xTr 12 2064.2548 1.3488 0.0723 or sensitive species because the dominant taxa were virtually the
Te(Pe)xLo(Tr) 48 1530.4526 2.1636 0.0001 same at the beginning and end of the experiment. In summary,
Residual 168 707.3747 – – the significant variations in the structure of local associations were
Total 251 – – – more related to the temporal variability than to the presence of oil
contaminants in the sediment. Our experimental approach demon-
strates that the macrobenthic associations in unvegetated tidal
cesses (selective dissolution, evaporation, photo-oxidation and flats of a subtropical bay may behave in a resilient fashion and
natural dispersion) that contribute to oil degradation (Readman be able to withstand minor oil spills or quickly recolonize oil dis-
et al., 2002; Kingston, 2002; Schratzberger et al., 2003; Lu and turbed sites.
Wu, 2006; Morales-Caselles et al., 2008b; Andersen et al. 2008).
According to Puente et al. (2009), the nature of the substrate, com-
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