effects of an experimental in situ

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

Keywords:

Diesel

Benthic macrofauna

In situ experiment

Recolonization

M-BACI

a b s t r a c t

The effects of diesel oil on benthic associations from unvegetated tidal flats in a subtropical estuary wereexperimentally evaluated using a Multivariate Before and After/Control and Impact Model (M-BACI).

Impacted treatments were contrasted with controls in 14 successive periods before and after the oil spill.An acute effect was recorded just after the impact, but the recovery to pre-disturbance population levelswas extremely fast. The increase in the total density of the benthic community after the disturbance was

the result of an increase in the densities of Heleobia australis, oligochaetes, and ostracods, observed inboth 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 benthicassociations, which were resilient or tolerant to oil disturbance at the temporal (147 days) and spatial

(cm2) scales used in the experiment. 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Oil spills are among the main sources of organic contaminationin 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 normalconditions, much of the oil is removed by physical forces such astidal 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 habitatsare relatively protected from strong winds and currents and tend

to gather fine grained sediments, which tend to accumulate andbind toxicants (Sanz-Lázaro and Marín, 2009), as it often happensin tidal flats, salt marshes and mangroves.

Estuaries are habitats to diversifiedbenthic populations that play

an important role as transfers of matter andenergybetween trophiclevels. Benthic organismslive in closeassociation 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 forthese organismsto avoid exposure to adverseconditions because of their relatively sedentary nature. Thus, ben-thic animals have beenfrequently 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 physicaland chemical parameters (Dauer, 1993; Poulton et al., 1998; Muniz

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 bychangingtheir abundanceand composition. Species that are tolerantand opportunistic become relatively more numerous after oil spills,

while those consideredsensitive become increasingly rare or disap-pear (Carman et al., 2000; Belan, 2003). Surviving or recolonizingspecies can form population patches after acute impacts in affectedareas, 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-disturbanceconditions (Glasby and Underwood, 1996).

Reliable biological indicators of environmental quality need todemonstrate logical connections between their responses and thevariables of interest and the idea that a taxonomic group or speciesis 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 widelyused for environmental risk assessment, both presenting specificadvantages and limitations (Carman et al., 2000; Bhattacharyya

et al., 2003; Schratzberger et al., 2003; Chung et al., 2004; Lu andWu, 2006). However, laboratory bioassays conducted under strictlycontrolled 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 fieldstudies (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 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.marpolbul.2012.10.007

⇑ Corresponding author. Tel./fax: +55 41 3511862.

E-mail address: [email protected] (A.G. Egres).

Marine Pollution Bulletin 64 (2012) 2681–2691

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(Hyland et al., 2005). In this context, the simulation of small- ormeso-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 andUnderwood, 1996).

These experimentalapproaches are verysimplefrom 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 throughkey 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 southernhemisphere (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, theport hosts the Transportation Terminal of Paranaguá (TEPAR)among its retro-port zone components, which operates refining,

storing, and transporting oil and derivatives. Intense ship trafficin the bay, together with smaller fishing and leisure boats, mustbe added as potential impact vectors. The most common fuel inmost 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, whichare most exposed to oil impacts. Local unvegetated tidal flats areresting and feeding areas for migratory birds and fishing areasfor 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 targetsof accidental oil spills (Noernberg and Lana, 2002).

In this context, this work aims: (i) to evaluate, through a fieldexperiment, the acute or immediate effects of diesel oil on thebenthic macrofauna from unvegetated subtropical tidal flats ex-pressed by the mortality rates and changes in vital signs of thenumerically dominant species; (ii) to assess the short-term effects

(weeks to months) of diesel oilexpressedby variations in total mac-rofaunal density, species numbers, and densities of the numericallydominant macrobenthicspecies; (iii) to assessrecovery speed to be-fore-impact population levels, e.g. local resilience. We hypothesize

that macrofaunalrecovery in intertidalareas of a subtropicalestuaryafter a small-scale oilspill will be fast and mostly determinedby themobility strategies of individual species.

2. Materials and methods

2.1. Study area

The Paranaguá Bay (25300S; 48250W), located in the coastalplain 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 bybanks of macro-algae or diatoms films, which are the main primaryproducers (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 ParanaguáBay, with about 15 km in extension, which receives the freshwater

input from the Maciel, Correias, Almeidas, Guaraguaçú, and Itiberêrivers (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 simulationof a single acute impact (January 29, 2010) using pre-establishedtemporal 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 andMapstone, 1997; Underwood, 2000; Downes et al., 2004).Three impacted treatments were contrasted with three controls

over 14 successive sampling times, seven before and seven afterthe spill (Table 1). Spatial and temporal sampling were replicatedto allow for a proper interpretation of the interactions betweentreatments (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, midsize ships, and to auxiliary engines in large carriers. The experi-mental spill was performed in three unvegetated tidal flats locatedbetween the mouths of the Maciel and Guaraguaçu rivers: Area 1

(2533003100S and 4825007400W); Area 2 (2532070600S and4825082600W); and Area 3 (2532055200S and 4827014300W) (Fig. 1).

Impacted treatments with diesel oil spill and the correspondingcontrol 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 centralizedsampling 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 fortheoil to percolateinto thesediment. Around2500 mlof dieselwere

dumped in each centralized sampling area. Immediately after thedischarge,the oil was containedby woodensquare artifacts properlyallocated to prevent the dispersionof 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 onthe benthic fauna from increasingoil volumes. In lesser oilvolumes,nomassivemortalityratesor loss ofvitalsigns, asindicatedby activelocomotion 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 eachof 14 sampling days (7 before and 7 after the oil spill), using a corer

of 10 cm indiameter and 8 cmin length. The samples were sorted inthe laboratory through 500 lm mesh sieves. The retained material

was fixed in 4% formaldehyde for 48 h and subsequently preservedin 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 theimmediate (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 mobilityand responses to mechanical or light stimuli.

2.4. Sampling of environmental variables

Sediment samples were collected in each treatment for particlesize analysis on the first and last day of the experiment (November

18, 2009 and April 13, 2010, respectively). Additional sedimentsamples were collected in each treatment to determine the

2682 A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691



aliphatic hydrocarbon concentrations and organic matter content

one day after and in the last sampling date after the experimentaloil spill (January 30, 2010).

The top 2 cm of undisturbed surface sediment was placed intopre-cleaned aluminum foil and plastic bags for hydrocarbons and

organic matter analyses, respectively, and stored at 20 C. Thesediments were oven dried (40 C), carefully homogenized with amortar and stored in clean glass bottles at room temperature untilorganic compounds analysis.

2.5. Analyses of aliphatic hydrocarbons

The analytical procedure described by Martins et al. (2007) wasadopted for sample preparation. Approximately 5 g of sedimentwas Soxhlet extracted over 8 h using 80 mL of a mixture of (1:1)

dichloromethane (DCM) and n-hexane. A mixture of surrogateswas 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 thealiphatic 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 wereperformed with an Agilent GC model 7890A equipped with a flame

ionization detector and an Agilent DB5 capillary fused silica col-

umn coated with 5% diphenyl/dimethylsiloxane (30 m, 0.25 mmID and 0.25 lm film thickness). Hydrogen was used as the carriergas. 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 wereindividually identified by matching retention times with resultsfrom standard mixtures of (C10–C40) n-alkanes within the range

of 0.25–10.0 lg L 1

.

2.6. Analyses of organic matter, grain size and water salinity

Organic matter content was measured by weight differenceafter the sediment was burned at 550 C for 1 h (Dean, 1974). For

sediment texture analyses, the samples were screened throughsieves ranging between 1.5 and 4.0 phi (phi = log2 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.

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 ( ).

A.G. Egres et al. / Marine Pollution Bulletin 64 (2012) 2681–2691 2683



2.7. Data processing

The significance of differences in total macrofaunal density (N ),

number of species (S ), and the density of the seven numericallydominant species between treatments were tested by ANOVAs, fol-lowing the routines described by Underwood (2000) and Downeset 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 fromsimilarity matrices using the Bray-Curtis index, was used tovisualize the variation trends of the benthic associations betweentreatments and periods. Average values of three treatment repli-

cates were considered for the n-MDS analysis. The significance of differences betweentreatments was testedby a permutationalmul-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 relativelyuniform in the three sampled areas during the studied period.

The sediment temperature ranged from 24.0 to 31.0 C, whereasthe 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 inthe impact treatment in the first (73) and last (+73) sampling dayin area 1 (Table 2).

3.2. Aliphatic hydrocarbons

The identification of oil contamination in impacted and controlareas, before and after spill experiment was based on the evalua-

tion of total aliphatic hydrocarbons, the presence of unresolvedcomplex 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 isbelow 10 lg.g1 (Maioli et al., 2011). Higher concentrations of totalaliphatic hydrocarbons (10.7–30.9 lg g1) were recorded in thesediments of impacted treatments, one day after the diesel oil spill

(Table 3; Fig. 3). Pre-impact concentrations were reestablishedafter 73 days, with the exception of the impacted treatment fromthe area 2, which still contained significant concentrations of totalaliphatic hydrocarbons, up to 13.7 lg g1 (Table 3; Fig. 3).

The unresolved complex mixture (UCM) is considered to be amixture of many structurally complex isomers and homologuesof branched and cyclic hydrocarbons that cannot be resolved bycapillary 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

Table 1

Schedule of sampling, adopting the M-BACI sampling design.

Before Day-73 Day-45 Day-32 Day-15 Day-3 Day-2 Day-1

18/11/09 16/12/09 29/12/09 15/01/10 26/01/10 27/01/10 28/01/10

Impact Experimental spill

29/01/10

After Day-1 Day-2 Day-3 Day-15 Day-32 Day-45 Day-73

30/01/10 31/01/10 01/02/10 12/02/10 01/03/10 15/03/10 13/04/10

Fig. 2. M-BACI sampling design used in this study; NB: number of replicates before oil spill; NA: number of replicates after the spill.




(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 fromnatural (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

between 5.6 and 6.4, indicating the predominance of recent higher

plants n-alkanes. However, one day after the experimental oil spill,the CPI values decreased to 2.4, 2.6 and 4.9 in the area 2, 3, and 1,respectively, which indicates some oil-genic influence (Table 3).

3.3. Macroinvertebrates

A total of 21,870 individuals were recorded, belonging to 90benthic taxa or morphotypes, including polychaetes (91.7% of spe-cies), crustaceans (6.03%), molluscs (1.73%), and insects (0.59%).The dominant taxa were two unidentified morphotypes of oligo-chaetes (25.1%), one morphotype of ostracod (21.2%), the gastro-

pods Heleobia australis (10.6%) and Bulla striata (5.1%), thepolychaetes Glycinde multidens (4.1%), Mediomastus sp. (3.7%),and Heteromastus sp. (3.2%), which contributed to 73.3% of the total

density of organisms.

3.4. Effects of the experimental spill

Considering the experiment as a whole, no significant statisticaldifferences 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 experimentalspill (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 fromtime to time within each period (TrTe(Pe), p < 0.05, Table 4).Oligochaete densities varied significantly between sites (i.e. tidal

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 20.3Fine sand 57.6 55.0 54.8 82.2 75.7 82.1 84.8 73.4 78.7 87.6 77.3 74.9Silt 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

Concentrations of aliphatic hydrocarbons (AHs) and unresolved complex mixture

(UCM), in lg g1, and Carbon Preferred Index (CPI) for the sediments analyzed.

Day Control Impact

AHs UCM CPI AHs UCM CPI

Area 1

(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

Area 2

(73) 2.77 ND 5.8 2.31 ND 6.4

(+1) 2.55 ND 5.8 30.9 20.9 2.4

(+73) 2.46 ND 6.0 13.7 10.6 5.6

Area 3

(73) 1.42 ND 5.9 2.67 ND 5.9

(+1) 1.84 ND 5.8 16.4 10.7 2.6

(+73) 0.73 ND 6.3 0.74 ND 5.5

ND – Not detectable.

Fig. 3. Concentrations of total aliphatics (lg g

1

) 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.




flats), within each treatment, and before and after the oil spill(Lo(Tr)Pe < 0.05, Table 4 and Fig. 4F).

The effects of oil on the densities of Bulla striata, Heleobiaaustralis, and Mediomastus sp. were expressed in the interactionof the smaller spatial and temporal scales analyzed, with variationsover time from one site to another (Lo(Tr)Te(Pe), p < 0.05, Table 4and Fig. 4C, E, and H).

Despite the absence of overall differences in benthic variablesbefore and after impact considering the experiment as a whole,acute exposure to oil-contaminated sediment caused high mortal-ity rates of the benthic macrofauna. The few remaining alive organ-

isms on the first day after the impact were not active and capitellidpolychaetes presented signs of hemolysis (personal observation).However, a quick recovery to pre-impact population levels was ob-served in the subsequent samplings (Fig. 4A–I). Although the poly-

chaete Glycinde multidens was sensitive and displayed high

mortality rates just after the impact, its pre-impact density wasrecovered after day 15 (Fig. 4D). The populations of the polychaete

Heteromastus sp. did not show significant variations in any of theinteractions (Table 4 and Fig. 4I). There was a marked decrease in

the density of oligochaetes shortly after the impact and a recoverytrend with a gradual increase from day 2, mainly in area 1 (Fig. 4F).

The similarity analysis indicated relative similarity between thebenthic associations in the impacted and control treatments, ex-

cept on the first day after the spill. More than between treatments,

major differences were observed in the distribution of replicates orsampling units, before and after the impact, indicating markedtemporal variability during the whole experiment (Fig. 5). The PER-

MANOVA indicated significant differences in the interactions at thesmallest spatial and temporal scales (Table 5) indicating that thebenthic associations varied before and after the impact, and be-tween periods in the different sites.

4. Discussion

Our working hypothesis was not refuted, and faunal recoverywas extremely fast, despite massive mortality just after the exper-imental spill. The in situ experimental approach allowed for a con-

sistent monitoring of the relationship between the presence of diesel oil and variation in total macrofaunal density, number of species, and densities of numerically dominant species. The M-BACI manipulative model (Keough and Mapstone, 1997; Under-

wood, 2000; Downes et al., 2004) indicated that the observed alter-ations were more related to the natural temporal variability in thebenthic community structure than to the oil impact itself.

Timescales to be used in planned impacts should be based on

the scales in which the local communities naturally vary. Other-wise, variations in macrobenthic associations might eventuallymask the adequate recognition of variability caused by the pres-ence of oil (Spellerberg, 2005). Hence, the timescale of 147 days

adopted in this experiment clearly incorporated processes thatare associated with the temporal dynamics of benthic associations.The increase in the total macrobenthic density after the distur-

bance was the result of a clear increase in the densities of Heleobiaaustralis, oligochaetes, and ostracods, observed in both impactedand control treatments, as a clear reflection of natural variabilityand not the presence of the contaminant.

The experimental oil spill had an immediate and acute effect of

the oil spill on the benthic macrofauna, evidenced by the high mor-tality of organisms, one day after the contamination. However, therecovery to pre-impact population levels was extremely fast, andrecolonization was already completed after day 2, as shown by

the elevated total macrofaunal density in comparison with pre-dis-turbance levels. This response pattern was defined by Glasby andUnderwood (1996) as a ‘‘pulse’’ disturbance, i.e. a short-term ef-

fect, with a sudden drop in density followed by a rapid recoveryin the absence of new disturbances.

Very fast recovery of benthic associations after small-scale dis-turbances has been previously reported (Faraco and Lana, 2003;

Negrello Filho et al., 2006) and variously associated to the migra-tion of juveniles and adults from adjacent populations (NegrelloFilho et al., 2006), larval recruitment (Carman et al., 2000), orhigh tolerance to toxic compounds by the recolonizing species

(Schratzberger et al., 2003). In our study, the main recolonizationvector was the active migration of adults from the surroundingsediments since their density began to increase on the secondday after the impact and few juveniles were recorded. This accel-

erated pattern of recolonization of intertidal macrobenthic spe-cies in a subtropical estuary is probably associated with theevolutionary history of adaptation to natural stressors such asdesiccation, high temperatures during tidal exposure, and a corre-

spondingly reduction in feeding and respiration times (Bolamet al., 2004).

Table 4

Summary of the ANOVAs for the M-BACI model for total macrofaunal density (N ),

number of species (S ) and densities of numerically dominant species. Source of

variation: treatment = T; period = Pe; time = T ; location = Lo. Significant interaction

( p < 0.05) indicated with.

Source of variation df Sq F P

N

TrPe 1 29143,25 9,734763 0,035525

TrTe(Pe) 12 4481,411 3,48322 0,00098

Lo(Tr)Pe 4 2993,73 2,326906 0,069616

Lo(Tr)Te(Pe) 48 1286,571 1,28812 0,123258

S

TrPe 1 114,6825 2,496545 0,189247

TrTe(Pe) 12 48,80423 4,780823 4,32E-05

Lo(Tr)Pe 4 45,93651 4,499903 0,003618

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

Lo(Tr)Pe 4 35,5 0,318197 0,864413

Lo(Tr)Te(Pe) 48 111,5661 2,45822 1,28E05

Glycinde multidens

TrPe 1 35,06349 7,33887 0,053591

TrTe(Pe) 12 13,11905 2,125134 0,032665

Lo(Tr)Pe 4 4,777778 0,773945 0,547574Lo(Tr)Te(Pe) 48 6,17328 1,409118 0,058649

Heleobia australis

TrPe 1 1325,73 4,315606 0,106335

TrTe(Pe) 12 185,4563 0,876424 0,575495

Lo(Tr)Pe 4 307,1944 1,45173 0,231624

Lo(Tr)Te(Pe) 48 211,6058 1,972796 0,000838

Oligochaeta

TrPe 1 38,11111 0,052423 0,830129

TrTe(Pe) 12 743,5807 2,743372 0,006522

Lo(Tr)Pe 4 726,9921 2,682169 0,042513

Lo(Tr)Te(Pe) 48 271,0463 1,346655 0,086954

Ostracoda

TrPe 1 8285,813 19,27058 0,011784

TrTe(Pe) 12 665,4153 3,720993 0,000541

Lo(Tr)Pe 4 429,9722 2,404398 0,062513

Lo(Tr)Te(Pe) 48 178,8274 1,419488 0,054828

Heteromastus sp.

TrPe 1 0,142857 0,026686 0,878157

TrTe(Pe) 12 7,205026 1,674197 0,102936

Lo(Tr)Pe 4 5,353175 1,243891 0,305077

Lo(Tr)Te(Pe) 48 4,303571 0,985909 0,507231

Mediomastus sp.

TrPe 1 3,813492 0,23577 0,652675

TrTe(Pe) 12 13,60053 1,069538 0,405706

Lo(Tr)Pe 4 16,1746 1,271961 0,294061

Lo(Tr)Te(Pe) 48 12,71627 1,544337 0,02341




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 inpopulation 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 studysite (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 insites 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 responddifferently to the same contaminants in distinct areas possibly be-cause of different histories of exposure to contamination ( Carman

et al., 2000).

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

Day

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

Day

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

Day

T o

t a l d e n s

i t y o

f m a c r o

b e n

t h o s

Control

Impact

A

N u m

b e r o

f s p e c

i e s

0 . 0

9 5 m −

2

0

B

D e n s

i t y o

f B . s t r i a t a 0

. 0 9 5 m −

2

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

5

1 0

1 5

2 0

2 5

0

1 0

2 0

3 0

4 0

5 0C

0 . 0

9 5 m −

2

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) Mediomast us sp. and (I) Heteromastus sp. before (73, 45, 32, 15, 3, 2, 1) and after impact (1, 2, 3, 15, 32, 45, 73) in thetreatments (impact and control).




The two morphotypes of oligochaetes proved tolerant or resil-

ient to the impact as shown by their high population densities afterday 2 in the impacted treatments. Similar results were reported byGomez 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 aresensitive 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 microcosmwith initial mortality followed by a later change in the trophicstructure. In our experimental analysis, live ostracods were not

found in the impacted treatments shortly after the spill, but highdensities 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 theprocesses that effectively controlled their distribution.

Descriptive or experimental analyses of the effects of oil spillson 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 reportincreases in densities of organisms. Chung et al. (2004), using

experimental mesocosms, observed that the density of deposit-feeders animals (both superficial or burrowers) decreased soonafter an oil spill. Some populations recovered pre-disturbance den-

sities only 42 days after the exposure. Lu and Wu (2006) recordedfast recolonization in oil contaminated sediments, but the domi-

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

Day

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

Day

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

Day

D e n s

i t y o

f G . m

u l t i d e n s

0 . 0

9 5 m −

2

D

D e n s

i t y o

f H . a u s t r a l i s 0 . 0 9 5 m −

2

E

D e n s

i t y o

f O l i g o c

h a e

t a 0

. 0 9 5

m −

2

0

2

4

6

8

1 0

1 2

0

2 0

4 0

6 0

0

2 0

4 0

6 0

8 0

1 0 0

F

Fig. 4. (continued)




nant species were very distinct from the control sediments for thefirst 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 sedimentsin Singapore. Andersen et al. (2008) observed that the abundanceand richness of the intertidal macrobenthos from impacted and

control sites were very similar just one month after an oil spill inGladstone (Australia). Ocon et al. (2008) recorded high densitiesof tolerant organisms in oil contaminated sites in the Río de la Plataestuary (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 intemperature, salinity, and other variables; and (9) type of affectedenvironment (Kennish, 1997). The hydrocarbon analyses indicated

that all impacted sediments were effectively contaminated by thediesel oil spillage. However, a trend of fast decrease in the totalconcentration of aliphatic hydrocarbons was observed during theexperimental period.

Significant changes in hydrocarbon composition occur due to

biological (biodegradation and bioturbation) and physical pro-

Day

D e n s

i t y o

f O s

t r a c o

d a

0 . 0

9 5 m −

2G

D e n s

i t y o

f M e d i o m a s t u s s p .

H

D e n s

i t y o

f H e t e r o m a s t u s s p .

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

Day

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

Day

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

0

2 0

4 0

6 0

8 0

0

5

1 0

1 5

2

0

2 5

0

2

4

6

8

1 0

I

0 . 0

9 5 m −

2

0 . 0

9 5 m −

2

Fig. 4. (continued)




cesses (selective dissolution, evaporation, photo-oxidation and

natural dispersion) that contribute to oil degradation (Readmanet al., 2002; Kingston, 2002; Schratzberger et al., 2003; Lu andWu, 2006; Morales-Caselles et al., 2008b; Andersen et al. 2008).According to Puente et al. (2009), the nature of the substrate, com-

posed of fine-grains, low permeability, and saturated sedimentsubstantially prevents oil penetration. Our study areas are underconstant tidal influence; and two of them (Site 1 and 3) are locatednear the mouth of rivers Maciel and Guaraguaçu, whose hydrody-

namics can further accelerate oil dilution and dispersion. Fast ratesof faunal recovery are known in areas with high tidal influence, asindicated by the presence of a large number of sensitive animals

(Ocon et al., 2008). Conversely, area 2, situated in a less exposedlocation and directly bordered by mangroves and plant debris (per-sonal observation), still contained detectable concentrations of to-tal aliphatics at the end of the experiment. Regardless of theseenvironmental differences and persistence of oil, significant

variations were recorded in the fauna among the three studiedareas. The IPC values confirmed that the n-alkanes present in thesediment of area 1 were not from diesel oil but of biogenic nature(Nishigima et al., 2001)

5. Conclusions

In general, the benthic macrofauna from a subtropical tidal flatwas resilient to the simulation of a heavy oil spill at the considered

spatial (cm2) and temporal scales (147 days). The initial recoloni-

zation and subsequent succession of the benthic community wasnot delayed in the oil contaminated sediments. There were no sig-nificant changes in overall abundance and number of species ex-cept just after the oil spill. The observed temporal variability was

not related to changes in the density and composition of tolerantor sensitive species because the dominant taxa were virtually thesame at the beginning and end of the experiment. In summary,

the significant variations in the structure of local associations weremore related to the temporal variability than to the presence of oilcontaminants in the sediment. Our experimental approach demon-strates that the macrobenthic associations in unvegetated tidalflats of a subtropical bay may behave in a resilient fashion and

be able to withstand minor oil spills or quickly recolonize oil dis-turbed sites.

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Table 5

PERMANOVA based on Bray-Curtis dissimilarities, without data transformation. p-

Values were obtained from 9999 permutations.

Source g MQ F p(MC)

PexTr 1 2016.4709 0.7012 0.6237PexLo(tr) 4 2875.7472 4.0654 10.0001

Te(Pe)xTr 12 2064.2548 1.3488 0.0723

Te(Pe)xLo(Tr) 48 1530.4526 2.1636 0.0001Residual 168 707.3747 – –

Total 251 – – –




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