Marine Pollution Bulletin 62 (2011) 1867–1876

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Marine Pollution Bulletin

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Recovery of macrobenthos in defaunated tropical estuarine sediments

Mônica L. Botter-Carvalho a,⇑, Paulo V.V.C. Carvalho b, Paulo J.P. Santos c

a Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irmãos 52171-900, Recife-PE, Brazilb Petrobras Transporte S/A (TRANSPETRO/DGN/GAS/TGN/NES/SMSOP), Rua Antônio Lumack Monte, 96, 4� andar 51020-905, Recife-PE, Brazilc Universidade Federal de Pernambuco, Av. Prof. Moraes Rêgo s/n, Cidade Universitária 50640-920, Recife-PE, Brazil

a r t i c l e i n f o

Keywords:BrazilEstuaryField experimentsIntertidalMacrozoobenthosMesoscale

0025-326X/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.marpolbul.2011.04.044

⇑ Corresponding author. Tel.: +55 21 33206321; faxE-mail address: monica.botter@db.ufrpe.br (M.L. B

a b s t r a c t

In the estuarine environment, hypoxia and/or anoxia have become a major cause of benthic defaunationand are strongly associated with increased eutrophication. Mesoscale field experiments were carried outto examine the recolonization and recovery time of macrobenthos after defaunation. Azoic sedimentswere achieved by covering four areas with polyethylene sheeting. Temporal changes and depth distribu-tion of macrobenthos within the defaunated sediments were compared with those in undisturbed naturalsediments at the same site. Within 3 days, annelids appeared as the first immigrants. After 153 days, theprocess of recovery had not yet been completed in terms of species richness. Whereas diversity and even-ness showed no significant differences between treatments during the entire experiment, multivariateanalyses proved that differences between treatments were still significant 93 days after the start of theexperiment.

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1. Introduction

A disturbance may be defined as any stochastic event thatcauses population changes, either due to density-independentmortality and/or to changes in the resource base of the community(Zajac and Whitlatch, 1982). Sediment disturbance is an importantforce structuring benthic communities (Diaz and Rosenberg, 1995),and is one of the most important ecological processes in maintain-ing the spatio-temporal heterogeneity of soft-bottom communities(Snelgrove et al., 1992). The effects of disturbance, recolonizationand succession on the community structure of sediment-dwellinginvertebrates have been a major focus in soft-bottom habitat stud-ies (Stocum and Plante, 2006). After disturbances, processes ofrecolonization followed by succession dynamics can re-establishthe structure of the macrofaunal community (Guerra-García andGarcía-Gomez, 2006).

In the estuarine ecosystem, the macrobenthos is one of thestructuring elements of the food web and plays an important rolein the system dynamics (Herman et al., 1999). Surveys of macro-zoobenthic communities are useful tools for assessing short- andlong-term changes in marine ecosystems, and thus for discriminat-ing between natural and man-induced disturbances.

The estuarine infaunal community is well adapted to naturalstress (e.g., tides and salinity). However, anthropogenic distur-bances have been an additional and growing cause of perturbations

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: +55 21 33206301.otter-Carvalho).

(Desprez et al., 1992) and frequently cause defaunation (Diaz andRosenberg, 1995). In the estuarine environment, hypoxia and/oranoxia have become a major cause of benthic defaunation andare strongly associated with increased eutrophication (Gallagheret al., 1983). Recolonization depends on the frequency and inten-sity of hypoxia events (Diaz and Rosenberg, 1995).

Experimental studies of recolonization, succession and recoveryin soft-sediment habitats have been conducted primarily at micro-scales (e.g., Gamenick et al., 1996; Hall and Frid, 1998; Bolam et al.,2004; Lu and Wu, 2006), and only a small number at mesoscales(e.g., Thrush et al., 1996; Beukema et al., 1999; Dernie et al.,2003; Ritter et al., 2005). Dernie et al. (2003) suggested that the re-sults of microscale experiments be extrapolated only to the small-est disturbance area, but even for a given scale, recovery times forany biological parameter vary strongly among studies, from oneday (Hall and Frid, 1998) to three months (Bolam et al., 2004) to re-cover macrofaunal abundance in microscale studies, for example.

Besides this limited background, it should be noted that exper-imental studies of zoobenthos recovery are particularly rare fortropical or even subtropical marine ecosystems; the microscalestudy in a subtropical estuary by Faraco and Lana (2003) is the sin-gle study carried out along the more than 7000 km-long Braziliancoast.

The aims of this study are to evaluate the recovery of the mac-rozoobenthos community in a tropical estuarine tidal flat aftercomplete mesoscale defaunation due to hypoxia/anoxia, and tocompare the main differences between tropical and temperateestuaries, considering the recovery times of the most commonlyevaluated biological parameters.

1868 M.L. Botter-Carvalho et al. / Marine Pollution Bulletin 62 (2011) 1867–1876

2. Materials and methods

2.1. Study site

The study area lies within a large estuarine complex composedby several small rivers connected to the Atlantic Ocean through theSanta Cruz Channel (Fig. 1). The experiment was conducted onItamaracá Island (northern Pernambuco State, Brazil) on a mud flat(7�46.184’S and 34�52.926’W) adjacent to the Santa Cruz Channel.The estuary receives organic wastes from small municipalities andfish farms, and inorganic nutrient inputs from agriculture andchemical effluents (CPRH, 2005). Near the study site, a five-yearmonitoring program (2001 to 2005) carried out in the Santa CruzChannel showed that dissolved oxygen concentrations range from2.5 to 6.5 mg/L and 4.4 to 6.4 mg/L in the dry and wet seasonsrespectively (CPRH, 2004/2006).

Regional climate is characterized by a mean annual precipita-tion of 2200 mm, of which approximately 80% falls during therainy season (April to August). The mean daily air temperatureand precipitation gradients between March and September 2005were obtained from the National Institute of Meteorology (INMET).Air temperature ranged from 22.9 �C (18 July 2005) to 28.4 �C(6 April 2005). Monthly precipitation ranged from 709.0 mm inJune 2005 to 45.3 mm in September 2005.

2.2. Experimental design and sampling

The experimental site area (upper intertidal zone of the mud-flat) is about 205 m2 and was divided into 2 experimental treat-ments (Defaunated and Control). Four replicate 2 x 2 m2 plots(experimental units) were delimited for each experimental treat-

Fig. 1. Location of the study area in the Santa Cruz Ch

ment. The Defaunated and Control plots were positioned about3 m from each other and about 200 m from low-tide level. Eachexperimental unit was subdivided into 64 smaller squares(0.25 x 0.25 m), termed sampling units (pseudo-replicates). Threesampling units for each experimental unit were randomly chosenin each survey, and their respective positions were located usinga numbered grid, ensuring that the same location within any plotwas not sampled more than once. Coupled biotic and abiotic sam-ples were taken within each sampling unit.

Defaunation was accomplished by covering the sediment sur-face with dark polyethylene sheeting for 70 days (adapted fromGamenick et al., 1996). Preliminary sampling was conducted to en-sure complete defaunation. The in situ recolonization experimentwas carried out from 5 April (day 0) to 5 September 2005 (day153).

Sediments were sampled for infauna with a cylindrical corer(7.2 cm inner diameter � 25 cm high) inserted to a depth of20 cm. In the field, each infaunal core was sectioned into 0–2, 2–5, 5–10 and 10–20 cm slices and fixed in 10% formalin-seawater.

In the laboratory, the samples were washed through a 0.3 mmmesh sieve and the retained organisms were preserved with 4%neutral formalin stained with Rose Bengal. Macrofauna weresorted out, identified to the lowest possible taxonomic level, andcounted, excluding nematodes and ostracods.

To quantify the top 5 cm, benthic microalgae biomass sampleswere collected using cores (1.2 cm inner diameter and 5 cm high),sliced into 0–2 and 2–5 cm fractions, transported on ice, and frozenat �20 �C until analysis. Chlorophyll-a (Chl-a) and total phaeopig-ments (Phaeo) concentrations were determined spectrophotomet-rically using the Lorenzen (1967) equations.

Redox profiles were measured following the methods outlinedby Pearson and Stanley (1979). Granulometry (silt–clay) was

annel and Itamaracá Island, Pernambuco, Brazil.

M.L. Botter-Carvalho et al. / Marine Pollution Bulletin 62 (2011) 1867–1876 1869

determined following Suguio (1973), and organic matter was ana-lyzed according to Wetzel and Likens (1990). These parameterswere obtained from sediment cores (4.6 inner diameter � 20 cmhigh) sliced into 0–2, 2–5, 5–10 and 10–20 cm strata.

2.3. Statistical Analysis

Data were analyzed using ANOVA, with factors: Experiment(levels: Defaunated and Control); Time (levels: 0, 1, 3, 5, 18, 35,93 and 153 days); and Depth (levels: 0–2, 2–5, 5–10 and 10–20 cm). After verifying homoscedasticity using the Cochran test,ANOVA was run using untransformed data. Fixed-factors 3-wayANOVA was carried out for each environmental variable. Tukey’spost hoc pair-wise comparison was used to distinguish means.All analyses used a significance level of 0.05 and followed the pro-cedures outlined by Zar (1996).

For macrobenthos, abundance values of sampling units wereaveraged to obtain a mean for each experimental unit of each differ-ent treatment. The total number of species (richness), total abun-dance, Shannon–Wiener index (H’ using loge), Pielou’s evennessindex (J’ using loge) and Taxonomic Distinctness index (D⁄) (War-wick and Clarke, 1995; Clarke and Warwick, 1998) were calculatedfor each sampling day. Only abundance data were logarithmicallytransformed to log(x + 1). As for the environmental data, 3-way AN-OVA was applied to each univariate community attribute. The Tu-key test was used for a posteriori comparisons. Analyses werecarried out using STATISTICA v.5 and PRIMER (Plymouth Routinesin Multivariate Ecological Research) version 6 (Clarke and Gorley,2006).

To prevent ‘‘noise’’ generated by species that show aggregateddistribution patterns, the Dispersion-based weighting procedure(Clarke et al., 2006) was applied to the abundance data. Before run-ning multivariate analyses and after Dispersion-based weighting,abundance data were transformed to log(x + 1) to down-weightconsistently abundant species relative to equally consistent butless numerous species.

Changes in macrofaunal community structure were analyzedusing Non-metric multidimensional scaling (MDS) (Clarke, 1993)

Fig. 2. Changes in redox potential (Eh) in each stratum during the expe

and Bray–Curtis similarity indices. The ANOSIM 2-way crossedprocedure (Clarke, 1993) was used to test for factor effects Exper-iment (Defaunated and Control) and Time (days). To assesswhether the assemblage development in the Defaunated and Con-trol areas followed similar trajectories, Spearman rank correlationswere calculated using the routine RELATE (Clarke and Gorley,2006).

SIMPER analysis of similarity percentages (Clarke, 1993) wasused to rank the macrofauna taxa that contributed most to theaverage Bray-Curtis dissimilarities. All calculations were per-formed using the PRIMER package, version 6.

3. Results

3.1. Chemical–physical parameters and sediment properties

Fig. 2 shows the depth profiles of the mean redox potentials inthe two experimental areas for each stratum. The redox potentialvalues indicated reduced sediments from day 0, in the Defaunatedand Control areas (Fig. 2). The sediment analysis revealed thatthere were significant differences of Eh (3-way ANOVA, F21,192 =2.5016; p < 0.001) among Times, Experiments and Depths. After5 days these differences were less apparent, and the redox poten-tial was similar between the Defaunated and Control areas.

The sediment analysis revealed that, at the start of the experi-ment, there were no significant differences in organic matter orsilt + clay contents (3-way ANOVA, p > 0.05) for both treatments.The mean values for organic matter varied from 4.75% to 5.77%in the Control area and from 4.39% to 5.60% in the Defaunated area.The silt + clay content varied from 43.34% to 42.02% in the Controlarea (days 5 and 153, respectively) and from 49.70% to 41.98% inthe Defaunated area (days 5 and 153, respectively).

3.2. Chlorophyll-a and Phaeopigments

Chlorophyll-a values ranged from 6.74 to 34.24 lg cm�2 in theControl area and from 3.55 to 21.64 lg cm�2 in the Defaunatedarea. Chl-a concentrations were significantly different among

riment. (mean ± SE, n = 4) (Tukey ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001).

1870 M.L. Botter-Carvalho et al. / Marine Pollution Bulletin 62 (2011) 1867–1876

Experiments (3-way ANOVA, F1,96 = 39.187; p < 0.001) and Depth(F1,96 = 186.465; p < 0.001). There was no significant change inchlorophyll-a between Time levels (p = 0.321), but the interactionbetween Time and Experiments was significant (F7,96 = 4.063;p < 0.001). Post-hoc comparisons showed that the concentrationof chlorophyll-a was significantly different among areas only dur-ing the first three days. On days 0, 3 and 5, chlorophyll-a concen-trations in the Defaunated area were 71%, 36% and 61% lowerthan in the control area (Tukey, p < 0.01, p < 0.01 and p < 0.05,respectively).

Phaeopigment values ranged from 15.32 to 32.53 lg cm�2 inthe Control area and from 13.55 to 31.18 lg cm�2 in the Defaunat-ed area. Phaeopigment concentrations were lower in the Defaunat-ed area, with significant differences between Experiments (3-wayANOVA; F1,96 = 15.106; p < 0.001) but without significant interac-tion between days, Experiment and Depth (3-way ANOVA,p > 0.05).

3.3. Colonization by Macrofauna

A total of 24 taxa and 7 phyla (3107 individuals and 17 taxa inthe Defaunated area and 15,500 individuals and 22 taxa in the Con-trol area) were collected. Seven taxa were exclusively found in thecontrol area, and two taxa were present only in the defaunatedarea. Initial colonization occurred quickly in the defaunated sedi-ment. Sphaerosyllis sp., Streblospio benedicti and Sigambra grubiiadults colonized the azoic sediments within 1, 3 and 5 days respec-tively in very low numbers, and their abundances were lower com-pared to control sites. Capitella spp. colonized defaunatedsediments within 18 days in the upper sediment.

3.4. Univariate Community indices and vertical recolonizatiom

Six taxa were considered sufficiently abundant for independentstatistical analysis. These were the polychaetes Capitella spp., Lae-

Table 1Results of three-way ANOVAs for the main effects: experiment (Defaunated and Control), deabundance (Log(x + 1)), species number (S), Shannon–Wiener Diversity (H0) and Taxonom⁄⁄⁄p < 0.0001).

Attributes Sources of variations Degrees of freedo

Abundance Day 7Experiment (Exp.) 1Depth 3Day � Exp. 7Day � Depth 21Exp. � Depth 3Day � Exp � Depth 21

S Day 7Experiment (Exp.) 1Depth 3Day � Exp. 7Day � Depth 21Exp. � Depth 3Day � Exp � Depth 21

H’ Day 7Experiment (Exp.) 1Depth 3Day � Exp. 7Day � Depth 21Exp. � Depth 3Day � Exp � Depth 21

D⁄ Day 7Experiment (Exp.) 1Depth 3Day � Exp. 7Day � Depth 21Exp. � Depth 3Day � Exp. � Depth 21

onereis acuta, S. grubii, Sphaerosyllis sp., S. benedicti and the oligo-chaetes Tubificidae spp. Twenty-two taxa were collected in theControl area during the experiments, and 8 occurred in the abun-dance class 1.0–1.5 (1–5 ind.). However, in the Defaunated area,only three taxa (Tubificidae spp., L. acuta and S. benedicti) reachedthe higher-abundance classes by the end of the experiment (Table3).

The vertical distributions of the most abundant species in theDefaunated and Control areas over time are shown in Fig. 4. Theupper 0–2 cm surface layer was dominated by polychaetes, andin deeper layers the oligochaetes Tubificidae spp. dominated.Approximately 80% of individuals of S. benedicti and L. acuta werepresent in the topmost 5 cm layer (Fig. 4). Capitella spp. and Sph-aerosyllis sp. occurred almost entirely in the 0–2 cm stratum(>80% and >50% total abundance, respectively) (Fig. 4).

Time, Experiment and Depth showed highly significant differ-ences in diversity, total abundance, species number and taxonomicdistinctness (3-way ANOVA, p < 0.001 for each effect). Only theinteraction between Time x Experiment was significant for abun-dance (p < 0.001), species number (p < 0.001) and taxonomicdistinctness (p < 0.001) (Table 1). The analysis for taxonomic dis-tinctness indicated an interaction between Experiment � Depth(p < 0.0033) and Time � Experiment � Depth (p < 0.0177) (see Ta-ble 2).

Taxonomic distinctness, species number and abundance stabi-lized after 18, 35 and 93 days, respectively (Tukey, p > 0.05)(Fig. 3). Although evenness and Shannon-Wiener were lower inthe Control sediments, the differences were non-significant overthe experimental period (Fig. 3).

Total abundance of macrozoobenthic animals within the Defau-nated plots was significantly below that of the Control areas. Therecolonization patterns in defaunated sediment were manifestedby a gradual increase in both total abundance and species richnessbetween days 0 and 18, declining after day 35 (Fig. 3) and increas-ing gradually until the end of the experiment (day 153).

pth (0–2, 2–5, 5–10, 10–20 cm) and days (0, 1, 3, 5, 18, 35, 93, 153), and interactions inic Distinctness index (Log(x + 1)) (D⁄) (ns: no significant effects; ⁄p < 0.05; ⁄⁄p < 0.01;

m (df) Variance ratio (F) Significance level (p)

11.0713 0.000⁄⁄⁄

380.8294 0.000⁄⁄⁄

84.1231 0.000⁄⁄⁄

16.2601 0.000⁄⁄⁄

2.1012 0.004⁄⁄

2.0097 0.031⁄

2.0722 0.005⁄⁄

4.3637 0.000⁄⁄⁄

142.9692 0.000⁄⁄⁄

57.8962 0.000⁄⁄⁄

9.9480 0.000⁄⁄⁄

0.7516 ns2.1053 ns2.2563 0.002⁄⁄

4.8480 0.000⁄⁄⁄

29.5119 0.000⁄⁄⁄6.3153 0.000⁄⁄⁄1.4760 ns1.5264 ns2.4398 ns1.5332 ns

7.5742 0.000⁄⁄⁄

122.4885 0.000⁄⁄⁄

19.4398 0.000⁄⁄⁄

6.8677 0.000⁄⁄⁄

1.1049 ns4.7254 0.003⁄⁄

1.8342 0.017⁄

Table 2Results of Tukey tests for differences between macrobenthic communities of theDefaunated (D) and Control (C) areas, considering days and Strata (ns: no significanteffects;⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.0001) (S: species number; D⁄: TaxonomicDistinctness Index).

Sources Day Strata

0–2 cm 2–5 cm 5–10 cm 10–20 cm

Abundance (D � C) 0 0.0000⁄⁄⁄ 0.0000⁄⁄⁄ 0.0000⁄⁄⁄ 0.0333⁄

1 0.0000⁄⁄⁄ 0.0000⁄⁄⁄ 0.0000⁄⁄⁄ ns3 0.0000⁄⁄⁄ 0.0000⁄⁄⁄ 0.0006⁄⁄ ns5 ns 0.0000⁄⁄⁄ 0.0018⁄⁄ ns

18 ns ns ns ns35 ns ns 0.0057⁄⁄ ns93 ns ns ns ns

153 ns ns ns ns

S (D � C) 0 0.0000⁄⁄⁄ ns ns ns1 0.0000⁄⁄⁄ ns ns ns3 0.0197⁄ ns ns ns5 ns ns ns ns

18 ns ns ns ns35 ns ns ns ns93 ns ns ns ns

153 ns ns ns ns

D⁄ (D � C) 0 0.0077⁄⁄ 0.0086⁄⁄ 0.0075⁄⁄ ns1 ns ns 0.0086⁄⁄ ns3 ns ns 0.0049⁄⁄ ns5 ns ns 0,0108⁄ ns

18 ns ns ns ns35 ns ns ns ns93 ns ns ns ns

153 ns ns ns ns

M.L. Botter-Carvalho et al. / Marine Pollution Bulletin 62 (2011) 1867–1876 1871

Abundance in the Control area showed the same pattern (Fig. 3).Diversity and evenness did not show temporal patterns.

Changes in evenness (J0) indicated an interaction betweenexperiments and days (3-way ANOVA, p < 0.000), but did not differsignificantly over depth (Tukey, p > 0.05).

3.5. Multivariate analyses

MDS ordination showed a clear difference between Experi-ments on all sampling days, and a conspicuous separation betweenControl and Defaunated temporal trajectories, with no intercept(Fig. 5). This pattern was reinforced by the ANOSIM test, whichindicated significant differences between the Defaunated and Con-trol areas (Global R = 0.548; p = 0.001%). The changes in the valuesof the R-statistic determined by ANOSIM tests showed strong signsof temporal convergence between the Defaunated and Controlcommunities (Fig. 5). The average dissimilarity decreased gradu-ally from 99.47% to 46.92% over the 5 months (SIMPER analysis).The temporal trajectories in the Defaunated and Control macrofa-unal communities were significantly related (RELATE:Rho = 0.615, p = 0.001), and indicated similar patterns betweenexperimental areas. Until sampling day 35, differences in commu-nity structure were observed along the horizontal line, and a sea-sonal pattern was evident on the vertical axis. MDS ordinationshowed that modifications in community structure over time weresimilar in both areas, and could be attributed to climate seasonality(days 35–153) (Fig. 5).

The four observed macrofaunal recolonization phases appear inthe MDS plot (Fig. 5). The first phase (days 0–3) suggests that fewchanges in community structure occurred in both the Defaunatedand Control areas. The second phase (days 3–18), with distanceincreasing between days 3–18, indicated rapid changes in the com-munity structure in the Defaunated area, while in the Control areathe community remained homogeneous. The third phase (days 18–35) was characterized by a directional change (horizontal to verti-cal) and with a parallel trajectory and decreasing dissimilarity

among Experiments. The last phase included days 35–153, whenthe dissimilarities continued to decrease.

SIMPER analysis revealed that differences between the Defau-nated and Control plots mainly occurred as a result of reducedabundance or absence of some species in the defaunated plots(e.g., S. grubii , Heteromastus sp. and Tubificidae spp.) (Table 3).The only exception to this was the high numbers of L. acuta, whichshowed a clear opportunistic behavior, with its abundance signifi-cantly increasing in the defaunated area (ANOVA day 35:F = 15.3465, p = 0.008; day 93: F = 120.5591, p = 0.0002), contribut-ing 10% of the dissimilarity between areas.

The average dissimilarity between the Experimental areas alongthe different strata increased with depth (0–2 cm: Global R = 0.275,p = 0.01%; 2–5 cm: Global R = 0.367, p = 0.01%; 5–10 cm: GlobalR = 0.438, p = 0.01%; 10–20 cm: Global R = 0.577, p = 0.01%). How-ever, the temporal trajectory was significantly correlated only inthe 0–2 and 2–5 cm strata (RELATE: 0–2 cm: Rho = 0.668,p = 0.13%; 2–5 cm: Rho = 0.554, p = 2.12%; 5–10 cm: Rho = -0.071,p = 59.09%; 10–20 cm: Rho = 0.384, p = 5.98%). The different phasesthat were previously suggested occurred only in the upper stratum(0–2 cm). Differences between the defaunated and control commu-nities along the vertical profile were due to the most abundantpolychaetes, including S. grubii, L. acuta and the oligochaetes Tub-ificidae spp. S. grubii dominated in the sub-surface sediment (80%between 2–10 cm) and contributed the most (>34%) to dissimilar-ity. The vertical stratification of L. acuta was well marked, withmore than 80% of the individuals in the top 2 cm in both areas(Fig. 4).

4. Discussion

The initial response of infauna to disturbance may be deter-mined by the availability of colonists, or by life-history featuresthat enable a species to preferentially settle into and exploit distur-bance habitats (Zajac and Whitlatch, 1982). The variability in theresponse of the macrofauna to disturbances emphasize the impor-tance of carrying out mesoscale studies, that will allow better-founded inferences and can be extrapolated to the naturalenvironment

After 30 days, 11 taxa were pioneers in the Defaunated area: L.acuta, Sphaerosyllis sp., Tubificidae spp., S. benedicti, Exogone sp., S.grubii, Eunicidae sp., Cerianthus sp., Gastropoda sp., Capitella spp.,Ceratopogonidae and Tellina sp. Of these, only Tubificidae spp.,Capitella spp., S. benedicti and Cerianthus sp. were considered asopportunistic species in other studies (Pearson and Rosenberg,1978; Zajac and Whitlatch, 1982; Morris and Keough, 2001), show-ing marked resistance to hypoxia (Levin, 1984; Diaz and Rosen-berg, 1995). In this study, Capitella spp., S. benedicti and theTubificidae spp. did not show opportunistic patterns, because thesetaxa occurred only in low abundance in the Defaunated area. In ac-cord with these observations, many studies have shown thatopportunism is not a common pattern for species of the Capitellacapitata-complex (Zajac and Whitlatch, 1982; Morris and Keough,2001).

Only the polychaete L. acuta was more abundant in the defau-nated area compared to the control after the first four weeks (untilday 35), showing the typical characteristics of an opportunisticspecies. This species is very common in southern and northeasternBrazil (Lana et al., 1989), as well as in areas polluted by organic(domestic) sewage (Dauer and Conner, 1980), sharing dominancewith C. capitata in sediments of degraded areas (Pagliosa, 2005).

Although several variables were analyzed, fine sediment (siltand clay) or organic-matter concentrations were not found toinfluence the recolonization, suggesting that differences amongsites were independent of sediment factors.

Table 3Temporal variation of macrofaunal taxa based on abundance classes (4th-root transformed data) for each treatment. Underline indicates the first occurrence of a taxon after thebeginning of the experiment.

Abundance class Defaunated (days) Control (days)

0 1 3 5 18 35 93 153 0 1 3 5 18 35 93 153

5.0–5.5 Tub

4.5–5.0 Tub Tub

4.0–4.5 Tub Tub

3.5–4.0 Lae Lae Tub Lae LaeTub

3.0–3.5 Tub

2.5–3.0 Lae Lae Tub Str

2.0–2.5 Tub Tub Tub Str Sph Lae Lae LaeLae Sig Str

Str

1.5–2.0 Lae Tub Str Sig Sph Cap Sig Sig Sig Sig StrCap Cap Sig Cap Sph SigCer Lae ExoStr Exo

Str

1.0–1.5 Tub Tub Tub Sph Exo Exo Sig Het Exo Exo Sph Exo Cap Sph Str CapLae Str Sph Het Sig Pol2 Cer Nem Cer Tel Exo SphStr Sph Sig Sph Sph Sph Lae Cap

Cap Boc2 TypCet Cer

TelCap

0.5–1.0 Lae Gas1 Sph Exo Sig Str Exo Nem Ano Nem Tel Tel Cer Eun Gam2 SphLae Cet Gas Cap Cap Cet Tel Pol Aca2 Ano Str Cer GamSph Str Tel Cer Eun Iso Cet Cer Luc Cet Tel TelEun Sig Cer Cyr1 Tip2 Tip Het Het Ano

Iso Luc2 LucTel Nem HetAno Tel

Typ2

1 – Taxa exclusive of Defaunated area; 2 – Taxa exclusive of Control area; Aca – Acarina; Gam – Gammaridae; Ano – Anomalocardia; Boc – Boccardia; Cap – Capitella; Cer –Ceratopogonidae; Cet – Cerianthus; Cyr – Cyrtoplax; Eun – Eunicidae; Exo – Exogone; Gas – Gastropoda; Het – Heteromastus; Iso – Isolda; Lae – Laeonereis; Tip – Tipulidae; Luc –Lucina; Nem – Nemertea; Pol – Polydora; Sig – Sigambra; Sph – Sphaerosyllis; Str – Streblospio; Tel – Tellina; Tub – Tubificidae; Typ – Typosyllis.

1872 M.L. Botter-Carvalho et al. / Marine Pollution Bulletin 62 (2011) 1867–1876

Tropical estuaries are subjected to extreme seasonal fluctua-tions, because of the reduction of river discharge in the dry periodsand increased flow during the rainy periods. Several studies haveemphasized the influence of seasonal climate variations on theprocess of recolonization and succession of macrofauna (Hall andFrid, 1998; Beukema et al., 1999; Lu and Wu, 2007). In the presentstudy, this influence could be ascertained by the similar temporalpatterns of variation in community structure in the experimentalareas (MDS ordination) after day 35. The distances that separatedthe Control and Defaunated areas from days 0–5 seem to be morerelated to different macrofauna community structures, whereasfrom day 35 to the end of the experiment, they seem to be dueto large-scale seasonal factors such as precipitation. These resultsreinforce the importance of natural factors such as air temperatureand/or precipitation in tropical estuarine environments, as statedabove.

The abundance values in the Control area reached their lowestpoint on day 35, possibly influenced by variations in rainfall. Theexception was S. grubii, which reached its highest abundance onday 35. In the Defaunated area, the recolonization rate was slowduring the initial 35 days. The start of the rainy season (after day35) benefited the growth of some populations, as observed after93 days, especially for S. benedicti, L. acuta and Tubificidae spp. Con-sequently, the differences between abundances in experimentalareas were reduced and were no longer significant after 93 daysof the experiment. This resulted from a sharp decline of the mostabundant species in the control area and increased abundances

of S. benedicti, L. acuta and Tubificidae spp. in the defaunated areaby the end of the experiment (153 days).

The recolonization processes of marine habitats have been con-sidered to be relatively rapid (Table 4), ranging from days tomonths and years. The time to complete recovery of defaunatedsediments depends on the attributes used (e.g., abundance, rich-ness, diversity, evenness). In the present mesoscale study, therecovery of the Defaunated area was reached in 18 days based onthe taxonomic distinctness index, later based on richness(35 days), and over the longest period (93 days) based on abun-dance. The conclusion that abundance was the attribute to recovermost slowly in the study area disagrees with results obtained byother investigators (e.g., Gamenick et al., 1996; Beukema et al.,1999; Faraco and Lana, 2003), whose results indicated that abun-dance recovered more rapidly.

The Shannon diversity index showed no significant differencesbetween the Control and Defaunated areas throughout the experi-ment, suggesting that it cannot be considered a good indicator ofrecovery. The nature of the Shannon index, which is independentof the identity of species and heavily dependent on the samplingeffort (Warwick and Clarke, 1991, 1995) makes it difficult to useas a measure of community recovery in the first days ofrecolonization.

As for the Shannon diversity index, the measure of a commu-nity’s taxonomic distinctness (D⁄) also cannot be considered agood tool to measure recovery in this type of system. The rapidmacrofauna recovery (18 days) in terms of taxonomic distinctness

Fig. 3. Comparison of univariate attributes between defaunated and control sediments during the study period (mean ± S.E). (n = 4; Tukey, ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001).Note non-linear time scale.

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may be related to the simplicity of the taxonomic structure in thestudy area (muddy tidal flat), a naturally stressed environment,closely analogous to polluted environments and therefore poor inspecies and dominated by the few phyla that are able to adapt tothe extremely variable conditions (Cognetti, 1992).

Some studies have shown higher richness in the natural areas atthe end of recolonization experiments, suggesting that the com-plete recovery of the defaunated area had been achieved (e.g.,Thrush et al., 1996; Lu and Wu, 2006). At the end of this experi-ment, the Control area contained seven exclusive species of a totalof 22, suggesting a lower total richness in the Defaunated area. Thisindicates that on day 153, the Defaunated area still contained aspecies-poor community, whose process of recovery had not yetbeen completed. In the Control and Defaunated areas, rare speciesaccounted for about 73% and 65% of the total richness, respectively.Ruth et al. (1994) observed in a recolonization experiment that

rare species accounted for 32% to 49% of all taxa in different sizesof defaunated microcosms. Thus, the importance of rare speciescannot be neglected, since they are often regarded as more threa-tened with extinction than the common ones and more sensitive todisturbance, making them the most reliable indicators of degrada-tion in aquatic environments (Cao et al., 1998).

Multivariate analysis of changes in the community structure ledto different conclusions from those indicated by the univariate in-dexes. This is due to the greater effectiveness of the multivariatemethods, which are dependent on species identity (e.g., MDS ordi-nation and ANOSIM), compared to the univariate indexes, whichare independent of the taxa involved (e.g., the Shannon index) indetecting changes in communities (Warwick and Clarke, 1991;Warwick and Clarke, 1993). Dernie et al. (2003) were also not suc-cessful in evaluating the recolonization of a defaunated area usingrichness, Shannon diversity and evenness in an experiment

Fig. 4. Temporal changes for the six most abundant species found in the defaunated and control areas over time. Note non-linear time scale. Error bars represent ±95%confidence intervals (n = 4).

Fig. 5. Non-parametric multi-dimensional scaling ordination based on meanabundance of species from each treatment. Letters indicate (C) Control area and(D) Defaunated area. Numbers refer to sampling days.

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simulating mesoscale physical disturbances. However, their resultsfrom a multivariate analysis (ANOSIM) indicated a communityrecovery between 64 and 208 days, depending on the intensity ofthe disturbance.

The present experiment clearly showed differences in therecovery rate of the different macrofaunal taxa in the differentsampling depths, showing that the rapidity of recolonization wasinversely proportional to the preferred depth. MDS and ANOSIMresults showed increased dissimilarities with increasing depth forboth the Defaunated and Control areas. The fact that the temporaltrajectories of recolonization in the 5–10 and 10–20 cm strata didnot show a significant correlation between the experimental areasmay be an indication that the influence of natural surroundings onthe colonization was not observed in the deeper strata until after5 months.

Table 4 presents the results obtained in different studies ofinfauna recolonization in estuarine sediments defaunated byhypoxia/anoxia, which have been carried out in temperate andsubtropical regions. In general, the recovery time of the estuarinemacrobenthos recorded in this study was more rapid whencompared with times observed for temperate and subtropicalcommunities.

Table 4Recovery times of the macrofauna of defaunated areas, for univariate community attributes, with respect to the different types of scale of disturbance (⁄tropical region;⁄⁄subtropical region; ⁄⁄⁄temperate region; H0: Shannon–Wiener Diversity Index; J0: Pielou’s Equitability Index).

Disturbance scale Reference Type of study Community recovery time

Abundance Richness H0 J0

Macroscale Dauer and Simon (1976)⁄⁄ Descriptive – 11 months – –Beukema et al. (1999)⁄⁄⁄ Experimental <6 months <6 months – –Lim et al. (2006)⁄⁄ Descriptive 1–4 months – – –Thrush et al. (1996)⁄⁄⁄ Experimental >9 months – – –

Mesoscale Moseman et al. (2004)⁄⁄ Experimental 14 months >19 months – –Ritter et al. (2005)⁄⁄ Experimental 2 weeks – – –Present Study⁄ Experimental 3 months 35 days – –

Microscale McCall (1977)⁄⁄⁄ Experimental – 10 days 3 months –Levin (1984)⁄⁄⁄ Experimental >40 days 8–21 days – –Reise (1984)⁄⁄⁄ Experimental >10 days – – –Smith and Brumsickle (1989)⁄⁄⁄ Experimental 8–41 days – – –Gamenick et al. (1996)⁄⁄⁄ Experimental 20 days 20 days 20 days 20 daysHall and Frid (1998)⁄⁄⁄ Experimental 1 day – – –Lu and Wu (2000)⁄⁄ Experimental 4–10 months 4–10 months 4–10 months –Faraco and Lana (2003)⁄⁄ Experimental >64 days >64 days – –Bolam et al. (2004)⁄⁄⁄ Experimental 3 months – – –

– – –Guerra-García and García-Gomez (2006)⁄⁄⁄ Experimental 15 days – – –

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Although we were able to reach some conclusions about the ra-pid recovery capacity of the infaunal estuarine community in thistropical mud flat, further investigations based on larger spatialscales and longer time series are needed in other, related sites be-fore these results can achieve a more universal value for experi-mental studies. Also, we infer that univariate measures ofcommunity structure and Taxonomic Distinctness Index were notsensitive enough to capture temporal differences of recovery ofmacrobenthos in a naturally stressed environment, while multivar-iate approaches were too robust to discriminate subtle changes.

Based on the preceding discussion, we conclude that results re-ported are useful for the management and protection of tropicalestuarine habitats endangered by nutrient over-enrichment.

Acknowledgements

The authors wish to express their thanks to the CNPq for thefinancial support n.477972/2004-7, for the research fellowship toPJP Santos n.305609/2004-1 and for the PhD scholarship awardedto M. L. Botter-Carvalho. The authors are grateful to Janet W. Reidfor English language revision.

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