Marine Pollution Bulletin 56 (2008) 2027–2036

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Marine Pollution Bulletin

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A general integrated ecotoxicological method for marine sediment qualityassessment: Application to sediments from littoral ecosystems onSouthern Spain’s Atlantic coast

José Usero a, José Morillo a, Hicham El Bakouri a,b,*

a Department of Chemical and Environmental Engineering, University of Seville, 41092 Seville, Spainb Department of Chemical Engineering, University of Abdelmalek Essaadi, 90000 Tangier, Morocco

a r t i c l e i n f o

Keywords:Integrated sediment quality indexAnthropogenic contaminationTrace metalsBioaccumulationSediment toxicityMacrobenthic community

0025-326X/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.marpolbul.2008.08.009

* Corresponding author. Address: Department of CEngineering, University of Seville, 41092 Seville, Spai

E-mail address: elbakouri@us.es (H. El Bakouri).

a b s t r a c t

In the last few decades, several integrated methods for marine sediment quality evaluation have beendeveloped and applied to many coastal sites. This paper describes the development of a general inte-grated method based on the determination of organic and inorganic sediment contamination, lethaland sublethal toxicity assessments, in situ benthonic community structure alteration and bioaccumula-tion of contaminants. The proposed method was applied to sediments from Southern Spain’s Atlanticcoast where six rivers (Guadiana, Carreras, Piedras, Tinto, Odiel and Guadalquivir) drain pollutants fromvarious anthropogenic sources. A series of experiments was conducted to determine the characteristics ofthe sediments: granulometry, organic matter content, etc. (1); metal contamination of sediments andChamelea gallina (bivalves widely distributed in the area studied) (2); lethal and sublethal toxicity by aseries of biological tests (3); and macrofauna distribution at the 16 stations included in the study (4).Ratio-to-reference values (RTR) calculated for each parameter were used to determine the organic andinorganic contamination indices, the lethal and sublethal toxicity indices, the index of bioavailabilityof metals in the organisms analyzed and the macrobenthic alteration index. The data were plotted onsix-axis plots to describe better the relations between the physical, chemical and biological propertiesstudied. A general integrated sediment quality index (ISQI) was determined and used as an indicatorof pollution-induced degradation. The ISQI values summarize the main characteristics of the environ-mental situation at each sampling point and can be used for purposes of comparison.

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1. Introduction

Marine sediment contamination by persistent anthropogenicchemicals poses one of the worst problems to coastal and estuarineecosystems around the world. The accumulation of urban, indus-trial and agricultural contaminants in marine sediments entersthe sea through river mouths or by direct discharge (Morillo andUsero, in press). Due to its ecological importance, the evaluationof marine sediment quality constitutes an important area of re-search (Silva et al., 2004).

Sediments may accumulate various kinds of hazardous andtoxic substances, including trace elements, to levels many timeshigher than water column concentrations, causing a serious prob-lem due to their toxicity and their ability to accumulate in the biota(Morillo et al., 2008). The most crucial property of heavy metals is

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that they are bioavailable and not biodegradable in the environ-ment. In aquatic ecosystems, sediments are the main sink for hea-vy metals (Ridgway and Shimmield, 2002). Metal fractionations insediments are strongly dependent on substrata geochemical condi-tions, such as pH and redox potential. When environmental condi-tions change, some of the sediment-bound contaminants canremobilize and be released back into the water, acting as a sourceof pollution that can have adverse effects on living organisms (Liet al., 2001; Horsfall and Spiff, 2002). The uptake of trace metalsby benthic organisms depends largely on their mobility, total con-centration and chemical forms (Morillo et al., 2008). Benthic inver-tebrates are an important link in the transfer of substances tohigher trophic levels because of their close association with sedi-ments and their ability to accumulate metals (Burgos and Rainbow,2001). An accurate way to assess the current bioavailability of sed-imentary contaminants is by measuring contaminants in aquaticorganisms such as bivalves, coincident with sediment measure-ments. Benthonic community structure also changes as a contin-uum on various spatial and temporal scales in accordance with

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both natural and anthropogenic gradients (Mucha et al., 2005).Therefore, macrobenthic organisms are good indicators for evalu-ating environmental change and contaminant impacts.

Studies on monitoring marine sediment quality (such as poros-ity, organic carbon and particle size) and the ecological systemhave been reported before (Berner, 1980; Tang et al., 2006). Otherstudies of sediment assessment combine chemical contaminationwith toxicity (Mucha et al., 2005; Cesar et al., 2007), and many eco-toxicological methods have been standardized for sediment toxic-ity testing using various species (Burton, 1992; US EPA, 1994;ASTM, 1997). Bivalves are one of the common test animals usedin sublethal sediment toxicity testing and various end points havebeen investigated, including mortality (Cheung and Wong, 1993;Naimo et al., 2000), species development (Chapman et al., 1992),histopathological changes (Bowmer et al., 1994) and behavioral re-sponses (Phelps, 1989; Roper and Hickey, 1994). Other microbialtoxicity tests using fish, daphnia, algae and luminescent bacteriahave been developed and compared for the determination of lethalsediment toxicity (Nendza, 2002; DelValls et al., 2004). The biolu-minescence assay using Vibrio fischeri is currently considered oneof the most suitable tests for sediment toxicity assessment. Thismicrobial biotest is highly sensitive, cost-effective and rapid (Olaj-ire et al., 2005). The use of sediment toxicity tests alone in sedi-ment quality assessment may result in lack of realism and/orgreat uncertainty (McCauley et al., 2000). However, the informa-tion obtained by integrating the chemical concentration in sedi-ments and from specific sediment bioassays (toxicity andbioaccumulation) is considered at the present time the best ap-proach for assessing sediment quality and for testing the toxicityand bioavailability to benthic organisms of chemical compoundsin sediments (Cesar et al., 2007; Chapman, 2007).

In this work, an integrated method for sediment quality evalu-ation was developed and applied to sediments from SouthernSpain’s Atlantic coast, where six rivers (the Guadiana, Carreras, Pie-dras, Tinto, Odiel and Guadalquivir) drain pollutants from variousanthropogenic sources. The proposed approach consists of six com-ponents: organic and inorganic sediment contamination, lethal andsublethal toxicity assessments, in situ benthonic community struc-ture alteration and bioaccumulation of contaminants. The levels ofanthropogenic contaminants were determined in sediments and inChamelea gallina (bivalve species from the Atlantic coast of south-ern Spain). The lethal and sublethal toxicities were determined bymeasuring the luminescence of the marine bacterium V. fischeriand the burrowing behavior of the Ruditapes philippinarum clam,respectively. The results were represented on a six-axis plot to de-scribe better the relations between the physical, chemical and bio-logical properties studied, and a general Integrated SedimentQuality Index (ISQI) was determined, summarizing the main char-acteristics of the environmental situation at each sampling point.

2. Area description

The study area is located on the Atlantic coast of Southern Spainand is limited by two large rivers: the Guadiana (in Huelva prov-ince) and the Guadalquivir (in Cadiz province). The other riversthat drain into this coastal area are the Carreras, Piedras, Tintoand Odiel. The last two rivers join and drain together, formingthe Huelva estuary, which is an important nature reserve. Partsof these rivers form the Punta Umbría estuary (Fig. 1).

The main sources of pollution on this 145 km coast are sewagefrom coastal towns, industrial dumps and pollution from rivers.The main contaminating rivers are the Tinto and the Odiel, bothof which pass through the Iberian Pyrite Belt, a region that hasbeen a rich source of minerals and metals from time immemorial(Usero et al., 2005). Both rivers are acidic (pH between 2 and 3)

and contain large amounts of metals from erosion and miningactivity (Nelson and Lamothe, 1993). Furthermore, sewage fromthe city of Huelva and wastes from numerous chemical plants inthe area’s industrial estates are discharged into the Huelva estuary.The amount of metal pollution from the other rivers that drain intothe coastal area is considerably less (Nelson and Lamothe, 1993;Van Geen et al., 1997).

3. Materials and methods

3.1. Sediment analysis

Fifteen stations and one reference site are distributed over thearea covered by this study at a distance of approximately 500 mfrom the coastline (Fig. 1). The reference site is situated in an areacharacterized by low contamination (Morillo et al., 2004). The sam-ples were taken at depths ranging from 3 m at P15 to 10 m at P3,except for the reference sample, which was taken at 18 m. Mostsampling points are around the Huelva estuary, where the Tintoand Odiel rivers merge. Five undisturbed surface sediment sampleswere collected from each sampling point using a Van Veen LMGgrab sampler with an area of 0.078 m2. Only grabs that achievedadequate penetration (2/3 of total volume) to collect the first5 cm of the sediment and that showed no evidence of leakage orsurface disturbance were retained, transferred and stored in a darkcooler at 4 �C.

In the laboratory, a representative sample from each stationwas prepared by mixing and homogenizing the five samples takenat each sampling point. Particle size profiles were determined bydry sieving the sediments (60 �C, 24 h) through a column of sieveswith different mesh sizes according to the Udden-Wentworth clas-sification (0–4 lm clay, 4–63 lm silt, 63–125 lm very fine sand,125–250 lm fine sand, 250–500 lm medium sand, 500–1000 lmcoarse sand, 1000–2000 lm very coarse sand) (Wentworth,1922). All conventional sediment parameters were expressed aspercentages of total dry sample weight.

For chemical and toxicological analysis, the sediment fractionwith a particle size of <63 lm was used (Rauret et al., 1988; Das-senakis et al., 2003). The Total Organic Carbon (TOC) was deter-mined in freeze-dried sediment samples using a TOC analyzer(Shimadzu, TOC–VCSH) connected to a solid sample module (Shi-madzu, Model SSM-5000 A). All analyses where replicated threetimes and the precision was up to 95%.

For heavy metal determination, each 0.5 g of sediment under-went acid digestion with a mixture of concentrated HNO3 (4 mL)and HClO4 (2 mL) in an automatic microwave digestion system at600 W for 1 h (Kingston and Jassie, 1988). CEM 3010 high-pressuredigestion bombs (consisting of a body made of a specific micro-wave-transparent polymer with a Teflon cup and cover) were usedfor sample digestion. These bombs are designed specifically formicrowave heating, are chemically inert and combine the advanta-ges of closed high-pressure (13.8 bar) and high-temperaturedigestion.

The analysis of Zn, Cu, Cr, Ni, Cd and Pb in the solutions ob-tained following sample digestion was carried out by atomicabsorption spectrophotometry (AAS) using a double-beam Per-kin–Elmer 2380 AAS with deuterium background correction and,in some cases (low concentrations), by graphite furnace atomicabsorption spectrometry (GFAAS) using a Perkin–Elmer 4110 ZLwith Zeeman background correction. The hydride-generation tech-nique was used to determine As, employing a Perkin–Elmer MSH-10 connected to a Perkin–Elmer 2380 spectrophotometer. Prior toAs analysis, an aliquot of the digested sample was treated withpotassium iodide and ascorbic acid to convert As(V) to As(III). Hgconcentrations were measured by cold vapor atomic absorption

Fig. 1. Location of the sampling stations.

J. Usero et al. / Marine Pollution Bulletin 56 (2008) 2027–2036 2029

spectrophotometry employing a Perkin–Elmer MSH-10 connectedto a Perkin–Elmer 2380 spectrophotometer. The solution wasanalyzed using sodium borohydride as reducing agent (3% NaBH4

in 1% NaOH) under the conditions recommended in the unit’smanual.

3.2. Bioaccumulation of metals

Samples of C. gallina (most abundant species of bivalve mollusksin the study area) were taken over a period of three days in October2007, a month in which no reproductive activity takes place in thisspecies. Individuals were selected for a standard shell size between25 and 30 mm. After collection, the clams were allowed to flushout undigested matter in filtered seawater from the sampling sitesfor 24 h (Sokolowski et al., 2002). The soft tissues of 60 individualscollected at each station (20 per replicate) were carefully removedby shelling the bivalves with a plastic knife; they were then freeze-dried (Chiu et al., 2000) and ground to a fine powder in a mortarbefore analysis (Szefer et al., 1997; Ruelas-Inzunza and Páez-Osu-na, 2000). The resulting pooled samples underwent microwave ni-tric acid (HNO3) digestion (Usero et al., 2005). Following aciddigestion, all the samples were analyzed for eight elements (Zn,Cu, Cr, Ni, Cd, Pb, As and Hg) by atomic absorption spectrometry(AAS) as described previously.

3.3. Sediment toxicity

Two separate bioassays were used to evaluate lethal and suble-thal sediment toxicity. Lethal toxicity was determined by using amarine luminescent bacterium, Vibrio phosphoreum (formerly Pho-tobacterium phosphoreum), naturally adapted to a saline environ-ment. The lethal toxicity tests on the sediments were performedaccording to the procedure described by Svenson et al. (1996). Asuspension of the sediment was prepared in a 2% NaCl solution;then a series of dilutions was made and bacteria were exposed tothese dilutions and to a blank (2% NaCl solution). Bioluminescenceat 15 �C was measured with a Microtox luminescence meter

(Microbics Corp., Carlsbad, CA) after 30 min of incubation. Inhibi-tion was expressed as the dilution of lowest percentage thatcaused a 50% reduction in the light emitted by the bacteria(EC50). Another way to express toxicity is in toxicity units(TU50), which are calculated as a percentage of the inverse ofEC50, providing a comparative scale on which the higher the valueof TU50 the higher the toxicity, contrary to EC50 values, which de-crease as toxicity increases (Van den Brink and Kater, 2006).

Sublethal toxicity was calculated using the R. philippinarumclam, a benthic species that has been used previously for biomon-itoring contaminated sites (DelValls et al., 2002; Shin et al., 2002).This bioassay consists of looking at the effects of potentially toxicpollutants contained in marine sediment on the clam’s burrowingbehavior. Higher sediment contamination levels could decreaseburrowing activity (McCloskey and Newman, 1995). Sixty individ-uals of R. philippinarum (20 per replicate) with shells 2.5–3 cm longpurchased from an aquaculture farm located at a clean coastal sitewere used in each bioassay. The individuals were acclimatized inthe laboratory at 15 �C for 3 d prior to being used. Filtered seawaterwas carefully added to the sediment matrix (solid/liquid ratio0.5 kg L�1) and placed in experimental tanks (50 � 50 � 16 cm).Experiments on burrowing behavior were conducted by placingclams on sediment and recording the time to complete burial,i.e., until no portion of the clam shell remained visible. Observationintervals were 5 min apart for the first 30 min, then 15 min to 2 h,then hourly for 6 h and then about every 12 h. The rate of burial ofthe clams was measured as the mean time for 50% of the clams toburrow (ET50).

3.4. Macrobenthic community

Five replicate samples for macrobenthic organism assessmentwere collected with a grab sampler and sieved in situ through a0.5-mm mesh. All samples were preserved in 10% formalin solutionand stained with buffered rose bengal stain (1 g of rose bengal in1 L of distilled water) for one week. Macrobenthic organisms weresorted, numbered and identified to the lowest possible taxon

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(usually to species level). Data on individuals per species were usedto calculate various biological parameters, such as abundance (A)(individuals/m�2), Margalef’s species richness index (R) (Margalef,1958), the Shannon diversity index (H’) (Shannon and Weaver,1963) and Pielou’s evenness index (J’) (Pielou, 1966).

3.5. Reagents and quality assurance

The nitric acid and perchloric acid used for digestion wereSuprapur (Merck). All other reagents used for testing were ofanalytical reagent grade (Merck). Stock solutions (Merck) of1000 mg L�1 of the different elements analyzed with certificatesof analysis traceable to NIST were used to prepare the calibrationstandards.

All the analyses were performed under the laboratory’s updatedrigorous quality control system (ISO/IEC, 2005). The digestion andanalytical procedures were checked by analysis of standard refer-ence materials in every batch digestion (sediment: CRM-277,Community Bureau of Reference; bivalves: CRM 278 R, M. edulismussel). Replicate analysis of these reference materials showedgood accuracy, with metal recovery rates between 94% and 105%for mussels and between 92% and 97% for sediment. Precision wasverified by analyzing a replicate sample in every batch digestion.

The percentage relation between the difference of the two val-ues obtained and the mean value of these was less than 10% inmost cases, and the precision was therefore considered satisfac-tory. Samples were evaluated for potential contamination by ana-lyzing one acid blank in every batch. The results of the blanks werealways below the method’s detection limit. Detection limits (calcu-lated on the basis of 10 determinations of the blanks as three timesthe standard deviation of the blank) in the bivalves and marinesediments are shown in Table 1.

3.6. Data analysis

The chemical contamination, toxicity and macrobenthic alter-ation indices were calculated for each sampling point by usingEq. (1) (Usero et al., 1997):

XYI ¼ ðVN1 � � �VN2 � � � � � VNnÞ1=n ð1Þ

in which X refers to O (organic), I (inorganic), L (lethal), S (suble-thal), M (macrobenthic) or B (bioavailability), and Y to C (contami-nation), T (toxicity) or A (alteration). VNi is the normalized value ofparameter i defined as the ratio of the value of parameter i at stationP to the parameter at reference sampling point R. Values for theseindices were close to one at stations with the least contamination,toxicity and macrobenthic alteration.

The various biological parameters of the macrobenthic commu-nity structure (abundance, species richness, diversity, evenness,etc.) were calculated using Biodiversity Professional software(McAleece, 1997).

Table 1Detection limits for metals analyzed (mg kg�1 dry mass)

Detection limit

Sediment Donax trunculus

Hg 0.01 0.01Cd 0.02 0.02As 0.20 0.20Zn 0.50 0.50Ni 1.00 0.06Cu 1.00 1.00Pb 2.00 0.06Cr 2.00 0.02

All clam metrics and heavy metal concentrations in surface sed-iments and clams were analyzed by one-way ANOVA to highlightdifferences between stations and clam exposures. Statistical analy-sis of the results was done with SPSS software (SPSS, 2006).

4. Results and discussion

4.1. Sediment analyses

The grain size of the marine sediments analyzed was verydifferent throughout the study area. Sediments from P1 and P2showed a high percentage (94%) of very fine sand (63 < Ø <125 lm) because of the fine particle size from the Guadiana river,whereas the central coastal zone (P5 to P10) was dominated bycoarse and medium sand (Table 2). In the east, near the Guadalqui-vir river, larger amounts of fine sand were observed near themouth of the Guadalquivir river. Table 2 shows that the selectioncoefficients defined by Trask (1950) were higher at P12 and P15and lower at the other sampling points. The lowest coefficient val-ues were at P4, P7 and P8. This indicates the heterogeneity of theanalyzed sediment grain size.

The amounts of total organic carbon (%) in the analyzed samplesare indicated in Table 2. The highest organic carbon content wasfound at the sampling points located in the proximity of the Gua-dalquivir (P12 to P15) and Guadiana (P1 and P2) rivers, character-ized by very fine sandy sediments. The pH of the samples rangedfrom 7.5 (at P15) to 8.0 (at P1 and P2).

The sampling points located in the central area of the South-western Spanish coast (P6 to P10) showed the highest levels ofCu, Cd, Pb, Zn and As in the marine sediments analyzed (Table 2).The highest concentrations of most of the metals analyzed werefound at P9 (Huelva estuary) and the second-highest values wereobtained near the Punta Umbría estuary (P7), strongly affectedby the acidic inputs from the Tinto and Odiel rivers, which haveone of the highest metal concentrations in Europe (Braungardtet al., 2003). The concentrations of Cu, Cd, Pb, Zn and As in the sed-iments from P9, P10 and P11 were higher than those reported forthe Barcelona coast (Northern Spain), the Athens Coast (SouthernGreece), and Algeciras bay (Southern Spain) (Savvides et al.,1995; Lopez-Sanchez et al., 1996; Benamar et al., 1999) andcoincide with those reported in the Policy Environmental Reportspublished by the Regional Government of Andalusia (Spain) (Useroet al., 2004).

Iron was the most abundant metal in the analyzed sedimentsand showed the highest concentrations in the central zone becauseof the high pyrite (FeS2) content beneath the Iberian Pyrite belt.The rest of the metals studied (Cr, Ni, Mn and Hg) showed a verydifferent spatial distribution and evidenced no significant increasein concentration in the central area. The highest values of Ni, Crand Mn were obtained beneath P1 and P15, located at the mouthsof the Guadiana and Guadalquivir rivers, respectively. The levels ofHg were similar in general to those found in areas of non-pollutedsediment (Ruiz, 2001).

4.2. Bioconcentration of metals

As in the case of the sediments, the highest metal concentra-tions in bivalves were found in the center of the study area (fromP7 to P9) (Table 2). This is in agreement with results obtained byBaumard et al. (1998), indicating that there is a good correlationbetween the metal concentrations in C. gallina and in the sedi-ments. Experimental results showed that the concentrations ofCu, Zn and Fe were of a higher order of magnitude than the othertrace elements analyzed. The Cu levels in this species can be con-sidered high if compared with those usually found for this element

Table 2Distribution of grain size, total organic carbon, heavy metal concentrations in sediments and bivalves and lethal and sublethal toxicity (N = 40) of the analyzed sediments (n = 3)

Sampling point RSDc %

R P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15

Granulometrya MS VFS VFS FS FS CS CS CS MS MS MS FS VFS VFS VFS VFSSelection coefficient (S0)b B B P P P B B P P B B B M B B MTotal organic carbon (‰) 0.20 0.41 0.38 0.27 0.23 0.22 0.24 0.24 0.25 0.29 0.28 0.29 0.39 0.56 0.73 0.98 4–22

Sediments metal contamination (mg/kg dry mass)Zn 156 155 204 165 210 244 357 552 430 649 599 340 314 284 204 191 6–14Cd 0.19 0.21 0.77 0.19 0.26 0.24 1.3 2.1 1.3 2.5 0.92 0.82 0.37 0.35 0.24 0.30 4–9Cu 41.2 53.0 71.5 44.5 41.9 78.6 133 286 185 336 250 125 98.2 91.4 68.0 60.2 4–11Pb 21.1 20.8 22.0 23.5 30.3 30.4 85.6 105 103 197 134 85.7 66.5 68.4 44.2 36.6 7–12Fe 16300 29600 28700 15900 22300 20100 34700 32100 35300 31300 32700 29500 21300 27700 25300 24700 9–17Ni 13.8 36.6 17.4 11.5 28.2 19.6 18.5 19.1 15.5 17.3 13.7 10.9 13.2 12.4 20.3 50.8 6–15Cr 34.5 82.3 67.2 32.3 68.2 38.3 74.3 67.4 63.5 46.4 57.5 64.3 35.4 62.5 46.2 92.3 5–12Mn 287 576 476 287 310 348 396 550 382 281 309 414 333 370 379 569 8–14As 18.4 15.3 17.1 18.1 19.2 24.2 23.3 45.4 43.4 61.5 47.4 36.5 31.4 22.5 16.3 14.1 11–19Hg 0.33 0.44 0.47 0.52 0.36 0.26 0.29 0.21 0.35 0.42 0.37 0.25 0.21 0.28 0.35 0.24 7–18

Metal concentration in bivalves (mg/kg wet mass)Zn 8.6 10.9 15.8 17.6 19.6 23.2 23.0 25.6 21.20 28.4 19.2 14.9 13.2 13.3 12.9 11.4 6–14Cd 0.15 0.22 0.33 0.39 0.49 0.55 0.70 0.67 0.46 0.52 0.49 0.47 0.41 0.38 0.36 0.35 4–11Cu 10.0 8.6 13.3 17.1 22.5 30.0 30.5 35.2 31.8 47.1 38.5 31.8 23.6 21.1 18.8 15.1 4–11Pb 0.47 0.54 0.57 0.54 0.58 0.67 0.84 0.89 1.08 2.18 1.17 1.06 0.93 0.84 0.76 0.74 7–12Fe 43.2 49.7 51.1 50.4 56.0 53.9 55.9 58.1 98.7 186 81.2 68.9 64.8 59.0 61.7 58.3 9–20Ni 0.17 0.39 0.37 0.35 0.30 0.28 0.35 0.34 0.42 0.33 0.31 0.27 0.34 0.35 0.36 0.35 6–15Cr 0.07 0.28 0.19 0.17 0.16 0.18 0.18 0.20 0.39 0.56 0.39 0.36 0.37 0.34 0.31 0.33 9–15Mn 0.90 1.23 1.21 1.33 1.36 1.50 2.41 2.18 1.86 2.58 1.79 1.69 1.88 1.95 2.05 2.15 11–15As 1.06 1.45 1.22 1.22 1.42 1.26 1.39 1.44 1.87 2.70 1.65 1.43 1.31 1.22 1.13 1.06 11–19Hg 0.03 0.03 0.06 0.05 0.07 0.07 0.08 0.09 0.15 0.17 0.12 0.12 0.08 0.06 0.06 0.05 7–18

Sediment toxicityLethal (TU50/g) 19 23 23 21 22 21 28 58 44 73 53 35 28 28 29 32 6–14Sublethal (ET50/min) 38 49 54 41 43 44 60 183 154 198 157 82 53 54 55 78 12–18

a VFS: very fine sand, FS: fine sand, MS: medium sand, CS: coarse sand.b M: moderate (1.20 < S0 < 1.35), P: poor (1.87 < S0 < 2.75), B: bad (2.75 < S0).c RSD: relative standard deviation (range).

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Table 3Abundance (ind. m�2) of the different species, number of species (S), Margalef’sspecies richness index (R), Shannon diversity index (H’) and Pielou’s evenness index(J’) measured at the sampling stations

Samplingpoint

Abundance Species richnessindex

Diversityindex

Evennessindex

R 3240 14.85 3.92 0.94P1 2276 15.52 1.98 0.52P2 2194 15.60 2.85 0.76P3 2449 15.38 3.00 0.75P4 2876 15.07 3.15 0.79P5 2898 15.05 3.27 0.80P6 3028 14.97 3.17 0.80P7 2593 15.27 2.16 0.62P8 2016 15.77 2.67 0.71P9 936 17.54 1.87 0.51P10 1612 16.25 2.71 0.78P11 1496 16.42 2.64 0.74P12 1704 16.13 1.94 0.54P13 1556 16.33 2.01 0.59P14 1892 15.90 1.92 0.52P15 2284 14.64 1.91 0.54

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in other bivalve studies (Baraj et al., 2003; Paulson et al., 2003). Theconcentrations of most of the metals in the bivalve studied variednotably depending on the location of the sampling sites. The high-est concentrations of Zn and Cu in bivalves were found near themouth of the Huelva estuary (P9), where the Tinto and Odiel riversdischarge larger amounts of metals. The concentrations of Cd re-corded in this study were similar to measurements previously re-ported by Usero et al. (2005) for the Atlantic coast of Spain. Theelement with the lowest mean concentrations was Hg. Besadaet al. (2002) reported similar concentrations for this element in bi-valves from the northern Atlantic coast of Spain.

The concentrations of Pb in C. gallina at P9 exceeded the maxi-mum limits established by the European Union in bivalve mollusksdestined for human consumption (Pb 1.5 mg/kg wet mass) (EEC,2001), and the bivalves analyzed are therefore not fit for consump-tion. The concentrations of Cd and Hg in all analyzed samples werelower than the maximum permissible concentration in the Euro-pean Union (Cd 1 mg/kg and Hg 0.5 mg/kg wet mass) (EEC, 2001,2002). The concentrations of Cu did not exceed the maximum limitpermitted by Spanish legislation (60 mg/kg wet mass). Maximumconcentrations of other metals are also regulated in other coun-tries. For example, the British Food Standards Agency states thatZn may not exceed 50 mg/kg wet mass in food (EGVM, 2003).The maximum concentration found in the bivalves analyzed was28.44 mg/kg wet mass; therefore, this metal does not represent ahigh risk for human health. There are no legislated levels for Crand Ni, and data relative to the effect on human or animal healthare not sufficient to derive a safe upper level for these metals.Anderson et al. (1997) indicate that 15 mg/kg chromium (as chro-mium chloride) was not associated with adverse effects in the rat.Based on this study, and allowing for uncertainty factors of 10 forinter-species variation and 10 for inter-individual variation, a totaldaily intake of about 0.15 mg/kg wet mass would be expected tohave no adverse health effects. Experimental results reported inthis study show that all bivalves analyzed exceed this concentra-tion. Therefore, it cannot be concluded with certainty that the ana-lyzed species are suitable for human consumption.

4.3. Sediment toxicity

The lethal toxicity data for the analyzed sediments are summa-rized in Table 2. Despite the obvious differences between the re-sponse spectra of P. phosphoreum from sampling points P7 to P10situated at the mouth of Huelva estuary, the levels of lethal toxicityobtained, expressed as toxicity units per gram of sediment (TU50/g), were mostly low and similar to those reported by Svenson et al.(1994) in the Faroe Islands (uncontaminated area in the AtlanticOcean).

The high lethal toxicity values obtained at the sampling pointsnear the Huelva estuary can be explained by the large amountsof heavy metals originating from erosion and mining activitiesand transported by the Tinto and Odiel rivers. The metals fromprecipitation after contact of acidic river water with seawater(pH 8.2 and high salinity), are weakly bound to the sedimentsand easily bioavailable (Morillo et al., 2005), and consequentlycan cause major damage to macrobenthic communities. A few lev-els of toxicity slightly higher than the rest of the samples were alsoobserved in the proximity of the Guadiana river (P1 and P2) andthe Guadalquivir river (P12 to 15), undoubtedly as a consequenceof pollution of these rivers by industrial and urban waste outflows,and because of the smaller particle size of the analyzed sediments,which increases the specific surface and, therefore, the contact be-tween bacteria and sediment.

The sublethal toxicity determined on the basis of the clams’burrowing capacity showed a pattern similar to the lethal toxicityof marine sediment samples (Table 2). The mean time for 50% of

the clams to burrow (ET50) increased when they were exposedto highly contaminated sediment. However, there was a significantdifference between the intensity of lethal and sublethal toxicity inthe central part of the coast (P7 to P10) due to wide variation in theburrowing behavior of R. philippinarum during the bioassay. Duringthe exposure period, there were no clam mortalities. The percent-age of clams buried after 24 h in reference sediment (n = 3) was100% compared to about 80% in sediments from the eastern andwestern zones and 60% at the sampling points located in the cen-tral coastal area (P7 to P10). During the three days of experimenta-tion, there was no significant variation in the number of clamsburied in all analyzed samples (p > 0.05), with the exception of sed-iment from P9 where the number of clams buried after 24 h was 2times lower (p < 0.05).

4.4. Macrofauna structure

A total of nine taxonomic groups of macrobenthic communitieswere found in sediments from the entire study area. The dominantgroups were crustaceans (39 families), mollusks (36 families) andannelids (35 families), followed by echinoderms (3 families) andcnidarians, nemertines, acranians, tunicates and platyhelminthswith only one family each. A very significant heterogeneity of mac-rofauna distribution at the different stations was observed (Table3) and could be attributed to the typical feature of a highly dis-turbed coastal zone such as the study area, characterized by highlevels of metal contamination.

The annelids are the dominant group, representing more than40% of the individuals found at each station. This predominanceof annelids is especially significant at P1 and P2 (close to the mouthof the Guadiana) and at P12 to P14 (near the mouth of the Guadal-quivir). The main factors that may explain this are the fine grainsize and the higher organic matter content in the sediments atthese stations, which facilitate the growth of this macrobenthicgroup, especially of families able to rapidly exploit organic-richsediments, such as Spionidae and/or Capitellidae.

The smallest number of families was recorded at samplingpoints P1, P9 and P15, located in the more disturbed areas (mouthsof the Guadiana, Tinto-Odiel and Guadalquivir rivers). This de-crease occurred especially at P9, undoubtedly because of the high-er levels of contamination in the sediments from the Huelvaestuary, which negatively affects the normal development of thebenthic community. However, it should be stressed that thestations in the western part of the coast have a larger number of

R P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15

1

2

3

4

5

6

ICI OCI BCI STI LTI MAI

Sedi

men

t Con

tam

inat

ion

Inde

x

Fig. 2. Evolution of the sediment quality index values in the study area: inorganiccontamination index (ICI), organic contamination index (OCI), bioavailabilitycontamination index (BCI), sublethal toxicity index (STI), lethal toxicity index(LTI), macrobenthic alteration index (MAI).

J. Usero et al. / Marine Pollution Bulletin 56 (2008) 2027–2036 2033

families. This is probably because sediments from the western areahave a heterogeneous structure and grain sizes are larger than atthe other stations. This significant difference in sediment grain sizecan be expected to lead to more structural heterogeneity andpotential niche space and therefore higher diversity in the westernsector of the study area.

The biological parameters in Table 3 show that the behavior ofspecies abundance is similar to that of species richness, previouslydiscussed. Nevertheless, a clear increase in abundance at samplingpoint P15 can be observed. This difference can be explained by thegreater abundance of the Semelidae (mollusk group) and Capitelli-dae (annelid group) families, facilitated by organic inputs from theGuadalquivir river and enhanced by the stands of macrovegetationfound in the area. In terms of diversity (H’) and evenness (J’), thetrends of these parameters are similar to those previouslydiscussed. The highest values were found in the western area ofthe coast, while lower values were recorded in the vicinity of themouths of the Guadiana and Guadalquivir rivers and near theHuelva estuary.

4.5. Integrated sediment quality assessment method

Ratio-to-reference values (RTR) were calculated for each of themeasurements or indicators found to be useful in assessing the im-pact status of the sites by dividing the mean value of each mea-surement at a site by the mean value of that measurement fromthe reference site (Lee et al., 2006). The RTR values calculated foreach component of the integrated sediment quality assessmentmethod are given in Table 4. These values provide a normalizedmeasurement of the degree to which a site differs from the refer-ence site and the other stations.

Fig. 2 shows that the site with the highest Inorganic Contamina-tion Index (ICI) was P9 (at the mouth of the Huelva estuary), show-ing the highest normalized values for Zn, Cd, Cu, Pb and As, whilethe area near the mouth of the Guadalquivir (P13–P15) showed thehighest Organic Contamination Index (OCI) and the highest nor-malized values for Ni. To the contrary, reference sampling pointsR and P3 had the lowest ICI and the lowest normalized values formost of the analyzed parameters. The values of the Lethal and Sub-lethal Toxicity Indices (LTI and STI) were similar to those obtainedfor ICI, and the most impacted sites were located in the Huelvaestuary. The Bioavailability Contamination Index (BCI) of metalsin the analyzed organisms showed the same pattern, and the mac-robenthic community alteration determined in situ showed thehighest Macrobenthic Alteration Index (MAI) at P9 and at the sta-tions at the mouths of the Guadalquivir (P12–P15) and the Guadi-ana (P1 and P2). This implies that the main source of alteration ofmacrofauna is the chemical contamination of these rivers.

To provide an overall view of the environmental quality of theanalyzed marine sediments from the southern Atlantic coast ofSpain, a six-axis plot was represented for each station (Fig. 3). A

Table 4Ratio-to-reference (RTR) values from each component of the ISQAM: inorganic contaminati(BCI), sublethal toxicity index (STI), lethal toxicity index (LTI), macrobenthic alteration ind

Sampling point

R P1 P2 P3 P4 P5 P6 P7

ICI 1.00 1.46 1.73 1.22 1.29 1.49 2.48 4.37OCI 1.00 2.05 1.90 1.35 1.15 1.10 1.20 1.20BCI 1.00 1.46 1.65 1.69 1.87 2.02 2.32 2.42STI 1.00 1.21 1.21 1.11 1.16 1.11 1.47 3.05LTI 1.00 1.29 1.42 1.08 1.13 1.16 1.58 4.82MAI 1.00 1.64 1.36 1.26 1.18 1.14 1.17 1.44ISQI 1.00 1.50 1.53 1.27 1.27 1.30 1.63 2.54

module vector equal to the value obtained for each of the indicesstudied was plotted on each axis (organic and inorganic sedimentcontamination, lethal and sublethal sediment toxicity, bioconcen-tration of metals and marine benthic community structure alter-ation). The polygon (hexagon) obtained by combining the valuesof the reference station indices is white (the values for this polygonare equal to one on each axis), and the polygons for each station areyellow. With this type of graph it is possible to determine the sta-tions with the highest degree of degradation at a single glance bythe sizes of the obtained polygons. In addition, the deformationof the polygons shows the main type of degradation in the samples.Plots for P1 and P2 are similar to those for the reference site, char-acterized by relatively low pollution-induced degradation indices(contamination indices close to one). Organic contamination ishighest at P1, undoubtedly as a result of insoluble organic matterdischarged from the Guadiana river. This phenomenon contributesto diminishing the sediment grain size, which leads to a higherMAI. P3, P4 and P5 are far from the main contamination sources,which is why the polygon areas are slightly larger than the areaof the reference site. This indicates that there is little environmen-tal alteration on this part of the coast.

The highly contaminated coastal area (from P7 to P10) has lar-ger polygon surface areas because of the high values for all themarine sediment indices determined (ICI, LTI, STI, MAI and BCI),except for OCI, which was relatively low. The highest values forICI, LTI, STI, MAI and BCI were found at P9 (next to the mouth ofthe Huelva estuary). P7 (closest to the mouth of the Punta Umbríaestuary) had the second highest values for ICI, LTI, STI and BCI. Con-trary to the reference site polygon, most of the polygon shapes at

on index (ICI), organic contamination index (OCI), bioavailability contamination indexex (MAI), integrated sediment quality index (ISQI)

P8 P9 P10 P11 P12 P13 P14 P15

3.45 4.97 3.38 2.38 1.74 1.69 1.40 1.641.25 1.45 1.40 1.45 1.95 2.80 3.65 4.902.83 3.86 2.66 2.36 2.17 2.01 1.93 1.822.32 3.84 2.79 1.84 1.47 1.47 1.53 1.684.05 5.21 4.13 2.16 1.39 1.42 1.45 2.051.46 2.34 1.62 1.64 1.84 1.82 1.78 1.752.35 3.30 2.48 1.94 1.74 1.82 1.84 2.11

Fig. 3. A six-axis plot of the sediment quality data with the integrated sediment quality index (ISQI) determined for each station: inorganic contamination index (ICI), organiccontamination index (OCI), bioavailability contamination index (BCI), sublethal toxicity index (STI), lethal toxicity index (LTI), macrobenthic alteration index (MAI), integratedsediment quality index (ISQI).

2034 J. Usero et al. / Marine Pollution Bulletin 56 (2008) 2027–2036

the sampling stations are deformed, giving relatively high valuesfor ICI, LTI and STI and low values for OCI. A progressive increasein OCI was observed between P11 and P15 (from 1.95 to 4.90). Thiscan be explained by inputs from the Guadalquivir river, which arethe main source of organic contamination in these marine sedi-ments (Belmonte Vega et al., 2005). The MAI levels for P12–P15were relatively high due to the decrease in the sediment grain sizeand the organic contamination from the Guadalquivir river.

This research proposes a general Integrated Sediment QualityIndex (ISQI), calculated using Eq. (2), which is derived from Eq.(1). The method used for calculating this index was similar to thatused for the indices discussed previously (see Eq. (1)). It couldtherefore be called an index of indices, which summarizes the maincharacteristics of the environmental situation of each samplingpoint. A higher index value indicates greater pollution-induceddegradation.

ISQI ¼ ðICI � OCI � LTI � STI �MAI � BCIÞ1=6 ð2Þ

Table 4 shows the integrated sediment quality index calculated foreach site analyzed. P1 and P2 show ISQI values around 1.5, higherthan those farther from the contamination sources (P3, P4 andP5), with ISQI values between 1.27 and 1.30. A considerable in-crease of the ISQI in the most contaminated marine area of theHuelva estuary (from P7 to P10) was observed, in particular at P9(3.30) and P7 (2.54). There was a clear decrease in the ISQI betweenP10 (2.48) and P11 (1.94), followed by an increase at P15. This lastincrease is due to effluents from the Guadalquivir river.

The sampling sites can be classified into three groups based onthe integrated sediment quality index obtained:

� Stations with low degradation (ISQI < 1.5): P3, P4 and P5.� Stations with moderate degradation (ISQI P 1.5 and <2): P1, P2,

P6, P11, P12, P13 and P14.� Stations with high degradation (ISQI P 2): P7, P8, P9 P10 and

P15.

The proposed ISQI intervals are not standardized and may bechanged in the future in terms of the number of studies carriedout and by using other data available in the scientific literature.This general index summarizes the information represented inthe six-axis plots. For this reason, the authors suggest adding theISQI values to each graphic representation.

Acknowledgements

This research was supported by the Environmental Council ofthe Junta de Andalucía (the Andalusian Governing Body), Spain.

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