Science of the Total Environment 605–606 (2017) 1055–1063

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Relationship of fish indices with sampling effort and land use change in alarge Mediterranean river

David Almeida a, Juan Diego Alcaraz-Hernández a, Roberto Merciai a, Lluís Benejam b, Emili García-Berthou a,⁎a GRECO, Institute of Aquatic Ecology, University of Girona, 17003 Girona, Catalonia, Spainb BETA Technology Centre, Aquatic Ecology Group, University of Vic – Central University of Catalonia, 08500 Vic, Catalonia, Spain

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Fish-based indices have been barelytested in lower reaches of largeEuropean rivers.

• Two fish-based indices (EFI+ andIBICAT2b) respond significantly to landuse change in the River Ebro.

• EFI+ also depends on sampling effort.• Existing standard protocols should becarefully followed for ecological assess-ment using fish.

E-mail address: emili.garcia@udg.edu (E. García-Berth

http://dx.doi.org/10.1016/j.scitotenv.2017.06.0250048-9697/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 April 2017Received in revised form 2 June 2017Accepted 3 June 2017Available online xxxx

Editor: D. Barcelo

Fish are invaluable ecological indicators in freshwater ecosystems but have been less used for ecological assess-ments in largeMediterranean rivers.We evaluated the effects of sampling effort (transect length) onfishmetrics,such as species richness and two fish indices (the new European Fish Index EFI+ and a regional index, IBICAT2b),in themainstemof a largeMediterranean river. For this purpose,we sampled byboat electrofishingfive sites eachwith 10 consecutive transects corresponding to a total length of 20 times the river width (European standard re-quired by theWater Framework Directive) andwe also analysed the effect of sampling area on previous surveys.Species accumulation curves and richness extrapolation estimates in general suggested that species richnesswasreasonably estimated with transect lengths of 10 times the river width or less. The EFI+ index was significantlyaffected by sampling area, both for our samplings and previous data. Surprisingly, EFI+ values in general de-creased with increasing sampling area, despite the higher observed richness, likely because the expected valuesofmetrics were higher. By contrast, the regional fish indexwas not dependent on sampling area, likely because itdoes not use a predictivemodel. Both fish indices, but particularly the EFI+, decreasedwith less forest cover per-centage, even within the smaller disturbance gradient in the river type studied (mainstem of a large Mediterra-nean river, where environmental pressures are more general). Although the two fish-based indices are verydifferent in terms of their development, methodology, and metrics used, they were significantly correlated andprovided a similar assessment of ecological status. Our results reinforce the importance of standardization ofsampling methods for bioassessment and suggest that predictive models that use sampling area as a predictormight be more affected by differences in sampling effort than simpler biotic indices.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Ecological statusEuropean fish index EFI +Human impactIndex of biotic integrityNon-native species

⁎ Corresponding author.

ou).

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1. Introduction

Freshwater ecosystems provide essential services to humanity (e.g.water provisioning, irrigation, energy production) and are thus severelydisrupted worldwide because of hydrologic alteration, pollution, riverfragmentation, and introduction of invasive species (Malmqvist andRundle, 2002; Nilsson et al., 2005; Gallardo et al., 2016). Consequently,fresh waters are among the most threatened habitats at the globalscale (Malmqvist and Rundle, 2002; Vörösmarty et al., 2010). Accurateassessments of the ecological status of these water resources are crucialfor environmental managers and to assist policy-makers in designingappropriate regulations (Boulton, 1999; Carballo et al., 2009). Such en-vironmental assessments are often undertaken by using Indexes of Biot-ic Integrity (IBIs), which can be based on a variety of biologicalcommunities from the aquatic ecosystem, such asmacrophytes, benthicinvertebrates, diatoms or fish (Karr, 1991; Hering et al., 2006). Fish arean excellent taxonomic group to estimate ecological integrity in rivers(Schiemer, 2000), as they are very sensitive to common anthropogenicdisturbances including hydrologic alteration, habitat degradation, lossof water quality or land-use change (Casatti et al., 2009; Aparicioet al., 2011). Fish have long lifespans and generation times and largehome ranges compared to most other aquatic taxa, making themmore appropriate to detect disturbances at large spatial and temporalscales, e.g. old disruptive events (Karr, 1981; Simon, 1999).

In the European Union (EU), theWater Framework Directive (WFD)regulates themanagement of water resources and requires theMemberStates to assess the ecological status of their water bodies (EC, EuropeanCommission, 2000; Schmutz et al., 2007). The WFD requires the use offish among other taxonomic groups for these assessments (Heringet al., 2006; Carballo et al., 2009; Vidal-Abarca et al., 2016). A recent re-view of aquatic bioassessmentmethods in Europe showed that their re-sponse with anthropogenic pressures is less tight in rivers than in othersystems such as lakes or coastal waters and also weaker with fish thanother taxonomic groups (Birk et al., 2012). One of the landmarks inthe development of a fish-based IBI was the European Fish Index (EFIhereafter), consisting of a multi-metric predictive model based on thedeviation between observed and predicted reference conditions forthe fish assemblages (Pont et al., 2006, 2007). A new version of thisindex, the EFI+, was later developed to overcome several limitations(e.g. low applicability to different eco-regions within Europe, better de-tection of anthropogenic disturbances) and thus, to provide a methodapplicable across nearly the whole range of European rivers (EFI+Consortium, 2009; Logez and Pont, 2011). However, the applicabilityof EFI+ in lowland reaches of large rivers is uncertain, as few referencesites under these environmental conditions were available for the de-velopment of predictive models (De Leeuw et al., 2007; EFI+Consortium, 2009). Because the EFI+ was developed at the Europeanscale, it is invaluable for comparison and intercalibration of indices butis expected to respond less well than methods developed at regionalscales (Segurado et al., 2014). An index developed for Catalonia (NESpain), IBICAT2b, has been shown to respond significantly to anthropo-genic pressure, particularly hydrologic alteration, in the Ebro River(García-Berthou and Bae, 2014; García-Berthou et al., 2016). In contrastto EFI+, IBICAT2b is a type-specificfish index that does not use a predic-tive modelling approach and consists of 4–8 different metrics depend-ing on the river type (García-Berthou et al., 2016). Therefore, theimplementation of fish as ecological indicators needs further study, par-ticularly in the lower reaches of large rivers. Fish respond better thanother taxa to hydrological and morphological deterioration (Birk et al.,2012), which is more important in the lower reaches of large riversdue to altered streambed and siltation, channelization and the loss ordisconnection of floodplains (Richter et al., 1997; Aarts et al., 2004).

Few studies have investigated the necessary sampling area and ef-fort for many of the methods used for the WFD (Birk et al., 2012) andparticularly fish in large rivers (Hughes et al., 2002; Meador, 2005).Too small sampling sizes produce inaccurate and imprecise estimates

of fish species occurrence and richness —key indicators in estimates offish assemblage integrity and diversity—, whereas oversampling causescosts higher than necessary (Hughes et al., 2002). Species richness risesmonotonically with sampling area (e.g., electrofishing transect length)to an asymptotic maximum number of species (Hughes et al., 2002;Meador, 2005). Typically, a smaller transect length might be expectedto be necessary to estimate relative abundance or species composition,whereas larger sampling efforts are needed to accurately estimate spe-cies richness (Dauwalter and Pert, 2003) and species abundance shouldbe more proportional to sampling size. The WFD states that methodsused for the monitoring of fish and other type parameters should con-form to the standards developed by the European Committee for Stan-dardization (CEN) or other national or international standards, whichensure the provision of data of an equivalent scientific quality and com-parability EC (European Commission) (2014)). For fish in particular, theEN 14962 European Standard (CEN, European Committee for Standard-ization, 2006) recommends electrofishing as one of the most suitablemethods for sampling in habitats b2 m deep of all kind of rivers. TheEN 14011 European Standard (CEN, European Committee for Standard-ization, 2003) provides detailed requirements on electrofishing, includ-ing that: i) for assessing species composition, abundance and agestructure in rivers, in general a river length of at least 20 times theriver width should be sampled; and ii) for large rivers (b30 m wide),where it is already known that the fish community is uniform, a lengthof 10 times the river width may be sufficient.

These CEN guidelines on sampling effort have not been systematical-ly followed in Spain and elsewhere, with surveys often by wading fromthe river bank during low flows and with river lengths much shorterthan required by the EN 14011 standard. The effects of electrofishing ef-fort in large European rivers have been barely examined (Flotemerschet al., 2011) and the applicability of EFI+ and other indices in largeMediterranean rivers is largely unknown. The objectives of our studyare: i) to evaluate the effects of sampling effort (transect length) onthe fish metrics, such as species richness and EFI+; and ii) to testwhether sampling effort affects the results of EFI+ and another regionalfish index (IBICAT2b) in a given site. We hypothesized that: i) as cus-tomary, species richness would increase with sampling effort, but lessthan in many other regions since local fish species richness is low inthe Iberian Peninsula; and ii) IBIs such as EFI+ and IBICAT2b wouldbe less affected than species richness by sampling effort, because theyalso rely on other metrics (species composition and size structure).

2. Materials and methods

2.1. Study area

The Ebro River (NE Spain) is the second largest catchment of the Ibe-rian Peninsula in terms of length (910 km) and drainage area(85,362 km2), flowing from the Cantabrian and Pyrenean ranges totheMediterranean Sea (Fig. 1). Climate in the river basin is very diverse:oceanic (Köppen classification) in much of the catchment, cold semi-arid in its centre, Mediterranean in the lowermost reaches and evenhumid continental in the uppermost reaches of the Pyrenees (AEMET-IM, 2011). Thus, temperature and precipitation vary greatly across thebasin, e.g. average annual temperatures ranging from 0.8 to 16.2 °C(López-Moreno et al., 2013) and precipitation from N1500 mm (meanannual precipitation) in the Pyrenees (with elevations N3000 m a.s.l.)to b400 mm in the semi-arid interior (AEMET-IM, 2011).

Mean annual discharge recorded at Tortosa town (the most down-stream gauge) is 452 m3/s (Solans and Poff, 2013) and mean annualrunoff is 13.4 hm3 (= 13.4 × 106 m3) (Batalla et al., 2004). The highestflows are generally in winter and spring, but the timing of the peak andseasonality vary spatially because contrasting climates and geology andheterogeneous topography generate high variability of the river re-gimes among different sub-basins (López-Moreno et al., 2013; Solansand Poff, 2013). Moreover, the Ebro river is regulated by over 187

Fig. 1.Map of the River Ebro basin, with elevation and location of the five sites sampled for this study (red circles) and previousfish samplings (black circles) in themainstem (official typeR-T17bis). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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reservoirs distributed throughout its watershed, which impound 57% ofthe mean annual runoff and have caused marked hydrologic alteration,in general with reduction in flood frequency and magnitude, decreasesin daily flow mean and variability and inversion of seasonal patterns,with increased flows in summer for irrigation purposes (Batalla et al.,2004).

Geology varies markedly in the basin, ranging from Palaeozoic plu-tonic rocks in the Central Pyrenees to dolomites and limestone in thePre-Pyrenean part, tertiary evaporites in the centre of the depression,and quaternary alluvial deposits in the valleys bottoms (Batalla andVericat, 2011). Land use is mainly agricultural and livestock farming,with cities mainly in the mainstem, where some industries and a fewnuclear energy plants are also present. Human population within thebasin is about 3.2 million people (year 2013, INE, Instituto Nacional deEstadística, 2016), mainly in themainstem (e.g. Zaragoza city). Detaileddata on physical and chemical water properties, phytoplankton assem-blages and fauna are available elsewhere (Sabater et al., 2008; Romaníet al., 2011). Native fish assemblages are dominated by endemic cypri-nids (e.g. Achondrostoma arcasii, Barbus haasi, Gobio lozanoi, Luciobarbusgraellsii, Parachondrostoma miegii, Phoxinus bigerri, and Squaliuslaietanus) although many introduced species (e.g. bleak Alburnusalburnus, common carp Cyprinus carpio, and European catfish Silurusglanis) thrive in mainstem reaches (García-Berthou and Bae, 2014).

Table 1Physiographical and limnological features of the five sites sampled during July and Septemberclosest gauging station (obtained from http://sig.magrama.es). Thewater quality data are the avwww.datossuperficiales.chebro.es).

Longitude(°W)

Latitude(°N)

Altitude(m a.s.l.)

Drainagebasin(km2)

Mean waterflow(m3/s)

Totalammo(mg/L

Castejón 1°41′41.69″

42°10′50.11”

271 24,317 229.3 0.160

Sobradiel 1°01′26.21″

41°44′45.42”

205 39,476 231.0 0.047

Pina de Ebro 0°32′06.97″

41°29′12.37”

158 46,033 247.6 0.282

Ascó 0°34′13.74″

41°11′08.18”

33 81,557 313.3 0.125

Xerta 0°30′11.34″

40°54′42.59”

13 83,166 426.5 0.123

2.2. Sampling

We sampled fish and assessed habitat from 20 July to 22 September2015 in five sites along the mainstem of the River Ebro (Fig. 1), corre-sponding to the official Spanish river type R-T17bis (‘Large Mediterra-nean watercourses’, formerly type 17 or 117). The sites were selectedin an upstream-downstream gradient along the whole river type forrepresentativeness and had good or very good chemical status (i.e.low concentrations of pollutants) but altered, regulated flow regime(Table 1). The drainage area increased markedly along this gradientbut mean water flow did not, because of the cumulative storage ofmany reservoirs and less precipitation in centre of the basin. Nutrientconcentrations are intermediate in the two most upstream sites andreached themaximum at the third site (Pina de Ebro), which has slight-ly less good water quality because it is ca. 40 km downstream of thelargest city in the basin (Zaragoza) (see also Sabater et al., 2008). Thetwo lowermost sites (Ascó and Xerta) are below two very large reser-voirs (Mequinenza and Riba-roja, of 1530 and 210 h m3 of capacity),after which phosphate and phytoplankton concentrations strongly de-crease (Sabater et al., 2008). Spatial and limnological features of everysampling site are given in Table 1.

Ten equal consecutive river transects were surveyed at each sam-pling site. In each site, every transect had lengths of twice the mean

2015. The water flow is the average for the period from 1 June 2014 to 1 June 2015 at theerages for the period 2002–2015 at the closest monitoring station (data available at http://

nium)

Conductivity(μS/cm)

Totalphosphorus(mg/L)

Nitrates(mg/L)

Dissolvedoxygen(mg/L)

pH Watertemperature(°C)

835 0.065 10.895 9.204 8.1 14.47

1161 0.047 5.689 9.380 8.0 16.07

1387 0.101 15.173 9.478 8.0 15.07

1035 0.065 9.977 9.031 8.1 18.01

1002 0.068 9.625 9.308 8.2 17.72

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river width (ca. 50–100 m), so a total of ca. 1000–2000 mwas sampledper site and the total surveyed river length at all siteswas 20× themeanriver width, as required by the EN 14011 European Standard (CEN,European Committee for Standardization, 2003). Only one bank wassurveyed in each transect, alternating the left and right margins alongthe site. Electrofishing was carried out from a boat (4.5-m aluminiumhull) by using a 2000 W DC generator at 1000 V and 16 A (Model:5.0-GPP Smith-Root Inc., Vancouver, WA, USA) with two anodesmounted on booms; a dip net (2.5 m long pole, 50 cm diameter net,10 mm mesh size) was used to collect the stunned fish. A single passwasmade in an upstream direction without using block nets, accordingto the EFI+ guidelines (EFI+ Consortium, 2009). In every transect, fishwere identified to the species level, counted, measured for fork length(FL, to the nearest 1 mm) (total length (TL) for fish without fork),weighed (wet body weight, ±0.1 g) and examined for diseases/abnor-malities. Habitat data were assessed per transect: wetted width (m),water depth (m), cover of riparian vegetation (%), and substratum com-position (boulder/rock, gravel/pebble/cobble, organic, sand, silt). Oxy-gen level (mg/L), conductivity (μS cm−1), pH and water temperature(°C) for themost neighbouring sites for the period 2002–2015were ob-tained from the Water Agency of the River Ebro Basin (in Spanish:‘Confederación Hidrográfica del Ebro’, ‘CHE’ hereafter).

After each transectwas concluded, non-native fishwere immediate-ly immersed in an overdose solution of anaesthetic (MS-222) for 15minto follow local regulations. Individuals of native fish specieswere kept ina tankwith supplied oxygen (two battery-operated aeratorswith porta-ble pump) until fully recovering from the anaesthetic and being re-leased to the river. All field procedures complied with animal use andcare regulations of Europe and Spain (specific licences were grantedfor Scientific Field Research in the River Ebro). Fish were collected bytrained personnel with all permits necessary.

2.3. Data compilation

Apart from the field sampling during 2015, data were also compiledfrom previous surveys (n = 41, 2003–2011) in this river type of theRiver Ebro (Fig. 1). Fish surveys were provided by the CHE. All suitabledata (e.g. having sampling area, fish lengths and other data requiredfor the computation of the indices) fromprevious electrofishing surveysin the R-T17bis river type were included. All these samplingscorresponded to wade electrofishing carried out between July–Octoberin 2003, 2005, 2007 or 2011 and in general had transect lengths shorter(length range: 10–200 m; sampled surface area: 28–3600 m2) thanthose requested by the EN 14011 standard. Data on environmental fea-tures and anthropogenic pressures for all sites were compiled andanalysed in detail elsewhere (García-Berthou and Bae, 2014; Du et al.,unpublishedmanuscript). In particular, for this studyweused an indica-tor of land use change, which is simply the first axis of a principal com-ponent axis of the percent covers of forest, agricultural and urban usesin the drainage upstream of the sampling site (higher values indicatemore agricultural and urban uses).

2.4. Data analyses

We assessed the role of electrofishing transect length on twomultimetric, guild-based fish indices of biotic integrity: the EuropeanFish Index, EFI+ (EFI+ Consortium, 2009) and IBICAT2b (García-Berthou et al., 2016). EFI+ is a site-specific pan-European fish indexthat uses a predictive modelling approach to compare the observedfish community with that expected from models for reference condi-tions (Logez and Pont, 2011; Segurado et al., 2014). EFI+ has beenshown to respond significantly to anthropogenic pressures throughoutEurope (EFI+ Consortium, 2009; Logez and Pont, 2011), including theIberian Peninsula (Segurado et al., 2014). Although EFI+ is essentiallya site-based index, the sites are classified prior to its computation intoeither salmonid or cyprinid zones, based on abiotic variables

(Segurado et al., 2014). These abiotic variables include altitude, flow re-gime, geomorphology, presence/absence of lakes upstream, watersource, flood plain, upstream drainage area, distance from source,river slope and air temperature, andwere obtainedwith Geographic In-formation Systems and available databases. All sites in river type R-T17bis corresponded to the EFI+ cyprinid zone, where two differentmetrics are computed by the EFI+ software: richness of species requir-ing rheophilic reproduction habitat and density of species requiringlithophilic reproduction habitat. EFI + does not explicitly considerwhether the species is native or alien although many of the former arerheophilic and litophilic in the Iberian Peninsula in contrast to mostalien species. Eachmetric ranges from zero (very impaired) to one (ref-erence conditions) and EFI+ is obtained as the average of both metrics.Although we use Doadrio et al. (2011) for recent changes in the taxon-omy of Iberian fish fauna, we considered older synonyms to use theEFI+ software (see Table S1 for details). The EFI+ and its two metricswere computed using specific software available at http://efi-plus.boku.ac.at/software/ (EFI+ Consortium, 2009) for the total area sam-pled per site and also accumulating the 10 different transects per site.

In contrast to EFI+, the IBICAT2b is a type-specific fish index, whichwas developed for Catalonia (including part of the Ebro) and does notuse a predictive modelling approach (García-Berthou et al., 2016).IBICAT2b uses the official river WFD typology of Spain and scores 4–8different metrics: number of native species; percentage of individualswith deformities, eroded fins, lesions or tumours (DELT) anomalies;percentage of introduced individuals (either exotic or translocatedwithin Spain); percentage of introduced species; and up to four addi-tional metrics depending on river types. For type R-T17bis, only thefour general metrics are used. IBICAT2b has been shown to respond sig-nificantly to anthropogenic pressure in Catalonia and throughout theEbro (García-Berthou and Bae, 2014; García-Berthou et al., 2016; Colinet al., 2016). IBICAT2b ranges from 1 (very impacted sites) to 5 (bestcondition) and was computed with unpublished software (HIBIM) de-veloped for the Spanish Ministry of Environment (see García-Berthouet al., 2016 for an alternative spreadsheet and further information). Toassess the effects of sampling area in previous monitoring not applyingthe EN standard, IBICAT2b and EFI+ were also computed for the com-piled fish data set, including the 2015 data (n = 46).

Generalized Additive Models (GAMs) were used to test for the ef-fects of sampling area and an indicator of environmental pressure(land use change) on the two indices and the two EFI+ metrics.GAMs were fitted with package ‘mgcv’ in the R software environment(R Core Team, 2015). Since EFI+ is a continuous metric ranging fromzero to one, we used the beta regression family in ‘mgcv’; IBICAT2bwas rescaled from 1 to 5 to 0–1 for analyses (but not for reporting itsvalues).

We also used species-accumulation curves (SACs) to examine the ef-fects and adequacy of sampling effort at the five sites. SACs show therate at which new species are found within a community when sam-pling effort increases and depend on local (alpha) and spatial (beta) di-versities (Colwell and Coddington, 1994). We used the real order oftransects to build the SACs, where in our study, the number of transectswas directly proportional to sampling area. Extrapolated fish richness(i.e. total number of species expected to be present in a particular site)was estimated by using the second-order jackknife estimator (Palmer,1991; Colwell and Coddington, 1994). SACs were obtained with the‘vegan’ package (Oksanen et al., 2015) in R.

3. Results

3.1. Species composition and relative abundance

Totalfish abundance peaked at the twomiddle reach sites (Sobradieland Pina de Ebro), where observed fish richness was lowest (b7 spe-cies) and percentage of introduced fish was highest (N95%) (Figs. 2and 3). Bleak was always the most abundant species except in the

Fig. 3. Species accumulation curves for the ten consecutive transects per sampling site.Each electrofishing transect had a length equal to twice thewidth of the river in the reach.

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lowermost site, where some native diadromous species (European eel,thinlip grey mullet, and sea bass) were common (Fig. 2). Only threealien invasive species (bleak, European catfish and common carp) werecaptured across the ten transects in Sobradiel, which had a similar speciescomposition than Pina de Ebro. Rheophilic, lithophilic species (Ebro bar-bel and Ebro nase) were only abundant in the most upstream site andnot captured in two of the middle reaches (Sobradiel and Pina de Ebro).

SACs generally showed considerable saturationwith about five tran-sects (i.e. lengths of 10 river widths) except for the most downstreamsite (Xerta) (Fig. 3). Except for this site, extrapolated richness(Table 2) confirmed that species richness was reasonably estimatedand that with five transects N50% of the estimated species pool hadbeen captured. In Xerta, with increasing sampling effort, additionalalien (e.g. zander, channel catfish, topmouth gudgeon, roach) and na-tive fishes (e.g. Pyrenean gudgeon, chub) were captured and conse-quently SACs barely flattened, and extrapolated richness wasconsiderably higher.

EFI+ scores (Fig. 4) were highest (corresponding to good ecologicalstatus) in the most upstream site because of the relative abundance oftwo (native) lithophilic and rheophilic species (barbel and nase)(Fig. 2) and lowest (corresponding to bad ecological status) in Pina deEbro, where no species of these guilds were captured. EFI+ scoreswere also low for Ascó and increased to poor ecological status inXerta, where although many more species were captured, fewcorresponded to the lithophilic or rheophilic guilds. The difference be-tween observed values and those expected from predictive modelswere lower for the lithophilic species density than for the rheophilicspecies richness (Fig. 4). The results for IBICAT2b were similar(Fig. S1) because all lithophilic or rheophilic species in the Ebro Riverare native, except for channel catfish (Table S1).

Correspondingly, when all available data were considered (Fig. S1),the two indices were significantly correlated (r = 0.55, P b 0.001) andagreed on the ecological status for 46% of the sites (21 of 46) although

Fig. 2.Number of fish captured (summer 2015) by species in each of the ten consecutive transectwice the width of the river in the reach (i.e. the ten transects amount to 20 times the width o

IBICAT2b yielded higher statuses (e.g. 11 of 26 of sites classified as‘poor’with IBICAT2b had ‘bad’ status with EFI+). Overall, EFI+ indicat-ed that the ecological status was bad or poor for themost of sites (31 of46),moderate for 10 sites and good for 5. According to IBICAT2b, the fre-quencies of ecological status were: 2 very good, 1 good, 11 moderate,and 32 poor. Both indices were decreased with land use change (i.e.

ts for the five sites sampled in this study. Each electrofishing transect had a length equal tof the river, as required by the EN 14011 European Standard).

Table 2Generalized additive models (GAM) of the EFI+ index and its two metrics (richness ofrheophilic species and density of lithophilic species) with the area of sampling as a predic-tor at the five sampling sites (Castejón, Sobradiel, Pina de Ebro, Ascó and Xerta). The P val-ue and adjusted determination coefficients (R2

adj) are shown. An estimate of theextrapolated richness using the jackknife estimator of second order is also shown.

Rheophilicspecies richness

Lithophilicspecies density

EFI+ Extrapolatedrichness

P R2adj P R2adj P R2adj

Castejón b0.001 0.88 0.001 0.93 b0.001 0.94 9.0Sobradiel 0.993 1.00 − 1.00 0.985 1.00 3.0Pina de Ebro 0.004 0.90 − 1.00 0.004 0.90 9.4Ascó b0.001 0.98 0.471 0.12 b0.001 0.96 13.0Xerta 0.001 0.79 0.011 0.96 0.009 0.88 22.8

Table 3Generalized additive models (beta regression family) of the EFI+ and IBICAT2b fish indi-ces with sampling area, with andwithout an indicator of land use change as an additionalsmooth term for 46 samples in the River Ebromainstem (official type R-T17bis). edf= ef-fective degrees of freedom.

Responsevariable

Predictor(s) edf χ2 P Deviance explained(%)

EFI+ Sampling area 6.234 25.1 0.0006 91EFI+ Land use change 1.000 9.3 0.0023 93

Sampling area 6.452 23.8 0.0056IBICAT2b Sampling area 1 0.36 0.55 2IBICAT2b Land use change 1.117 11.2 0.0021 22

Sampling area 1.000 0.09 0.77

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less forest cover andmore agricultural and urban cover percentages), al-though the percentage of variation explained was quite lower forIBICAT2b in this river type (Table 3, Fig. 5), which is more dominatedby invasive fishes than other parts of the river basin.

3.2. Effects of sampling effort on fish indices

The EFI+ values and its two metrics varied significantly with sam-pling area in four of the five sites sampled (not in Sobradiel, where spe-cies richness was lowest) (Table 2). In three of the sites, the index andparticularly one of its metrics (rheophilic species richness) decreasedwith increasing sampling area, but in Xerta it increased together withthe other metric (lithophilic species density) (Fig. 4).

The results when considering all previous available data showed thesamepattern,with significant decreases of the EFI+with sampling area(Table 3, Fig. 5). Although EFI+ significantly decreased with land usechange (Fig. 5), a significant effect of sampling area remained in themodel with both predictors (Table 3). Therefore, the effects of sampling

Fig. 4. Relationship of the EFI+ index and its twometrics (richness of rheophilic species, densit(by spatial order) per sampling site.

area on EFI+ are not due to more impaired ecological status down-stream (collinearity of sampling area and land use change). By contrast,IBICAT2b responded significantly but more variably to land use change,but did not depend significantly on sampling area (Table 3, Fig. 5).

4. Discussion

4.1. Comparison of fish indices

The fish community of the River Ebromainstem in the five samplingsites was dominated by introduced species, ranging from 40% of thetotal catches in themost downstreamsite and73% at themost upstreamsite to 100% in a middle reach site (Sobradiel). The most abundant fishspecies was bleak, which has been widely introduced in Iberian reser-voirs as a ‘forage’fish for piscivorous species (Vinyoles et al., 2007). Con-sequently, the IBICAT2b, which uses percentage of native species andnative individuals and number of native species as three of its metrics,displayed low values throughout the Ebromainstem, except in the low-ermost and uppermost reaches of this river type. Similarly, few of the

y of lithophilic species) with the cumulative sampling area of the ten consecutive transects

Fig. 5. Relationship of the EFI+ and IBICAT2b fish indices with sampling area (right) and land use change (left) of 46 samplings in the River Ebro mainstem (official type R-T17bis). Thefitted GAM function is also shown.

1061D. Almeida et al. / Science of the Total Environment 605–606 (2017) 1055–1063

individuals captured corresponded to the lithophilic or rheophilic guilds(maxima of 28% in Xerta and 26% in Castejón) and none in Sobradiel andPina de Ebro. Ebro barbel and Ebro nase, two native species withrheophilic and lithophilic reproduction habitats, were only abundantin themost upstream site (Castejón); Ebro chub, another native speciesof the same guild, was also abundant in the most downstream site(Xerta). Consequently, EFI+ was highest in the uppermost site, de-creased markedly in middle reaches (where only phytophilic,limnophilic introduced species were captured) and recovered slightlyin themost downstream site. Previous samplings (Fig. S1) showed sim-ilar patterns. Therefore, although EFI+ and IBICAT2b are very differentfish-based indices in terms of their development, methodology, andmetrics used (e.g. in contrast to IBICAT2b, EFI+ does not considerwhere species are native), they are significantly correlated and providea similar assessment of ecological status. Both indices were also nega-tively correlated with land use change (Fig. 5), i.e. decreased with in-creasing agricultural and urban land cover in the upstream drainage.Therefore, both indices seem to respond to environmental degradation,even when considering only the mainstem of this large Mediterraneanriver, where more preserved sites with less disturbances are much lessfrequent than in upstream reaches. However, less variation in land usechange was explained by IBICAT2b in this river type (Table 3, Fig. 5),which is more dominated by invasive alien species than other parts ofthe river basin. Although EFI+ has been developed using data fromthroughout Europe (EFI+ Consortium, 2009), few reference sites wereavailable for large rivers to develop the predictive models. Our study isone the few showing correlation of EFI+ with pressures in themainstem of large rivers (see Segurado et al., 2014 for similar results).

4.2. Effects of sampling effort

The relationship between species richness and sampling area isoften considered one of the main general rules in ecology (e.g.

Lomolino, 2000). This relationship and the adequacy of sampling effortwhen electrofishing in a multitude of conditions have been extensivelyinvestigated (e.g. Ebner et al., 2008; Benejam et al., 2012;Watkins et al.,2016). As far as we know, the effect of sampling effort on the EFI+ andfulfilling the CEN requirements has been much less investigated (butsee examples in Schmutz et al., 2007; Van Liefferinge et al., 2010). As hy-pothesized, SACs flattened in four out of five study sites, even with lessthan five transects (10 × wetted width), because Iberian streams andrivers generally have low local fish species richness (Doadrio et al.,2011). However, the lowermost reach (Xerta site) of the Ebro River(and probably other large Iberian rivers) was an exception and evensampling areas suggested by the CEN protocol (20 × wetted width)were not sufficient to reach the curve stabilization in fish richness.These results illustrate the importance of following the EN 14011 Stan-dard, carefully considering the sampling effort and not assuming a spe-cies is absent because it was not captured. This applies not only to fishrichness but also to metrics based on abundance estimations. Indeed,fish abundance is often much more dependent than richness on varia-tions in sampling effort (e.g. Benejam et al., 2012; Hangsleben et al.,2013; Samons, 2015). Our results showed that EFI+values virtually sta-bilized in the second half of the total surveyed area (fifth accumulatedriver transect) for every sampling site, which highlights the appropri-ateness of applying the CEN guidelines and that 10 times the riverwidth may often be appropriate in Iberian rivers. Therefore, our resultsalso confirm the hypothesis that fish indices are less affected than fishrichness because they also rely on other metrics such as species compo-sition or relative abundance. A relative homogeneity of ‘indicator’ spe-cies (i.e. those that increase EFI+ values such as rheophilic orlithophilic species) was observed in Castejón, Pina de Ebro and Ascósites across river transects. Therefore, observed patterns in EFI+ valuesmay be more likely due to higher expectations of predictive modelswith increasing sampling area. By contrast, IBICAT2b is not based on apredictive model, as this index only utilizes observed features of fish

1062 D. Almeida et al. / Science of the Total Environment 605–606 (2017) 1055–1063

assemblages, including species origin (García-Berthou et al., 2016). Al-though this index can also vary slightly across river transects under par-ticular conditions (e.g. Castejón site), it was not dependent on samplingarea, in contrast to EFI+. Overall, our findings reinforce that CEN guide-lines should be followed for ecological assessment usingfish and that 10times the river width might be adequate in many Iberian rivers. Resultsalso suggest the need to carefully consider the sampling protocol in in-tercalibration exercises and analyses using data from numerous teams(Beier et al., 2007; Benejamet al., 2012), particularlywhen using predic-tive models.

Acknowledgements

This research was funded by the ‘Confederación Hidrográfica delEbro’ (CHE) (code: 2015-PH-11.I). We thank Miguel Ángel García Veraand Patricia Navarro (CHE) for the supervision of the research projectand insightful comments. We also thank three anonymous reviewersfor helpful comments on the manuscript. Additional financial supportwas provided by the Spanish Ministry of Economy, Industry and Com-petitiveness (projects CGL2015-69311-REDT, CGL2016-80820-R andODYSSEUS PCIN-2016-168), and the Government of Catalonia (ref.2014 SGR 484). David Almeida held a postdoctoral fellowship fromthe ‘Beatriu de Pinós’programme, funded by theGeneralitat of Cataloniaand the Marie Curie COFUND.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in theonline version, at http://dx.doi.org/10.1016/j.scitotenv.2017.06.025.These data include the Google map of the most important areas de-scribed in this article.

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1

Table S1. Current scientific name of the species caught and synonymy used for calculation of

the index in EFI+ software (http://efi-plus.boku.ac.at/software), list of native and alien species

and breeding habitat requirement according to EFI+ (L, lithophilic; R, rheophilic) of the

species caught in 2015. Synonyms used in the EFI+ software are highlighted in red.

Current

scientific

name

Synonymy

used for the EFI+

software

Common

name

Species

origin

Breeding

habitat

requirement

according

EFI+

Alburnus alburnus Alburnus alburnus Bleak Alien

Ictalurus punctatus Ameiurus punctatus Channel

catfish Alien

L

Anguilla anguilla Anguilla anguilla European eel Native

Luciobarbus graellsii Barbus graellsii Ebro barbel Native L & R

Carassius auratus Carassius auratus Goldfish Alien

Parachondrostoma

miegii

Chondrostoma miegii Ebro nase Native

L & R

Cyprinus carpio Cyprinus carpio Common carp Alien

Dicentrarchus labrax Dicentrarchus labrax European

seabass Native

R

Gambusia holbrooki Gambusia holbrooki Eastern

mosquitofish Alien

Gobio lozanoi Gobio lozanoi Pyrenean

gudgeon Native

Lepomis gibbosus Lepomis gibbosus Pumpkinseed Alien

Squalius laietanus Leuciscus cephalus Chub Native L & R

Liza ramada Liza ramada Thinlip grey

mullet Native

Pseudorasbora parva Pseudorasbora parva Stone moroko Alien

Rutilus rutilus Rutilus rutilus Roach Alien

Sander lucioperca Sander lucioperca Pike-perch Alien

Silurus glanis Silurus glanis Wels catfish Alien

2

Fig. S1. Ecological status according to the EFI+ and IBICAT2b fish indices for 46 samplings

in the River Ebro mainstem (official type R-T17bis).