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Marine Pollution Bulletin 50 (2005) 374–387

Establishing the ecological quality status of soft-bottommining-impacted coastal water bodies in the scope of the

Water Framework Directive

Lazaro Marın-Guirao a,*, Augusto Cesar b, Arnaldo Marın a, Javier Lloret a, Ruben Vita a

a Departamento de Ecologıa e Hidrologıa, Facultad de Biologıa, Universidad de Murcia, 30100-Murcia, Spainb Departamento de Ecotoxicologıa, Universidade Santa Cecilia, Santos 11045-907, SP, Brazil

Abstract

The aim of the present study was to check the usefulness of the benthic biotic indices proposed for application in the European

water framework directive (WFD 2000/60/EC) for the ecological quality classification of coastal water bodies, together with some

other benthic methodologies used in different countries of the world. The different approaches were applied in two marine ecosys-

tems affected by the same heavy metal contamination source, coastal waters off Portman and the Mar Menor coastal lagoon, both in

SE Spain. Two marine biotic indices proposed for application in the Directive (AMBI and BENTIX) were used, together with com-

munity descriptors (abundance, Shannon–Wiener diversity, Margalef�s species richness, Pielou�s evenness and Simpson�s Domi-nance), the relative benthic index (RBI) and the abundance-biomass comparison method (ABC). Water-sediment interface

toxicity bioassays using sea urchin embryos and sediment metal analysis served to check the classifications obtained. The classical

community descriptors pointed to a progressive variation in benthic communities along the metal contamination gradient of Port-

man, but, they did not correctly characterize the environmental status of the lagoon stations. Although the RBI was the index that

best classified the sites according to their degree of pollution, the selection of indicator species can bias the results. Since the AMBI,

the BENTIX and the ABC method are based on the pollution resulting from organic enrichment, their application in the case of

purely toxic pollution may not be successful, as was found to be the case in these two mining-polluted ecosystems. Therefore,

the development of new indicator lists according to the type of pollutant may serve to improve the results obtained with organic

enrichment-based indices when studying other kinds of disturbance. Finally, we found the toxicity tests to be useful tools for the

environmental assessment of aquatic ecosystems, and recommend their inclusion in the WFD.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Water Framework Directive; Toxicity bioassays, Marine biotic indices, AMBI, BENTIX, RBI, ABC method; Mining wastes

1. Introduction

The European water framework directive (WFD2000/60/EC) establishes a framework for the protection

of all water bodies to prevent their deterioration and to

ensure the achievement of good quality status by 2015.

The Directive includes inland surface waters, ground

waters, transitional waters and coastal waters as catego-

0025-326X/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2004.11.019

* Corresponding author. Tel.: +34 968364977; fax: +34 968363963.

E-mail address: lamarin@um.es (L. Marın-Guirao).

ries of water bodies, and develops the concept of ecolog-

ical quality status (EcoQ), which is judged according to

five classes (High, Good,Moderate, Poor and Bad). ThisEcoQ will be based on the status of, among other fac-

tors, elements reflecting biological quality, including

the composition and abundance of benthic invertebrate

fauna. In May 2001, a ‘‘common implementation strat-

egy’’ (CIS) was agreed to assist with the implementation

of the WFD, with the COAST working group dealing

with coastal waters. To establish the ecological quality

of soft-bottom benthos and to classify coastal water

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 375

bodies, this group has proposed two marine biotic indi-

ces, the AMBI (Borja et al., 2000) and the BENTIX

(Simboura and Zenetos, 2002), which are based on the

sensitivity/tolerance of benthic fauna to stress gradients

mainly caused by organic enrichment pollution, declar-

ing that methods combining composition, abundanceand sensitivity are the most promising (Vincent et al.,

2002).

Great quantities of wastes are produced during min-

ing activities and these are sometimes discharged into

water-bodies, where they cause problems related with

metal pollution. Metals, in aquatic ecosystems are accu-

mulated in sediments, where they may reach concentra-

tions several orders of magnitude higher than in water(Bryan and Langston, 1992). Since sediments constitute

one of the most important compartments in aquatic eco-

systems, providing the habitat for many organisms

(Ingersoll, 1995), their contamination by metals would

cause deleterious effects on benthic organisms.

Historically, the Sierra of Cartagena-Portman (Mur-

cia, SE-Spain) was exploited to extract pyrite and lead

sulphide. During the 20th century, the mineral laundriesused floating techniques to extract metal, producing

great quantities of mining wastes. These muddy wastes

were discharged into the Portman Bay (Mediterranean

Sea) and the adjacent Mar Menor coastal lagoon, pro-

ducing a high degree of sediment metal contamination.

Mining operations pumped 3000–10,000tons of tailings

per day into Portman Bay from 1958 to 1991, first di-

rectly into the bay and later, when the bay was filledup through an outfall more than 2km in length. In total,

approximately 50 million tons of mine tailings were

dumped into Portman Bay during this period, including

heavy metals known to be toxic, such as cadmium, cop-

per, lead and zinc. For its part, the Mar Menor is the

biggest hypersaline coastal lagoon of Spain and one of

the biggest in Europe and the Mediterranean Sea. The

lagoon is relatively shallow with a mean depth of 3.5mand a maximum depth of just over 6m. Until the

1950s, wastes from the mining activities were discharged

into the coastal lagoon through the temporary streams

that exist in the area. Even now, when torrential rains

occur, the tailings that remain in the mountains continue

to enter the lagoon. Previous studies (Cesar et al., 2004;

DeLeon et al., 1982, 1984; Perez and Puente, 1989) have

reported high concentrations of Zn, Pb, and Cd in thesediments of both ecosystems.

In the present study, the biotic indices (AMBI and

BENTIX) were applied in both the above mentioned

heavy metal-polluted Mediterranean ecosystems to test

their applicability in metal pollution assessment. In

addition, we used classical community descriptive

parameters (abundance, Shannon–Wiener diversity,

Margalef�s species richness, Pielou�s evenness and Simp-son�s Dominance), the relative biotic index (RBI)(Anderson et al., 1998) developed for regional applica-

tion in North America estuarine habitats and adapted

to our conditions, and the widely used abundance bio-

mass comparison (ABC) method (Warwick, 1986). We

also employed water–sediment interface toxicity bioas-

says, using sea urchin embryos, and sediment physico-

chemical analysis to evaluate the EcoQ classificationobtained with the different indices since, in marine and

estuarine ecosystems, biological approaches may be

complemented by toxicity and chemical assessment

methods to serve as independent evaluations of the eco-

logical quality (Dauer, 1993; Elliot, 1994).

The aim of the present study was to examine the use-

ful application of the benthic biotic indices proposed in

the Directive 2000/60/EC for the classification of coastalwater bodies, together with some other benthic method-

ologies employed worldwide, in two marine ecosystems

affected by the same heavy metal contamination source

(Mediterranean Sea and Mar Menor lagoon).

2. Methods

2.1. Study areas and sampling points

The study took place close to the Spanish Mediterra-

nean coast off the Cartagena-Portman Sierra and in the

Mar Menor coastal lagoon (SE Spain), both corre-

sponding to the category of ‘‘coastal water bodies’’. Sed-

iment samples were collected off the Portman coast in

March 2002, along a spatial gradient in the same depthrange (10–15m). Six sampling stations ran from the

mine outlet in Portman Bay in the direction of the Mar-

ine Reserve of Cabo de Palos-Islas Hormigas (WSW-

ENE; Fig. 1). Sandy soft-bottoms characterized all these

stations, which were located in rocky exposed coastal

sections. A full description of both study areas can be

found in Calvın-Calvo (1999). In the Mar Menor la-

goon, six sampling stations were selected in the southernbasin of the lagoon, with similar salinity ranges to min-

imise possible differences in structure and composition

between populations due to the natural gradients char-

acteristic of coastal lagoons. Two of these stations,

BW and PW, were located at the mouth of two tempo-

rary streams through which the wastes were discharged

into the lagoon (Fig. 1). Sediment samples were col-

lected in May 2001. Four replicate sediment sampleswere collected by SCUBA divers from all points in both

Mediterranean ecosystems.

2.2. Community descriptive parameters

Benthic faunal samples were obtained by SCUBA di-

vers using a 0.09m2 hand grab and sieved through a

0.5mm mesh bag. The macroinvertebrates retained onthe screen were fixed with 4% buffered formalin, and

later washed and transferred to 70% isopropyl alcohol

Fig. 1. Map of the study area and location of sampling points: (a) Mar Menor coastal lagoon; (b) Portman coast. Black arrows indicate the discharge

points of the mining wastes.

376 L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387

prior to sorting and identifying. Organisms were sorted

and identified to the lowest possible taxon level (usually

species level) and then their biomass was determined

(AFDW). To describe the structure of the benthic com-

munities, classical descriptive parameters including theabundance, the Shannon–Wiener diversity index (H 0,

log2basis), Margalef species richness (S), Pielou�s even-ness (J) of distribution of individuals among species

and Simpson�s Dominance (D), were used.

2.3. Biological benthic indices

The AMBI (AZTI�s marine biotic index), as definedby Borja et al. (2000, 2003a), is a biotic index which pro-

vides a pollution classification of a particular site, repre-

senting benthic community health (sensu Grall and

Glemarec, 1997). It is based on the distribution of the

abundance of each species, into one of five ecological

groups (EG) (sensitive to pollution, indifferent, tolerant,

and second and first-order opportunistic species). The

distribution of these EG, according to their sensitivityto pollution stress, provides a biotic coefficient that

was recently adapted to the five EcoQ proposed by the

WFD (Borja et al., 2003b, 2004). For the index calcula-

tion we have used the AMBI� software (freely availableon http://www.azti.es).

In 2002, Simboura and Zenetos developed a new bio-

tic index (BENTIX) based on the initial idea of Borja

et al. (2000), the novelty of this index lying in treatingbenthic species as belonging to two wider EG (the sensi-

tive and the tolerant). The classification system appears

as a function of the BENTIX index, including the five

levels of ecological quality proposed by the WFD.

Another biological index used was the relative ben-

thic index (RBI), developed by Anderson et al. (1998,

2001), which is based on responses of marine benthic

communities to anthropogenic and natural distur-

bances. The RBI uses six categories (total number ofspecies, number of crustacean species, number of mol-

lusc species, number of crustacean individuals, and the

presence or absence of positive species and negative spe-

cies) and was developed for particular areas by selecting

different indicator species. To adapt the index to our

study areas and to the objectives of the present study,

the index, which comprises a range of values from 0 to

1 and which was originally divided into three thresholdvalues, was adapted to the five EcoQ categories estab-

lished in the WFD, corresponding to 0.00–0.20,

‘‘Bad’’; 0.21–0.40, ‘‘Poor’’; 0.41–0.60, ‘‘Moderate’’;

0.61–0.80, ‘‘Good’’ and 0.81–1.00 ‘‘High’’ EcoQ. We se-

lected our positive (sensitive) and negative (tolerant)

species in accordance with Hunt et al. (2001a), who indi-

cated that positive indicators are generally not found in

polluted habitats and are characteristic of regions whereanthropogenic and other severe disturbances do not

play a major role in structuring communities, while the

negative indicators are highly distributed in polluted sta-

tions and are not found in any unpolluted points. Final-

ly, for positive and negative species to be included in the

calculation of the RBI, they must have been identified as

sensitive or tolerant, respectively, in previous reported

species indicator lists (e.g. AMBI, BENTIX).The ABC (abundance-biomass comparison) method,

originally described by Warwick (1986), involves the

plotting of separate K-dominance curves for species

abundances and biomasses on the same graph. The

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 377

position of one curve with respect to the other identifies

three sediment conditions: undisturbed, moderately dis-

turbed and grossly disturbed. The W statistic of Clarke

(1990) measures the area between the two curves, values

close to zero indicating a moderately disturbed status,

while those far from zero (positive or negative) indicateundisturbed or grossly disturbed, respectively. The W-

statistics that goes from �1 to 1 were divided into fiveclasses, the boundaries between classes being set accord-

ing to the results of the graphs in the following way:

�1.00 <W < �0.50 ‘‘Bad’’ EcoQ, �0.49 <W < �0.15‘‘Poor’’ EcoQ, �0.14 <W < 0.14 ‘‘Moderate’’ EcoQ,

0.15 <W < 0.49 ‘‘Good’’ EcoQ and 0.50 <W < 1.00

‘‘High’’ EcoQ.

2.4. Toxicity tests

The toxicity of the sediment–water interface was

determined using the embryo larval development tests

with two sea urchin species, Arbacia lixula and Paracen-

trotus lividus, following the procedures described by

Cesar et al. (2004) and in accordance with the acceptedguidelines (Environment Canada, 1992; USEPA, 1995,

2002; CETESB, 1999). Two milliliters of the surface of

an intact (unhomogenized) sediment core were intro-

duced carefully through a syringe (5ml) with a cut tip,

and 8ml of dilution seawater (1 sediment/4 water) were

introduced carefully to minimize resuspension. Test

tubes were allowed to stabilize for 24h and then a circu-

lar mesh (100lm) with the same diameter as the testtube was placed carefully on the sediment–water inter-

face with the help of a clean glass rod. The duration

of sub-chronic tests was 28h for P. lividus and 38h for

A. lixula, after which times the number of normally

developed pluteus larvae was counted. Sediment sam-

ples from El Fraile Island (60km from Portman Bay),

where the sea urchins were collected, were used as con-

trol to validate the tests. To associate the five EcoQ withthe results obtained in the toxicity bioassays using sea

urchin embryos, the average of the results for both spe-

cies were calculated and the percentage of normally

developed pluteus obtained was divided into the

five EcoQ proposed by the WFD: 0–20% of Plu-

teus = ‘‘Bad’’ EcoQ, 21–40% = ‘‘Poor’’ EcoQ, 41–

60% = ‘‘Moderate’’ EcoQ, 61–80% = ‘‘Good’’ EcoQ

and 81–100% = ‘‘High’’ EcoQ. The established thresh-old values were tested against several sediment quality

attributes, such as metal concentrations or the molar

difference between the acid volatile sulfide concentra-

tions and the simultaneous extracted metals.

2.5. Physico-chemical analysis

Sediment particle size distribution was determined bymechanical dry sieving. The total organic carbon (TOC)

content was determined in the fine fraction (<63lm)

with a Carlo Erba Instruments (EA1108) elemental ana-

lyser following sample preparation with 1N HCl to

decompose the carbonate (Verdardo et al., 1990). The

percentage of organic matter in the samples was esti-

mated by the loss of weight on ignition at 450 �C for 6h.Sediment samples for the acid volatile sulphide (AVS)

and simultaneously extracted metals (SEM) were ana-

lyzed by a cold-acid purge-and-trap technique described

in detail by Allen et al. (1993). The hydrogen sulfide was

determined with an ion selective silver/sulfide electrode

(ThermoOrion, model 9616). The SEM (Zn, Cd, Pb

and Cu) were measured with an optical emission spec-

trometer (Optima 2000 DV—Perquin Elmer) in the case

of the Portman samples, and by anodic stripping vol-tammetry (Metrohm 646 VA Processor) with hanging

mercury drop for the sediments from the Mar Menor.

2.6. Data analysis

Benthic community analysis was performed using the

PRIMER v5 (Plymouth routines in marine ecological

research) software package (Clarke and Gorley, 2001).The Bray–Curtis similarity matrix was used on trans-

formed (double root) total invertebrate abundance data,

and classification was performed on the similarity ma-

trix using the multidimensional scaling (MDS) also pro-

posed by the CIS working group 2.4 (COAST), created

by the Common Implementation Strategy (Vincent

et al., 2002). To estimate the degree of association be-

tween the results obtained using the different methodol-ogies here described (biological benthic indices,

community descriptors and toxicity tests) and the load

of the pollutant content in the sediments (metal concen-

trations, SEM, SEM-AVS), the results were standar-

dised by subtracting the mean and dividing by the

standard deviation before running a Pearson correlation

analysis (computer application Statistica v4.5�).

3. Results

3.1. Physico-chemical analysis

Table 1 summarizes the results of the sediment phys-

ico-chemical analyses. The stations were mainly com-

posed of fine sands, although a few showed a relativesmall amount of silt-clay (CN, PN in Portman and

BW, CII in the Mar Menor), where the highest percent-

ages of organic matter and total organic carbon were re-

corded. As regards the sediment metal concentrations

off the Portman coast, PN and PG stations located both

sides of Portman Bay presented the highest metal con-

centrations, decreasing as the distance from the bay

increased (PG > PN > CM > CN > PL > PE). Cu wasonly detected in the first two stations, whereas Cd was

not detected in Portman. Only PL station presented a

Table1

Summaryofthesedimentphysico-chemicalanalyses

Stations

parametersPortman

MarMenor

PG

PN

CN

CM

PL

PE

UR

BW

PW

PH

CI

CII

%Silt-clay

4.19±0.11

4.32±2.74

0.23±0.17

2.98±1.05

0.72±0.07

0.41±0.11

0.45±0.19

10.53±5.29

0.58±0.10

0.18±0.12

0.16±0.02

8.39±0.58

%Sand

95.81±0.12

79.30±9.10

61.52±9.43

96.94±0.96

99.28±0.07

99.59±0.11

99.07±0.45

89.15±6.62

95.00±0.49

89.94±2.60

96.31±6.14

81.67±8.48

%OM

6.22±0.5

5.16±0.81

1.38±0.15

2.28±0.10

0.74±0.08

0.73±0.07

3.08±0.18

8.17±0.32

2.43±0.41

2.23±0.07

3.17±0.14

3.06±0.02

%TOC

18.32±3.32

16.67±0.53

0.58±0.02

1.49±0.18

0.29±0.007

0.12±0.004

0.85±0.02

1.60±0.24

1.40±0.03

0.90±0.02

1.17±0.05

1.29±0.61

NH4

0.003±0.00050.006±0.00

0.007±0.00050.005±0.0005

0.006±0.00

0.004±0.00050.005±0.001

0.010±0.0030.006±0.0010.005±0.0004

0.007±0.001

0.007±0.002

Zn

15.34±3.07

13.47±0.45

0.34±0.02

1.17±0.19

0.21±0.005

0.08±0.00

6.37±1.09

62.07±4.35

4.12±0.43

0.81±0.02

1.14±0.14

0.97±0.23

Pb

2.98±0.31

3.14±0.20

0.20±0.005

0.30±0.02

0.08±0.008

0.04±0.00

6.62±0.21

45.98±13.10

1.25±0.15

1.21±0.09

0.29±0.01

0.25±0.04

Cu

0.007±0.009

0.003±0.005BDL

BDL

BDL

BDL

0.06±0.02

0.55±0.02

0.18±0.03

0.02±0.004

0.02±0.004

0.03±0.008

Cd

BDL

BDL

BDL

BDL

BDL

BDL

0.01±0.001

0.02±0.01

0.005±0.0010.002±0.0005

0.003±0.0005

0.004±0.001

SEM

18.32±3.32

16.67±0.054

0.59±0.03

1.49±0.18

0.29±0.008

0.12±0.005

13.06±1.21

108.62±14.48

5.56±0.50

2.04±0.10

1.46±0.14

1.25±0.26

AVS

0.32±0.31

0.28±0.09

0.25±0.04

0.30±0.05

0.29±0.07

0.012±0.005

1.48±0.67

2.44±2.72

5.46±1.10

0.50±0.04

1.47±0.33

2.83±0.97

SEM-AVS

18.00±3.02

16.39±0.52

0.340±0.03

1.185±0.22

�0.005±0.06

0.105±0.01

11.58±0.81

106.18±5.18

0.10±0.08

1.54±0.14

�0.02±0.02

�1.58±0.78

Percentageofthefinefraction(%silt-clay),sand(%sand),organicmattercontent(%OM)andtotalorganiccarboncontent(%TOC),ammoniumconcentration(mg/l),heavymetalconcentrations(Zn,Pb,CuandCd),

simultaneouslyextractedmetals(SEM=Zn+Pb+Cu+Cd)andacidvolatilesulphideconcentration(AVS).Metals,SEMandAVSconcentrationsaregivenin(lmol/gdrysed.).BDL=belowdetectionlimits,forCuand

Cdare0.003and0.002lmol/gdrysed.,respectively.Meanvalues±S.D.

378 L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387

negative molar difference between the SEM and the

AVS analyzed, the rest of the stations showing a positive

difference. In PN and PG stations this positive difference

was high (>15lmol/g dry sed.), indicating the possiblebioavailability of metals. According to the classification

proposed by Long et al. (1995), PG and PN stationswere highly polluted by Zn and Pb, whereas the rest of

stations were classified as lowly polluted by the four

metals, except CM station, which was classified as mod-

erately polluted by Pb (Table 3). In the Mar Menor, the

highest metal concentrations were found in stations UR,

BW and PW, the two first presenting high molar differ-

ences between the SEM and the AVS concentration. For

the rest of the stations, the metal concentrations wererelatively low, both CI and CII stations presenting neg-

ative SEM-AVS molar differences. Every station in the

lagoon presented low pollution levels for Cu and Cd

(Long et al., 1995), whereas the Zn levels classified UR

and BW as highly polluted and PW station as moder-

ately polluted. Pb levels pointed to a high degree of pol-

lution in UR, BW, PW and PH stations and a moderate

degree of pollution in CI and CII (Table 3).

3.2. Toxicity tests

Both species of sea urchin larvae presented a similar

pattern of response in both ecosystems with the highest

toxicity effects being recorded in those sediments show-

ing a high metal content. These corresponded to sites

located close to the mine discharges (PG and PN inPortman coast and BW, PW and UR in the Mar Menor)

(Table 2). Control samples validated the results obtained

in the tests, since 89.7 ± 5.5 and 87.5 ± 5.3 percentage of

normally developed pluteus larvae were achieved for

A. lixula and P. lividus, respectively. The mean values

obtained by jointly considering the response of both spe-

cies and the established threshold values, classified the

stations in the following way: for Portman coast, highEcoQ in PE, good EcoQ in PL, moderate EcoQ in

CM, poor EcoQ in CN and a bad EcoQ for both PN

and PG stations. In the case of the Mar Menor coastal

lagoon, the toxicity results classified the CI station as

having a good EcoQ, as moderate EcoQ the PH station,

PW and UR stations corresponded to the poor EcoQ

while BW stations had a bad EcoQ (Table 4).

3.3. Community descriptive parameters and

multivariate analysis

In total, 83 species were found in Portman coast and

only four species, Ampelisca typica, Apseudes latreillei,

Photis longipes and Tellina donacina, were present in

every station. The minimum value of the abundance,

species richness S, diversity H and evenness J was at-tained at station PG, which is located close to the

mining wastes discharge, to the west side of Portman

Table2

Summaryofbenthicfaunaldescriptiveparameters:numberofspecies,abundance(N/m2),Margalefspeciesrichness(S),theShannon–Wienerdiversityindex(H

0 ,log2basis),Pielou�sevenness(J),

Simpson�sdominance(D);benthicfaunalindices:AMBI(bioticcoefficient,BC),BENTHIX,RBIandABC(W

-statistic);andseaurchinstoxicitytests(percentageofpluteus)

Stations

indices

Portman

MarMenor

PG

PN

CN

CM

PL

PE

UR

BW

PW

PH

CI

CII

No.species

7±1

15±3

8±2

18±2

19±4

26±2

7±1

4±1

7±4

5±2

9±2

11±2

No.individuals

(N/m2)

2956±7872416±672

450±144

3238±13452250±10262025±235

272±87

183±46

367±108

2731±827

4305±1852

1139±692

Richness(S)

1.38±0.303.51±0.822.86±0.39

3.86±0.37

4.25±0.86

5.87±0.48

1.81±0.161.10±0.561.77±1.03

0.63±0.40

1.42±0.33

2.31±0.14

Diversity

H0

(log2)

0.80±0.222.04±0.271.95±0.15

2.12±0.23

2.43±0.23

2.93±0.14

1.52±0.081.03±0.571.24±0.73

0.50±0.12

0.31±0.22

1.31±0.34

Evenness(J)

0.39±0.080.72±0.070.88±0.03

0.72±0.07

0.82±0.02

0.89±0.03

0.80±0.070.71±0.270.62±0.21

0.39±0.12

0.14±0.10

0.55±0.18

Dominance(D)

0.61±0.120.21±0.050.18±0.02

0.18±0.05

0.13±0.03

0.07±0.02

0.28±0.040.46±0.020.46±0.29

0.74±0.06

0.89±0.09

0.47±0.16

AMBI(BC)

2.67±0.151.20±0.311.15±0.49

2.39±1.19

1.14±0.53

0.98±0.28

0.35±0.160.99±1.011.74±1.54

0.01±0.03

0.12±0.09

0.73±0.39

BENTHIX

5.27±0.853.21±0.715.38±0.48

3.21±0.50

3.74±0.27

4.21±0.35

3.91±0.474.23±1.432.91±1.12

5.98±0.03

5.86±0.13

5.23±0.45

RBI

0.31±0.050.28±0.080.26±0.05

0.42±0.08

0.46±0.12

0.65±0.08

0.44±0.050.29±0.110.32±0.11

0.55±0.10

0.73±0.11

0.60±0.06

ABC(W)

�0.01±0.100.06±0.050.41±0.17

�0.01±0.05

0.19±0.14

0.08±0.070.121±0.060.22±0.270.16±0.14

�0.34±0.09

�0.21±0.18

�0.16±0.22

A.lixula

(%pluteus)

0±0

0±0

27.2±2.5

41.25±5.38

63.7±5.06

91.7±2.2

24.5±2.1

11.5±1.9

35.5±5.5

48±8.6

73.3±5.51N.A.

P.lividus

(%pluteus)

0±0

8.5±5.1

40.5±5.2

40.7±2.6

83±1.4

85±1.8

19.2±4.1

9.5±5.25

23±2.2

53.2±5.6

67.7±3.1

N.A.

Meanvalues±S.D.

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 379

Bay (Table 2). The highest dominance value corre-

sponded to this station, since it was clearly dominated

by just two species, the crustacean Apseudes latreillei

and one species of the polychaeta family Sabellidae,

the former reaching densities of 2225 indm�2 and the

latter 550 indm�2, approximately. These two organismswere also the dominant species in station PN (east side

of Portman Bay) with densities of 640 and 731 indm�2,

respectively.

Station CN, located close to Portman Bay, presented

the lowest abundance value and, with PG, the lowest

number of species. The richness and diversity values

were low, as was the dominance since neither of the spe-

cies was dominant.Station CM, located between the stations near the

emissary (PN, PG and CN) and those far from it (PE

and PL) presented the maximum faunal abundance,

with the most abundant species being the polychaeta

families Capitellidae (Capitella sp), Cirratulidae (Chae-

tozone setosa), Spionidae (Prionospio sp) and Syllidae,

some of them known to be indicators of environmental

instability (Simboura et al., 1995; Picard, 1965; Pearsonand Rosenberg, 1978), and the crustacean Monoculodes

carinatus, which is sensitive to pollutants (Borja et al.,

2000; Simboura and Zenetos, 2002).

The highest values of richness, diversity and evenness

were found in PE station. This was the only station,

where the sensitive indicator species Branchiostoma

lanceolatum, Lucinella divaricata, Erichthonius punctatus

and Apherusa alacris (Simboura and Zenetos, 2002)were found. PL station also presented a high number

of species and high diversity and evenness values, its fau-

nal composition being similar to the above mentioned

PE. Species common to these stations were the crusta-

ceans Bathyporeira guilliamsoniana, Harpinia sp, Phoxo-

cephalus aquosus, Urothoe grimaldii and the ophiura

Amphiura chiajei. Although the polychaeta faunal com-

position of PE and PL stations was similar to that ofCM, the crustacea and mollusca composition were gen-

erally different, except in the case ofM. carinatus, a crus-

tacean identified as sensitive to pollutants (Simboura

and Zenetos, 2002) and present in all three stations.

In Portman coast, the MDS ordination technique

(Fig. 2a) indicated a clearly defined gradient from PG

to PE station. Replicates of CN station were plotted to-

tally separated from the rest of the stations and off thedefined gradient.

In the Mar Menor coastal lagoon, a total of 31 spe-

cies were found, with only the amphipod Siphonoecetes

sabatieri being present in every station. The abundances

of this amphipod follows the descending order

CI > PH > CII > UR > BW > PW. Although, CI and

CII stations showed the highest number of species

(Table 2), CI presented the lowest diversity and highestdominance values due to the high abundance of

S. sabatieri (4131 ± 1852 indm�2). The sensitive species,

Table 3

Metal pollution classification of the stations in accordance with Long et al. (1995)

Stations metals Portman Mar Menor

PG PN CN CM PL PE UR BW PW PH CI CII

Zn H H L L L L H H M L L L

Pb H H L M L L H H H H M M

Cu L L L L L L L L L L L L

Cd L L L L L L L L L L L L

Low or absent of pollution (L), moderate pollution (M) and high pollution (H).

Table 4

Summary of the derived ecological quality status (EcoQ) calculated applying different biotic benthic indices (AMBI, BENTHIX and RBI), the

abundance-biomass comparison method (ABC) and the sea urchins toxicity tests (toxicity tests) in both Mediterranean ecosystems here studied:

Portman coast (Portman) and the Mar Menor coastal lagoon (Mar Menor)

Stations parameters Portman Mar Menor

PG PN CN CM PL PE UR BW PW PH CI CII

EcoQ-AMBI G G H G H H H H G H H H

EcoQ-BENTHIX H M H M G G G G M H H H

EcoQ-RBI P P P M M G M P P M G G

EcoQ-ABC (W-statistic) M M G M G M M G G P P P

EcoQ-toxicity tests B B P M G H P B P M G N.A.

Key: H—high EcoQ; G—good EcoQ; M—moderate EcoQ; P—poor EcoQ; B—bad EcoQ. N.A. data not available.

Fig. 2. MDS using group average linking of Bray-Curtis similarities

calculated on transformed (double root) abundance data: (a) Portman

coast; (b) Mar Menor coastal lagoon.

380 L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387

Cirrophorus sp and Venerupis aurea, and the Maldani-

dae polychaeta family were only found in station CII,

together with the tolerant species Lucinella divaricata.

PH station also showed low diversity values since thepolychaeta Scolopos armiger and the amphipod S. sabat-

ieri were clearly dominant at this station, attaining den-

sities of 363 ± 138 and 2308 ± 674 indm�2, respectively.

The highest diversity values were found in UR station;

although this station showed a medium number of spe-

cies and a low number of individuals, the even distribu-

tion of the individuals among the different species

improved the diversity value. In the same station, sensi-tive species, such as Cyclope neritea (also found in BW

and PW) and Tapes decussatus, and tolerant species such

as Turboella radiata and Loripes lacteus (72 indm�2)

were found. Mollusca was the dominant taxa.

Tolerant species exclusive to PW station were the

polychaeta, Cirriformia tentaculata (83 indm�2) and Pla-

tynereis dumerilii, while other tolerant species found in

low densities were Cerastoderma glaucum andMicrodeu-

topus gryllotalpa. On the other hand, sensitive species

were also observed in this station e.g. Tapes decussatus

and Sphaeromma serratum.

The minimum values as regards the number of species

and individuals were found in BW station, with a popu-

lation similar to that of PW but with the absence of Cirr-

iformia tentaculata and Tapes decussatus, the Crustacea

taxa only being represented by a few individuals of S.sabatieri.

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 381

The MDS ordination technique classified the coastal

lagoon stations into three groups according to their fau-

nal composition similarities (Fig. 2b). One group was

composed of PH, CI and CII stations, with a replicate

of the latter station (CII3) outside the group. UR station

formed another group with the four replicates wellgrouped, and both wadi stations (BW and PW), whose

replicates were mixed and dispersed to the right side of

the plot, formed the third group.

3.4. Biological benthic indices

3.4.1. AMBI

The application of the AMBI to the Mediterraneancoast of Portman ordered the stations in the following

way: PG > CM > PN > CN > PL > PE. PE, PL and

CN stations were identified as unpolluted, the last pre-

senting the greatest variance between replicates (Table

2). The biotic coefficient classified these stations as hav-

ing a high EcoQ (Table 4), pointing to an impoverished

benthic community health. On the other hand, PG, PN

and CM stations were classified as slightly polluted siteswith a good EcoQ, indicating an unbalanced benthic

community health. The first showed the highest biotic

coefficient values and highest concordance between rep-

licates, and the last the highest variability between repli-

cates (from unpolluted to mainly polluted). In every case

the percentage of individuals not assigned to any EG

was low (<5%).

In the case of the Mar Menor coastal lagoon, theAMBI classified all stations as unpolluted with a high

EcoQ, except PW which was classified as slightly pol-

luted (good EcoQ). The biotic index pointed to normal

benthic community health in PH and CI stations, an

unbalanced state of health in PW station and an impov-

erished benthic community health in the rest of the sta-

tions (UR, BW and CII). There was strong uniformity

among the classifications of the replicates in every sta-tion except PW, where unpolluted and meanly polluted

replicates were classified. The percentage of individuals

not assigned to any EG was low (<10%) in every station.

3.4.2. BENTIX

The results obtained applying the BENTIX to both

marine ecosystems are shown in Table 2. In the Portman

coastal stations the pollution classification indicatedthat CN and PG stations were pristine sites with a high

EcoQ. A good EcoQ rating was achieved in PE and PL

stations, with a slightly polluted classification. PN and

CM stations were classified as moderately polluted sites,

both corresponding to a moderate EcoQ (Table 4).

When the BENTIX was applied to the Mar Menor,

the stations were classified as follows. A pristine pollu-

tion classification and a high EcoQ was given to PH,CI and CII stations, with a high degree of concordance

between replicates. Although they showed some tolerant

species (Heteromastus filiformis, Cirriformia tentaculata

and Cerastoderma glaucum), the great abundance of

the amphipod S. sabatieri increased the percentage of

individuals belonging to the EGI (sensitive species) to

higher than 85%. UR and BW stations were classified

as slightly polluted or transitional sites with a good

EcoQ. The latter station presented a high degree of var-

iability between replicates, from high EcoQ (BEN-

TIX = 6) in one replicate where just one sensitive

organism was present (Cyclope neritea) to moderate

EcoQ (BENTIX = 2.5) due to the presence of some

H. filiformis and because 94% of its fauna, principally

S. armiger, were not assigned to any EG. PW station

was classified as moderately polluted, with replicatescorresponding to different EcoQ scores: poor EcoQ for

two replicates, mainly composed of H. filiformis and

C. tentaculata, and one replicate with a good EcoQ pre-

senting a few individuals of S. sabatieri, S. serratum and

C. neritea. The percentage of individuals not assigned to

any EG varies widely between stations, with the lowest

percentages corresponding to ‘‘pristine’’ stations

(<15%) and the highest to ‘‘polluted’’ stations (2.4–94%).

3.4.3. RBI

The values obtained applying the RBI to the two

Mediterranean ecosystems studied are shown in Table

2. In the case of Portman coast the positive/sensitive

indicator species selected belonged to the species Phoxo-

cephalus aquosus, Urothoe grimaldii (Amphipoda) andBranchiostoma lanceolatum (Branchiostomida), while

the negative/tolerant indicator species was the Tanaida-

cea Apseudes latreillei. When the index was divided into

the five ecological statuses proposed in the WFD, the

stations were classified in the following way: a good

EcoQ was given to PE station, located far from the

waste outlet, a moderate EcoQ to PL and CM stations

in the middle of the transect and a poor EcoQ to PN,PG and CN, the stations closest to the bay (Table 4).

Identified as positive/sensitive species in the Mar

Menor coastal lagoon were Siphonoecetes sabatieri

(Amphipoda) and Chamelea gallina (Bivalvia) and as

negative/tolerant the Tanaidacea, Leptochelia dubia. When

the RBI was applied in this ecosystem, CI and CII sta-

tions were classified as having a good EcoQ, UR and

PH stations a moderate EcoQ and those stations locatedin the mouth of the temporary streams, where the min-

ing wastes were historically discharged, a poor EcoQ.

The selected positive and negative species are in

accordance with the classification proposed by Simbo-

ura and Zenetos (2002) and Borja et al. (2000) and cor-

respond to sensitive and tolerant species, respectively.

3.4.4. Abundance-biomass comparison method

The application of the ABC method to the Mediter-

ranean coast of Portman (Fig. 3) and the values of the

Fig. 3. ABC plots.

382 L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387

W statistic obtained (Table 2) showed that stations PL

and CN, with both their abundance and biomass plots

clearly separated, could be classified as undisturbed.

TheW statistic obtained in these two stations was higher

than 0.15, indicating a good EcoQ, whereas in the rest of

stations (PE, CM, PN and PG) a moderate degree of

disturbance was found, since both curves were close

and crossed each other. The W statistic calculated

(�0.14 <W < 0.14) classified these stations as having amoderate EcoQ (Table 4).

In the Mar Menor lagoon, PH, CI and CII stations

seemed to be moderately to grossly disturbed as the

abundance curves lay above the biomass curves

(Fig. 3). The negative values of the W statistic

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 383

(�0.49 <W < �0.15) obtained in these stations (Table2) pointed to a poor EcoQ. The UR station showed

crossed curves and W values (�0.14 <W < 0.14) whichgave the station a moderate EcoQ. Both BW and PW

stations with 0.15 <W < 0.49 were classified as having

a good EcoQ, high variability existing betweenreplicates.

4. Discussion

The old mining activities developed in the Sierra of

Cartagena-Portman have caused metal contamination

in two adjacent Mediterranean aquatic ecosystems: theMediterranean Portman coast and the Mar Menor

coastal lagoon. A wide range of sediment metal concen-

trations was found in both ecosystems, the sites nearest

the mining waste discharges presenting the highest metal

concentrations, which decreased with increasing dis-

tance from the discharge points. These metal concentra-

tion gradients can be identified in both Mediterranean

ecosystems by mean of the sea urchin sediment–waterinterface toxicity tests. The stations were classified

through these toxicity tests according to the five EcoQ

proposed in the Directive, from bad to high EcoQ. Sea

urchin toxicity bioassays for sediment quality assess-

ment have previously been used in other Mediterranean

coastal areas and lagoons, where toxicological responses

generally fitted contamination levels to a satisfactory ex-

tent (Volpi Ghirardini et al., 2003; Cesar et al., 2004). Inour case, when the toxicity and chemical results from 11

stations (6 stations from Portman and 5 from Mar

Menor) with four replicates each (N = 44) were analyzed

with the Pearson correlation analysis, significant nega-

tive correlations were found (p < 0.001 for Zn,

r = �0.53; p < 0.01 for Pb, SEM and SEM-AVS,

r = �0.39, �0.46 and �0.47, respectively; p < 0.05 forCd and Cu, r = �0.32 and �0.35, respectively). Theexposure of developing sea urchin embryos to the inter-

face between sediment and water provides a more eco-

logically relevant bioassay for this species (Anderson

et al., 1996), and the results of the laboratory toxicity

tests could be considered predictive of ecological change

on a station-by-station basis because these are sub-

chronic toxicity tests and may reflect chronic impacts

in individual stations.The classical descriptive parameters (Margalef rich-

ness, Shannon–Wiener diversity, Pielou evenness and

Simpson dominance) showed a progressive variation in

benthic communities along the metal contamination

gradient of the Portman coast. Those stations with a de-

graded benthos (PG, PN and CN) had fewer species,

lower abundance, lower diversity and lower benthic

index scores than stations with a healthy benthos (PEand PL). In Portman coast, the ordination obtained by

means of the MDS grouped the stations in accordance

with this pollution-induced gradient from PG to PE sta-

tion, the stations were grouped in a consistent way with

the measured metals content in sediments and with the

classification obtained from the toxicity tests. Neverthe-

less, the classical indices did not correctly characterize

the environmental status of the lagoon stations, sinceUR, BW and PW stations, which are clearly polluted

by metals, showed higher diversity values than CI with

moderate metal-contamination. This inconsistency was

principally due to the high abundance (high dominance

values) of just one species (S. sabatieri) in CI, CII and

PH stations. Metal pollution probably has a deleterious

effect on this amphipod, decreasing its dominance and

thus increasing the diversity values of these contami-nated stations. A similar effect was recently reported

by Salas et al. (2004) in Mondego estuary (Portugal),

where the abundance of the small Crustacea Elminius

spp. caused a decrease in diversity values. As occurred

in naturally stressed environments (DelValls et al.,

1998; DelValls and Chapman, 1998; Drake et al.,

1999), the multivariate technique used (MDS) grouped

and classified the lagoon stations better than the classi-cal indices, as regards their ecological health. In the

Mar Menor lagoon, the MDS ordination technique pro-

vided more consistent conclusions than those obtained

with descriptive parameters and seems to be a useful

tool for evaluating the environmental quality of the

lagoon.

The ABC method (Warwick, 1986) did not seem to

work adequately in either of ecosystems polluted bytoxic metals. No significant correlations were found

between the results obtained with this method and the

sediment metal concentrations or toxicity test results.

Although, the ABC method is applicable to a wide vari-

ety of types of perturbation to marine macrobenthic

communities, its applicability to purely toxic pollution

(i.e. metal pollution) is uncertain (Warwick et al.,

1987; Anderlini and Wear, 1992). All the Portman coaststations were classified as having a good or moderate

EcoQ, a clearly disturbed station (CN) being given a

good EcoQ rating, while the undisturbed PE station, to-

gether with polluted stations, was classified as having

a moderate EcoQ. Although this method has been suc-

cessfully employed in Mediterranean coastal lagoons

organically enriched due to aquaculture practices (Reiz-

opouluo et al., 1996), its application to the Mar Menorcoastal lagoon classified the stations in the opposite way

to their pollution level, mainly due to the high abun-

dance of the small amphipod S. sabatieri in unpolluted

stations, where it reached 95% of the total abundance

in some replicates. A similar situation has been de-

scribed in two previous studies (Beukema, 1988; Salas

et al., 2004) where a high abundance of small species

led non-polluted areas to be classified as disturbed.The relative benthic index (RBI), which is used in

USA regional water quality control (Anderson et al.,

384 L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387

2001; Hunt et al., 2001a), was the index that best classi-

fied the sites according to their degree of pollution. The

results obtained with this index were highly positively

correlated with the toxicity results (p < 0.001; r = 0.69)

and negatively correlated with the chemical analysis

(p < 0.05 for Zn, SEM and SEM-AVS; r = �0.38,�0.34 and �0.33, respectively). The index is based ontoxicology and natural history, taking into account the

responses of marine benthic communities to anthropo-

genic and natural disturbances, and was developed for

particular areas by selecting different indicator species

(Anderson et al., 1996). The selection of indicator spe-

cies must be based on known responses to anthropo-

genic and other disturbances and related naturalhistory, such as life history traits and abundance pat-

terns along environmental gradients and between study

stations (Anderson et al., 1998). Accordingly, the selec-

tion of indicator species along an environmental gradi-

ent or between stations can bias the results obtained

with the RBI, since species are selected in relation to

their presence-abundance in both extremes of the gradi-

ent or in polluted-undisturbed stations. In this sense, be-fore selecting a species as being positive/sensitive or

negative/tolerant, we must be sure that this same species

has previously been cited as indicator in the same type of

pollution. Moreover, the RBI does not take into ac-

count the absence of truly unpolluted stations, as may

be the case in the Mar Menor lagoon, limiting the selec-

tion of positive and/or negative species and therefore its

applicability.One of the benthic biotic indices proposed by the CIS

working group 2.4 (Coast) (Vincent et al., 2002) for

application within the scope of the WFD is the Azti�smarine biotic index (AMBI) (Borja et al., 2000). When

the AMBI was employed in both Mediterranean ecosys-

tems, only high and good EcoQ classifications were ob-

tained and no significant correlations were found

between this index and the metal content of sediments.Although, the classification obtained with this index

did not seem to be very conservative since 9 out of 12

stations were classified as having a high EcoQ, in the

case of Portman coast the scores of the AMBI ordered

the stations in a more or less similar way to the ordina-

tion provided by the chemical results. In a previous

work, Borja et al. (2003a) analyzed three stations along

a deep-transect off Portman Bay, obtaining a good EcoQ(2.0 < BC < 2.3), a classification that we thought over

generous because Cesar et al. (2004) found high levels

of metals and toxicity in stations located near those

studied by Borja et al. (2003a). In this sense, we thought

that, since the indicative species list generated for the

AMBI is mainly based on organic-pollution literature

(Borja et al., 2000), when pure toxic substances, such

as metals, are responsible for the disturbance, the speciesresponse is not the same and the classification obtained

will not reflect the real ecological status. Something sim-

ilar may occur when employing the BENTIX (Simboura

and Zenetos, 2002), the other benthic biotic index pro-

posed by the CIS working group 2.4 (Vincent et al.,

2002), to communities affected by toxic pollutants. The

indicator species lists proposed by Simboura and Zene-

tos (2002), which depend on the type of community, arebased on organic pollution literature and, furthermore,

the index was validated in Saronikos, a Greek Gulf that

receives much organic pollution. Therefore, its appli-

cation in the case of purely toxic pollution will not

necessarily be successful. The BENTIX classification

obtained for both ecosystems studied here was not

correlated with the classification obtained with the tox-

icity tests, and only the sediment copper concentrationwas negatively correlated with the BENTIX results

(p < 0.05, r = �0.30). The failure of the index in theMar Menor coastal lagoon was not surprising, as limita-

tions in its use in estuaries and lagoons have been de-

scribed previously (Simboura and Zenetos, 2002).

We found the indicator species lists presented for the

BENTIX to be incomplete since a very small number of

Crustacea species were included, and it is known thatthis taxon is generally more sensitive to environmental

contaminants and other anthropogenic disturbances

than most other components of the infauna, particu-

larly polychaetes (Pearson and Rosenberg, 1978). The

list of indicator species proposed in the AMBI includes

a higher number of species and is more complete than

that of Simboura and Zenetos. Although the applica-

tion of both indices requires a high level of taxonomiceffort, the fact that many of the species listed in the

AMBI are also classified at a family level means that

any such effort using this index will be lower. When

both species lists (AMBI and BENTIX) are compared,

it can be seen that some species are classified into differ-

ent ecological groups; for example, Perinereis cultrifera

and Aonides oxycephala are classified by the AMBI as

species tolerant to excess organic matter enrichment(EGIII), whereas in the case of the BENTIX they are

classified as sensitive to disturbance in general (EG1).

The development of this type of environmental tool re-

quires the consensus of scientists in the assignation of

species to a particular ecological group. Recently con-

troversy arose concerning the two above mentioned

indices and their application as new monitoring tools

in the WFD (Borja et al., 2004; Simboura, 2004). Inour case, when the indices were applied in two different

ecosystems impacted by mining wastes, neither pro-

vided good results, and even species described as sensi-

tive to pollution were found in heavily polluted stations

(e.g. Cyclope neritea, in BW station). Although both

indices (AMBI and BENTIX) did not correctly charac-

terize the sites here studied, we support the philosophy

on which they are based (indicative species), but, asmany other authors have suggested (e.g. Washington,

1984), we accept that a biotic index is unlikely to be

L. Marın-Guirao et al. / Marine Pollution Bulletin 50 (2005) 374–387 385

universally applicable, since organisms are not equally

sensitive to all types of anthropogenic disturbance

and are likely to respond differently to different types

of perturbation. The above applied benthic biotic indi-

ces were developed for cases of organic enrichment pol-

lution and have been seen to clearly classify sitesaccordingly (Borja et al., 2000, 2003b; Salas et al.,

2004; Simboura and Zenetos, 2002). Therefore the

development of new indicator lists as a function of

the type of pollutant may serve to improve the results

of such indices when studying other kinds of distur-

bance. We consider the development of new indicative

species lists more specifically related to the type of con-

tamination (metals, pesticides, hydrocarbons, etc.) to bevery important. Indeed, one of the objectives in the

initial phase of the WFD is the development of ‘‘type-

specific’’ reference conditions, a description of the bio-

logical quality elements that exist, or would exist, in

waters of high biological status, i.e. with no or only

very minor disturbance from human activities (WFD

2000/60/EC). Those reference conditions and their bio-

logical values could be used for the species selection, asthe populations found in these conditions would be

composed of, among others, sensitive species and, in

contrast, tolerant species will be found only in polluted

stations. Therefore, these biological reference values

could support the creation of new indicator lists as a

function of the type of pollution. Other tools that

would help in the development of new species indicator

lists could be ecotoxicological tests; it is supposed thatwhen a site presents sediment toxicity, organisms living

in those sediments must be tolerant of the pollutants

that characterize the site and so will be included as

tolerant organisms in the required species lists. In fact,

Chapman (1995) affirmed that one of the primary

advantages of laboratory toxicity tests was their ability

to establish and to validate field bioindicators.

In conclusion, when establishing the ecological qual-ity status of soft-bottom aquatic ecosystems as regards

benthic measurements, not every type of pollution

should be treated in the same way, since different biolog-

ical responses arise depending on the type of pollution.

Hence, for biological benthic indices to be applied with-

in the Directive new indicator species lists for different

kinds of pollutants need to be developed as they seem

to be good environmental tools in the case of organicenrichment pollution. We consider toxicity tests to be

useful environmental tools for the environmental assess-

ment of aquatic ecosystems, as is demonstrated here and

in other studies (e.g. Bryn et al., 1998; Hunt et al.,

2001b; Cesar et al., 2004). They are technically well

developed (USEPA, 1994; ASTM, 1997) and are widely

accepted as useful tools for a wide variety of research

and regulatory purposes (Swartz, 1989; Burton, 1991;Luoma and Ho, 1993). As such, they should be taken

into account before applying the Directive in the same

way as other non-European countries have incorporated

them in their environmental monitoring policies (Envi-

ronment Canada, 1992; CETESB, 1999; Kiddon et al.,

2003; USEPA, 1995, 2002). In addition, the use of differ-

ent methodologies, such as toxicity tests, multivariate

ordination techniques and/or pollutant chemical analy-sis, is highly recommended before establishing the envi-

ronmental quality of an aquatic system (Dauer, 1993;

Elliot, 1994) since, they help to avoid inconsistencies

when classifying the sites.

Acknowledgments

We are grateful to the anonymous referee for his use-

ful comments and constructive suggestions. We wish to

thank Philip Thomas for checking the English of the

manuscript. This work was funded by the European Re-

gional Development Fund (ERDF), Consejerıa de Agri-

cultura, Agua y Medio Ambiente, Region de Murcia,

Programa Seneca 2001 (AGR/24/FS/02).

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