Marine Pollution Bulletin 60 (2010) 1040–1050

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Marine Pollution Bulletin

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Towards the application of the Water Framework Directive in Italy: Assessingthe potential of benthic tools in Adriatic coastal transitional ecosystems

Cristina Munari, Michele Mistri *

Department of Biology and Evolution, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy

a r t i c l e i n f o

Keywords:Water Framework DirectiveBenthic indicesEcological quality statusCoastal transitional ecosystemsAdriatic Sea

0025-326X/$ - see front matter � 2010 Elsevier Ltd.doi:10.1016/j.marpolbul.2010.01.022

* Corresponding author. Tel.: +39 0532 455736; faxE-mail address: michele.mistri@unife.it (M. Mistri

a b s t r a c t

The potential of four benthic indices (AMBI/M-AMBI, BENTIX, BITS) was assessed in Italian coastal tran-sitional ecosystems. The community composition showed a strong dominance of lagoonal, tolerant spe-cies, and out of more of 400 species found, only about 40 taxa were dominant. The full agreement of thefour indices on an undegraded (Good or better) or degraded (Moderate or worse) status occurred only in32.3% of stations. This study evidenced that BENTIX is inappropriate for eutrophic Adriatic lagoons, andthat in such environments M-AMBI classification is actually too much dependent on diversity and rich-ness, and seems unable to capture some peculiarities of benthic assemblages in transitional waters. AMBIand BITS gave similar classifications despite the different level of taxonomic identification needed. Theunmodified use of these indices might impair accurate assessment of ecological quality status and deci-sion-making on the managers’ point of view.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Italian coastal transitional ecosystems (CTEs) exhibit differentand peculiar characteristics depending on their geographical,hydrodynamic and ecological features, and are characterized byprogressive changes in several environmental variables, oftenmutually dependent or correlated. These variations generatecomposite gradients that involve salinity, marine water renewal(e.g. residence time), nutrients, turbidity and sediment structure.(Tagliapietra et al., 2009). CTEs also display distinctive features interms of their extraordinary history of environmental manage-ment, the importance of their productivity and associated econom-ical value, which is reflected on the peculiarity of their fauna(Cognetti and Maltagliati, 2008). Along Italian coasts there are al-most 170 CTEs, but 140 of them have a surface area <10 km2. Withthe exclusion of Orbetello Lagoon (Central Tyrrhenian Sea), all thelargest CTEs are located along the Western Adriatic coasts, and,apart the Apulian Lesina and Varano Lakes, they are all concen-trated in the Northern Adriatic area. These CTEs are ecologicallycomplex systems and therefore the identification of low-cost,routine indicators for description of ecological quality, as re-quested by the Directive 2000/60 (Water Framework Directive,WFD; European Community, 2000), is a difficult task. In this per-spective, indicators able to provide an efficient and realisticdescription of ecosystem quality status in such important areasand guidelines to their application strategy to Environmental

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: +39 0532 249761.).

Agencies are urgently needed. Indeed, Italy is the Member Statemost behind in Europe with the application of the WFD, and forthis reason has received three infringements from the EuropeanCommunities. No indicator is mentioned in the 152/06 Act, aimedto the application of the WFD in Italy, and methodological flaws areeven more evident (as also declared by the Act itself) for CTEs.Therefore, the application of the most recent ecological criteriafor the assessment of ecological quality in CTEs is a priority task,which is clearly needed by Italian legislators and the nationaland regional Environmental Protection Agencies.

An important step in the WFD development is the intercalibra-tion process (IC), aiming to achieve consistency and comparabilityof the classification system results developed by Member States(MS) for the biological quality elements. In the IC process, the Euro-pean maritime area was split in the three basic ecoregions on thebasis of latitude, longitude, tidal range and salinity (Atlantic/NorthSea; Baltic Sea, Mediterranean Sea). The first phase of the IC wasconcluded in 2007. The national methods used in the IC by MS fac-ing on the Mediterranean were: BENTIX (Simboura and Zenetos,2002) for Cyprus and Greece, M-AMBI (Muxika et al., 2007) forSlovenia; MEDOCC (not published yet) for Spain. Italy and Francetested different methods but at the present time no one has beenfinalized yet (GIG, 2008). CTEs were not considered at all by Italyin the first MED-GIG-IC phase (Mistri et al., 2009). Despite this, Ital-ian ‘‘creativity” led the development of a plethora of benthic indi-ces for the assessment of ecological status in coastal andtransitional ecosystems (e.g. Forni and Occhipinti-Ambrogi, 2007;Breber, 2008; Mistri et al., 2008; Marchini et al., 2008, 2009; Mistriand Munari, 2008; Magni et al., 2009; Tataranni et al., 2009). The

C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050 1041

attention was addressed both toward the structure of the benthiccommunity itself, investigating taxonomic aspects and towardthe analysis of existing relationships between benthic communityand the main sedimentological and hydrological factors. To a lesserextent, new methods were developed also for other biological ele-ments, e.g. macrophytes (Sfriso et al., 2007, 2009), and fish (Francoet al., 2009). This redundancy of new indices, however, made Ital-ian ministerial experts aware that domestic methods were avail-able. As a consequence, at the beginning of the second MED GIG-IC phase, Italy proposed the BITS index (Mistri and Munari, 2008)for benthos and the MaQI index (Sfriso et al., 2009) for macro-phytes to be tested in CTEs.

This study represents a contribution towards the assessment ofthe potential of benthic indices developed for the implementationof the WFD for environmental quality assessment of Italian CTEs.

2. Methods

2.1. Study areas and sampling procedures

We used soft-sediment benthic macrofaunal inventories gainedthrough several research programs that we carried out, between1999 and 2005, on six CTEs located in the Western Adriatic Sea(Fig. 1). These were: Venice Lagoon, Sacca di Scardovari, Sacca diGoro, Valle di Gorino, Valli di Comacchio, and Lesina Lagoon.Non-tidal CTEs were: Valli di Comacchio, and Lesina Lagoon, whilemicrotidal CTEs were: Venice Lagoon, Sacca di Scardovari, Sacca diGoro and Valle di Gorino.

The Venice lagoon is located at the northern end of the AdriaticSea. It extends for approximately 550 km2 and has an averagedepth of 1.2 m. On its east side, two long barrier islands separatethe lagoon from the Adriatic Sea. Water exchange occurs throughthree large entrances (Lido, Malamocco, and Chioggia). The lagoonis the most prolific clam farming ground in Europe and its averageannual yield of clams is more than 40,000 tonnes (Solidoro et al.,2000). Two sites that are representative of the main environmentalscenarios and different salinity (poly and euhaline) that occur inthe lagoon were sampled in summer 2005. One site was near theMalamocco inlet, and was strongly influenced by the sea. The othersite was on the northern side of the translagoon bridge, and wasstrongly influenced by the urban sewage inflow from the mainland.Although the number of sampling sites was small, our taxonomicinventory was comparable to those sampled from elsewhere inthe Venice lagoon (Tagliapietra et al., 1998; Volpi Ghirardiniet al., 1999; Sfriso et al., 2001).

The Sacca di Scardovari is a large embayment (32 km2) locatedbetween two branches of the Po River delta. The lagoon is con-nected to the Adriatic Sea through a wide mouth that is partly ob-structed by sand banks. It varies in depth from 0.5 to 2.8 m. Itsnorthern area receives nutrient-rich agricultural run-offs, whilethe southern area hosts extensive bivalve cultures of mostly clamsand mussels. Twelve sites were sampled in spring 2005.

The Sacca di Goro is a wide (26 km2) microtidal lagoon whosemaximum depth is 2.0 m. The lagoon receives nutrient-rich fresh-water, primarily from Po di Volano. The Sacca is the second largestclam farming ground in Europe, and its average annual yield is8000 tonnes (Solidoro et al., 2000). Six sites representative of dif-ferent microhabitats in the Sacca, were sampled in 2004 (May, July,November) and 2005 (February, May, July).

The Valle di Gorino (8 km2) is a cul-de-sac of the neighbouringSacca di Goro. It has a maximum depth of 1.5 m and receives fresh-water from the Po di Goro through a gate. Clams are cultured in itswesternmost portion. Three sites were established along the majoraxis of the Valle, and benthic fauna was sampled with monthly fre-quency from March 1999 to May 2000.

The Valli di Comacchio is the largest lagoon complex (approxi-mately 100 km2) of the Po River delta. The Valli has always been anarea of intensive commercial fishing. Sorokin et al. (1996) reportedthat the lagoon was in a state of hyper-eutrophication in the early1990s, but some recovery has taken place during the last few years(Munari et al., 2003). Four sites were quarterly sampled from 2004to 2005.

The Lesina lagoon (Apulia, southern Adriatic Sea) is one of thelargest (approximately 52 km2) lagoons in southern Italy. Thisshallow (maximum depth 0.8 m) basin is connected to the sea bytwo artificial narrow channels. Freshwater inflows are assured byseasonal streams, which are mostly located in the eastern area ofthe lagoon. Extensive fishing, mostly for sand smelt and eels, ispractised in the lagoon. Domestic waste water from the town ofLesina discharges into its south-western waters. Four sites, reflect-ing a gradient of impact decreasing from west to east were sam-pled in May and July 2004, then in April 2005.

Only soft-bottom macrofauna were considered due to the re-duced distribution of hard bottoms in Italian CTEs, where theyare mostly represented by wooden piles marking navigable canals.In Fig. 1 sites are identified according to sediment texture (sand ormud), and salinity (meso, poly, euhaline). Table 1 summarizes themain environmental characteristics of each site. The macrofaunawas collected with a Van Veen grab (area: 0.027 m2; volume: 4 l)in triplicate and sieved through 0.5 mm mesh. Taxonomic identifi-cation was carried out to the species level whenever possible.

2.2. Biotic indices

Abundance of species at each sample was averaged for each sta-tion at each sampling time, leading to a total number of 127 sta-tions. Four different biotic indices were calculated for each site,namely AMBI (Borja et al., 2000) and M-AMBI (Muxika et al.,2007), BENTIX (Simboura and Zenetos, 2002), and BITS (Mistriand Munari, 2008). These indices were chosen because they areproposed to be used in the WFD. Differently from AMBI, M-AMBIand BENTIX, that have been utilized both in coastal and transi-tional environments, BITS can be used only in CTEs. AMBI, M-AMBIand BENTIX indices are based on the classification of species intoseveral ecological groups representing species level of sensitivityto pollutions. The number of ecological groups varies accordingto each index (five for AMBI, two for BENTIX). The M-AMBI is cal-culated by factor analysis of AMBI, species richness (as number oftaxa) and Shannon diversity (H0 on log2 base) values. The BITSneeds the identification of animals to the family level instead ofthe species level, and recognises three groups (opportunistic, toler-ant and sensitive families). This broad categorization is possibleonly because the faunal complement in CTEs is reduced with re-spect to the sea.

AMBI and M-AMBI were calculated using the freeware programavailable on www.azti.es. For M-AMBI, reference conditionswere: status High, muddy habitats: AMBI = 1.67, Diversity = 3,Richness = 37; sandy habitats: AMBI = 1.54, Diversity = 3.93,Richness = 39; status Bad, all habitats: AMBI = 6, Diversity = 0,Richness = 0. BENTIX was calculated with the methodologyavailable at: http://www.hcmr.gr/listview3.php?id=1195. BITSwas calculated using the freeware program available onwww.bits.unife.it.

2.3. Data analysis

Agreement between AMBI, M-AMBI, BENTIX, and BITS wasassessed following two approaches: (i) match/mismatch betweenindices, and (ii) linear regression between indices. Match/mis-match between the five indices was determined by consideringonly two EcoQ status: ‘‘Undegraded” and ‘‘Degraded”. The

Fig. 1. Map of the studied sites showing their location along the Italian coast of the Adriatic Sea, and the sampled stations used in the dataset. A: Venice; B: Scardovari; C:Goro and Gorino; D: Comacchio; E: Lesina (square: mesohaline; circle: polyhaline; star: euhaline; black: muddy; white: sandy).

1042 C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050

undegraded status was determined for each index when the de-rived EcoQ status was High or Good, and scored as ‘‘1”.This means

that, on the managers point of view, no action has to be taken torestore the ecosystem. Degraded status corresponded to Moderate,

Table 1Main environmental characteristics at the study sites (Ve: Venice; SC: Scardovari; GO: Goro; GR: Gorino; CO: Comacchio; LE: Lesina).

Site Confinement Phanerogams Sediment Avg salinity

SMMVe Sea mouth Yes Sandy EuhalineSGVe Confined-reduced hydrodynamism No Muddy PolyhalineSC3 Confined-reduced hydrodynamism No Muddy PolyhalineSC5 Confined-reduced hydrodynamism No Muddy PolyhalineSC8 Medium confinement from sea No Sandy PolyhalineSC10 Sea mouth No Sandy PolyhalineSC15 Medium confinement from sea No Sandy PolyhalineSC18 Sea mouth No Sandy PolyhalineSC20 Sea mouth No Sandy PolyhalineSC23 Confined-reduced hydrodynamism No Muddy PolyhalineSC27 Confined-reduced hydrodynamism No Muddy PolyhalineSC30 Confined-reduced hydrodynamism No Muddy PolyhalineSC33 Confined-reduced hydrodynamism No Muddy PolyhalineSC38 Confined-reduced hydrodynamism No Muddy PolyhalineGOst1 Medium confinement from river No Muddy MesohalineGOst2 Medium confinement from sea No Muddy PolyhalineGOst3 Sea mouth No Sandy PolyhalineGOst4 Medium confinement from sea No Muddy PolyhalineGOst5 River mouth No Muddy MesohalineGOC Sea mouth No Sandy PolyhalineGR1 Medium confinement from river No Muddy PolyhalineGR2 Medium confinement from river No Muddy MesohalineGR3 Medium confinement from river No Muddy MesohalineCOst2 Confined-reduced hydrodynamism No Muddy EuhalineCOst4 Medium confinement from sea No Muddy EuhalineCOst5 Medium confinement from river No Muddy EuhalineCOst6 Confined-reduced hydrodynamism No Muddy EuhalineLEst1 Confined-reduced hydrodynamism No Muddy MesohalineLEst2 Medium confinement from sea No Muddy MesohalineLEst3 River mouth Yes Muddy MesohalineLEst4 River mouth Yes Muddy Mesohaline

C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050 1043

Poor or Bad EcoQ status, and was scored as ‘‘0”. When such an EcoQstatus is derived from the index, restoration measures are to be ta-ken in order to reach Good status by 2015. The scores given to eachof the four indices used were summed for each site (range: 0–4),and the sum of scores allowed us to define the level of match/mis-match between indices. The non-parametric Wilcoxon pairs testwas also used to assess agreement or disagreement between thedifferent indices on the undegraded and degraded status of sta-tions on a statistical basis. This non-parametric test was particu-larly adapted to our data as it allowed comparing related sampleclassifications based on nominal data (undegraded vs. degraded)and it is as powerful as the t-test (Siegel and Castellan, 1988).Regression between indices was performed not using the EcoQ sta-tus but the numerical score of each index in each station. Signifi-cance was assessed through regression ANOVA. Finally, therelationships between different indices and benthic communityattributes were also investigated by means of regression analysisand ANOVA.

3. Results

Because of the physiographical characteristics of Adriatic CTEs(closed lagoons have no sandy bottoms, while in semi-closed la-goons sand is found only in proximity of seamouths), the majorityof our stations were on mud (15 stations on sand out of 127). InAppendix I (sandy habitats) and II (muddy habitats), the macrofa-unal species dominating at each of the 127 stations are given, to-gether with (i) their percentage abundance, (ii) the number ofspecies and families, (iii) Shannon diversity (on log2 basis), (iv)assignment into ecological groups by BITS, AMBI and BENTIXcheck-lists, (v) ecological quality status from the four indices. Outof more of 400 species found in the 127 stations, only about 40 taxawere dominant (i.e. relative abundance >1% of the total abundancein that station) in the considered six Adriatic CTEs. Annelida largely

dominated in both habitats: Polydora ciliata, Capitella capitata, Het-eromastus filiformis, Streblospio shrubsolii, Prionospio caspersi, Spiodecoratus, Neanthes succinea and Tubificoides vestibulatus were themost common taxa. Among Crustacea, Corophium insidiosum wasoften recorded with elevated dominance at stations characterizedby freshwater inputs, and Gammaridea (Microdeutopus gryllotalpa,Gammarus insensibilis) were often found associated to macroalgae(mostly Gracilaria sp. and Ulva sp.). The snail Hydrobia ventrosawas often abundant on decaying macroalgal remnants.

In sandy habitats there were generally higher richness anddiversity, with a higher number of dominant taxa (even with somenoteworthy exceptions, e.g. GOCjul04, with 94.9% of Lentidiummediterraneum). Only in few cases there was complete agreementamong the four indices: at stations GOst3may05 on a degraded sit-uation, and at stations GOCjul04 and GOCnov04 on an undegradedclassification. Conversely, full agreement among BITS, AMBI andM-AMBI classification was obtained on 11 out of 15 stations (eightundegraded and three degraded situations). BENTIX was the mostsevere, giving only seven undegraded classifications. Even at sta-tion VeSMM, characterized by extremely high richness and diver-sity (the station was located at the Malamocco seamouth of theVenice Lagoon, on a luxuriant Nannozostera noltii meadow), BEN-TIX gave a Poor EcoQ.

On mud, richness and diversity were lower, and generally fewertaxa resulted as dominant. Again the BENTIX was extremely se-vere, assigning 107 degraded situations out of 112. Appendix IIshows that almost all taxa present on mud in Adriatic CTEs areclassified into EG II (tolerant + opportunists) by the BENTIXcheck-list. At station VeSG a community characterized by quite alow richness (S = 10) and diversity (H0 = 1.48), and a strong domi-nance by few (three taxa out of 10 made up 97.3% of the commu-nity abundance) opportunistic and tolerant taxa, rendered acomplete agreement (degraded situation) among all indices. Inthe same way, a community (COst5may04) characterized by amoderately high richness (S = 21) and diversity (H0 = 1.82), and

Table 2Level used for the measurement of agreement/disagreement between the four indicesfor each station.

Sum ofscores

Interpretation Number ofstations

0 Full agreement on moderate or worse EcoQ 381 Partial agreement (three out of four) on

Moderate or worse EcoQ26

2 Disagreement between the four indices 353 Partial agreement (three out of four) on Good

or higher EcoQ25

4 Full agreement on Good or higher EcoQ 3

Table 3Result of the non-parametric Wilcoxon sign test.

N T Z p-Level

BITS & M-AMBI 127 275 3.35 <0.001BITS & AMBI 127 168 0.50 nsBITS & BENTIX 127 124 5.90 <0.001M-AMBI & AMBI 127 255 3.69 <0.001M-AMBI & BENTIX 127 92.5 3.78 <0.001AMBI & BENTIX 127 248.5 5.82 <0.001

1044 C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050

by a dominance of tolerant taxa (with the presence of sensitivetaxa too) rendered a complete agreement between indices on aundegraded EcoQ. Stations SC3 and SC23 constituted a differentcase: there was agreement (degraded situation) among three outof four indices, with M-AMBI giving a better EcoQ status (unde-graded situation). BITS, AMBI and BENTIX gave more importanceto community composition considering only ecological groups(mostly opportunistic and tolerant taxa), whereas in M-AMBI cal-culation a substantial weight was given to richness (S = 20 and14, respectively) and diversity (H0 = 2.39 and 2.56, respectively).The opposite was registered when diversity was low due to domi-nance of one or two taxa: at COst4feb05, for example, despite therelatively high richness (S = 19), the low diversity (H0 = 0.85)caused by the dominance of C. insidiosum and T. vestibulatus (EGIII and V respectively, according to AMBI) made the M-AMBI clas-sification to drop to a degraded situation, while AMBI and BITSgave an undegraded classification. At stations LEst3jul04, LEst3-may04 and LEst4may04 the BITS EcoQ status was probably too se-vere. These stations, located on a N. noltii meadow, were classifiedas undegraded through another biological element (Sfriso et al.,2009). Stations were characterized by quite high benthic richnessand diversity, so AMBI and M-AMBI classified their EcoQ correctly.The strong dominance of Chironomus salinarius (an opportunistictaxa) made the BITS classification to drop.

Use of the different indices gave a different pattern of the over-all EcoQ of the investigated sites. These results are summarized inFig. 2 which shows the percentages of stations for each classes ofecological quality according to the benthic indices. The AMBI clas-sified a majority of stations (52%) as undegraded; in the same waythe BITS classified the stations as undegraded in 51.2% of cases.However, the AMBI classified all undegraded stations as Goodwhereas BITS classified stations as Good (35.4%) but also as High(15.7%) quality status. The proportion of stations classified as de-graded by the AMBI was similar to that of the BITS, with 48% and48.8% respectively of the stations classified as Moderate or worse.AMBI and BITS assessed Moderate status in 30.7% and 25.2% of sta-tions respectively. AMBI classified 0.8% and 16.5% of stations as Badand Poor respectively, whereas 5.5% and 18.1% of the stations wereclassified as Bad and Poor respectively by the BITS. The M-AMBIclassified 29.9% of stations as undegraded (24.4% as Good and5.5% as High), and 70.1% as degraded (0.8% as Bad, 24.4% as Poor,44.9% as Moderate). The classification of stations by the BENTIX in-dex was more severe with 90.6% of stations considered as degraded(3.9% as Bad, 77.2% as Poor, 9.4% as Moderate).

0%

25%

50%

75%

100%

BITS M-AMBI

Fig. 2. Percentage of stations classified as High, Good, Mode

The full agreement of the four indices on the undegraded (Goodor better) or degraded (Moderate or worse) status occurred in32.3% of stations (Table 2). Partial agreement (i.e. three indicesout of four agreed on undegraded or degraded status) occurred in40.2% of stations, whereas the different indices disagreed on thestatus of 27.6% of stations. The Wilcoxon paired sign test showedno significant disagreement only between BITS and AMBI classifi-cations (26 mismatch out of 127 cases; Table 3).

Regression between indices allowed us to assess whether thedifferent indices displayed similar tendency in the classificationof sites, i.e. it permitted to assess if two indices ranked the sitesfrom worst to best in the same way regardless of the precise clas-ses of EcoQ. Linear regression of BITS and AMBI/M-AMBI accountedfor 44% and 36% of the variability of the entire dataset, respectively,with quite a low degree of dispersion over the entire range of bothindices (Fig. 3). The ANOVA of both regressions was highly signifi-cant (Table 4). It meant that the BITS, AMBI and M-AMBI indicesbasically ranked stations in the same way from worst to best eco-logical condition. Despite ANOVAs’ significancy (due to the ele-vated number of points), the regressions among BENTIX and the

AMBI BENTIX

High

Good

Moderate

Poor

Bad

rate, Poor and Bad by BITS, AMBI, M-AMBI and BENTIX.

0

0.3

0.6

0.9

1.2

0 1 2 3 4

M-A

MBI

BITS

BITS vs M-AMBI

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6

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BITS vs AMBI

01234567

0 1 2 3 4

BEN

TIX

BITS

BITS vs BENTIX

01234567

0 0.3 0.6 0.9 1.2

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TIX

M-AMBI

M-AMBI vs BENTIX

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TIX

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AMBI vs BENTIX

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6

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M-AMBI

M-AMBI vs AMBI

Fig. 3. Linear regression between the four indices.

Table 4Regression ANOVA for the relationship between paired indices.

df F p-Level R

BITS vs. M-AMBI 1, 125 71.12 <0.001 0.602BITS vs. AMBI 99.20 <0.001 �0.670BITS vs. BENTIX 27.41 <0.001 0.424M-AMBI vs. AMBI 73.59 <0.001 �0.610M-AMBI vs. BENTIX 7.14 <0.01 0.233AMBI vs. BENTIX 3.20 0.08 �0.160

Table 5Regression ANOVA for the relationship between the four indices and diversity (H’) andrichness (S).

df F p-Level R

BITS vs. H0 1, 125 13.17 <0.001 0.309M-AMBI vs. H0 198.94 <0.0001 0.784AMBI vs. H0 4.72 <0.05 �0.19BENTIX vs. H0 2.67 0.105 0.144BITS vs. S 34.41 <0.001 0.465M-AMBI vs. S 267.94 <0.0001 0.826AMBI vs. S 11.38 <0.001 �0.289BENTIX vs. S 5.44 <0.05 0.204

C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050 1045

other indices showed a much higher degree of dispersion of points,with subsequently lower variability accounts (Fig. 3).

In Fig. 4, the relationship between Shannon diversity and differ-ent indices is shown, while Fig. 5 shows their relationship with

richness (as number of species). Table 5 summarizes regressionANOVAs. Notwithstanding the significancy of ANOVAs, only M-AMBI evidenced a very strong relationship with diversity and rich-ness, with a low degree of dispersion of points on the graphs. Thisobservation may seem quite trivial, since diversity and richness arethe two metrics, together with AMBI, used in the multivariate for-mulation of M-AMBI (Muxika et al., 2007; Borja et al., 2008a). Onthe other hand, BITS and, to a lesser extent, AMBI showed quite aclear ‘‘tendency” to increase (the former) and decrease (the latter)with increasing diversity and richness (Figs. 4 and 5).

4. Discussion

This study provides a good example from a comprehensive largedataset of the levels and ranges of benthic pattern which can beencountered in Adriatic coastal transitional ecosystems. The patternof ecological quality status of Adriatic CTEs obtained by applyingfour benthic indices was not always concordant, depending to theindex selected. In spite of their diversity, these indices are basedon the same paradigm: disturbances are generating secondary suc-cessions during which tolerant species are at first dominant andthen progressively replaced by sensitive species (Pearson andRosenberg, 1978). In CTEs, an index to be useful should display some‘‘plasticity” in considering anthropogenic or natural disturbance.

The BENTIX was the most severe assigning degraded EcoQ atthe majority of stations. However it is necessary to stress that BEN-

00.5

11.5

22.5

33.5

44.5

0 0.3 0.6 0.9 1.2

H'

M-AMBI

M-AMBI vs H'

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0 1 2 3 4

H'

BITS

BITS vs H'

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AMBI

AMBI vs H'

00.5

11.5

22.5

33.5

44.5

0 2 4 6 8

H'

BENTIX

BENTIX vs H'

Fig. 4. Relationships between Shannon diversity (H0) and the four indices.

00.5

11.5

22.5

33.5

4

0 20 40 60 80 100

BITS

Richness (S)

Richness vs BITS

0

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MBI

Richness (S)

Richness vs M-AMBI

0123456

0 20 40 60 80 100

AMBI

Richness (S)

Richness vs AMBI

01234567

0 20 40 60 80 100

BEN

TIX

Richness (S)

Richness vs BENTIX

Fig. 5. Relationships between richness (as species number) and the four indices.

1046 C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050

TIX was developed in the Eastern Mediterranean ecoregion, whichis an oligotrophic, euhaline and microtidal environment, where thebenthic fauna is usually very diverse and evenly distributed withno one species naturally dominating over 10% (Simboura and Reiz-opoulou, 2008). Results of this study suggest that BENTIX is not asuitable biotic index for eutrophic, poly/euhaline, Adriatic CTEs.According to AMBI, most stations should be considered as display-ing a undegraded EcoQ status (Good or better), while M-AMBI pro-vided a somewhat more degraded situation. The BITS response wasmore similar to AMBI. Despite the 5-category water quality classi-fication system requested by the WFD, the only really importantboundary is between Moderate (money has to be spent to makeit Good) and Good (no money needed to make it High). In this

study, taking into account the threshold between the classes Mod-erate and Good it appears that the percentage of stations classed asundegraded (Good or better) were 52% for AMBI, 51.2% for BITS,29.9% for M-AMBI, and only 9.4% for BENTIX. With such an ap-proach, the use of the four different indices to describe the EcoQseems to add some complexity, impairing the accurate assessmentof the EcoQ status of the benthic invertebrate communities. Such aproblem was also identified by Labrune et al. (2006), Quintino et al.(2006) and Blanchet et al. (2008). This study definitely shows thatthe classifications of EcoQ status derived from different indices dif-ferently agree on a managerial point of view, i.e. acceptable (unde-graded) versus not acceptable (degraded) situations, with thenoteworthy exception of the BENTIX. However, if numerical values,

C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050 1047

instead of the EcoQ status, are considered, correlations were gener-ally significant. It means that the BITS, AMBI, and M-AMBI indicesgenerally ranked stations in the same way but disagreed on theprecise level of EcoQ assessed to each station by the differentindices.

Several factors can be invoked to explain the differences in EcoQassessment with the four indices: (i) discordances on assignmentof ecological group to many species, e.g. P. caspersi which is classi-fied as sensitive (EG I) by BENTIX, tolerant (family Spionidae, EG II)by BITS, and opportunist (EG IV) by AMBI; (ii) the arrangement ofthe tolerant/opportunist taxa, which are weighted separately inAMBI/M-AMBI (three groups: EG III, IV and V) and BITS (2 groups:EG II and III), whereas BENTIX pools them in an unique group (EGII); (iii) incompleteness of check-lists, especially of BENTIX, withexclusion of taxa and sometimes quite large number of individualsin applying the index (e.g. Lekanesphaera hookeri, L. monodi, Micro-deutopus anomalus, Anadara inaequivalvis, etc.); (iv) habitat prefer-ence of some taxa (to which the indices assign different EG), e.g. C.insidiosum (BITS: EG II, AMBI: EG III, BENTIX: EG II) which can befound on both sandy and muddy habitats, and Corophium orientale(BITS: EG II, AMBI: EG III, BENTIX: EG II) found, in Adriatic CTEs,exclusively on muddy habitats despite the ecological status ofthe station (e.g. COst5may04, all indices: undegraded EcoQ; COst4-jul04, all indices: degraded EcoQ); (v) the ‘‘weight” of diversity(which, by the way, enters twice in the calculation, properly asH0 and, indirectly, as S) in M-AMBI calculation in areas which arenaturally characterized by low diversity (e.g. at the end of thesalinity gradient, as will be discussed later). As a matter of fact,M-AMBI classification seemed to depend more from diversity thanfrom community composition. More surprisingly was the correla-tion between BITS and diversity, even if with a higher scatteringof points on the graph, since neither diversity nor richness enterin the computational formula (number of families enters as a ra-tio). This finding, reflecting the fact that a reduced number of spe-cies lives in transitional systems (i.e. few species into a family),confirms the adequacy of using the taxonomic level of the familyin such environments.

CTEs are naturally organic enriched environments and thisstudy suggests that the use of indices based on the species toler-ance/sensitivity mainly due to organic pollution need to beadapted to such environments. The ‘‘paradox of estuarine quality”(Dauvin, 2007; Dauvin and Ruellet, 2009) has been recently ex-tended to the ‘‘paradox of transitional water” by Munari and Mistri(2008a): in these systems featured by low diversity and richness,and high abundance communities, it is extremely difficult distin-guish between natural or anthropogenic stresses. The M-AMBI iswidely and successfully utilized in assessing the EcoQ in marineenvironments (Borja et al., 2009), but its application in transitionalsystems would probably depend on a resettlement. As a matter offact, the application of AMBI/M-AMBI has achieved controversialresults in Mediterranean lagoons or enclosed basins (Ponti andAbbiati, 2004; Marìn-Guirao et al., 2005, Labrune et al., 2006, Pra-novi et al., 2007; Ponti et al., 2008). This is not surprising, since theaccuracy of methods for evaluating the ecological status of a waterbody is expected to decrease when they are applied at larger spa-tial scales, because no model (or method or index) can be simple,general and accurate at the same time. However, all those compar-isons have shown major discrepancies but have ignored their po-tential causes. Prato et al. (2009) compared the responses ofmacrobenthic indices in two small (surface area < 10 km2) Tyrrhe-nian CTEs, Caprolace and Fogliano Lakes. Their analysis demon-strated no evidence of high environmental disturbance on bothcoastal lakes, as shown by their chemical analysis and macroben-thic community composition. The Authors concluded that AMBIseems to be more appropriate than M-AMBI and BENTIX in reflect-ing ecological status of those brackish water ecosystems (Prato

et al., 2009). Munari et al. (2010) investigating the ecological qual-ity of Karavasta Lagoon (Albania), found that the AMBI/M-AMBIand BITS classifications gave very similar results in that hyperha-line environment. Conversely, Simboura and Reizopoulou (2008)observed that M-AMBI tends to underestimate the EcoQ in slightlyor moderately disturbed Hellenic lagoons. Ruellet and Dauvin(2007) argued that the inclusion of Shannon diversity and speciesrichness in M-AMBI computation gives too much weight to diver-sity. Results from this study confirm previous observations (Ruelletand Dauvin, 2007) in Adriatic CTEs, and add the hypothesis that M-AMBI robustness is reduced under low salinity conditions (e.g. sta-tions GOst1, GOst5, located in an area close to the mouth of the Podi Volano river, or stations GR2 and GR3, interested by the outflowof the Po di Goro river, where salinity oscillates between 3 and15 psu, depending on riverine hydraulic discharge). Conversely,AMBI and BITS, giving no weight to diversity but considering onlythe ecological meaning of species (or families) produced differentclassifications respect M-AMBI. The benthic communities at sta-tions GR2 and GR3, for example, were practically always domi-nated by two species: S. shrubsolii (Spionidae) and C. insidiosum(Corophiidae), together summing up over 80% of the total abun-dance, with a (low) complement of other taxa depending on theseason. Both taxa are classified as tolerant (EG II) by BITS and byAMBI (EG III). The other taxa ranged from EG I (sensitive, e.g. Gam-maridae) to EG III (opportunists, e.g. Capitellidae) in BITS classifica-tion, and from EG I (sensitive, e.g. G. insensibilis) to EG V (first orderopportunists, e.g. C. capitata, T. vestibulatus) in AMBI classification.As a matter of fact, BITS and AMBI gave the same classification 25times (five degraded, 20 undegraded) over 28. On the other hand,the reduced species complement and the quite low diversity(caused by the desalinization of the area which followed the open-ing of a flood gate on the Po di Goro mouth; Mistri et al., 2002)forced M-AMBI to classify the stations 24 times as degraded. Intransition environments, chemical–physical parameters can repre-sent limiting factors for species. In particular, salinity plays themost important role, since the distribution of organisms can beestablished in relation to isohalines (Cognetti and Maltagliati,2000). The steno and euhaline species living in these environmentsfollow a gradient of resistance to the increasing environmentalstress, and at a critical salinity level (5–10 psu) there is a sharp nu-meric drop in species richness (Kinne, 1971). The distribution pat-tern of the benthic fauna is similar to that found in polluted waters,since diversity and richness tend to decrease towards the source ofdisturbance, according to the Pearson and Rosenberg (1978) para-digm. But, as remarked by Cognetti and Maltagliati (2000) the dif-ference as compared to CTEs lies in the fact that the maximumcritical point in polluted waters corresponds to disappearance ofthe fauna, while in CTEs to a community made up of few taxa bet-ter adapted to low (or very variable) salinity (Munari and Mistri,2008b). Streblospio is reported to be dominant in low salinity zones(Mannino and Montagna, 1997; Maggiore and Keppel, 2007), aswell as C. insidiosum, characterized as a strictly paralic species(Guelorget and Perthuisot, 1992), with high dominance in lowsalinity areas (Kevrekidis, 2004).

BITS failed in classifying EcoQ status at some stations in muddyhabitats, e.g. in Lesina Lagoon on seagrass meadows, due to thedominance of Chironomidae found at LEst3jul04, LEst3may04and LEst4may04. Chironomidae benefits when labile matter fromdecaying seagrass leaves is delivered to the sediment surface (Bar-toli et al., 2008). From the ecological point of view, the family Chi-ronomidae is indicative of stagnant water and organic enrichment(Gascón et al., 2007), and BITS classifies it as opportunist (EG III). InBITS calculation huge amount of individuals, even though includedinto only one family, and belonging to EG III (the term fIII) makesthe BITS value to drop. It is however important to remember thatin transitional waters the ecological quality of an area can change

1048 C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050

very quickly (Blanchet et al., 2008) due to, e.g., massive recruit-ment, or transitory disturbance events (i.e. the opening of irriga-tion canal dams, canal dredging, sand nourishments, etc.). As amatter of fact, when Chironomidae reduced their dominance (e.g.LEst3apr05), BITS, AMBI and M-AMBI were concordant on an unde-graded EcoQ status.

Much of the mismatch between the indices that are based onmacrofauna, and proposed within the WFD, is related to spatialvariability in community structure measures and the type of hab-itat (Borja et al., 2008b). Adriatic CTEs, much more than the marineenvironment, are characterized by the presence of strong gradi-ents, and high variability in mesological parameters. The contribu-tion of different variables to gradients depends on the mainhydrodynamic energy source of the system. In environments withsensible fluvial energy (in this study: Valle di Gorino, eastern partof the Sacca di Goro, eastern part of Lesina Lagoon), the gradient isstructured mainly by the freshwater flows which dilute the seawa-ter. Conversely, in CTEs with weak river input (in this study: Saccadi Scardovari, Valli di Comacchio, central part of Sacca di Goro,eastern part of Lesina Lagoon) the component that mainly influ-ences the gradient is seawater renewal. These environmental gra-dients directly influence the progressive reduction in the numberof species when entering a water body, either from the sea or fromthe river. As summarized by Tagliapietra et al. (2009), the declinein species number and diversity along the gradient has been thesubject of various conceptualisations, each one emphasising a dif-ferent aspect of the gradient depending on the environment inves-tigated: salinity (e.g. Attrill, 2002), seawater renewal (e.g.Guelorget and Perthuisot, 1983) or sediment type (e.g. Thrushet al., 2003). So, and particularly for CTEs, a benthic index cannotbe universal, as different organisms are not equally sensitive toall different types of stress (anthropogenic or natural), and responddifferently to different types of environmental alteration or envi-ronmental gradients. This study confirms that different indicesbased on the same concept do not interpret the same informationin the same way. In similar cases, Ruellet and Dauvin (2007) statedthat two hypotheses are possible: either some indices are inappro-priate, or the EcoQ threshold values (i.e. the values signifying theboundaries between the EcoQ classes) of the indices must be mod-ified so that they produce the same or at least similar results for agiven station. This study confirms both hypotheses: BENTIX isinappropriate for eutrophic Adriatic CTEs (115 degraded classifica-tions over 127 cases), and the EcoQ threshold values of the otherthree indices might need to be modified (AMBI, M-AMBI and BITSgave different EcoQ, but their numerical values showed strong cor-relation, thus ranking stations in the same way from worst to bestecological condition). A third hypothesis that we advance is thatfour different typologies of water bodies (coastal, estuaries, macro-tidal, micro- and non-tidal lagoons) may need different indicesable to capture their own ecological and biogeographical peculiar-ities. The sensitivity/tolerance approach required by the WFDposes a certain degree of controversy within the scientific commu-nity, since the classification of the different taxa are subjectivelymade, and there is a lack of understanding of the links betweenthe effects of human activities and changes in populations of suchspecies, especially in CTEs. Hydrologic and geomorphic attributes,biogeographic constraints, diffuse and acute disturbances act syn-ergistically to shape CTE benthic communities. For these reasons,the ecological value of the benthic species may be different fromthat in coastal and marine ecosystems: Cognetti (1992) and Cog-netti and Maltagliati (2000, 2008) recognized that the ‘‘ecologicalbehavior” of many species is different when they have to adaptto lagoonal conditions. This makes it difficult to assign a sharp sen-sitivity/tolerance classification to each lagoonal species accordingto the five classes proposed by Grall and Glémarec (1997). In thedevelopment of BITS, considering microtidal and non-tidal lagoons

peculiarities, the sensitivity/tolerance classification of benthic fau-na was summarized into only three ecological groups, re-adaptingGray’s (1981) strategies to lagoonal conditions.

Finally, this study also shows that the EcoQ of a certain station(e.g. GOst1, GOst2) can noticeably vary (from Poor to Good) be-tween sampling periods. This illustrates the more or less predict-able changes in the main community structure parameters oflagoonal benthic assemblages, which fluctuate according to thetypical yearly cycle of coastal temperate waters (Gravina et al.1989), and to the frequency and intensity of disturbance events.In aquatic environments, disturbance may depends on several fac-tors, such as pollution, oxygen depletion, eutrophication and or-ganic enrichment (Wu, 1999; Gray et al., 2002; Hargrave et al.,2008). Eutrophication and organic enrichment are intrinsic charac-teristics of Northern Adriatic CTEs (Viaroli et al., 2001), with hyp-oxic events mainly caused by macroalgal masses decompositionrecurring quite regularly depending on seasonality (Giordaniet al. 1997), and with summer acute episodes (Viaroli et al.,2005) that often reshape the benthic assemblage (Mistri 2002).This temporal variability must have to be taken into account bythe Italian Environmental Agency when monitoring to providethe EcoQ status assessment of CTEs: as stressed by Dauvin andRuellet (2009) it might be more efficient to take only a mean valueof an index into account for each zone of a CTE to minimize thehigh variability of the index in such temporal and spatial heteroge-neous conditions.

Indices proposed for the implementation of the WFD and testedin this study are based on taxonomic identification at the species orfamily level. One problem with such indices is their use by thosewith real no expertise, and without any certitude that the taxaidentified are really the same. To overcome these problems, effortswere recently driven towards the development of rapid assess-ment protocols and the application of simpler indicators and indi-ces. Magni et al. (2009) demonstrated the robustness and globalapplicability of the total organic carbon (TOC) thresholds, identi-fied originally by Hyland et al. (2005), and confirmed TOC as a goodproxy indicator for the rapid assessment of environmental condi-tions potentially harmful to the benthos in CTEs. Reizopoulouand Nicolaidou (2007) proposed a benthos-biomass based index(Index of Size Distribution, ISD) for the ecological status assess-ment of Eastern Mediterranean lagoons. Breber (2008) proposedbenthic richness and bivalve biomass as a quality indicator forApulian CTEs. Despite the encouraging results obtained by thoseauthors, the application of these indicators has been so far negligi-ble (and in Italy it will probably remain) since the metrics neededare seldom considered in routine environmental monitoringprograms.

5. Conclusions

Three (AMBI, M-AMBI, BENTIX) out of the four benthic indicesinvestigated in this study are commonly used to define transitionalecosystem quality status, despite none of them were developedspecifically for such variable, anthropogenically-modified ecosys-tems. This study suggests that benthic indices developed in coastalwaters need to be adapted for their use in CTEs. The index BOPA(Dauvin and Ruellet, 2007), not considered in this study becausethe almost constant presence of macroalgae in Adriatic CTEs ren-ders its use ineffective (Munari et al., 2009a), was developed forFrench estuaries but was adapted to BO2A (Dauvin and Ruellet,2009) for its use in the freshwater zone of an estuary. The BITStoo, developed specifically for CTEs, needs more investigation andrefinement before becoming satisfactorily operational in suchenvironments, since in several cases its application demonstratedevident inconsistencies. It is also necessary to highlight that some

C. Munari, M. Mistri / Marine Pollution Bulletin 60 (2010) 1040–1050 1049

of the CTEs considered in this study (e.g. Goro and Gorino) aregoing to be classified as ‘‘Heavily Modified Water Bodies”, due tohydro-morphological alterations, combined with an high numberof anthropogenic pressures (Munari and Mistri, 2008a). As sug-gested by Borja and Elliott (2007), this situation should require aspecific treatment within the WFD, assessing the ‘‘ecologicalpotential” instead the ‘‘ecological status”, including further, addi-tional investigation.

This study also evidenced that BENTIX is inappropriate foreutrophic Adriatic CTEs, and that in such environments M-AMBIclassification is too much dependent on diversity and richness,and seems unable to capture some peculiarities of benthic assem-blages in transitional waters. On the other hand, AMBI and BITSgave often similar classifications, despite the different level of tax-onomic identification needed (at the species level for AMBI and atthe family level for BITS). Finally, as strongly remarked by Dauvinand Ruellet (2009) most benthic indices require the use of the spe-cies level as the level of identification. According to the taxonomicsufficiency principle it is possible to use the genus or the family le-vel of identification (see Dauvin et al., 2003; Chainho et al., 2007;Munari et al., 2009b), thus reducing the cost of obtaining resultsin routine monitoring programs. Despite the AMBI check-list con-tains the benthic taxa at the species level, the taxonomic suffi-ciency principle is already present in the list since it alsoprovides the ecological status of most of the genus, families andalso higher taxonomic levels (e.g. Hydroides dianthus, Hydroidessp., Serpulidae; Tubificoides swirencoides, Tubificoides sp., Tubifici-dae, Oligochaeta; Heterotanais oerstedi, Heterotanais sp., Tanaida-cea; etc.). A genus or family level of identification for the WFDimplementation in the benthic compartment of Italian CTEs mightbe sufficient for evaluating the status of such water bodies.

Acknowledgements

An anonymous Reviewer is acknowledged for constructive crit-icism that greatly improved this manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.marpolbul.2010.01.022.

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