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Marine Environmental Research 84 (2013) 31e42

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Marine Environmental Research

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Measuring restoration in intertidal macrophyte assemblages following sewagetreatment upgrade

I. Díez*, A. Santolaria, N. Muguerza, J.M. GorostiagaDepartment of Plant Biology and Ecology, University of the Basque Country UPV/EHU, PO Box 644, 48080 Bilbao, Spain

a r t i c l e i n f o

Article history:Received 13 August 2012Received in revised form15 November 2012Accepted 18 November 2012

Keywords:OutfallsRecoveryFunctional groupsSpecies richnessDiversitySpatial variabilityMacroalgal community

* Corresponding author. Tel.: þ34 94 6015355; fax:E-mail address: isabel.diez@ehu.es (I. Díez).

0141-1136/$ e see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.marenvres.2012.11.006

a b s t r a c t

Understanding the effectiveness of pollution mitigation actions in terms of biological recovery isessential if the environmental protection goals of management policies are to be achieved. Few studies,however, have evaluated the restoration of seaweed assemblages following pollution abatement. Thisstudy aimed to investigate the response of macroalgal vegetation to the upgrade of a wastewatertreatment plant using a "Beyond BACI" experimental design. Temporal differences in vegetation structurebetween the outfall and two control locations over a 10-year period were assessed. Improvement insewage treatment was found to lead to increases in diversity, cover of morphologically complex algaeand spatial heterogeneity. The multivariate composition of assemblages at the outfall location becamemore similar to that at the controls; however, their complete recovery may depend on factors other thanpollution removal. Our findings also suggest that the extent of restoration and the time required to detectit are largely predetermined by the response variables we choose to assess recovery.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The biodiversity and ecological processes of coastal ecosystemsare severely threatened by increasing human pressure (Airoldiet al., 2008). One prominent concern is the sewage effluents asso-ciated with urbanization developments, which chemically andphysically modify coastal environments by causing nutrientenrichment, toxic contamination, water turbidity and high siltationrates (Terlizzi et al., 2005; Azzurro et al., 2010). Drastic changes inthe patterns of distribution of marine assemblages in time andspace have been reported worldwide as a consequence of sewagedischarges (Tewari and Joshi, 1988; Roberts et al., 1998; Smith et al.,1999). The decline and even local disappearance of large perennialalgae have been indicated among their major impacts (Brown et al.,1990; Bellgrove et al., 1997; Benedetti-Cecchi et al., 2001; Erikssonet al., 2002; Gorgula and Connell, 2004; Thibaut et al., 2005; Pinedoet al., 2007; Coleman et al., 2008; Connell et al., 2008; Mangialajoet al., 2007). The loss of canopy seaweeds is presumably exacer-bated by other concomitant effects of sewage such as theenhancement of opportunistic algae and suspension feeders that,once established, inhibit the recruitment of large macrophytes(Airoldi, 1998; Kraufvelin, 2007). The nutrient uptake efficiency and

þ34 94 6013500.

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fast growth of annual filamentous and sheet-like algae favouredthese algae over thick algae at higher nutrient levels (Bellgroveet al., 1997; Johansson et al., 1998; Eriksson et al., 2002). Filter-feeding invertebrates can also outcompete seaweeds, takingadvantage of increased suspended particulate organic matter(Hindell and Quinn, 2000). Under the stress of pollution, the shift incommunity structure is frequently parallelled by a decline indiversity (Brown et al., 1990; Fairweather, 1990; Munda, 1993; Díezet al., 2010). The scale of sewage impact seems to be determined bythe volume, level of treatment of sewage and by the type of biotainvolved (Smith et al., 1999).

The Urban Wastewater Treatment Directive (91/271/EEC)(UWWTD) was established to protect the environment from theadverse effects of sewage discharges within the framework of theEuropean Union. In this sense, great strides have been made incontrolling municipal wastewaters in terms of the proportion ofpopulation connected to sanitation systems and the technologyinvolved. In the last ten years efforts in wastewater treatment haveactually intensified, since the full implementation of the UWWTD isa prerequisite for accomplishing the integrated water managementaddressed by the Water Framework Directive (WFD, 2000/60/EC).However, restoration of degraded habitats does not end with theconstruction of facilities and infrastructures which it is hoped willimprove the environment: the success or failure of these actions interms of improving the ecological structure and functioning of therestored habitat must also be assessed (Chapman, 1999). The

Intertidal outfall

Fig. 1. Map of the study area showing the putatively impacted location (I), the controllocations (C1, C2) and the intertidal sewage outfall (*).

I. Díez et al. / Marine Environmental Research 84 (2013) 31e4232

European Union’s water policy needs scientific support to evaluatethe effectiveness of mitigation measures in terms of biologicalrecovery in order to make decisions and take actions for achievingenvironmental protection goals. Efforts to evaluate the effective-ness of mitigation measures should be made on both large-scalesewage disposal and moderately sized outfalls, since most of theworld’s near-coastal population live in relatively densely populatedrural areas and small-to-medium towns rather than in large cities(Small and Nicholls, 2003).

Macroalgal assemblage recovery processes following waterquality improvement have been documented as including increasesin species richness and diversity (Bonk et al., 1996; Soltan et al.,2001), recolonization of habitats where seaweeds had dis-appeared (Hardy et al., 1993), decreases in the abundance of greenalgae (Archambault et al., 2001), development of morphologicallycomplex species (Díez et al., 2009a), and increases in communityvertical layering (Gorostiaga and Díez, 1996). Nevertheless, theexisting knowledge on recovery processes of hard bottom assem-blages resulting from pollution mitigation actions is far fromcomprehensive. There are few studies dealing with this topic andmost of them lack an appropriate experimental design with pre-action data and multiple control locations to distinguish restora-tion from natural variability (Archambault et al., 2001).

The upgrade of a wastewater treatment plant (WWTP) in themunicipality of Gorliz (northern Spain) provided an opportunity fora field experiment to investigate how seaweed communitiesrespond to pollution mitigation measures. The WWTP came intooperation in 1998 with the application of physical and chemicaltreatment followed by a second stage (BIOFOR: biological aeratedfiltration system plus nitrificationedenitrification process) from2006 onwards. The purpose of this study was to assess whetherintertidal macrophyte assemblages recovered in response to theonset of the secondary treatment. The potential changes in therestored community against a variable background were distin-guished by applying a ‘Beyond BACI’ experimental design(Underwood, 1994). Asymmetric analyses of variance were used todetect temporal differences in assemblage variables between theoutfall and two control locations over a 10-year period. We pre-dicted that the assemblages at the putatively impacted locationwould become more similar to those at the reference conditionsafter the set up of the secondary wastewater treatment. Hence,restoration was measured as a significant interaction in time andspace between the putatively impacted location and the controls.We expected significant changes from before to after the treatmentupgrade in the differences between the outfall and control loca-tions in relation to multivariate structure (i.e. quali-quantitativespecies composition), spatial variability (in terms of homogeneityof multivariate dispersion) and several community descriptors(species richness, diversity, evenness, morpho-functional groupcover) of intertidal seaweed assemblages.

2. Methods

2.1. Study locations and sampling design

This study was conducted in ‘Bahía de Plentzia’, an inlet on theBasque coast (northern Spain) (Fig. 1). Wastewaters emanatingfrom three municipalities (Barrika, Plentzia, and Gorliz witha combined population of 8000 inhabitants inwinter and 20,000 insummer) are discharged into an intertidal area with an averagedaily flow of 4.7 Ml d�1. Raw sewage was dumped into the inlet forover 40 years before physicalechemical treatment was introducedin 1998. This primary treatment consisted in the removal of sus-pended solids bymeans of chemically assisted sedimentation. Eightyears later, in October 2006, the sewage treatment was improved

with the addition of a biological treatment (BIOFOR: BiologicalAerated Filtration System) that included a nitrificationedenitrification process. Physico-chemical characteristics of thesewage effluent before and after the onset of the biological treat-ment are showed in Table 1.

Six surveys were carried out over the period 2001e2011: threesamplings were conducted when wastewaters received onlyprimary treatment (2001, 2003, and 2005) and the rest after theimplementation of the biological treatment (2007, 2009, and 2011).In order to avoid seasonal variability in macrophyte abundancessampling surveys were always performed in summer (from lateJune to August) (Díez et al., 2009b). The assemblages were sampledat three locations, one putatively impacted (30 m from the point ofdischarge) and two controls (Fig. 1). Each location was 50 m longand represented by a set of three randomly selected sites separatedby at least 10 m. At each site two bathymetric levels (0.5 m and1.2 m above the mean low tide level) were established and a set ofthree random replicated quadrats, separated by at least 1 m, wassampled on stable substrates (continuous bedrock) with slight tomoderate slope (<30�). A non-destructive sampling strategy wasused which consisted of visually assessed estimates of seaweedcover in 40� 40 cm quadrats using the BrauneBlanquet coverscale: þ(1%), 1 (1e5%), 2 (5e25%), 3 (25e50%), 4 (50e75%) and 5(75e100%). Mean cover per species was calculated for each quadratusing the median of each range. A total of 162 sampling units wereobtained for each tidal level: 6 times, 3 locations, 3 sites and 3quadrats. Data on the 1.2-m assemblages at the outfall locationcollected in 2001 were not considered in the present study becausethere were some problems with the habitat selection.

2.2. Statistical analysis

A Beyond BACI experimental design (Underwood, 1992, 1994)was employed to assess the effects of pollution abatement onmacroalgal assemblages. For each tidal level a five-factor permu-tational multivariate analysis of variance (PERMANOVA) was per-formed to test hypotheses on spatial and temporal variation in thephytobenthic assemblages (Anderson et al., 2008). The factors

Table 1Annual mean and standard deviation of physicochemical parameters of the sewage effluent before (2001, 2003, 2005) and after (2007, 2009, 2011) the onset of the biologicaltreatment. EC: Electrical conductivity at 20 �C; COD: Chemical oxygen demand; TSS: Total suspended solids dried at 105 �C; NH3: Ammonia; NO3

�: Nitrate; NO2�: Nitrite; PO4

�:Orthophosphate and pH. Data from the local water supply and sewerage authority Consorcio de Aguas Bilbao Bizkaia.

Primary treatment Secondary treatment

2001 2003 2005 2007 2009 2011

EC (mS/cm) 1934.7� 1265.8 2346� 909.4 1660.6� 1355.3 2009.7� 1298.2 1683.9� 949.8 1803.5� 1188.4COD (mg/l) 151.34� 69.33 155.36� 76.42 188.78� 75.69 49.18� 25.35 52.40� 28.05 36.22� 10.56TSS (mg/l) 44.76� 16.23 36.98� 16.18 47.11� 17.51 11.64� 5.15 15.41� 5.02 14.37� 8.41NH3 (mg/l) 20.49� 9.14 22.20� 11.52 24.60� 11.04 0.86� 0.69 0.84� 0.61 1.35� 1.69NO2

� (mg/l) 0.09� 0.10 0.20� 0.18 0.08� 0.09 0.09� 0.06 0.11� 0.06 0.11� 0.06NO3

� (mg/l) 0.85� 1.32 1.03� 0.76 1.61� 0.68 9.72� 2.72 12.12� 3.73 13.64� 3.45PO4

� (mg/l) 0.64� 1.36 1.05� 2.75 1.01� 1.01 1.02� 1.08 0.73� 0.52 0.72� 0.61pH 7.41� 0.24 7.38� 0.20 7.36� 0.19 7.38� 0.21 7.38� 0.16 7.25� 0.24

I. Díez et al. / Marine Environmental Research 84 (2013) 31e42 33

considered were: (1) Before vs After (BA: two levels, fixed); (2)Controls vs Impacted (CsI: two levels, fixed and orthogonal); (3)Time (T: six levels, random and nested in BA); (4) Location (L: threelevels, random and nested in CsI); and (5) Site (S: three levels,random and nested in L), with n¼ 3. This asymmetrical analysis ofvariance provided an orthogonal contrast between a single outfalland two control sites sampled before and after the onset of thesewage treatment. Variations from before to after treatmentupgrade that occur just at the outfall location and that make theoutfall location assemblages more like those of the control loca-tions may be considered as signs of recovery. Such variations can beexpressed as a significant Before-vs-After� Controls-vs-Impacted(BA�CsI) interaction, but also as significant CsI� T(BA) if the effectof the treatment upgrade is delayed. These significant terms rele-vant to the hypothesis were examined using post hoc pairwisecomparisons with the PERMANOVA t statistics and 9999 permu-tations. Spatio-temporal multivariate patterns of variation werevisualized by non-metric multidimensional scaling (nMDS) plots ofyear� location centroids based on BrayeCurtis dissimilaritiescalculated on raw data.

SIMPER analysis of similarity percentages (Clarke, 1993) wasused to rank the contribution ðdiÞ that each species made to thetotal average BrayeCurtis dissimilarity d between the putativelyimpacted location and the average of the controls Before and Afterthe onset of the wastewater secondary treatment. Species wereselected as ‘most discriminant’ if they exceeded an arbitrarilythreshold value of percentage contribution �1% to the averagedissimilarity between groups before the treatment upgrading. Theratio di=SDðdiÞ was also calculated to measure how consistentlyeach taxon contributed to dissimilarity across pairs of samples.

The effect of the setting up of the secondary treatment on thespatial heterogeneity of seaweed assemblage composition was alsoexamined. Changes in heterogeneity were explored for both tidallevels and two different spatial scales (metres: replicates; tens ofmetres: sites) by means of applying the permutational test forhomogeneity of multivariate dispersions (PERMDISP) developed byAnderson et al. (2006). The analysis calculates an F-statistic tocompare the average distance of the observation units to theirgroup centroid defined, in this case, by Jaccard dissimilaritymeasures. The P-value was obtained by 9999 random permutationsof least-square residuals. Post hoc pairwise comparisons wereperformed to compare differences in dispersion between theoutfall and the controls before and after the sewage treatmentimprovement.

PERMANOVA analyses based on Euclidean distance were per-formed to test the null hypothesis that there were no differences inthe average number of species (richness), ShannoneWiener index(H’ using loge), Pielou’s evenness index (J’ using loge), and the meanpercentage of cumulative algal cover using the same multi-factorexperimental design applied in multivariate analyses. Post hoc

pairwise comparisons using PERMANOVA t statistic and 9999 wereperformed for relevant terms. In addition, for each replicate,intertidal level and sampling site species cover data were aggre-gated into three morphologies: calcareous red algae (articulatedplus crustose species), simple forms (uniseriate, polysiphonous,foliose non-corticated and slightly corticated: cortex with oneetwolayers) andmorphologically complex algae (corticated algae: cortexwith more than two layers, plus leathery macrophytes) to test forspatio-temporal differences in morpho-functional groups.Cochran’s C-test was performed before the analyses to check forhomogeneity of variance and data were square root-transformedwhen necessary.

Analyses were performed using the PRIMER V. 6. PERMANOVApackage (Clarke and Gorley, 2006; Anderson et al., 2008).

3. Results

3.1. Community multivariate structure

PERMANOVA analyses on multivariate data detected a signifi-cant variability over time at the scale of site for both 1.2-m and 0.5-m assemblages, as evidenced by the significant T(BA)� S(L(CsI))interactions (Table 2). Differences between locations also variedfrom time to time as indicated by the significant T(BA)� L(CsI)term. These differences between locations in the temporal path aregraphically illustrated by the patterns in the non-metric multidi-mensional scaling (nMDS) plots (Fig. 2). Distance betweencentroids of macroalgal assemblages at the outfall location and atthe control locations decreased noticeably from 2007 onwards.

The SIMPER procedure identified 26 taxa as being the mostimportant in differentiating assemblages located at the controlsfrom those at the outfall location (Table 3). These taxa showedindividual contribution values in excess of an arbitrarily chosenthreshold of �1% before the treatment upgrading. Values of theratio di=SDðdiÞ were >1 in most cases, which denotes that theircontribution to the average Cs vs I dissimilarity was consistentacross pairs of samples. The abundance, temporal changes andrelative contribution of the discriminating taxa to the Cs vs Idissimilarity varied according to the tidal level.

For the 1.2-m level, Corallina elongata was the most importantspecies in discriminating between the controls and the outfall(Table 3). Following the wastewater treatment upgrading, thiscalcareous alga increased at the outfall location and its relativecontribution to the average Cs vs I dissimilarity decreased. Manyspecies, including Ceramium ciliatum, Mesophyllum lichenoides,Chondria coerulescens, Gelidium attenuatum, Chondracanthus aci-cularis, Lithophyllum incrustans, Halopteris scoparia, Gelidium spi-nosum, Ceramium echionotum and Hypnea musciformis, exhibitedhigher cover values at the controls than at the outfall prior to theonset of the biological treatment, whereas only Caulacanthus

Table 2PERMANOVA results and pairwise comparisons based on BrayeCurtis dissimilarities of raw data from assemblages at 1.2 m and 0.5 m tidal levels. B: Before; A: After; I:Putatively impacted location; Cs: Control locations; T: Time; L: Location; S: Site and n¼ 3. P-values were obtained using 9999 permutations on given permutable units.Macroalgal assemblage structure e PERMANOVA table of results.

Source 1.2-m assemblages 0.5-m assemblages

df MS Pseudo-F P (perm) df MS Pseudo-F P (perm)

BA 1 10,164 1.4684 0.2057 1 15,809 0.6341 0.7867CsI 1 19,387 0.9813 0.4873 1 71,472 0.7080 0.6481T(BA) 4 4117.4 0.94405 0.5699 4 6228.9 1.491 0.1641L (CsI) 1 24,415 3.8321 0.0033 1 101,950 14.246 0.0002BA� CsI 1 6213.6 1.1151 0.4152 1 12,787 0.5618 0.8491S(L(CsI)) 6 2155.3 2.0125 0.0022 6 3082 2.0987 0.0042BA� L(CsI) 1 6151.7 1.1386 0.325 1 25,293 4.3966 0.0006CsI� T(BA)a 3 3780 0.86669 0.6035 4 4906 1.1743 0.3296BA� S(L(CsI)) 6 1912.5 1.7858 0.0114 6 1909.1 1.3 0.1544T(BA)� L(CsI) 4 4361.4 4.0725 0.0001 4 4177.7 2.8448 0.0003T(BA)� S(L(CsI)) 22 1070.9 2.5826 0.0001 24 1468.6 2.9361 0.0001Res 102 414.67 108 500.17Total 152 161

a For the 1.2-m assemblages, this term lacks data corresponding to the 2001 survey at the putatively impacted location.

I. Díez et al. / Marine Environmental Research 84 (2013) 31e4234

ustulatus, Ulva rigida and Gelidium pulchellumwere more abundantat the outfall. Differences between the controls and the outfall inthe abundance of these discriminating species narrowed after thesetting up of the biological treatment in most cases and, as a result,their relative contribution to the average Cs vs I dissimilaritydecreased (Table 3).

At the 0.5 m level, the macrophytes H. scoparia, Codium decorti-catum,U. rigidaandG. attenuatumweremore abundantat the controlsthan at the outfall before the wastewater treatment upgrading,whereas the calcareous algae C. elongata and M. lichenoides, and thefilamentous C. echionotum showed higher cover values at the outfall(Table 3). Following the onset of the secondary treatment, the relativecontributionof these taxa to theaverageCsvs I dissimilaritydecreasedin most cases. Vegetation at the outfall location underwent anincrease in the abundance of H. scoparia and G. attenuatum, anda decrease in C. elongata, M. lichenoides and C. echionotum.

The improvement in water treatment also affected patterns ofspatial heterogeneity of seaweed assemblage composition assessedin terms of dispersion of the sampling units around their centroidin the multivariate space. Vegetation was more homogeneous atthe outfall location than at the controls (Fig. 3). PERMDISP revealedthat multivariate dispersion of quadrats (scale of metres) and sites(tens of metres) increased at the supposedly impacted locationfollowing the implementation of the secondary treatment (Fig. 3).Pairwise comparison tests indicated that before the treatmentupgrading there were significant differences between the controlsand the outfall location in the spatial heterogeneity at both spatialscales studied (Table 4). After the onset of the biological treatment

Control 2Control 1

Outfall location

0909

09

07

07

07

05

05

05

03

03

03

01

01

11

11

11

Kruskal Stress: 0.14

1.2 m tidal level assemblages

K

Fig. 2. Non-metric MDS ordination based on the BrayeCurtis dissimilarity coefficient applietreatment improvement at 1.2 m (left) and 0.5 m (right) tidal levels. The lines reflect the dispinitial positions.

these differences were no longer significant for the communities at0.5 m tidal height, and only remained significant at the scale ofmetres for 1.2-m communities.

3.2. Species richness and diversity measures

Ninety-seven taxa of algae were identified during this study,distributed as follows: 65 Rhodophyceae, 17 Chlorophyceae, and 15Phaeophyceae.

For assemblages at 1.2 m tidal level the mean number of speciessignificantly increased (p ¼ 0.04) at the outfall location followingwastewater treatment upgrade, whereas no change (p ¼ 0.89) wasdetected at the controls (Fig. 4). However, this pattern of variationin outfall vs control locations from before to after was not detectedby PERMANOVA analysis since the BA�CsI interaction is notsignificant (p ¼ 0.09) (Table 5). This may be a consequence of thetemporal variability at the scale of site evidenced by the significantT(BA)� S(L(CsI)) interaction (Table 5), which is likely to decreasethe statistical power for detecting the BA�CsI interaction. Therewas similar pattern of spatio-temporal variance at site level forShannon (H’) diversity measures (Table 5), but PERMANOVA anal-ysis revealed a significant BA�CsI interaction, indicating that theeffect of the treatment upgrade was over and above the naturalspatial and temporal variability. Pairwise tests showed that differ-ences in Shannon (H’) diversity between the putatively impactedlocation and the controls were significant before the onset of thesecondary treatment (p ¼ 0.02) but ceased to be so following thetreatment upgrade (p ¼ 0.54). Evenness also increased over time at

Control 2Control 1

Outfall location

09

09

09

0707

07

05

05

0503

03

03

01

0101

11

11

11

ruskal Stress: 0.09

0.5 m tidal level assemblages

d to untransformed data of centroids of each location before and after the wastewaterlacement of centroids of macroalgal assemblages at each location with respect to their

Table 3Summary of SIMPER analyses identifying contributions ðdiÞ from the most important taxa (individual contribution >1%) to the average BrayeCurtis dissimilarity (on raw data)between macroalgal assemblages at the control locations (Cs) and the outfall (I). SDðdiÞ standard deviation of individual species contribution; di % percentage of contribution.Taxa average cover in % at the controls ðCsÞ and at the outfall location ðIÞ, before (B) and after (A) the onset of the secondary treatment.

Species Before After

CsB IB di SDðdiÞ di

SDðdiÞdi% CsA IA di SDðdiÞ di

SDðdiÞdi%

1.2 m tidal levelCorallina elongata J. Ellis et Sol. 58.1 43.8 11.4 1.3 8.9 20.4 69.9 61.6 7.0 1.1 6.3 14.7Caulacanthus ustulatus (Turner) Kütz. 10.3 26.5 8.4 1.3 6.7 15.0 21.4 19.9 6.8 1.3 5.5 14.4Ulva rigida C. Agardh 9.0 19.7 5.8 1.0 5.8 10.4 3.7 11.6 3.3 1.6 2.0 6.8Ceramium ciliatum (J. Ellis) Ducluz. 15.2 2.0 5.5 1.0 5.3 9.8 1.6 2.9 1.1 0.7 1.5 2.3Mesophyllum lichenoides (J. Ellis et Sol.) Me. Lemoine 6.8 0.2 2.8 0.7 4.2 5.1 2.6 0.4 0.8 0.6 1.4 1.8Chondria coerulescens (J. Agardh) Falkenb. 5.9 0.2 2.3 0.7 3.4 4.0 1.3 0.6 0.5 0.5 0.9 1.0Gelidium attenuatum (Turn.) Thuret 5.3 0.0 1.9 0.5 3.6 3.4 1.3 1.0 0.6 0.6 1.1 1.3Chondracanthus acicularis (Roth) Frederiq 4.5 0.0 1.7 0.5 3.4 3.1 8.0 0.6 2.7 0.7 3.9 5.7Lithophyllum incrustans Phil. 3.8 0.3 1.5 0.5 2.7 2.6 5.2 1.7 1.6 0.9 1.9 3.4Gelidium pulchellum (Turner) Kütz. 2.1 3.1 1.4 0.7 2.1 2.5 3.0 3.3 1.6 0.8 2.0 3.3Halopteris scoparia (L.) Sauv. 3.4 0.0 1.4 0.6 2.3 2.4 2.9 1.6 1.3 0.5 2.9 2.8Gelidium spinosum (S.G. Gmelin) P.C. Silva 3.4 0.0 1.4 0.5 2.6 2.4 0.4 0.1 0.2 0.5 0.4 0.3Ceramium echionotum J. Agardh 3.4 0.0 1.3 0.3 3.8 2.2 0.7 0.9 0.5 0.4 1.1 1.0Hypnea musciformis (Wulfen) J.V. Lamour. 3.1 0.0 1.1 0.4 3.0 2.0 0.4 0.0 0.2 0.5 0.3 0.3Bifurcaria bifurcata R. Ross 2.7 0.0 1.0 0.4 2.5 1.8 1.9 0.6 0.8 0.4 2.1 1.7Ulva clathrata (Roth) C. Agardh 2.2 0.0 0.9 0.3 2.6 1.6 4.0 0.1 1.3 0.6 2.3 2.8Falkenbergia rufolanosa (Harv.) F. Schmitz 1.8 0.5 0.8 0.5 1.6 1.4 0.9 3.7 1.6 0.7 2.3 3.3Dictyota dichotoma (Huds.) J.V. Lamour. 0.5 0.0 0.2 0.5 0.4 0.4 2.4 0.0 0.8 0.5 1.5 1.6Chaetomorpha ligustica (Kütz.) Kütz. 0.5 0.3 0.3 0.6 0.4 0.5 0.4 1.5 0.5 0.6 0.9 1.0Phymatolithon lenormandii (Aresch.) W.H. Adey 0.4 0.3 0.2 0.6 0.3 0.4 1.3 0.6 0.5 0.5 0.9 1.0Cladostephus spongiosus (Huds.) C. Agardh 0.0 0.1 0.1 0.6 0.2 0.1 0.1 1.4 0.5 0.5 1.0 1.0

0.5 m tidal levelCorallina elongata J. Ellis et Sol. 19.5 79.2 21.2 2.3 9.3 27.1 26.2 70.0 15.3 1.6 9.6 22.2Halopteris scoparia (L.) Sauv. 34.2 0.0 12.5 0.9 14.7 16.0 23.0 8.4 7.5 0.9 8.4 10.9Codium decorticatum (Woodw.) M. Howe 30.7 1.3 10.2 0.9 11.6 13.1 0.0 0.1 0.0 0.5 0.0 0.1Mesophyllum lichenoides (J. Ellis et Sol.) Me. Lemoine 7.7 22.8 6.5 1.4 4.8 8.3 8.7 9.2 2.8 1.1 2.7 4.0Ceramium echionotum J. Agardh 0.4 12.7 4.3 0.7 6.6 5.5 1.4 0.5 0.5 0.5 1.1 0.7Ulva rigida C. Agardh 6.8 1.3 2.3 0.7 3.2 2.9 3.1 4.0 1.2 0.8 1.5 1.8Gelidium attenuatum (Turn.) Thuret 6.0 0.6 2.1 0.6 3.8 2.7 8.6 10.2 3.5 0.9 3.7 5.0Falkenbergia rufolanosa (Harv.) F. Schmitz 1.7 5.4 1.8 0.7 2.6 2.3 0.6 2.9 1.0 0.6 1.6 1.4Gelidium spinosum (S.G. Gmelin) P.C. Silva 5.1 0.6 1.7 0.5 3.3 2.2 1.4 3.4 1.1 0.8 1.3 1.5Lithophyllum incrustans Phil. 2.5 4.7 1.6 0.8 1.9 2.1 5.0 7.4 2.1 1.0 2.2 3.1Pterosiphonia complanata (Clemente) Falkenb. 2.6 3.7 1.6 0.6 2.6 2.1 1.4 1.7 0.5 0.7 0.7 0.8Ceramium ciliatum (J. Ellis) Ducluz. 3.3 2.5 1.5 0.7 2.2 1.9 1.8 4.1 1.3 0.8 1.7 1.9Chondria coerulescens (J. Agardh) Falkenb. 3.9 0.8 1.3 0.7 1.8 1.6 3.7 6.2 2.0 0.7 2.7 2.9Bifurcaria bifurcata R. Ross 3.9 0.0 1.3 0.3 4.2 1.6 17.5 0.0 5.1 0.7 7.5 7.4Ceramium botryocarpum A.W. Griffiths ex Harv. 0.1 3.4 1.1 0.4 2.5 1.4 0.2 0.5 0.2 0.6 0.3 0.2Chondracanthus acicularis (Roth) Frederiq 2.0 0.9 0.9 0.5 1.6 1.1 5.3 3.6 2.2 0.6 3.8 3.2Chondracanthus teedei (Mertens ex Roth) Kütz. 0.0 2.0 0.7 0.5 1.4 0.8 0.6 2.7 0.9 0.6 1.5 1.2Acrosorium ciliolatum (Harv.) Kylin 1.1 1.4 0.6 0.6 1.0 0.7 2.1 2.2 0.8 0.7 1.1 1.2Hypnea musciformis (Wulfen) J.V. Lamour. 0.3 0.0 0.1 0.4 0.3 0.1 4.0 0.2 1.2 0.6 1.9 1.7Gelidium pulchellum (Turner) Kütz. 0.1 0.2 0.1 0.5 0.2 0.1 1.9 1.7 1.0 0.5 2.2 1.5

I. Díez et al. / Marine Environmental Research 84 (2013) 31e42 35

the outfall location after the setting up of the secondary treatment,whereas the controls showed a different temporal pattern of vari-ation (Fig. 4). Although not significant, the pseudo-F ratio of thecontrast of outfall vs control locations from before to after was thelargest of all the sources of variation (Table 5). PERMANOVA anal-ysis probably did not detect this BA�CsI interaction becausedifferences between sites and between locations significantlyvaried over time, as evidenced by the significant T(BA)� L(CsI) andT(BA)� S(L(CsI)) interactions (Table 5). For assemblages at thelowest tidal level (0.5 m), PERMANOVA on species richness, diver-sity and evenness showed significant temporal variability at sitelevel (Table 5). The analyses detected no significant effect of thewastewater treatment improvement.

3.3. Functional groups

The cover of morphologically complex algae at the 1.2 m tidallevel showed significant temporal fluctuations at site and locationlevels (Table 6). PERMANOVA analysis also revealed a significanteffect of sewage treatment upgrade, as indicated by the significant

BA�CsI interaction. The cover of complex algae increased at theoutfall location following water quality improvement (Fig. 5),though post hoc pairwise comparisons showed no significantdifferences (p ¼ 0.08) from before to after. The MCA group showedthe highest abundance at the 0.5 m tidal level (Fig. 5). At this tidallevel, complex algae also increased in the surroundings of theoutfall following the sewage treatment upgrade, though PERMA-NOVA analysis did not detect any significant BA�CsI interaction;however, the significant CsI� T(BA) interaction suggests a delayedeffect of the treatment upgrade since variation over time at theoutfall location differed from the temporal changes that occurredon average at the controls. Pairwise comparisons showed a signifi-cant increase (p¼ 0.02) in MCA from the 2007 to 2011 samplings atthe outfall location. With respect to the cover of calcareous algae(CA) at the 1.2 m tidal level, PERMANOVA showed that there wassignificant temporal variation at the scale of both site and location,but no significant differences related to treatment upgrade weredetected (Table 6). The 0.5-m assemblages at the outfall locationwere dominated by calcareous algae. This group exhibiteda significant temporal variability at site level (Table 6). Despite this

Fig. 3. Average distance from centroid (�SE) of the controls (Cs) and the putatively impacted (I) assemblages before (light bars) and after (dark bars) the setting up of thewastewater secondary treatment on the basis of Jaccard dissimilarity measures.

I. Díez et al. / Marine Environmental Research 84 (2013) 31e4236

variability, PERMANOVA revealed a significant CsI� T(BA) interac-tion, indicating effects of sewage discharge that were significantbut variable over time (Table 6). Post hoc pairwise tests showed thatCA significantly decreased from the 2007 to 2011 samplings. Thepatterns of distribution of morphologically simple species werehighly variable in space and in time (Fig. 5, Table 6). There was noevidence linking pollution abatement to the variability in abun-dance of simple forms (Table 6).

4. Discussion

This study detected a significant recovery of macroalgalassemblages in the vicinity of an urban sewage discharge after theupgrade of the WWTP. In general terms, our results indicate thatpollution abatement led to an increase in species richness anddiversity, promoted the development of morphologically complexalgae and increased the spatial heterogeneity of macrophytevegetation. Differences in the multivariate composition of assem-blages between the putatively impacted location and the controlsdecreased after the treatment improvement, but 5 years after theimplementation of the secondary treatment some differences stillpersisted. Similarly, Bustamante et al. (2012) found symptoms of

Table 4PERMDISP results and associated pairwise comparisons for intertidal seaweed assemblagesite centroids. P-values were obtained using 9999 permutations. Bold numbers correspo

PERMDISP summary of results 1.2-m assemblages

Quadrats Sites

Deviation from centroid F P(perm) F7.979 0.0004 9.0014

Pairwise comparisons t P(perm) tBeforeControls vs. Impacted 2.6789 0.0156 5.7855

AfterControls vs. Impacted 2.4886 0.0365 1.426

recovery of rocky macrofaunal assemblages in the surroundings ofthis outfall.

Another relevant finding that emerges from this study is thatintertidal seaweed assemblages show high spatio-temporal vari-ability. For some of the response variables studied, such variabilitymay have precluded the detection of the effects of the sewagetreatment upgrade. It is worth noting here that natural variabilityshould be taken into account when drawing up sampling designs tomeasure the effects of human mitigation interventions. In thissense, Pérez-Ruzafa et al. (2007) pointed out that sources of vari-ability should be controlled by sampling replicates at the lowestsignificant scale.

The multivariate composition of assemblages at the outfalllocation noticeably shifted from 2007 onwards. More speciesincreased than decreased in abundance. The macrophytes H. sco-paria and G. attenuatum showed an increase from before to aftersewage treatment upgrade at the 0.5 m tidal level, as did C. elongataat 1.2 m. Changes in species cover in the neighbourhood of thesewage discharge resulted in a zonation pattern more similar tothat observed at the controls. The perennial H. scoparia, a poten-tially dominant macrophyte along exposed to semi-exposed inter-tidal areas of the Basque coast (Borja et al., 2004), has been reported

data sets at 1.2 m and 0.5 m tidal levels using Jaccard similarities from quadrats andnd to significant values of p.

0.5-m assemblages

Quadrats Sites

P(perm) F P(perm) F P(perm)0.0007 7.0675 0.0005 4.3401 0.0264P(perm) t P(perm) t P(perm)

0.0003 3.757 0.0016 3.1079 0.0186

0.2227 1.524 0.1701 1.7858 0.1422

Fig. 4. Mean and standard error of species richness S, Shannon diversity H0 , and Pielou’s evenness J0 of 1.2 m and 0.5 m phytobenthic assemblages at the controls (Cs) and at theputatively impacted location (I) over time (light and dark bars correspond to sampling times before and after the onset of the secondary treatment, respectively).

I. Díez et al. / Marine Environmental Research 84 (2013) 31e42 37

as an indicator species in the recovery processes of phytobenthiccommunities following pollution abatement (Díez et al., 2009a). Itis important to note that the genus Cystoseira was absent from thewhole study area. Given that species of Cystoseira are highlysensitive to pollution (Sales et al., 2011), this fact may be related tothe occurrence of sources of pollution other than the effluentdischarge, e.g. the Butrón river that flows into Plentzia Bay. Thedredging of the river channel, urban development on the banks andinsufficient wastewater treatment by some industries and munic-ipalities dumping into its basin may substantially alter the river’swater quality and, in turn, the environmental conditions of the inletstudied.

Our study indicates that water quality improvement also led toan increase in the spatial heterogeneity of intertidal macrophyteassemblages at the scale of meters (small-scale patchiness) andtens of meters. Changes in spatial variability of community struc-ture have been associated with anthropogenic impacts (Warwickand Clarke, 1993; Chapman et al., 1995; Bustamante et al., 2012).Thus, a number of other studies (Fairweather, 1990; Rodríguez-Prieto and Polo, 1996; Fraschetti et al., 2006; Echavarri-Erasunet al., 2007; Bevilacqua et al., 2012) have reported lower hetero-geneity in rocky reef communities close to sewage outfalls. Thecomplex interactions between many environmental-stress gradi-ents and biotic factors that structure undisturbed algal communi-ties (Menge and Sutherland, 1987) are simplified in disturbedenvironments because the impact of pollution exerts a prevailingpressure on intertidal organisms, making communities more

homogeneous (Martins et al., 2012). Nevertheless, different find-ings have been reported by other authors (Warwick and Clarke,1993; Wear and Tanner, 2007) who have found greater multivar-iate dispersion among replicates under impacted conditions than inunaltered areas.

According to Johnston and Roberts (2009), anthropogeniccontamination of marine habitats is frequently associated withreductions in diversity. With regard to macroalgal assemblages, theresults of previous studies range from no detectable effects ofpollution upon diversity (Underwood and Chapman, 1996; Terlizziet al., 2002) to large-scale effects, both declines (Borowitzka, 1972;Littler and Murray, 1975; Brown et al., 1990; Fairweather, 1990;Munda, 1993) and increases (Reopanichkul et al., 2009). Suchdiscrepancies in community responses may be related to themagnitude of the disturbance in terms of the volume and quality ofthe effluent. Wastewater impact may also depend on the initialnatural productivity of the system. Thus, sewage nutrient enrich-ment in oligotrophic waters can result in more macrophytes(Reopanichkul et al., 2009). In our case, assemblages at the 1.2 mtidal level experienced significant increases in both species rich-ness and diversity associated with water quality improvement.These results are in agreement with most previous studies thathave examined the responses of phytobenthic assemblages tosewage mitigation actions (Hardy et al., 1993; Bonk et al., 1996;Archambault et al., 2001; Soltan et al., 2001; Díez et al., 2009a).Our results also show that macrophytes at the outfall locationbecome more evenly distributed after sewage treatment upgrade.

Table 5PERMANOVA results and pairwise comparisons showing the effect of sewage treatment upgrading on species richness S, Shannon diversity H0 , and Pielou’s evenness J0 fromassemblages at 1.2 m and 0.5 m tidal levels. Legends as in Table 1.

PERMANOVA results ofdiversity measures

1.2-m assemblages 0.5-m assemblages

df MS Pseudo-F P (perm) df MS Pseudo-F P (perm)

SSourceBA 1 323.57 4.1721 0.0966 1 2346.9 11.855 0.0147CsI 1 371.71 2.374 0.2246 1 4 0.0563 0.9941T(BA) 4 54.651 2.5673 0.1987 4 163.83 9.4617 0.0269L(CsI) 1 166.26 5.2307 0.0164 1 255.15 7.1371 0.0065BA� CsI 1 282.08 4.602 0.0948 1 64 0.5118 0.7349S(L(CsI)) 6 14.595 0.5817 0.7427 6 23.475 0.6525 0.6901BA� L(CsI) 1 42.815 1.5158 0.2395 1 35.593 1.1638 0.3553CsI� T(BA)a 3 30.745 1.4443 0.3411 4 123.3 7.1209 0.039BA� S(L(CsI)) 6 23.047 0.9185 0.4996 6 44.179 1.228 0.3284T(BA)� L(CsI) 4 21.287 0.8484 0.505 4 17.315 0.4813 0.7481T(BA)� S(L(CsI)) 22 25.091 3.8466 0.0001 24 35.975 7.5688 0.0001Res 102 6.5229 108 4.7531Total 152 161Cochran’s C-test C ¼ 0.1483 C ¼ 0.0675Transformation None None

H0

SourceBA 1 0.7075 0.7209 0.5918 1 11.342 3.7169 0.1158CsI 1 1.0478 7.705 0.0484 1 0.8903 0.8936 0.5426T(BA) 4 1.031 4.5652 0.0823 4 2.8606 9.9853 0.0224L(CsI) 1 0.0392 0.6194 0.7049 1 0.6633 1.0642 0.3992BA� CsI 1 3.2072 7.7991 0.0424 1 0.1231 0.4444 0.7728S(L(CsI)) 6 0.0484 0.3905 0.8776 6 0.5447 2.4621 0.0532BA� L(CsI) 1 0.3829 1.7144 0.1929 1 0.2680 0.5523 0.7492CsI� T(BA) a 3 0.1281 0.5673 0.6674 4 0.6536 2.2816 0.2136BA� S(L(CsI)) 6 0.0700 0.5643 0.7489 6 0.5994 2.7095 0.0367T(BA)� L(CsI) 4 0.2258 1.8215 0.1651 4 0.2865 1.295 0.2977T(BA)� S(L(CsI)) 22 0.1240 2.9004 0.0002 24 0.2212 4.7564 0.0001Res 102 0.0428 108 0.0465Total 152 161Cochran’s C-test C ¼ 0.1100 C ¼ 0.1070Transformation None None

J0

SourceBA 1 0.0058 0.2462 0.885 1 0.4496 1.8506 0.2841CsI 1 0.0135 1.6542 0.3125 1 0.1352 0.6913 0.6314T(BA) 4 0.1015 3.3341 0.1392 4 0.2219 9.9458 0.0227L(CsI) 1 0.0056 0.3741 0.9048 1 0.1868 3.1036 0.066BA� CsI 1 0.1757 5.5529 0.0737 1 0.0005 0.3082 0.8597S(L(CsI)) 6 0.0089 1.0169 0.4413 6 0.0439 2.3219 0.0642BA� L(CsI) 1 0.0220 0.8729 0.5071 1 0.0331 0.6946 0.6245CsI� T(BA) a 3 0.0184 0.6050 0.6459 4 0.0410 1.8381 0.2767BA� S(L(CsI)) 6 0.0051 0.5864 0.739 6 0.0526 2.775 0.0351T(BA)� L(CsI) 4 0.0304 3.487 0.0248 4 0.0223 1.1782 0.3456T(BA)� S(L(CsI)) 22 0.0087 1.9601 0.0111 24 0.0189 4.6423 0.0001Res 102 0.0045 108 0.0048Total 152 161Cochran’s C-test C ¼ 0.1319 C ¼ 0.0797Transformation None None

a For the 1.2-m assemblages. this term lacks data corresponding to the 2001 survey at the putatively impacted location.

I. Díez et al. / Marine Environmental Research 84 (2013) 31e4238

This finding is consistent with the review and meta-analysis ofJohnston and Roberts (2009), who conclude that evenness tends tobe low under the stress of pollution because of the increaseddominance of a few pollution-tolerant species.

Morpho-functional group classifications have been proposed asa suitable surrogate for species composition in reflecting changes inmacroalgal community patterns (Littler and Littler, 1984; Steneckand Dethier, 1994; Balata et al., 2011). Therefore, we expected therecovery process to involve changes in the percentage cover ofdifferent morphologies regardless of their taxonomic identity. Ourresults are consistent with these predictions and suggest thatpollution abatement leads to increases in the percentage cover ofmorphologically complex algae. This finding supports previousstudies that propose the abundance of corticated forms asa community-level measure to assess water quality (Wells et al.,

2007; Rubal et al., 2011). In contrast, coralline turfs are consid-ered tolerant to intermediate levels of pollution (Borowitzka, 1972;Littler and Murray, 1975; May, 1985; Bellgrove et al., 1997; Arévaloet al., 2007; Díez et al., 2009a). Thus, the replacement of canopyspecies by articulate coralline algae under the effects of domesticsewage and close to urbanizations is now becoming a majorconcern (Kindig and Littler, 1980; Brown et al., 1990; Benedetti-Cecchi et al., 2001; Arévalo et al., 2007; Mangialajo et al., 2007).In our study we found that the abundance of coralline algae wasonly higher at the outfall location than at the controls at the lowesttidal level. Following the onset of the secondary treatment,calcareous red algae decreased at the lowest tidal level, but theywere still the predominant component of vegetation at the end ofstudy. Experimental studies (Bellgrove et al., 2010) have providedevidence that competitive exclusion by coralline turfs may limit the

Table 6PERMANOVA results and pairwise comparisons showing the effect of sewage treatment upgrading on percentage cover of morphologically complex algae (MCA), calcareousred algae (CA), and simple forms (SF) from assemblages at 1.2 m and 0.5 m tidal levels. Legends as in Table 1.

PERMANOVA table of resultsof cover measures

1.2-m assemblages 0.5-m assemblages

df MS Pseudo-F P (perm) df MS Pseudo-F P (perm)

MCASourceBA 1 18.903 2.2241 0.2187 1 10,655 1.9143 0.277CsI 1 82.105 2.3806 0.2167 1 46,848 0.5421 0.7092T(BA) 4 15.535 0.8735 0.5487 4 5592.1 93.781 0.0003L(CsI) 1 44.346 1.7475 0.1967 1 83,167 148.94 0.0001BA� CsI 1 35.031 9.0283 0.0342 1 4268.4 1.2844 0.4094S(L(CsI)) 6 8.5924 3.2608 0.019 6 500.5 1.9146 0.1179BA� L(CsI) 1 0.5839 0.1471 0.9988 1 4.8981 1.3832 0.2806CsI� T(BA)a 3 4.9891 0.2805 0.8441 4 3364.9 56.43 0.0009BA� S(L(CsI)) 6 5.0255 1.9072 0.1273 6 132.9 0.5084 0.7982T(BA)� L(CsI) 4 17.786 6.7497 0.0009 4 59.63 0.2281 0.9193T(BA)� S(L(CsI)) 22 2.6351 1.9385 0.0129 24 261.41 0.9428 0.5454Res 102 1.3594 108 277.27Total 152 161Cochran’s C-test C ¼ 0.0968 C ¼ 0.0884Transformation Sqrt(x) None

CASourceBA 1 27.773 7.4657 0.0385 1 0.2212 0.0707 0.9911CsI 1 40.979 3.7056 0.1277 1 633.32 8.1596 0.0284T(BA) 4 4.8761 0.6405 0.6559 4 4.145 6.4852 0.0449L(CsI) 1 6.0128 0.6844 0.6432 1 66.328 7.133 0.0068BA� CsI 1 2.7 1.0829 0.4557 1 45.507 2.3805 0.217S(L(CsI)) 6 4.841 1.8882 0.1254 6 9.1895 2.4312 0.0578BA� L(CsI) 1 0.2817 0.2847 0.963 1 8.0188 1.2296 0.3324CsI� T(BA)a 3 7.8923 1.0367 0.4777 4 11.367 17.784 0.0073BA� S(L(CsI)) 6 2.8244 1.1016 0.3884 6 8.9568 2.3696 0.0611T(BA)� L(CsI) 4 7.6132 2.9694 0.0394 4 0.6392 0.1691 0.9526T(BA)� S(L(CsI)) 22 2.5639 1.8605 0.0235 24 3.7798 2.6875 0.0004Res 102 1.378 108 1.4065Total 152 161Cochran’s C-test C ¼ 0.1430 C ¼ 0.1116Transformation Sqrt(x) Sqrt(x)

SFSourceBA 1 1272 0.2588 0.8653 1 530.28 0.0512 0.9944CsI 1 416.58 0.5206 0.7109 1 6324.7 0.2280 0.9053T(BA) 4 7543.6 6.9119 0.0395 4 10,036 12.886 0.0153L (CsI) 1 110.01 0.3855 0.8996 1 29,222 25.509 0.0001BA� CsI 1 3637.5 1.2451 0.4068 1 1238.6 0.1156 0.9715S(L(CsI)) 6 1073.4 1.525 0.2184 6 393.55 0.5749 0.7518BA� L(CsI) 1 1541.3 1.3774 0.2833 1 15,516 12.248 0.001CsI� T(BA)a 3 2389.5 2.1894 0.2303 4 1937.6 2.4878 0.189BA� S(L(CsI)) 6 536.83 0.7627 0.6058 6 543.93 0.7946 0.5834T(BA)� L(CsI) 4 1091.4 1.5505 0.2287 4 778.83 1.1378 0.36T(BA)� S(L(CsI)) 22 703.88 1.9947 0.0113 24 684.52 2.6633 0.0003Res 102 352.88 108 257.02Total 152 161Cochran’s C-test C ¼ 0.1352 C ¼ 0.1009Transformation None None

a For the 1.2-m assemblages. this term lacks data corresponding to the 2001 survey at the putatively impacted location.

I. Díez et al. / Marine Environmental Research 84 (2013) 31e42 39

successful restoration of large fucoids to shores affected by sewageeffluent.

Our study shows no evidence linking pollution to variability inthe abundance of morphologically simple forms. This finding is incontrast with many earlier studies on the effects of sewage onintertidal assemblages, which report the proliferation of filamen-tous and sheet-like algae in polluted environments (Fairweather,1990; Schramm and Nienhuis, 1996; Bellgrove et al., 1997;Eriksson et al., 2002; Martins et al., 2012). Likewise, investiga-tions of the recovery processes following outfall decommissionshave documented declines in the abundance of ephemeral greenalgae (Smith et al., 1981; Archambault et al., 2001). These changesin the distribution patterns of simple forms have been mainlyassociated with changes in nutrient availability (Martins et al.,2012). However, synergistic effects with other disturbance factors

such as the addition of fresh water could be determining theabundance of green algae close to the outfalls. Experimentalresearch has provided evidence that green algae are more tolerantto low salinity than red algae (Larsen and Sand-Jensen, 2006). Inour study the average daily flow of sewage was low and waters atthe outfall location did not show reduced salinity (unpublisheddata). With intermediate nutrient enrichment and full marineconditions, coralline algae may outcompete ephemeral algae byusing different mechanisms, including synthesis of antifoulingcompounds (Jeong et al., 2000; Kim et al., 2004) and providingrefuge for grazers (Karez et al., 2004).

In summary, this study provides evidence that the wastewatertreatment upgrade significantly reduced the impact of sewage onmacroalgal vegetation. It also concludes the choice of variablesused to assess community recovery largely determines the degree

Fig. 5. Mean and standard errors of percentage cover of morphologically complex algae (MCA), calcareous red algae (CA), and simple forms (SF) of 1.2 m and 0.5 m phytobenthicassemblages at the controls (Cs) and at the putatively impacted location (I) over time (light and dark bars correspond to sampling times before and after the onset of the secondarytreatment, respectively).

I. Díez et al. / Marine Environmental Research 84 (2013) 31e4240

of restoration and the time involved in detecting it. As an example,there were no differences in diversity between the outfall and thecontrol locations 3 years after the onset of the secondary treatment,whilst differences in cover of calcareous algae still persisted at theend of the study. Seaweed vegetation seems to take a long time torecover in response to water quality improvement. Moreover, thesuccessful recolonisation of lost species into remediated areas mayalso depend on factors other than pollution removal. Long distancesto be travelled by potential colonists, low dispersal ability andbiological interactions with turf-forming species that currentlydominate outfall location may hinder complete assemblagerecovery (Bellgrove et al., 2010). Further manipulative experimentson macrophyte recruitment should be conducted to determinewhether remediated shores can be naturally restored or whetheralternative management measures, such as algal transplants, needto be included (Sales et al., 2011).

Acknowledgements

We sincerely thank the local water supply and sewerageauthority ‘Consorcio de Aguas Bilbao Bizkaia’ (www.consorciodeaguas.com) for supporting this research. We are alsomost grateful to our zoologist colleagues of the marine benthosresearch group at the University of the Basque Country, and to Dr.Antonio Secilla for his assistance in species identification andfieldwork during the first stage of this investigation.

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