Estuarine, Coastal and Shelf Science 71 (2007) 278e286www.elsevier.com/locate/ecss

Estuarine sediment toxicity tests on diatoms: Sensitivitycomparison for three species

Ignacio Moreno-Garrido a,*, Luis M. Lubian a, Begona Jimenez b,Amadeu M.V.M. Soares c, Julian Blasco a

a Institute of Marine Sciences of Andalucia (CSIC), Campus Rio San Pedro, s/n Apartado Oficial, 11510 Puerto Real, Cadiz, Spainb Department of Instrumental Analysis and Environmental Chemistry, Institute of General Organic Chemistry Lora Tamayo,

c/ Juan de la Cierva n� 3, 28006 Madrid, Spainc CESAM & Department of Biology, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal

Received 10 May 2006; accepted 3 August 2006

Available online 11 September 2006

Abstract

Experimental populations of three marine and estuarine diatoms were exposed to sediments with different levels of pollutants, collected fromthe Aveiro Lagoon (NW of Portugal). The species selected were Cylindrotheca closterium, Phaeodactylum tricornutum and Navicula sp. Previousexperiments were designed to determine the influence of the sediment particle size distribution on growth of the species assayed. Percentage ofsilt-sized sediment affect to growth of the selected species in the experimental conditions: the higher percentage of silt-sized sediment, the lowergrowth. In any case, percentages of silt-sized sediment less than 10% did not affect growth. In general, C. closterium seems to be slightly moresensitive to the selected sediments than the other two species. Two groups of sediment samples were determined as a function of the generalresponse of the exposed microalgal populations: three of the six samples used were more toxic than the other three. Chemical analysis of thesamples was carried out in order to determine the specific cause of differences in toxicity. After a statistical analysis, concentrations of Sn, Zn,Hg, Cu and Cr (among all physico-chemical analyzed parameters), in order of importance, were the most important factors that divided the twogroups of samples (more and less toxic samples). Benthic diatoms seem to be sensitive organisms in sediment toxicity tests. Toxicity data frombioassays involving microphytobentos should be taken into account when environmental risks are calculated.� 2006 Elsevier Ltd. All rights reserved.

Keywords: bioassays; sediment; toxicity tests; diatoms; microphytobenthos

1. Introduction

Toxicity testing in aquatic marine environments presentsseveral difficulties. High dilution capacity and mobility ofocean water masses could make it very difficult to detect theresponse of organisms to toxicants. Salinity (and often pHvalues) changes in water when inland effluents meet the ocean,mainly in estuaries or coastal lagoons. This usually causesa quick precipitation of many xenobiotics to sediments.Thus, measurement of toxicity in coastal sediments becomes

* Corresponding author.

E-mail address: ignacio.moreno@icman.csic.es (I. Moreno-Garrido).

0272-7714/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ecss.2006.08.003

a very important topic. Chemical analyses provide onlymeasured concentrations of a given chemical in an environ-mental compartment. Actual toxicity information is availableonly from bioassays (Munawar and Munawar, 1987). Hence,bioassays have been designed to determinate the effectof potentially toxic samples to biota. The key role ofmicrophytobenthos on intertidal mudflats has been pointedout (Absil and Van Scheppingen, 1996), as those microorgan-isms (mainly diatoms and cyanobacteria) are major contribu-tors to the primary productivity in mudflats and the basis ofthe trophic chain (Hillebrand and Kahlert, 2002). Additionally,the importance of microphytobenthos in the maintenance ofthe structure and protection against erosion of upper sedimenthas been demonstrated (Riethmuller et al., 2000; Widdows and

279I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

Brinsley, 2002). Thus, perturbations in the structure of micro-phytobenthic populations may propagate to the structure ofpopulations of organisms situated in upper trophic levels andmay affect to the physical and chemical sediment structure.

Toxicity tests involving sediments and microphytobenthicorganisms are extremely scarce and recent (Moreno-Garridoet al., 2003a,b, 2006; Adams and Stauber, 2004). There areno standardized protocols for sediment bioassays involvingmicrophytobenthos in the literature (SETAC, 1993). The useof microorganisms (Bitton and Dutka, 1986) presents manyadvantages relative to the use of higher (multi-cellular) organ-isms. Microorganisms are more sensitive to toxicants becausetheir surface/volume ratio is quite higher and thus expositionto toxicants is more direct than in the case of multi-cellular or-ganisms. The fast growth of microorganisms allows develop-ment of acute and chronic bioassays involving many lifecycles, which permits faster and less expensive assays. Micro-organisms play a key role in different biocenoses, being pri-mary producers or organisms involved in the nutrientcycling. Ethic sensitivity of animal suffering in laboratory testsis avoided using microorganisms. Disadvantages include thatsome toxicants (i.e., neurotoxins or certain heavy metals)seem to be more toxic to higher organisms than to microorgan-isms, as the latter lack target tissues or organs affected forthese substances. Additionally, short life cycles of microorgan-isms do not permit the design of chronic bioassays that focuson toxicant accumulation. In highly polluted areas often theonly organisms that can survive are microorganisms. This isdue to the ability of such organisms to develop resistance totoxicants (by selection of strains or acquisition of resistanceplasmids). However, this is not a handicap when populationsof non-exposed organisms are continuously cultured in opti-mal conditions and used in toxicity tests.

Most of the standard bioassays have been designed forfreshwater environments (OECD, 1998) but there is a recentincrease in the interest on developing standard toxicity bio-assays for marine environments, some of them focused onmarine microalgae (APHA-AWWA-WPCF, 1989; ISO, 1995;Moreno-Garrido et al., 2001). Importance of sediment toxicitytests in marine, estuarine and freshwater environments hasbeen pointed out in the last decade (Van deer Kooij et al.,1991). Acute bioassays for whole marine sediments havealso been designed for amphipods, cumaceans, copepods,shrimps, bivalves, polychaetes and fishes (Lamberson et al.,1992). In this work, a toxicity test involving benthic microal-gae has been designed in order to detect and compare toxicityof estuarine surface sediments.

2. Materials and methods

2.1. Sampling sites

Sediments were collected from the Aveiro Lagoon (Fig. 1),a shallow vertically homogeneous lagoon located in the north-west of Portugal, connected to the Atlantic Ocean througha narrow artificial channel (San Jacinto Channel) and suppliedwith freshwater by two major rivers, Antu~a and Vouga (Dias

et al., 2000). Six locations were chosen near to the mouth ofthe lagoon, in order to obtain surface (first 0.5 cm) sediment.Samples I and II were taken from the shore near Barra, a smalltown between Costa Nova Village and the connecting channelof the lagoon and the ocean. Sample I was around 400 mnearer the mouth channel than sample II. Sample III was lo-cated near the Fort of Barra, in the junction of San JacintoChannel and the Ria of Aveiro, beside a dock for outboard mo-tor launches. Sample IV was located near an ancient bridgeover a small water branch and sample V was located at theshore in the inner part of a small bay formed by the same waterbranch. Sample VI was located off Lavacos beach, in front ofthe Vila Channel, in the upper part of the mouth of the lagoon.All samples were taken at low tide. As all locations werechosen near the mouth of the Lagoon, salinity of supernatantwater was 35.

2.2. Sediment sample preparation

Sediment samples were stored in ice at collection and fro-zen at �4 �C within 2 h. For analysis, samples were de-frostedand dried for 24 h at 60 �C. Higher temperatures were not usedin order to prevent loss of volatile compounds, such as Hg(Loring and Rantala, 1992). In any case, storage treatmenteliminates the natural population of micro- and meio-faunaand flora in the sediments, which can change the behavior ofthe experimental microalgal population (if grazers or local mi-croflora appears). Resultant dry sediment was stored in her-metically closed plastic vials at room temperature.

2.3. Organisms and culture media

Three pennate diatoms (Bacillariophyta, order Pennales)were used in this study: Phaedactylum tricornutum Bohlin, Cy-lindrotheca closterium (Ehremberg) Lewin and Reimann (for-merly Nitzschia closterium (Ehremberg) W. Smith) andNavicula sp. All the species were obtained from the collectionCCMM (BIOCISE) of the Institute of Marine Sciences of An-dalucıa (CSIC). All the species assayed can be found in the up-per part of the sediments, but P. tricornutum is not strictlyconsidered a microphytobenthic but a planktonic species. Se-lection of this latter organism was done because of the litera-ture on its use as ‘‘standard’’ organism in marine toxicity tests(ISO, 1995). The strains were cultured in artificial seawater(ASTM, 1975) for at least 6 months before the beginning ofthe tests. Microalgal stocks were routinely cultured in f/2 me-dium (Guillard and Ryther, 1962). Artificial seawater (ASTM,1975) was also used in the tests, but medium was enrichedwith low nutrient levels (Moreno-Garrido et al., 2003a), ofphosphate, nitrate and silica concentrations similar to thosefound in natural locations (Establier et al., 1990). This avoidedchelators or trace metals (present in Guillard’s f/2 medium)that might react with substances present in the sediments.Growth of the species used during at least 72 h was assessedbefore the test in flasks without sediments. Three-day-old mi-croalgal cultures of each diatom were used in the tests in orderto ensure a microalgal population in exponential growth phase.

280 I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

Fig. 1. Sampling sites at the mouth of the Aveiro Lagoon. Light gray, water; dark gray, mudflats that remain submerged in low tide; white, emerged land.

Aliquots of these cultures were centrifuged in a desktop cen-trifuge model MSE Micro Centaur (12 s pulses at10,000 rpm) and re-suspended in clean medium before thestart of the experiments.

2.4. Sediment analysis

Grain size analyses for each sample was determined by drysieving. Total carbon and nitrogen were analyzed using a CarloErba Analyzer 1106. Heavy metals were determined accordingto the protocol described by Loring and Rantala (1992). Theanalyses were carried out by graphite furnace atomic absorp-tion spectrophotometer (GFAAS) with background Zeemancorrection (Perkin Elmer, 4100ZL) or by ICP optical emissionspectrophotometer (Perkin Elmer Optima 2000DV), except Snand Hg, which were measured by a FIAS-MHS system cou-pled to GFAAS 4100ZL. PCBs and DDTs were analyzed byGC-ECD, model GC Agilent 6890 Series II, after extractionin a Soxhlet device.

2.5. Study of the influence of particle-size distribution

Sediment containing different silt and sand size distribu-tions were prepared in triplicates. For all species assayed,50 ml of culture media containing 104 cells ml�1 were addedto each flask. Flasks were shaken daily, and cellular densitywas measured after 72 h. The study of the influence of grain

size distribution on sediment bioassays developed on micro-phytobenthos is important as this parameter could have influ-ence on microalgae population growth. This paper suggests anexperimental design in order to avoid this influence.

2.6. Toxicity tests

Acid-washed Erlenmeyer flasks of 125 ml capacity filledwith 50 ml of medium and topped with synthetic cotton (Per-lon) were used in the tests. Each flask contained 5 g of totalsediment, which is the minimum amount of material necessaryto cover the base of the flask. Sediment obtained from fieldsamples was mixed at different percentages with acid-washedbeach sand obtained from a non-polluted area in the south-west of Spain (Moreno-Garrido et al., 2003a), in order tokeep silt-sized sediment percentage under 10% in all flasks(4.5 g washed sand plus 0.5 g of mixtures of ground cleansand and natural sediments). The flasks containing 100% ofwashed sand and 0% of sampled sediment were consideredcontrols. Experiments were carried out in triplicates. Initialcellular density for the tests was 104 cells ml�1, which meansaround 18 � 103 cells cm�2 when settled at the bottom of theflask. This cellular concentration in surface of sediments issimilar to those found in natural locations (Delgado, 1989).Cells were sampled after shaking and were counted daily ina Neubauer chamber, using a microscope (Olympus BH-2).When difficulties in discriminating cells from sediment

281I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

particles occurred, fluorescence emission was used in the mi-croscopy. The use of blue excitation light and a barrier filter of530 nm resulted in an observable bright-red fluorescence ofthe chlorophyll contained in chloroplasts of the diatoms,thus being easy to discriminate cells and to count the cells.Growth curves of microalgal populations were plotted after72 h, area under the growth curves calculated and comparedwith area under growth curves of control cultures, as describedin OECD (1998). Average inhibition of data was fitted to a sig-moid function (Hampel et al., 2001) in order to obtain EC50

values (percentage of natural sediment able to inhibit thegrowth of the microalgal population to 50% when comparedwith controldall sanddcultures). This model (eq. (1)) isbased in the arctangent:

y¼ 50þ�

100

parctg

�x� T

W

��ð1Þ

Parameters W and T were automatically fitted by iterations(Sigma Plot Graphic Software), operated in radians mode.Arctg means arctangent. W relates with the slope of the sig-moid curve. T is EC50%, because

x ¼ T þWtg

�y� 50

100

�ð2Þ

and the second term of the addition is 0 when y ¼ 50, i.e. wheninhibition is 50% (in this case, tg means tangent).

2.7. Statistical analysis

Statistical analysis of the results was performed by usingthe Plymouth Routine in Multivariate Ecological Research(PRIMER), statistical computer program developed by theMarine Science and Technology Board of the Plymouth Ma-rine Laboratory (Devon, UK). Similarity matrices were per-formed measuring Euclidean distance between variables.Cluster diagrams based in these similarity matrices. Simperanalysis between two groups of samples (determined by previ-ous similarity analysis) was performed in order to determinatethe influence of each variable in the dissimilarity of eachgroup of samples (Clarke and Warwick, 1994). Simper analy-sis was performed in order to establish which parameters,among all analyzed, have more influence in the different re-sponse of growth inhibition. Cluster diagrams were createdin order to separate different types of samples (more andless toxic to diatoms).

3. Results

3.1. Effect of particle-size distributionon microalgal growth

The analysis of particle size distribution in samples re-vealed that percentage of silt-sized particles in analyzed sedi-ments was never higher than 10%. This means that sampleswere mainly sandy. Previous assays demonstrated that

inhibition of growth due to the presence of silt-sized sedimentfor the three microalgal species assayed only happens at per-centages of silt-sized sediment higher than 10% (Fig. 2). Phae-dactylum tricornutum is the most sensitive among the assayedspecies to the particle-size distribution (differences with con-trolsdtaken as 100% sand-sized sedimentdbegan to appearat 12% silt-sized sediments) although it is the smaller speciesof the three species assayed (and thus sedimentation speedmust be slower than those of Cylindrotheca closterium and Na-vicula sp.). Cultures of C. closterium began to show differ-ences respect to the controls at silt-sized sedimentpercentages of 15%. Cultures of Navicula sp. began to showdifferences respect to the controls at silt-sized sediment per-centages of 37%. In the case of this study, sediment particledistribution has no influence in the response of the algae. Infact, in the similarity analysis differences between the twogroups of samples (more and less toxic sites) due to sedimentparticle distribution suppose less than 3% of total differences.

3.2. Chemical analyses of sediment samples

All analyzed variables (Fe, Mn, Cu, Zn, Pb, Cr, Cd, Hg andSn concentrations in sediment; average of sand and silt-sizedparticles; total PCB and DDT concentrations and percentagesof C and N in sediments), for sediments obtained from the sixsample sites are shown in Table 1.

Phaeodactylum tricornutum

0

20

40

60

80

100

Cylindrotheca closterium

% Silt-sized sediment0 20 40 60 80 100

0

20

40

60

80

100

Navicula sp.

% In

hibi

tion

(72

h)

0

20

40

60

80

100

Fig. 2. Effect on growth of particle-size distribution of sediments. Silt size and

sand size sediment were mixed at different percentages. Cellular densities after

3 days have been plotted. Error bars mean standard deviation between three

replicates.

282 I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

Table 1

Physico-chemical analysis of surface sediment sampled in sites IeVI

Parameter measured Sample sites

I II III IV V VI

Percentage >63 mm (sand) 96 96 98 90 92 96

Percentage <63 mm (silt) 4 4 2 10 8 4

% C 0.2368 0.2413 0.8293 0.1856 0.6224 0.1831

% N 0.0028 0.0124 0.0060 0.0007 0.0155 0.0000

Fe (mg g�1) 2.091 2.821 5.846 5.052 5.167 4.052

Cu (mg g�1) 1.794 1.682 14.423 2.879 9.407 0.379

Zn (mg g�1) 10.363 11.121 23.155 66.918 23.565 14.110

Pb (mg g�1) 7.4 19.5 12.7 8.1 11.2 8.0

Cr (mg g�1) 4.583 6.262 13.834 9.134 12.219 10.890

Cd (ng g�1) 21.4 43.0 44.7 39.7 70.4 28.9

Hg (ng g�1) 4.89 16.62 14.40 26.40 32.40 3.15

Sn (ng g�1) 99.6 93.5 245.3 148.9 145.5 94.7

Total PCBs (ng g�1) 4.991 1.896 2.798 1.220 2.472 3.184

Total DDTs (ng g�1) 0.170 0.073 0.509 0.168 0.103 0.132

3.3. Toxicity tests

Growth curves for each microalgal population when ex-posed to the different percentages of sampled sediment wereplotted and area under growth curves calculated. Inhibition av-erages resulting from the comparison of area under the controland problem growth curves are shown in Fig. 3. From thesefigures EC50 values were calculated. Results from these calcu-lations are shown in Table 2. Values exceeding 100% were ex-pressed as 100%. Values higher than 100% are produced whengrowth inhibition due to exposure to whole (100%) sampledsediment do not reach 50% with respect to the controls: Equa-tion (1), employed in fitting the sigmoid curves, was designedto fit curves obtained by tests where at least 50% of inhibitionis reached. Data in Table 1 were introduced in the statisticalprogram (PRIMER) and a matrix of similarity (consideringEuclidean distances between values of variables in each sam-ple) was created. From this matrix a cluster diagram was cre-ated (Fig. 4a). When a similarity matrix was created fromtoxicity data (Table 3), the resulting cluster diagram fromthis matrix was quite similar (Fig. 4b). In order to determinatethe influence of each measured variable in the dissimilarity ofsediments from samples sites in the two groups of samples,a Simper analysis was performed.

4. Discussion

4.1. Effect of particle-size distributionon microalgal growth

Mixture of clean sand with studied sediment was main-tained in order to keep conditions able to be used in future ex-periments and thus permitting comparisons. In this case, sandwas taken from an unpolluted beach and acid washed, butstandard clean sand could be acquired from commercial brandsuppliers if a standard procedure is intended to be designed.Sensitivity of benthic microalgae to particle size compositionof experimental media in batch cultures and in the experimen-tal conditions should be due to a ‘‘shadow effect’’: after

shaking the culture, fine sediment particles lay on the cells.Two of the species assayed (Cylindrotheca closterium and Na-vicula sp.) are mobile, while Phaedactylum tricornutum is not.This can explain why this latter is the most sensitive species tofine grain sediments: mobile species would spend energy inunbury from covering sediment, while non-mobile cells cannotdo this: if cells are covered with sediment, they will remain ina non-photic zone. In the case of this study, sediment particledistribution has not influence in the response of the algae. Infact, in the similarity analysis (Table 3) it can be seen that dif-ferences between the two groups of samples (more and lesstoxic sites) due to sediment particle distribution suppose lessthan 3% of total differences.

Four cellular counts in 72 h (time 0 and after every 24 h) isa standard in toxicity tests involving phytoplankton (APHA-AWWA-WPCF, 1989; ISO, 1995; OECD, 1998). Recent ex-periments (unpublished data) suggest the avoidance of the‘‘shadow effect’’ in sediment toxicity tests if cells are gentlyadded on flask with settled sediment. If a single count is per-formed at the end of the test (not daily shaken), cells remainon the sediment surface all the experimental time and no effectdue to grain size is observed. This could be an improvement ofthe test in order to avoid limitations due to grain size distribu-tion although it supposes a change respect to growth inhibitiontests for microalgae described in most of the guidelines.

4.2. Chemical analyses of sediment samples

Total PCBs and DDTs are present in low concentrations,even two orders of magnitude below levels considered as‘‘moderately polluted’’ (Manyin and Rowe, 2006). PCBs con-tribute in near 1% to the chemical differences between the twosample groups. DDTs contributed in 0.1% to the dissimilar-ities between the two groups.

Organotin compounds were not measured in this study. Tinconcentration in sediments, which could indicate the presenceof those substances, is the main factor splitting two groups ofsamples (more and less toxic samples). Tin concentration con-tributes to more than 55% to the dissimilarities between the

283I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

I

% In

hibi

tion,

Na

vic

ula

sp

.

0

20

40

60

80

100 II

IV

0

20

40

60

80

100 VIV

III

VI

I

0

20

40

60

80

100 II

IV

0

20

40

60

80

100

III

% In

hibi

tion,

C. clo

steriu

m

V

IV

100

% In

hibi

tion,

P. tric

orn

utum

0

20

40

60

80

100

I

0

20

40

60

80

100

V

0 20 40 60 80 0 20 40 60 80 100

II III

VI

0 20 40 60 80 100% of sampled sediment

Fig. 3. Growth inhibition versus percentage of sampled sediment from the six sample sites, for the three microalgal species assayed. Data have been fitted with

a sigmoid function.

284 I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

two samples group. The presence of this element in sedimentsis probably due to its use in anti-fouling paints (and thus con-centration of Sn was expected to be higher in sample III, neara dock of outboard motor boats, as indeed it is).

Bulk chemical analyses do not provide information abouttoxicity, except in extreme cases (O’Connor and Paul, 2000).Thus, bioassays are necessary to establish pollution risk insediments. It is not easy to determine which part of the Sn,Zn, Hg Cu, Cr or any other element or substance concentration(or any combination of two or more of them) is responsible ofthe different algal growth response, as there is not a clear ideaabout bioavailability of those substances from sediments.

4.3. Toxicity tests

Sample III seems to be the most toxic (in general) to micro-phytobenthos. Importance of sediment particle distribution inmudflats (under and above the silt limit, 0.63 mm) has been re-cently pointed out by Riethmuller et al. (2000). Re-suspensionof cells and sediment particles in mudflats due to wind is animportant item to be taken into account when understandinginteractions between microphytobenthos and physical mediain natural scenarios (de Brower et al., 2000). These consider-ations should be taken into account in order to find optimal

Table 2

EC50%b 72 h (sediment percentage, mixed with clean experimental sand, that

implies a growth inhibitiondreferred biomass, measured as cellular densityd

of 50% with respect to the controls) obtained from the sediment toxicity tests

involving three benthic diatom species

Algal species Sample sites

I II III IV V VI

Phaeodactylum tricornutum 100 100 27 51 62 100

Cylindrotheca closterium 92 90 62 31 61 100

Navicula sp. 100 100 64 71 65 100

Fig. 4. Cluster diagrams expressing (a) the distance of the sample sites attend-

ing their physicochemical properties and (b) the similarity attending to the

toxic response of the three microalgal species assayed.

organisms to the proposed bioassay: Among the three assayedtaxa, Navicula sp. demonstrated resistance to a wider range ofparticle-size distribution, but at the same time it is the leastsensitive of the species used. Correction of the percentage ofsilt-sized sediment has been avoided in bioassays with sedi-ments spiked in the laboratory by the use of ground cleansand (Moreno-Garrido et al., 2003a,b).

Drying sediment at 60 �C does not alter most of presentcontaminants. Loring and Rantala (1992) suggest this treat-ment in geochemical analyses of marine sediments. Neverthe-less, freeze-drying techniques can also be used in order to getsediments free of potential grazers or local flora.

Cluster analysis of the samples obtained from each sam-pling site discriminates two groups of samples: Sites I, IIand VI are different from sites III, IV and V (Fig. 4a). Theseresults agree with toxicity data obtained from sediment exper-iments, which are shown in Table 2 and Fig. 4b. Measurementof all possible potential xenobiotics in natural sediments is notpossible. But agreement between these two cluster diagrams inFig. 4 led us to suspect that xenobiotics responsible for toxic-ity in our samples are among the variables measured, or, atleast, that contamination gradient is the same for other toxi-cants. In order to determine which variables are responsiblefor the dissimilarity between the two groups of samplesdandprobably are responsible of the higher or lower toxicitydaSimper analysis was performed. This analysis provided infor-mation about similarity inside each of the defined groups (con-sidering as group one the sampling sites I, II and VI, and grouptwo the sampling sites III, IV and V), as well as informationabout the dissimilarity between the two groups. Dissimilaritybetween the two groups of samples is shown in Table 3.This table confirms that concentrations of Sn, Zn, Hg, Cuand Cr contributed to more than 90% of the dissimilarity be-tween the two groups of samples.

Tin (Sn) forms part of tributyltin (TBT). This is a biocideemployed in the anti-fouling paints. The higher concentrationof Sn in samples III, IV and V indicates contamination associ-ated with nautical activities (DelValls et al., 1998). Sample IIIcontains the highest concentration of this metal. This is not

Table 3

Simper analysis comparing the two groups of sample sites determined by sim-

ilarity matrices. Group 1 is composed of sample sites I, II and VI, while group

2 is composed of sites III, IV and V

Variable Group 1 Group 2 Dissim. Contrib. % Accumul. %

Average Average

Sn ng g�1 95.95 179.90 13.53 55.28 55.28

Zn mg g�1 11.86 37.88 4.39 17.93 73.21

Hg ng g�1 8.22 24.40 2.86 11.68 84.90

Cu mg g�1 1.28 890 1.24 5.07 89.96

Cr mg g�1 7.24 11.73 0.80 3.28 93.25

Part. size dist. 96.00 93.33 0.68 2.78 96.02

Fe mg g�1 2.99 5.36 0.39 1.60 97.63

Total PCBs ng g�1 3.36 2.16 0.26 1.05 98.67

Ni mg g�1 4.28 5.72 0.24 0.99 99.66

% C 0.22 0.55 0.06 0.23 99.90

Total DDTs ng g�1 0.12 0.26 0.02 0.10 100.00

% N 0.01 0.01 0.00 0.00 100.00

285I. Moreno-Garrido et al. / Estuarine, Coastal and Shelf Science 71 (2007) 278e286

surprising, as outboard launches moor in a dock beside thissample site. Zinc is used in treatments against corrosion. Sam-ples III, IV and V (mainly sample IV) present higher concen-trations of this metal. Although it is not considered highlytoxic (in comparison with other metals), toxicity of this ele-ment must exist, as Zn is usually associated with metallothio-neins under basal conditions (Newman and Unger, 2003).There are reports about the concentration of Hg in fishes, ma-terial collected in plankton nets and surface sediments in theinner parts of the Aveiro Lagoon (Abreu et al., 2000). Theseauthors also reported high concentrations of Hg in the upperpart of the Lagoon, due to discharges from a chlor-alkali plant.Mercury is a non-essential heavy metal which biomagnifiesand can cause damage at different levels in marine biota (Ken-nish, 1996). At the sample sites of this study, levels of Hg arenot so high when compared with the levels reported by Abreuet al. (2000). It is possible that high salinity of the zones stud-ied implies an early precipitation of this metal to the sedimentin parts of the lagoon far away from the mouth. Cu is known tobe a very toxic metal to phytoplankton, as it affects photosyn-thesis (Moreno-Garrido et al., 1999). It is also used in anti-fouling paints (Newman and Unger, 2003), and this explainsthe high level in sample III, for the same reason that explainsthe high level of Sn. Chromium is known to be carcinogenic toanimals (Shaw and Chadwick, 1998), and is used in alloys,pigments and wood preservatives (Newman and Unger, 2003).

Anthropogenic inputs of heavy metals in the Aveiro Lagoonhave been increased with the growth of the population sur-rounding the Lagoon (nearly 300,000 inhabitants in 1997)(Dias et al., 1999). In spite of this, quite high levels of heavymetals in sediments from Cadiz Bay (SW Spain) (23.4 mgSn kg�1; 107 mg Zn kg�1; 0.14 mg Hg kg�1; 49.4 mgCu kg�1; 64.1 mg Sn kg�1) were considered as ‘‘low toxic’’in a sediment toxicity test using juvenile stages of the fish Spa-rus aurata (DelValls et al., 1998). Sediment toxicity bioassaysinvolving microphytobenthos could be more sensitive thanthose designed for other organisms. In consequence, thistype of bioassay should be taken into account when environ-mental risk limit concentrations are intended to be calculatedfor xenobiotics in sediments.

The study of sediment toxicity in estuarine and coastal la-goon environments is important, as benthos in these transitionzones receives a great part of the diluted xenobiotics from thefreshwater effluents. Sediments in these zones act as reservoirsand future sources of potential toxicants. Sediment toxicitytests involving benthic microalgae are cheap, quick and easy.In spite of all this (adding the sensitivity), there are no guide-lines on similar tests provided by any environmental protect-ing organizations. Literature on these types of tests is veryscarce and recent (Moreno-Garrido et al., 2003a,b, 2006;Adams and Stauber, 2004), and do not permit clear compari-sons of results with other marine microalgal bioassays becausethere is a lack of standardization. Thus, serious efforts shouldbe made by researchers and protecting organizations in orderto standardize methods and develop toxicity testing involvingmicrophytobenthos, due to the advantages that this key biolog-ical group present in this unexplored field of research.

5. Conclusion

Microphytobenthic diatoms are suitable organisms to de-velop sediment toxicity tests. Particle-size distribution mustbe taken into account when a bioassay is designed. In thisstudy, there are two different types of samples (more andless toxic) attending to the sensitivity of the three microalgalspecies used in the described bioassay. Chemical differencebetween the two types of samples is mainly due to concentra-tions of five trace metals: Sn, Zn, Hg, Cu and Cr (in order ofimportance). Among the microalgal species used, Cylindro-theca closterium seems to be a little more sensitive, althoughpopulations of all assayed species show comparable pattern re-sponses when exposed to the different sampled sediments.

Acknowledgment

Thanks must be given to Dr. Clemente Moreno-Garrido forthe design of the sigmoid arctangent-based equation. Thanksmust be also given to Dr. Francisco Baldo Martınez for hisunvaluable help in the statistical treatment of the data. Thiswork has been granted by the Spanish National Plan of Scien-tific and Technique Investigation, project ‘‘A new ecologicallyrelevant tool in environmental toxicology: microphytobenthosfor the environmental quality assessment of estuarine andcoastal sediments (MECASEC)’’ (CTM2006-01473/MAR).

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