Marine Environmental Research 48 (1999) 213±224

www.elsevier.com/locate/marenvrev

Brown algae species as biomonitors of Zn and

Cd at Sepetiba Bay, Rio de Janeiro, Brazil
G.M. Amado Filho a,*, L.R. Andrade b, C.S. Karez c,
M. Farina b, W.C. Pfei€er d
a
Programa Zona Costeira, Instituto de Pesquisas Jardim BotaÃnico do Rio de Janeiro,
Rua Pacheco LeaÄo 915, 22460-030 Rio de Janeiro, Brazil
b
Departmento de Anatomia, Instituto de CieÃncias BiomeÂdicas, CCS/UFRJ, Cidade UniversitaÂria,
21941-590, Rio de Janeiro, Brazil
c
UNESCO Oce, ROSTLAC, Montevideo 11300, Uruguay
d
LaboratoÂrio de RadioisoÂtopos, Instituto de BiofiÂsica Carlos Chagas Filho, CCS/UFRJ,
Cidade UniversitaÂria, 21949-900 Rio de Janeiro, Brazil

Received 14 September 1998; received in revised form 17 February 1999; accepted 15 March 1999

Abstract
In order to contribute to monitoring heavy metal contamination of Sepetiba Bay, Rio de
Janeiro, Brazil, long term evaluation of Zn and Cd concentration was performed in two
brown algae species, Padina gymnospora and Sargassum stenophyllum. In relation to Sepetiba
Bay macroalgae community, these species were the most abundant in substrate cover. The
algae metal concentration variation from 1990 to 1997 should be related to the inputs of
metals released into the bay by industrial process of Zn production in the area. In situ uptake
and release transplant experiments with Padina between Sepetiba Bay and a near uncontami-
nated area showed that the species could re¯ect the variation in metal environment availability.
A lower reduction in metal concentration by plants transplanted to the uncontaminated area
was observed. Analytical electron microscopy showed that the Zn in P. gymnospora from
Sepetiba Bay was present as small deposits distributed in the cell walls. # 1999 Elsevier Science
Ltd. All rights reserved.
Keywords: Pollution monitoring; Sepetiba Bay; Heavy metals; Brown algae, bioaccumulation; Epibenthic
community; Transplant experiment; Subcellular localisation

* Corresponding author. Tel. and fax: +55-21-2947526.

E-mail address: g®lho@jbr.gov.br (G.M. Amado Filho)

0141-1136/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0141-1136(99)00042-2
214 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224

1. Introduction

Long-term determination of critical pollutants in speci®c compartments of eco-

systems is an essential component of environmental monitoring programs. In the
last 15 years Cd and Zn have been identi®ed as the main contaminants in sediments
and biota of Sepetiba Bay, Rio de Janeiro, Brazil (Barcellos & Lacerda, 1994; Kur-
ita & Pfei€er, 1991; Lacerda, Carvalho, & Gomes, 1989; Lima, Lacerda, Pfei€er, &
Fiszman, 1986; Lacerda, Pfei€er, & Fizman, 1985, 1987, 1989; Pfei€er, Lacerda,
Fizman, & Lima, 1985). However, few studies have quanti®ed (Barcellos, 1995;
Rezende, Lacerda & Pfei€er, 1991) the contamination status in recent years. Lack of
long-term follow-up information makes the interpretation of available data and the
application of mitigation decisions dicult.
Heavy metal levels have been analysed in organisms from di€erent taxonomic
groups in Sepetiba Bay: ®shes, crustaceans, molluscs, seagrasses and algae (Karez,
MagalhaÄes, Pfei€er, & Amado Filho, 1994; Lacerda & Rezende, 1986; Lima et al.,
1986; Pfei€er et al., 1985). Among these groups, the highest levels of Zn and Cd were
found in oysters (Crassostrea brasiliana), followed by macroalgae. Among 10 mac-
roalgae analysed, the brown alga Padina gymnospora (Dictyotales) presented the
highest metal concentrations (Karez et al., 1994). The concentrations of Zn and Cd
in P. gymnospora from Sepetiba Bay were up to 25 and four times higher, respec-
tively, than individuals from a nearby uncontaminated area.
Knowledge of factors a€ecting the rate of accumulation and release in brown
algae is of special value when using them to biomonitor heavy metal pollution (Eide,
Myklestad & Melson, 1980; Rainbow, 1995). In laboratory assays, Zn accumulated
by P. gymnospora increased in direct proportion to its concentrations in seawater
over 21 days experimental time (Amado Filho, Karez, Andrade, Yoneshigue-
Valentin, & Pfei€er, 1997). In short-term experiment of 65Zn kinetics, the release of
Zn by P. gymnospora was insigni®cant, suggesting that the metal could be strongly
bound into cellular sites (Karez et al., 1994). Although these results indicated the
great ability of P. gymnospora to accumulate Zn, data on uptake and release of the
metal in situ were lacking.
Two mechanisms have been associated with the capability of some brown algae to
accumulate high metal concentrations: the presence of cell wall-speci®c polyanionic
polysaccharides that provide the sequestration and precipitation of metals in the cell
walls (Crist et al., 1992; Lignell, Roomans & Pederson, 1982; Mariani, Tolomio, &
Braghetta, 1985; Volesky & Holan, 1995) and the anity between metals and poly-
phenols (Ragan & Glombitza, 1986; Ragan, Smidsrod, & Larsen, 1979). Karez and
Pereira (1995) showed a low percentage association of Zn and Cd with polyphenolic
fraction of P. gymnospora from Sepetiba Bay and suggested that the high Zn and Cd
found in whole algae from polluted areas (like Sepetiba Bay) might thus be greater
than the capability of polyphenols to bind these metals. Analytical electron micro-
scopy of P. gymnospora, submitted to high Zn concentrations under laboratory
experimental conditions, showed the presence of Zn in dense granules along the cell
walls indicating that these walls could play the main role in the Zn accumulation
(Amado Filho, Karez, Pfei€er, Yoneshigue-Valentin, & Farina, 1996).
 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224 215

In this work, the abundance of macroalgae in the epibenthic community of two

studied sites in Sepetiba Bay is described and the Zn and Cd levels in populations
of brown algae from 1990 to 1997 is evaluated. Also, the accumulation and release
of metals was determined in the ®eld by transplanting experiments. Metals were
localised at the subcellular level in individuals of P. gymnospora from Sepetiba
Bay.

2. Materials and Methods

Sepetiba Bay (SB) is a semi-enclosed lagoon, 447 km2 in area, situated 60 km west
of Rio de Janeiro City (Fig. 1). In order to describe the abundance of macroalgae,
20 m-long transects were placed horizontally at about 1 m below lower astronomic
tide (LAT) at two sites in SB (Ilha do Gato [IG] and Praia Grande [PG]; Fig. 1), in
February, April, June and September 1997. Percentage cover of macroalgae species
were recorded using 10 0.25 m2 quadrats randomly positioned (a table of random
numbers was used) along the transects.
Zn and Cd analyses were conducted on two selected brown algae species,
P. gymnospora and S. stenophyllum, collected at the sublittoral zone in the two
sites of SB. The collections were performed from April 1990 to November 1993
(some results were published in Karez et al., 1994) and from August 1995 to July
1997.
Transplanting experiments were carried out with 25 whole-adult individuals (8±12
cm long) of P. gymnospora. The plants were collected from the sublittoral zone of
SB (PG), ®xed by their holdfast in nylon nets and immediately transported and ®xed
to rocky blocks at the same littoral zone (similar depth) in the uncontaminated area,
Ribeira Bay (RB) situated 60 km west of SB (Fig. 1). The same procedure was used
to transfer individuals of P. gymnospora from RB to SB. These experiments were
done in three seasons: September 1996, March 1997 and June 1997. After 30 days of
transplantation, the individuals were collected for metal analysis and the results
were compared with data from natural populations of PG.
Only whole, healthy and adult plants were analysed. Samples were initially
cleaned of epiphytes, washed in seawater from the sites of collection and thereafter
in distilled water, dried at 50 C, homogenised and ashed at 420 C. Fractions of 0.5 g
(dry wt) were digested in 10 ml concentrated HNO3 (65% wt). The resulting solution
was evaporated and redissolved in 0.1 N HCl. Metal concentrations were measured
by ¯ame atomic absorption spectrophotometry (Varian AA-1475) in triplicates.
Results are given in mg. gÿ1 of dry weight. Analytical procedures were tested by
comparative analysis of International Atomic Energy Agency (IAEA) reference
material IAEA-140 (sea plant homogenate, Fucus reference values: Zn=47.3 mg. gÿ1,
Cd=0.537 mg. gÿ1: mean value obtained in this study: Zn=45.8 mg. gÿ1, Cd=0.510
mg. gÿ1).
Specimens of P. gymnospora were prepared for transmission electron microscopy
according to Amado Filho et al. (1996). The localisation of metals was obtained by
electron spectroscopic imaging (ESI) performed with a ZEISS CEM 902 analytical
216 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224

electron microscope, operating at 80 KV. An imaging ®lter integrated in the columm

of the microspope allows for the selection of electrons with particular energy losses
to form an electron spectroscopic image. An energy selective slit of variable width
determines the energy resolution (~; in the present work ~=30 eV; for details see
Bauer, 1988).

Fig.1. Location of sampling areas and collection sites. *Rio de Janeiro City; PG, Praia Grande; and IG,
Ilha do Gato. Arrow indicates the main metal source.
 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224 217

3. Results

3.1. Macroalgal community

The macroalgae abundance and community structure were di€erent in the two
studied sites. The mean percentage cover of macroalgae at PG was 61.8% while at
IG it was 22.7%, although seasonal changes were observed in a range varying from
51.6 (April) to 72.4% (June) in PG and from 40.0% (February) to 10.8% (June) in
IG (Table 1).
The higher abundance and community diversity of macroalgae in PG, relative to
IG, was due mainly to the presence and abundance of Hypnea musciformis
(mean=10.8%) and algal turfs (composed mainly by the red algae species, i.e. Cen-
troceras clavulatum, Gigartina teedii, Gymnogongrus gri€thsiae, Gelidiopsis sp.,
Spyridia clavata, Grateloupia sp.) with a mean percentage cover of 23.5%. P. gym-
nospora and S. stenophyllum were the only two species found in both sites. Popula-
tions of P. gymnospora were found throughout the sampling study period, showing a
mean percentage cover of 8.5% at PG and 12.8% at IG. Populations of S. steno-
phyllum were not represented in one sampled period (February 1997) at IG when it
was replaced by ®lamentous cyanophytes and a higher abundance of P. gymnospora
(30.8%; Table 1).

3.2. Zn and Cd accumulation

Tables 2 and 3, respectively, show the temporal variation of Zn and Cd con-

centrations in the selected species at the two collection sites. At all sampling dates,
the concentrations of both metals were signi®cantly higher in SB (analysis of var-
iance [ANOVA], p<0.001) than found in the uncontaminated area (RB). The mean
concentrations at RB (12 analysed samples along the period) were 23‹8 mg Zn. gÿ1,
0.36‹0.09 mg Cd. gÿ1 in P. gymnospora and 17‹4 mg Zn. gÿ1, 0.20‹0.06 mg Cd. gÿ1
in S. stenophyllum. The ranges of metal accumulation in SB during the studied period
varied from 101‹13 to 899‹134 mg. gÿ1 of Zn, 0.61‹0.13 to 2.70‹ 0.42 mg. gÿ1 of
Cd in P. gymnospora; and from 76‹21 to 580‹68 mg. gÿ1 of Zn, 0.30‹0.07 to
2.01‹0.12 mg. gÿ1 of Cd in S. stenophyllum (Tables 2 and 3).
The highest levels of Zn at SB were observed in February 1996 in the two species
at both sites. In the next sampled period (August 1996) zinc levels were reduced at
least four times in P. gymnospora and 3.2 times in S. stenophyllum. Like Zn, ele-
vated Cd levels were found in February 1996. However, the highest level of this
metal was found in P. gymnospora at IG in May 1991 (Table 3).
In relation to distance from the main SB source of metal, the seaweed metal levels
at IG, situated nearest the Zn factory, were signi®cantly higher (ANOVA, p<0.05)
than the levels found in PG (Tables 2 and 3).
At PG, P. gymnospora presented signi®cantly higher metal levels (ANOVA,
p<0.05 to Zn and p<0.001 to Cd) than S. stenophyllum. A positive correlation
(correlation coecient=r) in metal content between species was detected during the
studied period (Zn, r=0.89; Cd, r=0.64). At IG, signi®cant di€erences were not
 218

Table 1
Mean percentage cover (standard error in parentheses) of macroalgae rocky shores in the two Sepetiba Bay sample sites (Praia Grande and Ilha do Gato) from
February 1997 to September 1997

Species or groups

Sample sites and Padina gymnospora Sargassum stenophyllum Hypnea musciformis Algal turfs Filamentous cyanophytes Total macroalgae
collection dates cover
(month/year)

Praia Grande
02/97 9.1 (1.1) 9.1 (1.4) 13.2 (1.9) 36.6 (3.9) ± 68.0 (4.0)
04/97 8.4 (1.6) 13.6 (2.5) 13.6 (3.5) 16 (2.2) ± 51.6 (3.4)
06/97 9.2 (1.5) 28.8 (2.3) 1.2 (0.6) 33.2 (4.9) ± 72.4 (4.2)
09/97 7.2 (2.1) 24.4 (3.8) 15.2 (4.6) 8.4 (2.5) ± 55.2 (5.3)
Mean 8.5 (0.5) 19.0 (4.6) 10.8 (3.2) 23.5 (6.8) ± 61.8 (5.0)

Ilha do Gato
02/97 30.8 (3.2) 0 ± ± 9.1 (2.6) 40.0 (4.5)
04/97 8.8 (3.5) 18.8 (2.5) ± ± 0 27.6 (4.1)
06/97 5.2 (1.9) 5.6 (2.3) ± ± 0 10.8 (4.0)
09/97 8.4 (2.5) 6.0 (2.8) ± ± 0 12.4 (5.2)
Mean 12.8 (6.1) 7.6 (4.0) ± ± 2.3 (2.3) 22.7 (6.9)
G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224
 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224 219

Table 2
Mean Zn concentration in mg gÿ1 dry weight (standard deviation in parentheses) in the two species in the
two collection sites of Sepetiba Bay (PG,=Praia Grande; IG, Ilha do Gato) from April 1990 to July 1997

Species
Padina gymnospora Sargassum stenophyllum

Collection dates (month/year) Collection site

PG IG PG IG

04/90 240 (70) 470 (79) 120 (13) 127 (33)

05/91 315 (36) 465 (41) 114 (6) 366 (20)
11/93 414 (64) 541 (219) 215 (15) 450 (63)
08/95 490 (28) 580 (105) * *
02/96 610 (35) 899 (134) 509 (36) 580 (68)
08/96 147 (22) 166 (16) 128 (3) 180 (16)
09/96 220 (25) 310 (45) 164 (12) 296 (18)
10/96 200 (14) 290 (40) 96 (10) 209 (25)
03/97 170 (20) 320 (20) 136 (22) 360 (40)
04/97 101 (13) 269 (47) 76 (21) 399 (73)
06/97 278 (8) 294 (34) 176 (13) 344 (76)
07/97 290 (15) 380 (50) 148 (16) 320 (29)

*Not determined.

observed between species in metal levels, and only for Zn was a positive correlation
(r=0.67) between species observed during the period.

3.3. Transplant experiment

Increased uptake of Zn and Cd by P. gymnospora transferred from RB to SB (PG)

was detected after the 1 month transplanting period. Zn accumulation varied from
70‹14 to 283‹34 mg. gÿ1 in transplanted samples, corresponding to a range of
69.3±97.5 % (Table 4) of the levels of samples from natural population of PG col-
lected when each transplant experiment ended (Table 2). Cd accumulation levels
varied from 0.37‹0.10 to 0.70‹0.01 mg. gÿ1 (Table 4) and the uptake of Cd by
transplanted samples was in a range from 46.2 to 77.3% of the levels of the PG
population (Table 3).
On the other hand, the samples transferred from SB to RB presented the metal
levels in a range of 82.7±89.2% of Zn and 62.3±86.2% of Cd (Table 4) in relation to
the metal levels of the PG population samples collected at the same time each
experiment began (Tables 2 and 3).

3.4. Subcellular localisation

The observations of longitudinal ultrathin sections of a mature thallus of P. gym-

nospora by transmission electron microscopy showed the ultrastructural features of
the brown algae cells with the typical physodes in the cytoplasm (Fig. 2), which were
shown by Amado Filho et al. (1996). In the cell walls of individuals collected in the
220 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224

Table 3
Mean Cd concentration in mg. gÿ1 dry weight (standard deviation in parentheses) in the two species in the
two collection sites of Sepetiba Bay (PG, Praia Grande; IG, Ilha do Gato) from April 1990 to July 1997

Species
Padina gymnospora Sargassum stenophyllum

Collection dates (month/year) Collection site

PG IG PG IG

04/90 0.76 (0.26) 1.33 (0.22) 0.37 (0.08) 0.62 (0.18)

05/91 0.87 (0.09) 2.70 (0.42) 0.30 (0.07) 0.42 (0.12)
11/93 0.61 (0.13) 1.63 (0.53) 0.33 (0.06) 0.80 (0.11)
08/95 0.90 (0.26) 1.6 (0.50) * *
02/96 1.20 (0.30) 1.82 (0.70) 1.01 (0.12) 2.01 (0.33)
08/96 0.76 (0.08) 1.22 (0.10) 0.48 (0.08) 0.58 (0.10)
09/96 0.98 (0,15) 1.51 (0.12) 0.55 (0.07) 0.96 (0.12)
10/96 0,80 (0,09) 1.10 (0.08) 0.50 (0.06) 0.88 (0.16)
03/97 0.94 (0.13) 1.05 (0.16) 0.41 (0.05) 1.12 (0.26)
04/97 0.96 (0.05) 1.23 (0.09) 0.40 (0.06) 0.90 (0.21)
06/97 0.75 (0.06) 0.98 (0.05) 0.66 (0.14) 1.10 (0.15)
07/97 0.88 (0.06) 1.06 (0.08) 0.58(0.10) 0.92 (0.11)

*Not determined.

Table 4
Mean Cd and Zn concentrations (standard deviation in parentheses) of Padina gymnospora after 1 month,
transplanted from the uncontaminated area (Ribeira Bay, RB) to Praia Grande (Sepetiba Bay, SB) and
vice-versa

Metal (mg. gÿ1 dry weight)

Cd Zn
a b
Periods of transplanting (month/year) RB to SB SB to RB RB to SBa SB to RBb

09/96 to 10/96 0.37 (0.10) 0.61 (0.22) 153 (23) 194 (25)
46.2% 66.3% 76.5% 89.2%
03/97 to 04/97 0.70 (0.01) 0.81 (0.10) 70 (14) 142 (11)
72.9% 86.2% 69.3% 83.2%
06/97 to 07/97 0.68 (0.02) 0.54 (0.04) 283 (34) 230 (24)
77.3% 72.0% 97.5% 82.7%
a
Mean percentage (in bold) of transplanted sample metal levels relative to the levels of natural popu-
lation of SB ( Tables 2 and 3) found at the same time of the end of each experiment.
b
Mean percentage (in bold) of transplanted sample metal levels relative to the levels of natural popu-
lation of SB ( Tables 2 and 3) found at the same time of the beginning of each experiment.

most contaminated site of SB (IG, July 1997) small granules were dispersed along
the cell wall layers, mainly in the external walls of cortical cells (Fig. 3). No granules
were seen in the cytoplasm or organelles. The ESI technique detected Zn as the main
component of these granules and allowed us to map the metal distribution by using
inelastically scattered electrons corresponding to the Zn L2,3 ionisation edge (Fig. 3).
 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224 221

Fig. 2. General view of a typical cortical cell of Padina gymnospora by transmission electron microscopy.
Nucleus (N), physodes (P) and chloroplasts (arrows) are seen (Magni®cation 2000).

A control image was obtained with electrons with energy losses below the Zn L2,3
edge (Fig. 3) where no granules are seen.

4. Discussion and conclusion

Ideal biomonitors should be sedentary, easy to identify, abundant, long lived,

available for sampling throughout the year, large enough to provide sucient tissue
for analysis, resistant to handling stress caused by laboratory studies of metal
kinetics and/or ®eld transplantation, and tolerant of exposure to environmental
variations in physico-chemical parameters such as salinity. Moreover, they should
be net accumulators of the metal in question with a simple correlation between
metal concentration in tissue and average ambient bioavailable metal concentration
over a recent time period (Rainbow, 1995).
Among the three main ¯eshy algae groups, the brown species were one of the most
abundant (in substrate cover) at the two SB sites during the sampling period,
although clear di€erences in algal community diversity between sites were observed.
The lower diversity of macroalgal ¯ora at IG and the absence of red species, like H.
musciformis, could be related to di€erent tolerance levels presented by algae species and
the higher availability of metals in this area. Amado Filho et al. (1997) showed that
among six seaweed tested under laboratory experimental conditions, H. musciformis
was the most sensitive species to increased Zn concentration in seawater. The most
222 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224

Fig. 3.. (A). Electron spectroscopic image (ESI) of the external cell wall of Padina gymnospora obtained
with energy loss electrons below the carbon K edge. Non-carbon structures appear in white in this image.
The arrow indicates a possible Zn deposit. (B) ESI of the same area of A obtained with energy loss elec-
trons above the Zn L2,3 edge. Structure in white represent Zn deposits (arrow). (C) ESI of the same area of
(A) obtained with energy loss electrons below the Zn L2,3 edge. This image corresponds to non-Zn struc-
tures. Granules indicated by arrow in A and B absent (Magni®cation 9000).

resistant species were P. gymnospora and Sargassum ®lipendula. Thus its possible that
contamination prevented the colonisation of species with low metal tolerance in SB.
It has been reported that seasonal variations in the metal content of brown sea-
weeds from temperate regions could be related to metabolic processes, with faster
growth in summer and slower growth in winter (Fuge & James, 1973; Munda, 1986;
Riget, Johansen, & Asmund, 1995). No data is available from tropical species, but
no seasonal variation would be expected in metal levels because of the small varia-
tions in active growth between summer and winter plants (Luning, 1990). The tem-
poral variation of Zn and Cd contents in the two analysed species from SB is
probably related to the additional inputs of the metals released from industrial
process of Zn production. The higher levels of seaweed Zn and Cd found in Feb-
ruary 1996 could be caused by an unexpected metal over¯ow to the bay waters of
the tailing disposal site of the Zn factory.
A general trend of higher metal levels in P. gymnospora than S. stenophyllum was
observed during the studied period, in spite of higher accumulation levels presented
by S. stenophyllum in some periods at IG. The di€erentiation of the thallus of
Sargassum in to vegetative and reproductive parts could a€ect the total tissue metal
 G.M. Amado Filho et al. / Marine Environmental Research 48 (1999) 213±224 223

levels. Munda (1986) showed in Fucus spiralis (Fucales, same taxonomic order of
Sargassum) that heavy metal accumulation is lower in receptacles (reproductive
structures) than in vegetative parts of the thalli and, hence, responsible for dimin-
ished metal accumulation in fucoids. The relative proportion of vegetative and
reproductive parts biomass of S. stenophyllum could have contributed to the varia-
tion in metal levels, especially in the most contaminated site (IG) of SB.
The results of the transplant experiments show that P. gymnospora is able to
accumulate the studied metals in a short period of time (1 month) re¯ecting the
ambient bioavailability. The changes in metal level observed among transplanted
periods could be related to the variation of available metals in seawater since, espe-
cially in the case of Zn, the metal levels in plants transplanted to SB followed the
metal levels variation found in natural population. Eide et al. (1980), in a similar
transplant experiment with the temperate brown alga Ascophyllum nodusum, con-
cluded that a decrease or increase of heavy metal concentration in the water will give a
de®nitive change in the heavy metal accumulation pattern of the plants. The release of
metals by brown algae has been frequently associated with exudation of metal chelated
to polyphenolic compounds known to be present in this algal group (Eide et al.,
1980; Ragan & Glombitza, 1986). The reduction in metal levels observed in P.
gymnospora transplanted to the uncontaminated area could be related, not to a tissue
metal loss (or release) to seawater, but to a dilution of metal tissue concentration asso-
ciated with the 1-month growth in a seawater free of higher metal levels. This postula-
tion is due to the fact that the metals in P. gymnospora were not detected through
analytical electron microscopy in physodes (Amado Filho et al., 1996, and present study).
The localisation of Zn in cell walls of individuals from SB, in addition to previous
in-vitro results (Amado Filho et al. 1996), con®rm that cell walls play a main role in
metal accumulation in the case of P. gymnospora. Although Cd was not detected in
SB individuals, recent in-vitro experiments performed with P. gymnospora submitted
to high Cd concentration in seawater (Andrade, 1998), suggested that cell walls also
play the main role in Cd accumulation.

Acknowledgements

Dr. Wanderley de Souza of the LaboratoÂrio de Ultraestrutura Celular Hertha

Meyer, IBCCF/UFRJ, kindly provided the electron microscopes for the ultra-
structural observation and analytical electron microscopy. This work was supported
by Instituto de Pesquisas Jardim BotaÃnico/MMA, PRONEX/MCT, and a research
grant of Conselho Nacional de Desenvolvimento Cienti®co e TecnoloÂgicoÐCNPq
No. 521688/96-5 OcÐto G.M. Amado Filho.

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