Integrative assessment of marine pollution in Galician estuaries using sediment chemistry, mussel...

Chemosphere 52 (2003) 1209–1224

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Integrative assessment of marine pollution in Galicianestuaries using sediment chemistry, mussel

bioaccumulation, and embryo-larval toxicity bioassays

R. Beiras a,*, N. Fern�aandez a, J. Bellas a, V. Besada b,A. Gonz�aalez-Quijano b, T. Nunes b

a Departamento de Ecolox�ııa e Biolox�ııa Animal, Universidade de Vigo, E-36200 Vigo, Galicia, Spainb Instituto Espa~nnol de Oceanograf�ııa (IEO), Cabo Estay, Canido, E-36200 Vigo, Galicia, Spain

Received 13 March 2002; received in revised form 10 January 2003; accepted 28 March 2003

Abstract

An integrative assessment of environmental quality was carried out in selected sites along the Galician coast (NW

Iberian Peninsula) combining analytical chemistry of seawater and sediments, bioaccumulation in the marine mussel,

and embryo-larval sediment toxicity bioassays, in order to link biological and chemical criteria for the assessment of

coastal pollution. Maximum values of Hg and Cu in seawater, sediment and mussels, were found in the inner part of

Ria of Pontevedra, while maximum levels of organics (polychlorinated biphenyls, hexachlorobenzene and aldrin) were

found in mussels from A Coru~nna. Outstanding values of Cu, Pb and Zn have been found in seawater and sediment from

a single site, P3, which also was the most toxic in the embryo-larval bioassays performed with four different phyla of

marine organisms: mollusks, echinoderms, arthropods and chordates. Sediment quality effects range-median values

provided a valuable reference to predict biological effects from sediment chemistry data, while effects range-low values

were too conservative. Sediment toxicity could also be predicted by using a toxic-unit model based on published EC50

values for trace metals and mobilization factors independently obtained from measurements of metal contents in

sediments and their elutriates. When chemical and toxicological data are independently used to arrange sampling sites

by using non-metric multidimensional scaling, a remarkable degree of concordance between both types of configura-

tions could be observed.

� 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Paracentrotus lividus; Mytilus galloprovincialis; Ciona intestinalis; Trace metals; Chlorinated organics; Sediment toxicity

bioassays

1. Introduction

Marine sediments and mussels are commonly used as

environmental matrices in chemical monitoring pro-

grams because they accumulate persistent pollutants at

concentrations orders of magnitude above those in the

*Corresponding author. Tel.: +34-986-812648; fax: +34-986-

812556.

E-mail address: [email protected] (R. Beiras).

0045-6535/03/$ - see front matter � 2003 Elsevier Science Ltd. All ri

doi:10.1016/S0045-6535(03)00364-3

water (e.g. OSPAR Commission, 2000). Mussels have

been selected as indicator organisms world wide because

of their abundance, ubiquity, sessile nature, long life

span, high filtration rates, and especially, because of

their high bioconcentration factor for most pollutants of

concern in the sea (Whitfield, 2001). Biological assays

are currently used as a rapid and cost-effective screening

tool to detect polluted sites where thorough analytical

chemistry may be subsequently performed. In addi-

tion, bioassays respond to the bioavailable fraction

of the toxicants, thus providing ecologically relevant

ghts reserved.

mail to: [email protected]

Fig. 1. Map of the sampling sites in the Galician Rias, and

location of the Rias in the Atlantic coast of Europe (insert).

1210 R. Beiras et al. / Chemosphere 52 (2003) 1209–1224

information. Because of their high sensitivity, early life

stages of marine invertebrates are particularly suitable

for marine environmental bioassays. First reported by

Wilson (1951), sea-urchin embryo development may be

impaired by waterborne toxicants, indicating a poor

biological quality of that water sample. This concept is

the basis of later assessment of coastal environmental

quality using sea-urchin (e.g. Kobayashi, 1971, 1991;

Bougis et al., 1979; Vashchenko and Zhadan, 1993; Carr

et al., 1996; Beiras et al., 2001) and bivalve (e.g. Woelke,

1961; Cardwell et al., 1977; Thain, 1992; His and Beiras,

1995) embryogenesis and larval development (for a re-

view see His et al., 2000). Recently, tunicates were also

proposed in order to detect toxicants which may act

selectively on chordates (Bellas et al., 2001).

In order to extract meaningful information from the

large and heterogeneous bulk of data generated by the

chemical analyses and toxicological assays, multivariate

statistical methods are currently employed (e.g. Shin and

Fong, 1999; Nascimento et al., 2001). Among the mul-

tivariate techniques, non-parametric tests are particu-

larly suitable to deal with non-linear responses, such as

those recorded in toxicological assays, and with highly

variable variables that contain only noise, which may be

the case for the levels of certain chemicals. Non-metric

methods do not rely on data transformation or unwar-

ranted assumptions on their theoretical distribution, and

their use have been recommended within the field of

ecotoxicology (Landis et al., 1997).

The goal of the present study was to assess coastal

marine pollution in the main Galician estuaries by

combining chemical and toxicological data in order to

compare and integrate both approaches. Toxicity bio-

assays provide ecologically meaningful information on

the threat posed by pollution, while analytical chemistry

contributes to the interpretation and explanation of

toxicity patterns. The information acquired will allow

ordination of the sampling stations by using non-metric

multidimensional scaling (MDS) and classification into

clusters based on similarity of both chemical and toxi-

cological data.

2. Materials and methods

2.1. Field sampling, environmental parameters and ana-

lytical chemistry

2.1.1. Sampling sites

On spring 1997 and 1998 we sampled three sites in

each of the three major Galician Rias (NW Iberian

Peninsula): Ria of Vigo (V 1–3), Ria of Pontevedra (P 1–

3), and Ria of Arousa (A 1–3). The sites (Fig. 1) were

chosen at the inner part of the Southern coast of each

estuary, representing some of the most polluted places in

the Rias according to previous data, and the time rep-

resented the seasonal maxima of pollutants in mussels

for this area (Fumega et al., 1984). On 1998 we sampled

in addition two sites at a fourth estuary, Ria of O Burgo

(B 1–2), in the vicinity of a major Galician city (A

Coru~nna). Sampling dates and locations are shown in

Table 1.

2.1.2. Water samples

Triplicate samples of 250 ml were taken in poly-

propylene flasks and stored at )20 �C for analysis of

phosphate. These flasks were previously washed with

diluted HCl and bleach, and rinsed with distilled water.

Triplicate pairs of samples of 140 ml were taken in dark

bottles for the measurement of biological oxygen de-

mand (BOD5) from the oxygen consumption after 5 d

incubation at 20 �C. These bottles were previously wa-

shed with diluted nitric acid. Dissolved oxygen was

measured by the Winkler method (APHA-AWWA-

WPCF, 1992) The detection of the titration endpoint

was visual in 1997 and potentiometric, by using a 721

NET Titrino (Metrohm), in 1998. Triplicate samples of

1 l were taken in polypropylene flasks, acidified in situ

with 1 ml/l of ultrapure nitric acid, and stored at room

temperature for analysis of Cd, Cu, Pb and Zn. These

Table 1

Locations and dates of sampling

Estuary Site Location (N–W) Sampling dates

Ria of Vigo V1 42�1702100–8�3702800

08/04/97 26/03/98

V2 42�1701200–8�3705900

V3 42�1702500–8�3802500

Ria of Pontevedra P1 42�2505700–8�3805400

08/05/97 27/03/98

P2 42�2402900–8�4005700

P3 42�2402500–8�4101700

Ria of Arousa A1 42�3802500–8�4503700

22/05/97 31/03/98

A2 42�3703000–8�4601000

A3 42�370000–8�4602900

Ria of O Burgo B1 43�1805900–8�2103000

– 30/03/98

B2 43�1904800–8�2202000

B sites were sampled in 1998 only.

R. Beiras et al. / Chemosphere 52 (2003) 1209–1224 1211

flasks were previously washed with ultrapure acids.

Surface water temperature, salinity, pH and dissolved

oxygen were recorded in situ by means of Orion and

Hanna electrodes.

2.1.3. Sediment samples

The surface layer (2 cm) of the intertidal sediments

was collected with wooden palettes at low tide, placed

into sealed polyethylene bags, carried to the laboratory

on ice and stored at 4 �C in the dark. Samples intended

for chemical analyses were stored freeze-dried in glass

flasks. Redox potential at 1, 3 and 5 cm depth was re-

corded in situ by means of a Crison electrode. Organic

matter content was measured from triplicate sediment

samples by drying at 80 �C until constant weight, and

incineration in furnace at 450 �C for 24 h. Salt weight

was discounted from the sediment dry and ash weight.

Mean particle diameter was calculated from the dry

weight of the fractions after sieving at 4, 2, 1, 0.5, 0.25,

0.125 and 0.063 mm (Restch, AS2000 model).

2.1.4. Mussel samples

Mussels (Mytilus galloprovincialis) of approximately

4 cm long were sampled from each site (with a single

exception, P1, where the fresh water prevented the oc-

currence of this species), placed into sealed polyethylene

bags and stored at )20 �C.

2.1.5. Analysis of metals in seawater

Heavy metal solutions were prepared from 1 g/l (in

0.5 N HNO3) Atomic Absorption Standards. For Cu,

Zn, Cd and Pb voltammetric measurements were carried

out by means of a GSTAT10 (Eco-Chemie B.V.) po-

tentiostat coupled to a VA Stand 663 (Metrohm). A

hanging mercury drop electrode of 0.33 mm2 of area was

used as working electrode, and potentials were measured

versus a saturated calomel reference electrode. The

voltammetric procedure consisted of anodic stripping

voltammetry (ASV). A differential pulse scanning mode

was chosen with a scan speed of 10 mV s�1 and a su-

perimposed pulse amplitude of 50 mV. Purge time,

stirring, deposition time, and other typical variables

were controlled with a General Purpose Electrochemical

System version 4.2 software (Eco Chemie B.V.). Nitro-

gen (N50) was used to deaerate solutions. The electro-

chemical cell was constructed of borosilicate and had

been previously siliconised. Before each analysis, the cell

was thoroughly washed with a sulfonitric solution con-

taining potassium dichromate to saturation. Afterwards

it was fully flushed with plenty of Milli-Q50 (Millipore,

Waters) purified water. Natural water samples were fil-

tered through a 0.45 lm cellulose acetate filter. Hydro-

gen peroxide 30% (Analysis grade), and nitric acid 60%

(Analysis grade) were used for digestion of organic

matter. An in-line UV photochemical digestion of

samples was achieved by means of a low-pressure 14 W

mercury lamp that axially irradiated a 15 ml quartz coil,

through which the sample flowed, forced by a peristaltic

pump. Manipulation of samples, filtration procedures,

reagent additions and cell solution preparation were

carried under laminar flow of air in a hood (Crumair)

provided with a pre-filter and an ULPA 0.2 mm absolute

final filter.

2.1.6. Analysis of metals in sediment and mussels

The digestion of total sediment (fraction <2 mm;

ICES, 2000) were digested with a mixture of HNO3–

HCl–HF in teflon digestion bombs by means of a mic-

rowave oven. The excess of HF was neutralized with

HBO3. The Cu, Zn, Ni, Fe and Li were determined by

atomic absorption spectrometry (Perkin–Elmer Zeeman

5000 with HGA 400) with air–acetylene flame and Pb,

Cd, As and Cr in graphite furnace with Zeeman back-

ground correction (Besada and Schultze, 1999). Mussel

samples were digested with HNO3 in teflon bombs and

analyzed by atomic absorption spectrometry with the

same equipment as for sediments. Mercury was also

analyzed in the seawater, sediment and mussels; the


methods and results were reported elsewhere (Beiras

et al., 2002), and those data will be used here only for the

correlations with other pollutants and multivariate

analysis.

2.1.7. Analysis of organics in sediments and mussels

Analytical methods for individual polychlorinated

biphenyls (PCBs), hexachlorobenzene (HCB), aldrin

and 4,40DDE followed for biota De Boer (1988) and

Gonz�aalez-Quijano and Fumega (1996), and for sedi-

ments (fraction <2 mm) Smedes and De Boer (1997),

and Nunes et al. (1997). Samples were extracted in a

Soxhlet apparatus with a mixture pentane–dichloro-

methane (1:1 vol.). The extracts were purified in an alu-

mina column eluted with pentane, and fractionated in a

silica gel column sequentially eluted with iso-octane

and iso-octane/diethylether. In addition, sulfur was

previously eliminated from sediment samples by adding

an aqueous solution of sodium sulfite and tetrabutyl-

ammonium (Jensen et al., 1977; Nylund et al., 1992).

The purified extracts were analyzed by gas chromato-

graphy with electron capture detector (Perkin–Elmer

PE 8500 and Autosystem) using capillary columns (50

m, 0.22 mm i.d. and 0.33 lm film) and H2 as carrier

gas. The following seven individual PCBs were ana-

lyzed: IUPAC numbers 28, 52, 101, 118, 138, 153 and

180, considered as priority marine pollutants by inter-

national agencies.

2.1.8. Quality control

The quality of the chemical analyses at IEO-Vigo was

controlled at two levels: (i) Internally by using certified

reference materials along with each series of samples.

For sediments, HS-1, BCSS-1 and BEST-1 reference

materials were obtained from the National Research

Council of Canada. For mussels, the reference materials

CRM-278, CRM-349 and CRM-350 were obtained

from the BCR (Boureau Communautaire de Reference,

EU). (ii) Externally, by participation in intercalibration

exercises promoted by international institutions, namely

the International Atomic Energy Agency (IAEA), (Co-

query et al., 2000; Villeneuve and de Mora, 2000), the

QUASIMEME programme (Quality Assurance of In-

formation for Marine Environmental Monitoring in

Europe) from 1993 to 1996 (Wells, 1996), QUASI-

MEME Laboratory Performance Studies from 1996 to

date (QUASIMEME, 2001), and QUASH (Quality

Assurance of Sample Handling) from 1997 to 2000

(Smedes et al., 2000). IEO-Vigo was Reference Labo-

ratory for the metal analyses in sediments within

QUASH.

2.2. Embryo-larval bioassays

The toxicity of the sediments from all sampling sites

was assessed in 1997 and 1998 by means of sediment

elutriate bioassays with embryos of the echinoderm

Paracentrotus lividus. Additional bioassays with selected

samples were performed using early developmental

stages of the bivalve M. galloprovincialis, the crustacean

Palaemon serratus, and the chordate Ciona intestinalis.

These species are hereafter referred to as sea-urchin,

mussel, prawn, and ascidian, respectively.

Elutriates were obtained according to methods pre-

viously described (Beiras and His, 1995; Beiras, 2001),

consisting of magnetic stirring (100 g sediment in 400 ml

seawater) for 30 min and decantation for 12 h approxi-

mately, at 20 �C. Long decantation times allowed testing

unfiltered elutriates. Elutriates from each sediment

sample were tested undiluted (1:4 sediment:water w:w),

and after dilution to 1:16 and 1:40 sediment:water (w:w).

Water for dilutions and blanks was 0.22 lm-filtered

oceanic seawater in 1997 and artificial seawater (His

et al., 1997) in 1998. Temperature, salinity, pH and

dissolved oxygen in the elutriates was recorded prior to

the bioassays. When the presence of hydrogen sulfide in

the elutriates was suspected, its concentration was also

measured by means of a commercial colorimetric test

(Merk, Aquaquant), after 5 times dilution of elutriate

samples in distilled water.

Sea-urchin bioassays were based on the methods

described by Fern�aandez and Beiras (2001). Gametes

were obtained by dissecting mature individuals collected

from natural populations inhabiting the outer part of

the Ria of Vigo. Gamete quality (round eggs and motile

sperm) was checked under the microscope and one fe-

male and one male showing optimal condition were se-

lected. Eggs from the selected female were transferred

into a 100 ml measuring cylinder containing 0.2 lm fil-

tered seawater, a few ll of undiluted sperm from the

selected male were added and the contents of the cylin-

der were gently stirred with a plunger for 2 min ap-

proximately to allow fertilization. Four 50-ll samples

were then taken by pipette and observed under the mi-

croscope in order to record the number of eggs and

percentage of fertilization. Fertilization success (indi-

cated by the presence of a fertilization membrane) was

generally higher than 98% in all the batches used for the

bioassays. Within 30 min after fertilization volumes

corresponding to 600 fertilized eggs were transferred

into 20 ml vials containing the elutriates. Five replicates

of each experimental treatment, including the blank,

were used (except Pontevedra 1997, where n ¼ 3). The

vials were incubated for 48 h at 20 �C, fixed by adding

two drops of 40% formalin, and directly observed under

an inverted microscope. Embryogenesis success, evalu-

ated as percentage of 4-arm pluteus larvae, and larval

length were recorded.

Mussel bioassays followed His et al. (1997), and

consisted of incubating 30 min-old fertilized eggs (25 per

ml) in the experimental solutions at 20 �C for 48 h. The

biological response recorded was percentage of D-larvae

Fig. 2. Embryogenesis success for sea-urchin exposed to elutriates from sediments sampled in Vigo (a), Pontevedra (b), Arousa (c) and

O Burgo (d) in 1998. Symbols indicate sites 1 (diamonds), 2 (squares) and 3 (triangles).


with normal shell. This excludes shell abnormalities ((b),

(c) and (d) in Fig. 2, His et al., 1997) but not protruding

mantle ((e) in Fig. 1 op. cit.). Ascidian bioassays fol-

lowed Bellas et al. (2001); 1 h-old fertilized embryos (2

cell stage) were incubated at 20 �C and a density of 15

per ml for 24 h, and percentage of normal tadpole larvae

was recorded. Prawn bioassays followed Mari~nno-Balsa

et al. (2000); newly hatched larvae (zoea I) were incu-

bated at 18 �C and a density of 10 individuals in 25 ml

for 72 h, and mortality (assessed by lack of motility) was

recorded.

2.3. Statistical analysis

Statistical analyses of toxicity data were performed

according to Newman (1995). Percentages of positive

response (embryogenesis success or survival) in each

vial (P ) were corrected by the mean response in

the control vials (Pc) according to Abbot�s formula,

which for positive responses takes the form: P 0 ¼100P=Pc.

For statistical analysis, the P 0 values were trans-

formed to the arcsine of the square root, and the values

were back-transformed for presentation.

Correlation analysis was performed according to Zar

(1984), and MDS followed Kruskal and Wish (1978).

All tests were performed using SPSS statistical soft-

ware.

3. Results

3.1. Environmental parameters

A profile of the environmental characteristics of the

sampling sites is reflected in Table 2. Despite high tem-

poral and microgeographical variability, some regula-

rities can be identified. The P1 and V2 sites are at

sampling time (low tide) under the influence of fresh

water inputs, while all other points show salinity higher

than 20 ppt. This is also the reason why no mussels are

present at P1. Seawater may be considered to be pol-

luted by organic matter when BOD5 > 4 mg/l. P1 and

particularly P2 and P3 exceeded this threshold. P1 and

P2 were also the sites with highest phosphate content, an

indicator of urban wastewater input (see Table 2).

Concerning the sediment, V2, B2 and particularly P3

where sandy sites, while all other samples consisted

mainly of silt. Consistently, P3 site showed positive

redox potential values, in contrast with all other sam-

ples, composed by finer sediment. The P1 site showed

the highest values of organic content and, along with V3

and P2, the lowest redox potentials.

3.2. Analytical chemistry

Table 3 summarizes the results from the chemical

analyses performed with the water, sediment and biota

Table 2

Physical and chemical parameters recorded in the water and sediment samples taken from the Galician Rias, and in the elutriates obtained from these sediments, in 1997 (first value)

and 1998 (second value)

Water Sediment Elutriate

T(�C)

S(ppt)

pH O2

(mg/l)

BOD5

(mg/l)

PO4

(lM)

Eh (mV) OM

(%)

63 lm(%)

T(�C)

S(ppt)

pH O2

(mg/l)

H2S

(mg/l)1 cm 3 cm 5 cm

V1 16.2 n.m. 8.0 8.7 1.01� 0.636 1.59 )83 )194 )311 3.3� 0.1 57.3 21.4 n.m. 7.0 4.0 n.m.

15.0 34.6 7.8 7.7 3.02� 0.867 1.10 )156 )212 )269 7.6� 0.60 46.4 22.1 32.5 7.2 6.2 <0.1

V2 15.7 n.m. 7.2 7.8 0.25� 0.233 1.09 )182 )313 )358 9.1� 1.4 12.2 21.2 n.m. 7.2 4.3 n.m.

15.5 6.3 7.7 9.7 2.61� 2.12 0.76 )70 )105 )167 4.59� 0.134 23.3 22.1 31.5 7.3 6.6 n.m.

V3 16.3 n.m. 7.3 8.4 0.35� 0.262 1.05 )278 )346 )356 2.23� 0.25 54.5 21.3 n.m. 6.7 3.6 n.m.

17.0 n.m. 7.7 8.7 0.65� 0.177 1.14 )249 )286 )311 5.31� 0.229 31.6 22.1 36.6 7.5 6.5 n.m.

P1 13.3 n.m. 6.8 8.7 3.29� 0.057 6.21 )352 )376 )393 12.2� 0.2 48.6 20.0 n.m. 7.1 0.0 2–2.5

13.8 17.0 7.1 6.4 4.57� 1.807 2.90 )215 )243 )278 11.6� 0.35 39.8 22.2 31.6 6.5 5.6 <0.1

P2 17.3 n.m. 7.3 5.4 9.50a 5.30 )240 )318 )347 4.9� 0.5 19.2 20.1 n.m. 7.4 1.5 <0.1

15.4 33.0 7.6 9.3 6.99� 0.194 7.55 )303 )329 )324 8.4� 0.34 24.7 22.2 32.3 7.2 1.1 <0.1

P3 17.1 n.m. 7.6 10.4 2.48� 0.340 1.97 109 98 86 2.6� 0.03 7.6 20.0 n.m. 7.7 4.5 <0.1

15.9 36.5 7.5 14.7 7.74� 3.682 0.90 134 137 78 0.69� 0.079 2.3 22.3 32.5 7.7 7.7 n.m.

A1 15.6 n.m. 8.2 12.0 2.07� 0.750 0.51 0.3 )136 )211 1.20� 0.191 15.9 21.8 n.m. 7.4 3.7 <0.1

16.1 24.8 8.5 3.00� 0.374 1.41 )131 )226 )325 7.73� 0.230 24.2 21.2 32.4 7.3 3.9 n.m.

A2 19.9 n.m. 8.1 12.0 3.41� 0.919 1.49 )295 )307 )325 2.10� 0.038 52.2 21.7 n.m. 7.0 3.8 <0.1

16.9 32.5 7.7 9.3 2.39� 0.572 1.02 )120 )220 )303 8.4� 0.37 65.9 21.4 31.9 7.8 4.1 n.m.

A3 19.5 n.m. 7.8 8.8 1.78� 0.113 0.74 )340 )362 )368 2.67� 0.27 29.1 21.8 n.m. 7.3 3.0 <0.1

15.2 31.9 7.4 7.7 1.82� 0.461 0.85 )92 )241 )334 9.29� 0.272 23.2 21.5 32.6 7.5 3.8 n.m.

B1 13.3 21.5 7.4 7.5 1.92� 0.382 1.60 )194 )227 )278 5.81� 0.155 63.2 20.9 32.4 7.7 5.1 n.m.

B2 16.7 30.7 7.5 10.2 2.63� 0.293 2.82 )67 )126 )279 2.67� 0.130 13.5 20.9 33.2 7.7 5.3 n.m.

B sites were sampled in 1998 only. T : temperature; S: salinity; O2: dissolved oxygen; BOD5: biological oxygen demand after 5 days (mean� s.d., n ¼ 3); PO4: phosphate (mean� s.d.,

n ¼ 3); Eh: redox potential at three depths (mean of three measurements per depth); OM: organic matter (mean� s.d., n ¼ 3). Annual maxima for BOD5 and PO4 are marked in

bold.aOriginal samples not measurable, extra sample taken on 3/10/97, n ¼ 1.

1214

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52

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1209–1224

Table 3

Concentration of pollutants in the environmental samples taken from the Galician Rias in 1997 (first value) and 1998 (second value)

Water Sediment Mussels

Metals (nM) Metals (mg/kg dw) (lg/kg dw) Metals (mg/kg dw) Organics (lg/kg ww)

Cu Pb Cd Zn Cu Pb Cd ZnP

7PCB Cu Pb Cd ZnP

7PCB DDE HCB Aldrin

V1 20 25 0.24 60 43.1 73.5 0.20 164.5 4.2 7.15 6.39 0.478 249 6.1 0.53 <0.05 0.13

43.4 93.7 0.27 177.6 7.0 7.76 5.24 0.625 234 7.5 0.58 <0.05 0.14

V2 14 13 0.29 104 24.9 48.3 0.09 81.4 2.2 6.45 4.44 0.427 231 6.2 0.48 <0.05 0.16

28.1 53.1 0.07 136.4 4.5 6.92 4.55 0.831 284 7.2 0.59 <0.05 0.12

V3 27 36 0.20 105 236.1 173.7 0.35 196.9 16.2 8.74 8.79 0.547 291 8.3 0.50 <0.05 0.15

76.1 76.0 0.27 175.2 28.9 9.02 7.61 0.809 277 9.1 0.70 <0.05

P1 38 9 0.14 95 51.0 83.8 0.26 182.3 9.8 – – – – – – – –

48.4 75.0 0.31 197.7 18.6 – – – – – – – –

P2 1306 37 0.62 455 91.8 106.6 0.20 219.2 17.5 12.82 4.31 0.300 177 10 1.57 nd nd

206.0 99.3 0.24 393.8 31.9 8.39 2.72 0.400 218 9.8 1.39 <0.05 0.16

P3 3010 215 1.01 4819 3887 1423 3.45 17 609 155 27.43 11.68 0.487 362 14 3.07 nd nd

4282 2302 9.57 44 957 132 25.38 8.93 0.614 453 13 2.20 <0.05 0.20

A1 45 9 0.31 74 15.4 30.8 0.09 57.9 0.8 10.76 1.59 0.992 233 4.6 0.99 <0.05 0.28

32.3 32.1 0.13 82.0 1.4 11.25 1.73 0.814 171 5.3 0.87 <0.05 0.21

A2 28 11 0.28 51 51.3 47.8 0.39 136.5 2.3 9.89 2.99 0.891 312 2.6 0.74 <0.05 0.19

78.6 48.8 0.55 180.5 4.4 11.41 3.24 1.045 316 3.4 1.02 <0.05 0.17

A3 27 12 0.21 36 89.9 128.6 0.35 178.4 16.4 11.34 4.78 0.500 259 4.8 1.15 <0.05 0.25

90.9 143.1 0.40 217.7 12.9 12.53 5.00 0.701 304 5.4 0.89 <0.05 0.18

B1 – – – – – – – – – – – – – – – – –

95 28 1.74 992 201.0 161.4 2.00 1053.7 46.2 14.31 17.49 0.617 467 33 1.70 0.071 0.27

B2 – – – – – – – – – – – – – – – – –

75 32 0.51 479 15.1 40.4 0.09 88.8 27.1 11.55 8.24 0.584 447 21 0.95 <0.05 0.24

C 18 26 0.15 82

B sites were sampled in 1998 only. Annual maxima are marked in bold. dw: dry weight; ww: wet weight.P

7PCBs: sum of seven individual congeners (see text).

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collected at the different sites two consecutive years. In

1997, P3 site showed maximum values for all pollutants

in all environmental matrices analyzed except for cad-

mium in the mussels (maximum in Ria of Arousa,

probably due to upwelling conditions) and Hg in sedi-

ment and mussels (maximum in P2 nearby). In 1998 two

additional sites were included in the monitoring. For

sediments P3 showed again the highest values, but B1

replaced P3 in maximum contents for Pb, Zn and or-

ganics in the mussels. Comparing the concentrations

showed in Table 2 with the levels usual in the coastal

environment (see Section 4), two groups of sampling

sites could be identified. The P3, P2, B1, and V3 sites can

be considered as chemically polluted, whereas the re-

maining sites showed in general low concentrations of

pollutants. The concentrations of Cu, Pb, Cd, Zn and

PCBs in sediments from P3 are particularly remarkable.

Logarithmic transformation normalized most toxi-

cant concentration data, except for Cu, Pb, Cd and Zn

in sediments, and Cu and Zn in water, due to the ex-

tremely high values at site P3. Non-metric correlation

(Spearman) was additionally performed when these data

were involved. According to the correlations (Table 4),

three distinct groups of pollutants could be identified: (i)

metals other than Hg, (ii) Hg and PCBs, (iii) orga-

nochlorine pesticides.

Comparing matrices, the contents of Hg, Cu, Pb, and

PCBs showed significant correlations between mussels

and sediment, and mussels and seawater (see Table 4).

The correlations between sediment and seawater though

were generally poor. For these pollutants, mussels seem

a suitable monitoring species. In contrast Cd showed

very low or even negative correlations between different

matrices.

3.3. Embryo-larval bioassays

Fig. 2 shows the embryogenesis success in sea-urchin

fertilized eggs exposed to sediment elutriates from the

sites sampled in 1998. The results of the sea-urchin

bioassays conducted with the 1997 sediments (not

shown) were fully consistent and showed the same pat-

tern. All sites from the Rias of Vigo (Fig. 2(a)), Arousa

(Fig. 2(c)), and O Burgo (Fig. 2(d)) showed no toxicity

to sea-urchin embryos. In contrast, P1 from Ria of

Pontevedra (Fig. 2(b), diamonds) showed moderate

toxicity, and P3 (triangles) showed extreme toxicity,

completely inhibiting embryogenesis even after 10 times

dilution in control seawater. Fig. 3 shows the same data

obtained in 1998 with mussels. Again sediment elutriates

from Ria of Vigo showed no embryotoxicity, while

elutriates from site P1 were moderately toxic and those

from P3 extremely toxic. For this species P2, A1 and A3

also showed moderate toxicity, inhibiting embryogenesis

when undiluted. Fig. 4 illustrates the data obtained for

ascidian bioasays in 1998. In full agreement with sea-

urchin data, sediment elutriates from all sites of Vigo,

Arousa and O Burgo were non-toxic, while sites from

Pontevedra were toxic. Again P3 showed higher toxicity

than the other sites of this Ria, although in this case the

highest dilution of the elutriate did suppress toxicity.

The toxicity of the sediment elutriates from the Pont-

evedra sites was also tested on prawn zoea larvae and

the data obtained are reflected in Fig. 5. Although these

larvae showed lower sensitivity than sea-urchin, mussel

and ascidian embryos, the pattern of response was

similar, with P3 being the most toxic site, and in 1997

elutriates from P1 and P2 showing toxicity when undi-

luted.

4. Discussion

Despite the high socioeconomical value of the Gali-

cian Rias derived from extensive aquaculture and coastal

fisheries, comprehensive studies concerning chemical

pollution in these ecosystems are scarce. Concerning

seawater, only Guerrero P�eerez et al. (1988) published

data for some metals, thus we have sampled in this study

a control site of oceanic characteristics (see C site in

Table 3). Based on these references, seawater from sites

P3, P2, B2 and B1 (in this order) showed marked metal

pollution, while the other sites are comparable to

background levels, except for Cu in A1 and P1.

Trace metal pollution in coastal sediments from

Galician Rias is more documented (Barreiro et al., 1988;

Nombela et al., 1994; Rubio et al., 1995, 1996, 2000;

Belzunce-Segarra et al., 1997a,b; Besada et al., 1997b,

1999; Carballeira et al., 1997). Despite the method-

ological variability in sample fractionation, digestion

and analysis, a consistent overall picture emerges from

the inspection of the data available for the geographical

area covered in the present study. Metal contents are

generally close to background levels except for localized

areas. High levels of Pb have been measured in the inner

part of Ria of Vigo, Cu in particular locations of the Ria

of Pontevedra, and Zn in O Burgo. There is no signifi-

cant Cd pollution in the Galician r�ııas. The case of Hg

has been discussed elsewhere (Beiras et al., 2002).

Accumulation of trace metals in mussels from the

Galician coast have been reported by Besada et al.

(1997a, 2002); PROVIGO (2000, 2001, metal analyses

by Besada), and Carballeira et al. (1997). In general, the

values found here are comparable with those previously

reported by Besada and coworkers, and markedly lower

than those from Carballeira et al. (1997).

For PCB and organochlorine pesticides previous

data are fragmentary compared to the geographic range

covered in the present study, but there are remarkable

concordances. For sediments, the range of variation inP7PCBs reported by OSPAR Commission (2000) in

some Galician Rias was 1.0–37 lg/kg dw, very similar to

Table 4

Matrix of correlation coefficients among the concentrations of pollutants in the sediments (Sed.), mussels (Mus.) and seawater (Wat.), significance level, and sample size

Sed. Mus. Wat.

Hg Cu Cd Pb ZnP

7PCB Hg Cu Cd Pb ZnP

7PCB DDE HCB Aldrin Hg Cu Cd Pb Zn

Sed. Hg 0.500� 0.250 0.549� 0.556� 0.573�� 0.512� )0.004 )0.482� 0.478� 0.088 0.478� 0.174 0.372 0.025 0.215 0.291 308 0.246 0.300

0.025 0.287 0.012 0.011 0.008 0.030 0.988 0.043 0.045 0.727 0.045 0.490 0.156 0.929 0.406 0.385 0.357 0.465 0.370

20 20 20 20 20 18 18 18 18 18 18 18 16 15 17 11 11 11 11

Cu 0.793�� 0.880�� 0.545 0.680�� 0.486� 0.519� )0.342 0.441 0.329 0.401 0.558� 0.471 )0.039 0.386 0.364 0.146 0.573 0.364

0.000 0.000 0.083 0.001 0.041 0.027 0.165 0.067 0.182 0.099 0.016 0.066 0.889 0.126 0.272 0.669 0.066 0.272

20 20 11 20 18 18 18 18 18 18 18 16 15 17 11 11 11 11

Cd 0.685�� 0.737�� 0.512� 0.112 0.625�� 0.049 0.490� 0.632�� 0.150 0.514� 0.564� 0.221 0.216 0.318 0.099 0.290 0.189

0.001 0.000 0.021 0.659 0.006 0.848 0.039 0.005 0.552 0.029 0.023 0.429 0.405 0.341 0.771 0.386 0.578

20 20 20 18 18 18 18 18 18 18 16 15 17 11 11 11 11

Pb 0.655� 0.735�� 0.494� 0.445 )0.445 0.684�� 0.391 0.531� 0.420 0.218 )0.177 0.431 0.255 0.082 0.618� 0.418

0.029 0.000 0.037 0.064 0.064 0.002 0.108 0.023 0.083 0.418 0.528 0.084 0.450 0.811 0.043 0.201

11 20 18 18 18 18 18 18 18 16 15 17 11 11 11 11

Zn 0.791�� 0.536� 0.595�� )0.364 0.521� 0.418 0.529� 0.651�� 0.477 )0.041 0.393 0.545 0.269 0.655� 0.545

0.000 0.022 0.009 0.137 0.027 0.084 0.024 0.003 0.062 0.884 0.118 0.083 0.424 0.029 0.083

20 18 18 18 18 18 18 18 16 15 17 11 11 11 11

PCBs 0.694�� 0.647�� )0.429 0.769�� 0.575� 0.723�� 0.668�� 0.412 0.114 0.606�� 0.673� 0.598 0.789�� 0.709�

0.001 0.004 0.076 0.000 0.013 0.001 0.002 0.112 0.686 0.010 0.023 0.052 0.004 0.015

18 18 18 18 18 18 18 16 15 17 11 11 11 11

Mus. Hg 0.336 )0.574� 0.442 0.158 0.562� 0.471� 0.085 )0.309 0.573� 0.612 0.518 0.793�� 0.794��

0.173 0.013 0.066 0.531 0.015 0.049 0.754 0.263 0.026 0.060 0.125 0.006 0.006

18 18 18 18 18 18 16 15 15 10 10 10 10

Cu )0.041 0.373 0.507� 0.390 0.892�� 0.592� 0.611� 0.383 0.915�� 0.716� 0.718� 0.648�

0.871 0.128 0.032 0.110 0.000 0.016 0.016 0.158 0.000 0.020 0.019 0.043

18 18 18 18 18 16 15 15 10 10 10 10

Cd )0.308 0.211 )0.410 )0.222 0.027 0.198 )0.395 0.139 )0.161 )0.436 )0.1150.214 0.400 0.091 0.375 0.920 0.479 0.146 0.701 0.656 0.208 0.751

18 18 18 18 16 15 15 10 10 10 10

Pb 0.710�� 0.742�� 0.267 0.294 )0.012 0.710�� 0.273 0.599 0.680� 0.648�

0.001 0.000 0.284 0.270 0.967 0.003 0.446 0.067 0.031 0.043

18 18 18 16 15 15 10 10 10 10

Zn 0.510� 0.357 0.512� 0.303 0.623� 0.382 0.507 0.323 0.430

0.030 0.146 0.042 0.272 0.013 0.276 0.134 0.362 0.214

18 18 16 15 15 10 10 10 10

PCBs 0.465 0.465 0.242 0.814�� 0.564 0.821�� 0.591 0.903��

0.052 0.052 0.384 0.000 0.090 0.004 0.072 0.000

18 18 15 15 10 10 10 10

DDE 0.796�� 0.628� 0.477 0.830�� 0.785�� 0.601 0.479

0.000 0.012 0.072 0.003 0.007 0.066 0.162

16 15 15 10 10 10 10

HCB 0.657�� 0.348 0.667 0.820� 0.024 0.048

0.008 0.243 0.071 0.013 0.955 0.911

15 13 8 8 8 8

Aldrin 0.032 0.667 0.532 )0.293 0.119

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1209–1224

1217

Table

4(c

ontinued)

Sed

.M

us.

Wat.

Hg

Cu

Cd

Pb

Zn

P7PCB

Hg

Cu

Cd

Pb

Zn

P7PCB

DDE

HCB

Aldrin

Hg

Cu

Cd

Pb

Zn

0.918

0.071

0.175

0.481

0.779

13

88

88

Wat.

Hg

0.242

0.742��

0.749��

0.699�

0.473

0.009

0.008

0.017

11

11

11

11

Cu

0.697�

0.464

0.709�

0.017

0.151

0.015

11

11

11

Cd

0.626�

0.697�

0.039

0.017

11

11

Pb

0.745��

Zn

0.008

11

Spea

rman

correlation

coeffi

cien

tsare

shown

forco

rrelationsinvolvingnon-n

orm

allydistributed

variables,

and

Pea

rson

correlation

coeffi

cien

tsoth

erwise(see

text).

� p<

0:05;��p<

0:01;�� p

<0:001.


the range found in this study (0.8–46 lg/kg dw, except

for the highly polluted site P3). Franco et al. (1984) and

Nunes et al. (1997) reported PCB levels in sediments

from inner Ria of Vigo within the 10–40 lg/kg dw range,

comparable to the values found here in V1,2 and 3. For

wild mussels from Ria of Vigo,P

7PCBs ranged, except

for a single highly polluted site, from 2 to 15 lg/kg ww

(Gonz�aalez-Quijano et al., 1997, 1999), which agrees with

the present study (see Table 3). Our finding of maximum

PCBs accumulation in mussels close to A Coru~nna (33 lg/kg ww) is also in line with previous data (46 lg/kg ww;

OSPAR Commission, 2000). Finally, instances of PCB

and DDT pollution in certain sites of Ria of Pontevedra

(P3) had also been previously reported (Fumega et al.,

1984).

Water quality standards are intended to protect the

aquatic ecosystems vis a vis threats to their beneficial

uses. One of the main uses of water in the Galician Rias

is the shellfish culture. According to the European leg-

islation (Anon., 1979) the levels of pollutants in these

waters should not exceed those causing deleterious ef-

fects on the adult mollusks or their larvae. The LC50

values impairing bivalve embryogenesis in marine biv-

alve species have been reviewed by His et al. (2000), and

the geometric means are 40 lg/l for Cu, 320 lg/l for Zn,

968 lg/l for Pb and 2219 lg/l for Cd. Cu content in P2

and P3 seawater are above these values. Zn content in

P3 seawater is also very close to average LC50. There-

fore, current levels of Cu and Zn pollution in these

particular sites pose a threat to this important economic

activity.

The main goal of the present study was to link sedi-

ment toxicity bioassays with analytical chemistry. Sedi-

ments are preferred to water as environmental matrix

for both chemical and biological monitoring because

pollutant concentrations in sediments are much higher

and less variable in time and space (e.g. Beiras et al.,

2002). A qualitative approach will be first discussed.

Long et al. (1995) published biologically meaningful

sediment quality guidelines based on compilation of

large toxicity databases for marine organisms. The ef-

fects range-median (ERM) and effects range-low (ERL)

levels correspond to the 50 and 10 percentile of the

distribution of effective concentrations. Pollutant con-

centrations above ERM indicate that adverse effects on

the marine fauna would often occur. In Southern Spain,

DelValls and Chapman (1998) have established for some

pollutants site-specific sediment quality values on the

basis of integrative environmental quality assessment.

The comparison of the levels of pollutants found in the

Galician Rias with ERM values stress the high level of

pollution found in site P3. The contents of Cu, Pb and

Zn in P3 sediments both years, and the Zn in B1, exceed

ERM. In particular Cu content in P3 was 15 times ERM

and Zn content was 40–100 times ERM. These metals

are thus very likely responsible for the high toxicity

Fig. 3. Embryogenesis success for mussel exposed to elutriates from sediments sampled in Vigo (a), Pontevedra (b) and Arousa (c) in

1998. Symbols indicate sites 1 (diamonds), 2 (squares) and 3 (triangles).

Fig. 4. Embryogenesis success for ascidia exposed to elutriates from sediments sampled in Vigo (a), Pontevedra (b) and Arousa (c) in

1998. Symbols indicate sites 1 (diamonds), 2 (squares) and 3 (triangles).


shown by the sediment samples from this site. No other

sampling sites show levels of pollutants in the sediment

exceeding ERM. This is consistent with the toxicological

data obtained with marine invertebrate embryos and

larvae, where P3 is the only site showing high toxicity

disregarding dilution or sampling date. Therefore, this

study supports the use of ERM values when biological

effects are not directly tested and are to be predicted

from sediment chemistry. In contrast, ERL values

were too conservative, and they are exceeded in many

Fig. 5. Survival of prawn larvae exposed to elutriates from

sediments sampled in Pontevedra in 1997 (a) and 1998 (b).

Symbols indicate site 1 (diamonds), 2 (squares) and 3 (trian-

gles).


instances with no biological effects on the marine in-

vertebrate embryos associated. ERL values may still be

valuable to predict effects on infaunal organisms living

in contact with the sediment.

P1 was the only other site showing toxicity on sea-

urchin embryos, though to a much lesser extent. In 1997

this toxicity may be attributable to hydrogen sulfide, a

natural component of highly reduced sediments whose

concentration greatly increases with time of storage in

anaerobic conditions. The concentration of H2S mea-

sured in the P1 elutriate used for sea-urchin bioassay

was above 2 mg/l, whilst toxic effects were observed by

Knezovich et al. (1996) above 0.12 mg/l. However, in

1998 H2S content was negligible and the elutriates still

caused a certain degree of toxicity when undiluted. We

will further discuss this case below.

Secondly, we may try to establish a posteriori a

quantitative link between sediment toxicity and sedi-

ment chemistry. This falls beyond the aim of the ex-

perimental design of the present study, and it is

attempted here only for discussion purpose.

We have previously reported additive effects of metal

combinations on sea-urchin embryos (Fern�aandez and

Beiras, 2001). The present data allow the comparison of

the actual toxicity of the elutriates recorded in the sea-

urchin bioassays with the theoretical toxicity expected

according to an additive model for all the metals mea-

sured. With this aim, we have calculated the theoretical

toxicity units (TU) present in the elutriates attributable

to each metal analyzed. IfP

TU > 1 impaired larval

development is expected. The fraction of the metal

content of the sediment that is mobilized into the elu-

triated water has been measured independently in more

recent experiments with sediments and elutriates from

Ria of Pontevedra by using ASV (Lorenzo et al., 2002).

The average mobilization factors (lg/l in elutriate di-

vided by mg/kg DW in sediment) ranged between 10 and

150� 10�3 for Hg, Cu, Zn, Cd and Pb. Metal concen-

trations in the elutriates of the present study (½M �), es-timated using those data, were transformed into TU

following the expression;

TU ¼ ½M �=CL50M

where CL50M is the median effective concentration of

the metal on sea-urchin embryos, taken from Nacci et al.

(1986) for Zn and Fern�aandez and Beiras (2001) for the

other metals. The summation of the toxic units was 3.9

for P3 in 1997, 6.9 for P3 in 1998 and lower than 0.3 for

all other sites and sampling dates. Therefore, the cal-

culations made on the basis of an additive model with

the chemical data available predict that only P3 should

show toxicity on the sea-urchin embryos. This prediction

is thoroughly supported by the toxicological data with a

single exception: undiluted elutriates from P1 in 1998

were also toxic. The very simple additive model used

above must be refined in the future on the basis of more

comprehensive chemical and toxicological data. For

instance, it does not take into account the effects of sa-

linity on metal toxicity. As mentioned above, the P1 site

is affected by a fluvial input, and its elutriate shows

comparatively low pH and salinity (see Table 2), con-

ditions that enhance Cu and Zn toxicity. The effects of

pH and acid-volatile sulfides need also to be taken into

account. Alternatively, non-measured organic pollutants

may be responsible for the toxicity of 1998 P1 sediments.

A pleasure boats marina is located a few hundred meters

away from this site.

When multiple and heterogeneous information is

available for each site, ordination of sampling sites ac-

cording to the degree of pollution may be performed by

using multivariate techniques like MDS. This method

produces a graphical representation based on the ma-

trices of similarities of each site with every other site for

all the variables measured. When various potential

pollutants are measured in a set of sampling sites, data

are likely to include variables oscillating within very

different ranges, variables non-normally distributed, and

variables with no biological effects that only bring noise

into the model. Non-linear analyses are advocated for

these cases (Landis et al., 1997). By using non-linear

MDS, we have obtained the spatial configuration of the

sampling sites, for each sampling year independently, on

the basis of (i) the analytical chemistry data (Fig. 6(a)

and (c)), and (ii) the toxicological data obtained in the

(a) (b)

(c) (d)

Fig. 6. Spatial configuration of the sampling sites on the basis of analytical chemistry (a, c) and toxicological data (b, d) by using non-

linear MDS. (a, b) and (c, d) show the configurations for 1997 and 1998 data respectively.


embryo-larval bioassays (Fig. 6(b) and (d)). A marked

degree of concordance between both types of configu-

rations can be observed. In 1997 sampling station P3,

and to a lesser extent P2, are discriminated from the

others in both the chemistry-based configuration and

the toxicology configuration, whilst P1 is separated in

the toxicological configuration only. This may be due

to the high levels of H2S and anoxic conditions recorded

in this elutriate. In 1998 again station P3 is markedly

separated from the others according to both chemical

and toxicological data. The other stations segregated in

the toxicological configuration is P1, although in this

case O2 and H2S were within the range allowing normal

embryo development, indicating that additional chemi-

cals not measured were responsible for the toxicity

found. Further, in the configuration based on analytical

chemistry (Fig. 6(c)) the sampling stations are arranged

according to their degree of pollution, from less polluted

(A1, A2, V2) on the upper right corner to the most

polluted (B1, P2, V3) on the bottom.

The pattern of pollution described above is further

supported by the data on bioaccumulation by the wild

mussels. On the basis of the OSPAR Commission (2000)

background levels (upper limit), and considering high an


enrichment factor (measured level/background level) >5

and very high a factor >10, mussels from inner Ria of

Pontevedra showed very high levels of Hg and instances

of high Pb and PCBs, those close to A Coru~nna very high

levels of Pb and PCBs and high Hg, and mussels from

inner Ria of Vigo high levels of Pb and Hg. Mussels

from Arousa did not show high levels of any of these

pollutants.

Concerning the choice of species for bioassays, a re-

markable concordance in the patterns of response

among test species from very distant phylogenic groups

can be observed, suggesting that the toxicants mobilized

from the sediment act through non-selective universal

mechanisms. This is consistent with pointing at Cu and

Zn as presumably responsible for the toxicity observed.

However, the additional presence of very specific toxi-

cants cannot be discarded, and the use of a multispecies

battery of bioassays is still recommended, including

representatives of the main taxonomical groups of sea

resources to be protected. Along with the widely used

sea-urchins and bivalves, ascidians may cover neuro-

toxic compounds specifically toxic to chordates (Bellas

et al., 2001). A marine arthropod is also needed to detect

the presence of highly selective toxicants such as insec-

ticides. However, the use of prawn larvae assayed here

by first time showed important limitations, namely

limited biological material causing low replication, and

time-consuming experimental set-up. Prawn larvae are

also known to show low sensitivity to copper (Mari~nno-Balsa et al., 2000). Adult amphipods may be advisable,

and they are also suitable to test the toxicity of whole

sediment. Finally, a photosynthetic organism would

provide the bioassay with sensitivity against herbicides,

and it will be included in the battery of test species in the

future.

Acknowledgements

Authors are grateful with all the staff who lend a

helpful hand over these five years of intense work. J.I.

Lorenzo made the BOD and organic matter measure-

ments; J.C. Mari~nno-Balsa conducted the prawn

bioassays, A. Cobelo analyzed metals in seawater, L.

Pombar, Z. Romero, F. Schultze, B. Cambeiro, A.

Garc�ııa, I. Alves and M. Cerqueira provided technical

assistance.

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