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Are eulittoral assemblages suitable for detectingthe effects of sewage discharges in Atlantic andMediterranean coastal areas?J. Cabral-Oliveiraa, S. Bevilacquab, A. Terlizzib & M.A. Pardalaa CFE – Centre for Functional Ecology, Department of Life Sciences, University of Coimbra,Coimbra, Portugalb Laboratory of Zoology and Marine Biology, Department of Biological and EnvironmentalSciences and Technologies, University of Salento, Lecce, ItalyPublished online: 14 Aug 2014.

To cite this article: J. Cabral-Oliveira, S. Bevilacqua, A. Terlizzi & M.A. Pardal (2014) Are eulittoral assemblages suitablefor detecting the effects of sewage discharges in Atlantic and Mediterranean coastal areas?, Italian Journal of Zoology, 81:4,584-592, DOI: 10.1080/11250003.2014.947336

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Are eulittoral assemblages suitable for detecting the effects of sewagedischarges in Atlantic and Mediterranean coastal areas?

J. CABRAL-OLIVEIRA1*, S. BEVILACQUA2, A. TERLIZZI2, & M.A. PARDAL1

1CFE – Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal, and2Laboratory of Zoology and Marine Biology, Department of Biological and Environmental Sciences and Technologies,University of Salento, Lecce, Italy

(Received 5 November 2013; accepted 14 July 2014)

AbstractThe search for cost-effective indicators able to detect anthropogenic impacts is an essential prerequisite to plan mitigationinterventions, which can prevent further deterioration of coastal areas. Eulittoral assemblages are accessible for sampling,and their dominant species are easy to identify and widely distributed. This study assessed the potential of eulittoralassemblages to reflect environmental perturbations related to sewage discharge across different environmental and geogra-phical locations. Sampling occurred in impacted (near sewage discharge) and control areas, on both Atlantic andMediterranean shores. Multivariate analyses showed that the structure of eulittoral invertebrate assemblages differedsignificantly between control and impacted areas for the Mediterranean, but not for the Atlantic case study. Macroalgaeresponses to sewage discharge were found on the Mediterranean shore with a significantly higher abundance of Chlorophytanear the outfall. Limpet populations of the genus Patella appear to be affected by sewage discharge. The response of limpetpopulations differed between Atlantic and Mediterranean shores, probably due to among-species differences of ecologicaltraits. Our results suggest that the response of eulittoral assemblages and limpets to sewage discharge might be strictlycontext-dependent, thus limiting their potential to serve as general indicators in monitoring the effects of this source ofhuman impact over large spatial scales.

Keywords: Nutrient enrichment, ecological indicators, eulittoral assemblages, rocky shores

Introduction

Coastal areas are constantly under the threat of anthro-pogenic activities, being one of the marine ecosystemsmost affected by human disturbance (Crowe et al.2000; Halpern et al. 2008). As a result, several initia-tives at the European scale, such as the WaterFramework Directive (WFD, 2000/60/EC) and theMarine Strategy Framework Directive (MSFD, 2008/56/EC), have been enforced to manage marineresources, to assess human impacts on marine ecosys-tems and to implement cost-effective methods forlarge-scale monitoring of coastal areas. Both directivesare based on the same management principle: asses-sing the health of aquatic ecosystems by determiningthe deviation from the expected control conditionsand, in case of degradation, intervening to recover agood environmental status (Van Hoey et al. 2010).

Benthic invertebrates play an important role in thisevaluation, being used worldwide to assess the statusof marine, estuarine or freshwater ecosystems. Theyhave been proved to respond to several types of anthro-pogenic impacts, from physical disturbance to chemi-cal contamination or nutrient enrichment (Teixeiraet al. 2010 and references therein). Moreover, theirrole as ecological indicators is widely accepted due totheir sedentarism, long lives and easy sampling, and tothe existence of an extensive literature on their distri-bution in specific environments and on their responseto different environmental stresses (Simboura &Zenetos 2002; Fano et al. 2003; Borja & Dauer 2008;Dauvin et al. 2010).One of the most widespread and harmful sources

of human disturbance in coastal areas is nutrientenrichment due to sewage discharges (Fraschettiet al. 2006; Halpern et al. 2007). In Europe, sewage

*Correspondence: J. Cabral-Oliveira, CFE – Centre of Functional Ecology, Department of Life Sciences, University of Coimbra, 3001–401 Coimbra, Portugal.Tel: +35 1962359168. Email: joanaco@ci.uc.pt

Italian Journal of Zoology, 2014, 584–592Vol. 81, No. 4, http://dx.doi.org/10.1080/11250003.2014.947336

© 2014 Unione Zoologica Italiana

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can receive secondary (organic matter removed) ortertiary (nutrients and bacteria removed) treatment,and then be released directly near shore, or at somedistance through pipeline systems. Sewage outfallsdischarging near rocky shores affect subtidal andintertidal hard-bottom communities located nearby.At the community level, changes in the structure ofepifaunal assemblages were observed, with the repla-cement of sensitive species by tolerant ones (Littler& Murray 1975; Archambault et al. 2001; Bishopet al. 2002; Cabral-Oliveira et al. 2014), but thereis no consensus about the effect on the species rich-ness. Polychaete assemblages seem to increase theirabundances and species richness near sewagedischarges, due to opportunistic species fromfamilies like Capitellidae or Spionidae (Dauer &Conner 1980; Elias et al. 2006). Crustacean andbivalve filter-feeders (e.g. Balanidae, Mytilidae)have been reported to be more abundant near sew-age discharges due to the higher amount of sus-pended solids (López-Gappa et al. 1993; Pinedoet al. 2007). However, amphipods seem to be verysensitive to environmental changes produced by thepresence of sewage discharges (Dauvin & Ruellet2007). A general decrease in abundance and thedisappearance of sensitive species near sewage dis-charges is known for gastropod assemblages (Terlizziet al. 2005b; Atalah & Crowe 2012). Gastropods alsoseem to reflect the diversity patterns of the wholecommunity (Smith 2005), which demonstrates theirpotential as bioindicators of anthropogenic impacts.Furthermore, previous studies on the effects of sew-age pollution on limpets have noticed a reduction inthe number of species near the outfall (e.g. Espinosaet al. 2007), suggesting that limpet species might begood indicators of nutrient enrichment associatedwith sewage effluents. Other studies reported asignificant increase in the density of limpets (e.g.Patelloida lastritigata) in intertidal areas impacted bysewage discharges with respect to unaffected areas(Bishop et al. 2002).

Intertidal areas of rocky shores, including eulit-toral assemblages, are often exposed to sewage dis-charges (Ashton & Richardson 1995; Smith et al.2000; Fraschetti et al. 2006; Bustamante et al.2012; Garaffo et al. 2012). Eulittoral assemblagesare generally accessible to sample, and the speciespresent have widespread distributions and are easy toidentify at high taxonomic resolution. Moreover,eulittoral assemblages usually present lower speciesrichness than assemblages characterizing the sublit-toral fringe. These features make eulittoral assem-blages potentially cost-effective in discerningbetween disturbed and undisturbed environmentalconditions across different geographical regions.

Nevertheless, to our knowledge, no attempt hasbeen made to understand whether eulittoral assem-blages may represent a suitable indicator in monitor-ing human impacts related to sewage discharges,able to consistently respond over large spatial scales.This paper aims to evaluate the potential of eulittoral

assemblages to reflect the effects of sewage pollution incoastal areas from different regions. For this purpose,eulittoral assemblages were studied in two differentenvironmental contexts (near sewage discharges, andin control conditions) and also in different geographi-cal regions (Atlantic andMediterranean shores). At thesame time, the response of the most common andabundant taxa (i.e. barnacles, Melarhaphe neritoides,Patella spp., Rhodophyta, Chlorophyta, Phaeophyta)was evaluated individually, in order to assess whetherspecific organisms can be used as effective indicators ofenvironmental changes induced by sewage discharges.

Materials and methods

Study areas

The study areas were located on the central WesternPortuguese shore (east Atlantic) and on the south-east Italian shore (Mediterranean Sea) (Figure 1). Inthe Mediterranean Sea the mean temperature of sur-face waters varies between 14–16°C in winter and22–28°C in summer. Tidal amplitudes are small(max 50 cm). Evaporation greatly exceeds precipita-tion and river runoff, so the Mediterranean Sea ischaracterised by very high salinity (≈38‰) (Barale2008). On the Atlantic shores (Iberian Peninsula)the temperature of the sea surface varies between13°C and 15°C during winter and 20–22°C duringsummer. The extreme spring tides range from 3.5 to4 m. Surface salinity varies between 35 and 36‰(Boaventura et al. 2002).On both shores (Atlantic and Mediterranean),

impacted locations were characterized by the presenceof an outfall discharging effluents directly on therocky shore in the intertidal zone. Previous studieshave already reported the impact of sewage dischargeson macrofauna, meiofauna and fish assemblages inthese areas (Guidetti et al. 2003; Terlizzi et al.2005b; Fraschetti et al. 2006; Cabral-Oliveira et al.2009). Both outfalls release secondary treated efflu-ents from a human population of 30,000 to 40,000.

Sampling procedures

Assemblages were sampled at the eulittoral, middlezone of the shore, daily covered and uncoveredby tides.On sheltered shores, the eulittoral is dominatedby large macroalgae, but barnacles and limpets are

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also present. In exposed shores, the eulittoral isusually covered by barnacles, limpets and mussels(Hawkins & Jones 1992). The upper limit is set bythe presence of barnacles, while the lower limit ismarked by the top of the filamentous red or brownalgae (Lewis 1964). At each of the two shores(Atlantic and Mediterranean), sampling was under-taken in three locations: the outfall (Imp) and twocontrol locations (C1 and C2) (Figure 1). All loca-tions presented comparable environmental conditions(slope, orientation, wave exposure and type of sub-strate). Three sites (approximately 100–300 m apart)were selected in each location, and at each site, fiverandomly located quadrats (20 × 20 cm) weresampled. Each quadrat was divided into 25 sub-quad-rats (4 × 4 cm), to help quantify the percent cover.Sampling was repeated on two random dates betweenJune and December 2012 (June and October in theMediterranean, and September and December in theAtlantic). The percent cover values of algae were

estimated and the number of animals was countedin the field, for each quadrat. It was set at an arbitraryvalue of 0.5 for the percent cover whenever the algaefilled less than 1/4 of one sub-quadrat (Dethier et al.1993). Final values were expressed as percentages foralgae and density for invertebrates.

Statistical analyses

An asymmetrical permutational multivariate analysisof variance (PERMANOVA; Anderson 2001) wascarried out separately for each region (Atlantic andMediterranean shores) to test for differences in theeulittoral assemblage (fauna and flora) structurebetween impacted and control conditions (Terlizziet al. 2005a). The model consisted of three factors:Time (Ti, two levels, random), Location (Lo, threelevels, random), Site (Si (Lo), three levels, nested inLo, random). All analyses were based on Bray-Curtissimilarity on untransformed data, and each term in

Figure 1. Study areas in (a) Portugal and (b) Italy. Imp = impact; C1, C2 = control locations 1 and 2. Three sites (●) were chosen in each area.

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the analysis was tested by 4999 random permuta-tions of appropriate units. To visualize multivariatepatterns, non-metric multidimensional scaling(nMDS) ordinations were done on the basis of aBray-Curtis similarity matrix.

Asymmetrical analysis of variance (ANOVA),based in the same model described above, was car-ried out separately for each region (Atlantic andMediterranean) to test for differences in the densitiesof barnacles (Chthamalus spp. and Balanus spp.),limpets (i.e. Patella spp.) and littorinids (i.e.Melarhaphe neritoides) between impact versus controlareas (Imp-v-Cs). ANOVA was also employed to testfor differences in algal cover in both the Atlantic andthe Mediterranean shores. For all statistical tests, thesignificance level was set at p < 0.05. The analyseswere performed using the Plymouth Routines inMultivariate Ecological Research version six(PRIMER v.6) software package (Clarke & Gorley2006).

Results

PERMANOVA showed a significant Imp-v-Csinteraction for the multivariate structure of eulittoralassemblages in the Mediterranean shores, but not forthe Atlantic shores (Tables I and II). Additionally,on both shores, analyses showed a significant Ti × Si(Lo) interaction, highlighting a highly significantspatial and temporal variability at the site scale(Tables I and II). These patterns were illustrated inthe corresponding nMDS plots (Figure 2).

ANOVA on the average abundance of inverte-brates revealed no significant differences in Imp-v-Cs for barnacles and M. neritoides in either theAtlantic or Mediterranean data (Table III). In con-trast, Patella spp. showed a significant effect of sew-age discharge that was consistent in time for bothshores (Table III). However, for the Atlantic, themean abundance of Patella spp. was lower at theimpact than at control locations whilst the oppositepattern was found for the Mediterranean shore(Figure 3).

Total algal cover can be observed in Figure 4. Onthe Atlantic shore, Rhodophyta and Chlorophytawere more abundant near the sewage outfall(Figure 4). However, ANOVA results showed thatsuch patterns were not consistent in time as a sig-nificant Ti × Imp-v-Cs interaction was detected(Table IV). On the Mediterranean shores,Chlorophyta seems to be more abundant near theoutfall, while the opposite was noticed forPhaeophyta (Figure 4). ANOVA on total algalcover showed a significant effect of sewage dischargefor both Chlorophyta and Phaeophyta but not for

Rhodophyta (Table IV). For Phaeophyta this effectwas not consistent in time.

Discussion

The urgency to find efficient tools allowing the pre-vention of the deterioration of coastal areas has led tothe search for indicators able to detect the effects ofanthropogenic impacts on those habitats (Juaneset al. 2008).

Table I. Atlantic shore. PERMANOVA testing for differences inassemblage structure between impacted (Imp) and reference loca-tions (Cs) (see text for further details). Significant results are givenin bold.

Source ofvariability df SS MS Pseudo-F P (perm)

Time = Ti 1 4174.5 4174.5 2.28 0.125Location = Lo 2 2710.4 1355.2 0.83 0.655Imp-v-Cs 1 1878.4 1878.4 1.11 0.429Cs 1 831.9 831.95 0.75 0.645Site (Lo) = Si (Lo) 6 15612.0 2602.0 1.42 0.257Si (Imp) 2 3063.0 1531.5 1.90 0.280Si (Cs) 4 12549.0 3137.3 1.34 0.334Ti × Lo 2 2463.1 1231.5 0.67 0.654Ti × Imp-v-Cs 1 1355.3 1355.3 0.79 0.507Ti × Cs 1 1107.8 1107.8 0.47 0.625Ti × Si (Lo) 6 10985.0 1830.8 3.39 0.000Ti × Si (Imp) 2 1608.6 804.3 1.19 0.295Ti × Si (Cs) 4 9376.3 2344.1 4.96 0.000Res 72 38908.0 540.4Res Imp 24 16216.0 675.7Res Cs 48 22692.0 472.7

Table II. Mediterranean shore. Asymmetric PERMANOVA test-ing for differences in assemblage structure between impacted(Imp) and references locations (Cs) (see text for further details).Significant results are given in bold.

Source ofvariability df SS MS Pseudo-F P (perm)

Time = Ti 1 8020.4 8020.4 2.25 0.139Location = Lo 2 49718.0 24859.0 4.87 0.008Imp-v-Cs 1 9779.9 9779.9 4.41 0.045Cs 1 39939.0 39939.0 4.95 0.081Site (Lo) = Si (Lo) 6 4980.9 830.1 0.23 0.999Si (Imp) 2 2379.2 1189.6 0.14 0.963Si (Cs) 4 2601.7 650.4 0.54 0.852Ti × Lo 2 10007.0 5003.7 1.40 0.259Ti × Imp-v-Cs 1 2341.9 2341.9 0.56 0.567Ti × Cs 1 7665.5 7665.5 6.34 0.024Ti × Si (Lo) 6 21377.0 3562.9 6.16 0.000Ti × Si (Imp) 2 16561.0 8280.7 11.23 0.000Ti × Si (Cs) 4 4815.9 1204.0 2.42 0.002Res 72 41621.0 578.1Res Imp 24 17692.0 737.1Res Cs 48 23929.0 498.5

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Our results showed that the multivariate structureof eulittoral assemblages seems to be differentlyaffected by sewage discharges in the two bioregions.No significant Imp-v-Cs effect was found for theAtlantic shore, although on the Mediterraneanshore there was a significant effect. Previous studies(e.g. Archambault et al. 2001; Bustamante et al.2012; O’Connor 2013) found no changes in thestructure, abundance or species richness of eulittoralassemblages exposed to sewage discharge. Theseauthors suggested that eulittoral assemblages werenot affected since waves, tides and currents removedthe effluents rapidly from the areas (Underwood &Chapman 1996; Archambault et al. 2001). Thus, thedifferent responses of eulittoral assemblages inAtlantic and Mediterranean shores may be poten-tially due to variations in the tidal regimes andhydrodynamics between the two regions.Furthermore, assemblages from rocky shores arenaturally highly variable and therefore make it moredifficult to detect the effects of a given impact(Bustamante et al. 2012). Finally, this intertidalzone is exposed daily to high level of natural distur-bance caused by tidal regimes, desiccation and waveaction, being thus characterized by species that

might be better adapted to tolerate sharp environ-mental variations (Lüning 1990) such as thosepotentially induced by sewage discharge (e.g. organicenrichment, freshwater inputs).Concerning macroalgae, the structure of the

assemblages was quite different comparing the outfalland control locations in both shores. For instance,green filamentous algae were more abundant at theimpact areas (although not consistently in time in theAtlantic shores), while control locations were mostlycharacterized by encrusting and filamentous redalgae. This is a well-known outcome of sewage dis-charges in the intertidal zone that often leads toincreased organic matter, which, in turn, triggersthe proliferation of opportunistic green algae(Raffaelli & Hawkins 1996). Nevertheless, on theAtlantic shore, the differences in the abundance ofChlorophyta varied in time. This could depend onseasonal variations in the environmental parameters(Archambault et al. 2001). Also, the responses of thedominant invertebrate species were relevant, espe-cially Patella spp., whose abundances were signifi-cantly different between control and impacted areas,on both Atlantic and Mediterranean shores. Atalahand Crowe (2012) have associated the presence of

Table III. Asymmetric analysis of variance (ANOVA) testing for differences between impacted (Imp) andreference locations (Cs) (see text for further details). Significant results are given in bold.

Barnacles M. neritoides Patella spp.

Source F P F P F PAtlantic Imp-v-Cs 1.03 0.492 0.21 0.919 8.48 0.038

Ti × Imp-v-Cs 0.01 0.896 7.95 0.059 3.17 0.157Mediterranean Imp-v-Cs 1.95 0.279 2.30 0.220 23.92 0.011

Ti × Imp-v-Cs 1.15 0.327 2.73 0.175 0.05 0.808

Figure 2. Non-metric multidimensional scaling ordination (nMDS) of site centroids for (a) Atlantic and (b) Mediterranean eulittoralassemblages. Numbers indicate the time of sampling. Imp = impact; C1, C2 = control locations 1 and 2.

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sewage discharges with changes in the abundanceand structure of gastropod assemblages, highlightingthe value of such assemblages as indicators of nutri-ent enrichment. Furthermore, previous studies havesuggested that limpet species could be good indica-tors of sewage pollution (e.g. Espinosa et al. 2007).

In this study, populations of limpets of the genusPatella were demonstrated to be significantly affectedby sewage discharge on both Atlantic andMediterranean shores. However, responses variedacross shores. In the Atlantic, limpet density waslower at the outfall than at control locations, whilethe opposite pattern occurred in the Mediterranean.This opposite pattern may be related to differencesin the natural environment of Atlantic andMediterranean shores. Variations in the tidal regimesthat produce diverse emersion times, and the differ-ent hydrodynamics, may influence the communitystructure and composition. A possible explanationof such contrasting patterns, therefore, may rely onthe fact that the species of limpets are different

between the two investigated shores. Species belong-ing to the genus Patella reported for the centralPortuguese shores are P. depressa and P. ulyssiponensis(Boaventura et al. 2002), with P. depressa being mostcommon in the eulittoral zone (Silva et al. 2003).More species are present on Mediterranean shores:P. rustica, P. ulyssiponensis, P. caerulea, P. ferrugineaand P. nigra (Giannuzzi-Savelli et al. 1994). P. ulys-siponensis, P. rustica and P. caerulea, in particular, arethe most common species in the Mediterraneaneulittoral zone (Simunovic 1970; Della-Santinaet al. 1993). Such species might respond differentlyto sources of disturbance related to sewage dis-charge, such as nutrient enrichment or increasedcontaminants, showing different levels of tolerance.For instance, P. caerulea is more tolerant than theother species to environmental changes induced bysewage discharge (Espinosa et al. 2007), whereas P.ulyssiponensis may tolerate an increased amount ofpollutants (e.g. Bebianno et al. 2003). Therefore,discrepancies between the two shores concerning

Figure 3. Density (ind/m2 ± standard error, SE) of Patella spp., Melarhaphe neritoides and barnacles in Atlantic and Mediterranean shores.Imp = impact; C1, C2 = control locations.

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Figure 4. Rhodophyta, Chlorophyta and Phaeophyta cover (%) on Atlantic and Mediterranean shores, for each time of sampling.Imp = impact; C1, C2 = control locations 1 and 2.

Table IV. Analysis of variance (ANOVA) testing for differences in cover algae between regions: Atlantic and Mediterranean shores (see textfor further details). Significant results are given in bold, and results of corresponding pairwise comparisons are marked with asterisks.

Rhodophyta Chlorophyta Phaeophyta

Source F P F P F P

Atlantic Imp-v-Cs 0.64 0.678 1.119 0.472 – –

Ti × Imp-v-Cs 17.57 0.014* 14.17 0.019** – –

Pairwise: *T1: Imp > CsT2: Imp = Cs

**T1: Imp > CsT2: Imp = Cs

Mediterranean Imp-v-Cs 3.83 0.107 7.04 0.044 1.25 0.436Ti × Imp-v-Cs 0.25 0.654 0.09 0.769 115.4 0.001*

Pairwise: *T1: Imp < CsPairwise: T2: Imp = Cs

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the effect of sewage discharge on patterns of limpetabundance might depend on the species-specificresponses to the pollution and on region-specificenvironmental conditions. In fact, the MarineStrategy Framework Directive has considered theAtlantic Ocean and the Mediterranean Sea differentbioregions, due to the important ecological varianceswithin each area (Borja 2006). Our results suggestthat the response of eulittoral assemblages and lim-pets to sewage discharge might depend on species-specific responses and bioregion characteristics.Although further investigations are needed to assessthe consistency of the observed patterns within eachbioregion, this suggests a potential limit of limpetpopulations and eulittoral assemblages to serve asgeneral indicators in monitoring the effects of thissource of human impact over wide spatial scales.

Acknowledgements

We wish to thank all the colleagues who helped in thefield and laboratory work. This work was supportedby the Fundação para a Ciência e Tecnologia (FCT)through a PhD grant to J. Cabral-Oliveira (SFRH/BD/48874/2008), with funds from the PortugueseOperational Human Potential Program (POPH), thePortuguese National Strategic Reference Framework(QREN Portugal) and the Portuguese Ministry ofScience, Technology, and Higher Education(MCTES). Analyses were conducted during a visitby JCO to the Department of Biological andEnvironmental Sciences and Technologies at theUniversity of Salento, Lecce, Italy.

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