Metal accumulation by Halodule wrightii

populations

Gilberto M. Amado Filhoa,*, Joel C. Creedb,Leonardo R. Andradec, Wofgang C. Pfeifferd

aPrograma Zona Costeira, Instituto de Pesquisas Jardim Botanico do Rio de Janeiro/MMA,

Rua Pacheco Leao 915, 22460-030 Rio de Janeiro, RJ, BrazilbDepartamento de Biologia Animal e Vegetal—Rua Sao Francisco Xavier 524,

PHLC Sala 220, Setor de Ecologia, 20559-900 Rio de Janeiro, RJ, BrazilcLaboratorio de Biomineralizacao, Departamento de Histologia e Embriologia,

ICB, UFRJ, 21941-590 Rio de Janeiro, RJ, BrazildLaboratorio de Radioisotopos Eduardo Penna Franca, Instituto de Biofısica Carlos

Chagas Filho, CCS, UFRJ, 21949-900 Rio de Janeiro, RJ, Brazil

Received 7 August 2003; received in revised form 13 July 2004; accepted 13 July 2004

Abstract

Metal concentrations and population parameters of the seagrass Halodule wrightii were deter-

mined at three locations at Rio de Janeiro State, Brazil. The possible increase of metal availability in

one of these areas, Sepetiba Bay, as a result of dredging of contaminated bottom sediments which

ocurred, was evaluated by analyses of Al, Cd, Cr, Cu, Fe, Ni, Pb and Zn in root, rhizome and shoots.

In addition, analyses were carried out in H. wrightii populations from non-contaminated areas located

at northwestern (Cabo Frio) and southeastern (Angra do Reis) regions of Rio de Janeiro State.

Concurrently, abundance and density data of the seagrass populations were obtained. It was found

that concentration from Sepetiba Bay samples up to 1.6 � 0.4 mg g�1 of Cd, 12 � 1.0 mg g�1 of Cr,

27 � 2.4 mg g�1 of Pb, 291 � 47 mg g�1 of Mn, 128 � 23 mg g�1 of Zn were significantly higher

than that from two other collection sites. An increase in Cd and Zn concentration was observed in H.

wrightii from Sepetiba Bay indicating that metal mobilization from contaminated sediments through

dredging activities were, at least in part, transferred to the biotic compartment via accumulation by

the seagrass. The populations of seagrass within the region demonstrated quite substantial changes in

biomass data but not in shoot or rhizome density during the study. Such changes in biomass are to be

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Aquatic Botany 80 (2004) 241–251

* Corresponding author. Tel.: +55 21 2294 7526; fax: +55 21 2294 7526.

E-mail address: gfilho@jbrj.gov.br (G.M.A. Filho).

0304-3770/$ – see front matter # 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquabot.2004.07.011

expected, as these dynamics are typical of the small, isolated monospecific populations of H. wrightii

along the Rio de Janeiro coast.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Seagrass; Pollution; Metal concentration; Plant compartment; Population dynamics

1. Introduction

During the last 35 years, the Sepetiba Bay has received effluents from mining and

smelting industries and the metals Cd and Zn have been identified as the main

contaminants in local sediments and biota (see Amado Filho et al., 1999a for a review). In

1997, during a governmental program to increase the capacity of the Port of Sepetiba, an

estimated 20.86 million m3 of bottom sediment was dredged. Most of the dredged

sediment was highly contaminated by heavy metals (Amado Filho et al., 1999b) due

to its proximity to the metal industry’s activities. Under strongly reducing anoxic

conditions, metals such as Cd and Zn may be de-mobilized or ‘fixed’ by the formation of

sulfides. However, changes in Eh or pH due to disturbance or burial (or even

microbial activity) may remobilize the metals (Rees et al., 1998). The dredged sediment

was disposed of within the bay, entering the water column and causing such

remobilization.

Seagrasses, which are important contributors to primary production (Klumpp and e Van

der Valk, 1984), take up metals from both water through leaf surfaces and from sediment

and interstitial water through their roots (Brinkhuis et al., 1980; Ward, 1987; Malea and

Haritonidis, 1995; Caccia et al., 2003; Ferrat et al., 2003). In certain areas seagrasses may

represent the greatest heavy metal reservoir, and this biotic compartment can remobilize

metals by litter production and in food chains. This contrasts with the relative

immobilization, which occurs in unvegetated sediments (Pulich, 1980). Metals sequestered

by seagrasses may be transferred to higher trophic level consumers, including the green

turtle Chelonia midas (Lanyon et al., 1989), which occurs at Sepetiba Bay.

Halodule wrightii is the most common seagrass species in Brazil and this species

approaches its southern distributional limit along the Rio de Janeiro state coast (Creed,

1997,1999). Lacerda and Rezende (1986) determined the concentration of Zn, Cu, Mn, Fe

and Pb in one Sepetiba Bay population during a growing season and pointed out that heavy

metal content can be very high, and that these plants can play an important role in their

cycling. Although the site was probably contaminated at this time, they did not compare the

obtained results with results of populations from uncontaminated areas.

In order to evaluate the role of seagrass H. wrightii populations in metal accumulation

during period of sediment dredging activity, heavy metal analyses were carried out in

plants from two Sepetiba Bay populations in 1997 and 1998. In addition, two other H.

wrightii populations of Rio de Janeiro State from northwestern (Cabo Frio) and

southeastern (Angra do Reis) regions were evaluated as to their metal content.

Concurrently, abundance and density data of the seagrass populations were obtained,

as until now we know almost nothing about the growth and biomass characteristics of these

southern seagrass populations or how they influence metal accumulation.

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251242

2. Material and methods

2.1. Study area and sampling

Sepetiba Bay is a semi-enclosed lagoon, 447 km2, situated 60 km west of Rio de Janeiro

city. Two main H. wrightii populations were recognized, one at Itacuruca Island

(2285700500S and 4385402800W) and other at Jaguanum Island (2380001600S and

4385503700W), the latter situated near to the dredged sediment disposal area. These sites

were visited in 1997 (September) and 1998 (August), respectively. The locations of the

other two sampling sites are: Saco de Piraquara (2285900000 4482600400 Angra do Reis, Ilha

Grande Bay) southward of Sepetiba Bay; and Araruama Lagoon (2285205900 4280004000

Cabo Frio) northward from Sepetiba Bay. All sampling procedures were carried out with

scuba diving as the seagrass populations were found between 1 and 3 m depth.

For heavy metal analysis, several plants samples were hand collected and lifted into

1 mm mesh collecting bags. These were repeatedly shaken underwater to remove most of

the sediment and then transported to the laboratory in plastic bags containing seawater.

Thereafter, the plants were cleaned and washed with filtered local seawater (0.45 mm) and

then washed in distilled water. The effectiveness of the cleaning method to remove

particles adsorbed to the plant surfaces was confirmed by observation of root samples

under light microscopy (Fig. 1). Leaves, rhizomes and roots were separated and were dried

at 50 8C until constant weight.

2.2. Heavy metal analyses

Fractions of 0.5 g d.w. (dry weight) of each plant compartment were digested in 15 mL

concentrated HNO3 (65% w/v) in Teflon flasks and exposed to microwaves (CEM-MDS/

2000) for 25 min. The resulting solution was evaporated and re-dissolved in 0.1N HCl.

Metal concentrations were measured by flame atomic absorption spectrophotometry

(VARIAN AA-1475) in triplicates and the results are given in mg g�1. Analytical

procedures were tested by comparative analysis of International Atomic Energy Agency

(IAEA) reference material IAEA-140 (sea plant homogenate). The existence of significant

differences between metal concentration in different sites and plant compartment was

tested by two-way analysis of variance (ANOVA) and by pos-hoc Tukey multiple

comparisons test.

2.3. Seagrass population characteristics

A scuba diver took n = 5 samples (a number previously established as adequate by

Creed, 1999) from the centre of the study sites. Quadrats were haphazardly positioned in

the centre of patches of H. wrightii although bio-perturbed areas were rejected. To sample,

a knife was drawn down the sides of the 30 cm � 30 cm quadrats so that straddling

rhizomes were cut, and the entire ‘turf’ samples were lifted into 1mm mesh collecting bags.

These were shaken underwater to remove most of the sediment before the samples were

bagged with formalin for transport, as refrigeration facilities were not available. Samples

were processed according to Creed (1999). The formalin was rinsed off and the seagrass

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251 243

separated from sediment and other organisms. Fifty (or all if n < 50) rhizome fragments

were randomly drawn from the sample. For each fragment the number of short shoots was

noted, they were parted and the shoots were pooled. Of these pooled shoots 50 were

randomly selected and their lengths (to the longest leaf tip) were measured. The roots were

separated from the sub-sampled rhizomes. The sub-sampled roots, shoots, rhizomes and

remaining seagrass were dried in an oven at 60 8C to constant final mass before weighing.

From these measures the following characteristics of the populations could be determined:

biomasses, and proportional biomass as short shoots, rhizomes, and roots; the density and

mean length of short (vertical) shoots; the length of rhizome per area sampled—a measure

of rhizome ‘density’. Statistical analyses employed were ANOVA and Tukey multiple

comparisons test (Zar, 1996).

3. Results

3.1. Metals concentration

The analyses of the nine metals in H. wrightii population from Rio de Janeiro State

showed three main features, differences among plant compartments (roots, rhizomes and

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251244

Fig. 1. Light microscopy image (dark field) showing detail of a root cleaned of particles. Root hairs can be seen.

shoots), differences among collecting sites and differences within collecting sites

(ANOVA, p < 0.05).

In relation to plant compartments, a significant difference in metal concentration was

observed between roots and rhizomes/shoots for all analyzed metals, and between

rhizomes and shoots for Cu, Fe, Mn, Pb, and Zn (Table 1). Seven metals (Al, Cd, Cr, Cu, Fe,

Ni and Pb) presented a trend of higher concentration in roots than rhizomes and shoots; one

metal (Mn) presented higher concentration in shoots than rhizomes and roots; and one

metal (Zn) presents a trend of higher concentrations in both roots and shoots (Fig. 2).

Comparing the sample sites, it was found that concentration of Cd, Cr, Pb (roots), Zn

(roots and shoots) and Mn (shoots) were significantly higher in samples from Sepetiba Bay

(at least in one of the two sample sites) than that of two other collected sites. The

concentrations of Cu, Fe and Ni were not significantly different between Sepetiba Bay and

Angra dos Reis. Significantly lower concentrations of eight metals (Cd, Cr, Cu, Fe, Mn, Ni,

Pb and Zn) were found at Cabo Frio. No differences were seen in Al concentrations among

the four sites.

Variation of metal concentration at Sepetiba Bay between the samples from 1997 and

1998 were observed in the following metals and plant compartments: Cd and Zn

concentration in roots and Mn in shoots were significantly higher in 1997 than 1998

samples (for both Sepetiba sites for Zn, for Jaguanum Island for Cd and for Itacuruca Island

for Mn); in contrast, Pb was still higher in roots sampled from 1998 than 1997 (Fig. 2).

3.2. Characteristics of the seagrass populations

In the studied populations at Rio de Janeiro, the mean total mass of H. wrightii varied

from 0.5–10 g quadrat�1 (Fig. 3). Most of the mass was shoot (19–80%) or rhizome (13–

69%) material, and roots made up a lower proportion (6–20%) of the mass (Fig. 3). The

populations at Itacuruca and Cabo Frio had greater masses of seagrass than those at

Jaguanum or Angra dos Reis in April 1997 (Fig. 3a) and a similar pattern was found when

the plant parts were considered separately (Fig. 3b–d). At this time, the population at Angra

dos Reis had proportionally more shoot, and less rhizome and root biomass than Cabo Frio.

In August 1998, Cabo Frio had proportionally more root and less seagrass rhizome mass

than the Itacuruca population. During the study the total mass of seagrass, and shoot, root

and rhizome fractions decreased at Itacuruca (Fig. 3a–d) and during this time the mass

of shoots became proportionally smaller and rhizomes proportionally greater (Fig. 3e–f).

The proportion of mass as roots was constant for the Itacuruca population during the study

(Fig. 3g).

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251 245

Table 1

Comparison ANOVAs of the concentrations of metals between plant compartments

Plant compartments Metals

Al Cd Cr Cu Fe Mn Ni Pb Zn

Root/rhizome 0.001** 0.001** 0.001** 0.001** 0.001** 0.001** 0.005** 0.001** 0.001**

Root/shoot 0.001** 0.001** 0.001** 0.009** 0.001** 0.001** 0.001** 0.015** 0.086

Rhizome/shoot 0.379 0.788 0.966 0.025* 0.001** 0.001** 0.629 0.001** 0.042**

Significant levels: *p < 0.05, **p < 0.01; number of samples (n) for each metal = 18.

G.M

.A.

Filh

oet

al./A

qu

atic

Bo

tan

y8

0(2

00

4)

24

1–

25

12

46

Fig. 2. Metal concentrations in root (&), rhizome ( ) and shoots (&) of H. wrightii from the Rio de Janeiro collected sites. Sepetiba Bay sites: ITA/97, Itacuruca Island,

September 1997; ITA/98, Itacuruca Island, August 1998; JAG/97, Jaguanum Island, September 1997; JAG/98, Jaguanum Island, August 1998; ANG/97, Angra dos Reis,

September 1997; CAB/97, Cabo Frio, Sepetember 1997.

G.M

.A.

Filh

oet

al./A

qu

atic

Bo

tan

y8

0(2

00

4)

24

1–

25

12

47

Fig. 3. Biomass of the seagrass H. wrightii in four populations at Rio de Janeiro, Brazil, and at four sampling times during 1997–1998. (a–d) Mean mass (total dry weight

and of parts of the plants); (e–g) mean proportional mass of the plant parts. Data are means, bars are S.E., n = 5 except Cabo Frio when n = 4.

At Jaguanum, the seagrass mass (total and parts) was greater at the two sample times in

1997 than those in 1998 (Fig. 3a–d). As in the Itacuruca population, at Jaguanum the mass

of shoots became proportionally smaller and of rhizomes proportionally greater during the

monitoring period (Fig. 3e–f), while the proportion of mass as roots was constant (Fig. 3g).

Considering the Jaguanum and Itacuruca populations together, clear changes in the

proportion of shoot and rhizome fractions were observed over time, but not between

populations, and there seemed to be no time–population interaction. Neither time nor

population seemed to affect the proportion of biomass as roots.

The mean density of shoots of H. wrightii in the populations varied from 180–585

shoots quadrat�1 during the study period. Mean rhizome density of the seagrass (estimated

as the total length of rhizomes in a quadrat area) varied from 3.2–12.3 m quadrat�1. As for

biomass, in April 1997 at Cabo Frio and Itacuruca the densities of shoots and rhizomes

were higher than at Angra dos Reis and Jaguanum. At the last sample time in August 1998,

shoot density was lower at Jaguanum than Itacuruca (Fig. 4a) and rhizome density lower at

Jaguanum than Cabo Frio (Fig. 4b). In fact, comparing Jaguanum and Itacuruca, shoot

density varied only between populations—the sample time apparently had little effect on

shoot density (Fig. 4a). This was not the case for rhizome density, because although

rhizomes were half as dense at Jaguanum, an overall temporal difference in rhizome

density was detected (Fig. 4b).

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251248

Fig. 4. Density of the seagrass H. wrightii in four populations at Rio de Janeiro, Brazil, and at four sampling times

during 1997–1998. (a) Mean shoot density; (b) mean rhizome density (total length of rhizomes per area of

quadrat). Data are means, bars are S.E., n = 5 except Cabo Frio when n = 4.

4. Discussion

It is to be expected that local fluctuations in heavy metal levels nearby seagrass beds will

be reflected in levels in the seagrasses themselves and in sediments (Caccia et al., 2003) and

that these levels will be influenced by intrinsic attributes of the species and populations.

The results of metals (Cu, Mn, Pb and Zn) concentrations determined by Lacerda and

Rezende (1986) in H. wrightii populations from Jaguanum Island, were in general similar

to the results found in our study for the same Sepetiba Bay collection site. These results

indicate that concentrations of above cited metals have not changed in a period of 15 years

(1983–1997/1998). Only for Fe, an increase of tissue concentration was seen along the

period, in spite of no differences between Sepetiba Bay and the two other populations in

our work. Although Lacerda and Rezende (1986) did not compare their results with results

from other Rio de Janeiro State coastal areas, we confirm the significantly higher

concentration of Cd, Cr, Mn, Pb and Zn from H. wrightii Sepetiba Bay populations in

relation to the two other Rio de Janeiro State analyzed population. Among these metals,

Cd, Cr, Pb and Zn had been recognized as historical contaminants of Sepetiba Bay

(Lacerda et al., 1987).

The populations of seagrass within the region demonstrated quite substantial changes in

biomass, but not in shoot or rhizome density during the study. Such changes in biomass are

to be expected, as these dynamics are typical of the small, isolated monospecific

populations of H. wrightii, along the Rio de Janeiro coast (Creed, 1999). Seagrasses, in

general, and these populations in particular, demonstrate extremely plastic morphologies

(Creed, 1997). H. wrightii is considered to be a pioneer species in many Caribbean seagrass

successions because it thrives in disturbed environments (Williams, 1990). Such fast

growing species are adapted to frequent perturbations in the shallow coastal environment.

The populations demonstrated a quite significant shift in biomass allocation from shoots in

1997 to rhizomes in 1998. However, contaminated and control sites showed similar

patterns in biomass allocation which leads us to believe that the shifts were related to

region-wide interannual variation and not to the effects of metals. These shifts were due to

reduced leaf biomass rather than reduced shoot density or increase in rhizome biomass.

As seven of nine analyzed metals presented a trend of higher concentration in roots than

rhizomes and shoot, we can suggest that H. wrightii roots are the main compartment for

metal accumulation, reflecting the metal concentration and availability in the sediment

pore waters. On the other hand, the two metals (Mn and Zn) that presented elevated

concentration in shoots, have been cited for others seagrass species as metals that tend to be

accumulated in higher degree in shoots, as was pointed out by Malea (1994), Sanchiz et al.

(1999) and Prange and Dennison (2000). The observed differences among seagrass species

in relation to the main compartment for metal accumulation, roots or shoots, indicates a

difference in these species in their physiology of absorption, accumulation and/or

translocation of these elements (Sanchiz et al., 1999).

In a short-term experiment of Mn uptake by H. wrightii in bathed seawater, Pulich

(1987) described that leaves and roots, display similar uptake kinetics at low concentration,

but above 0.1 mM Mn, leaves accumulate Mn much faster than roots. In another uptake

experiment of 54Mn added to sediment cores at a depth of 8 cm, Pulich (1987) shows that54Mn is detected in newly-produced leaves before it is measured in surface sediment layers

G.M.A. Filho et al. / Aquatic Botany 80 (2004) 241–251 249

or overlying seawater, indicating that H. wrightii absorbs 54Mn through the roots, and then

translocates it to the leaves. Under this experimental condition, accumulation of 54Mn in

leaves is significantly greater than in roots and it was concluded that the amount of Mn

accumulated by roots appears low, but the translocation process is apparently rapid when

plants are rooted in sediment. From the experiments of 54Mn uptake kinetics conducted by

Pulich (1987) in addition to the in situ results, we concluded that the pattern of Mn

accumulation by H. wrightii in shoots could be explained by both a great ability of leaves to

take up these metals directly from the surrounding water and to an acropetal translocation.

The antagonistic relationship patterns of Mn and Fe distribution between shoots and roots

described for H. wrightii by Pulich (1982) was confirmed by results of Rio de Janeiro

populations. While Mn accumulation is higher in shoots, roots are the main compartment

for Fe accumulation. Al also presents a characteristic preferential distribution in roots.

Concerning Zn, in addition to elevated shoot concentrations in Sepetiba Bay

populations observed by us and by Lacerda and Rezende (1986), an increase of root

concentration was detected in populations sampled during 1997. These results probably

relate to an increase in Zn concentration in surface sediments during the period of the

intensive dredging activities. Analysis of Zn in the surface sediment (total fraction) from

the area of the Sepetiba Bay seagrass beds reveals that in September 1997 the

concentrations were in the range of 1.4–1.6 (356 � 18 mg g�1 d.w. at Itacuruca Island and

114 � 6 mg g�1 d.w. at Jaguanum Island) times higher than that from sediments analyzed

in August 1998 (213 � 22 mg g�1 d.w. at Itacuruca Island and 78 � 12 mg g�1 d.w. at

Jaguanum Island). As was observed for Zn, elevated Cd concentrations in surface

sediments during the period of intense bottom sediment dredging of Sepetiba Bay,

especially at Jaguanum Island (1997, 1.25 � 0.08 mg g�1; 1998, 0.60 � 0.10 mg g�1)

should explain the higher Cd concentrations (1.66 � 0.44 mg g�1) in roots observed in

1997 populations from Jaguanum Island. The highest Cd level, and all of Zn data observed

in H. wrightii from Sepetiba Bay are in the same range of those found by Pulich (1982) for

the H. wrightii populations from contaminated metal sites of Corpus Christi Bay, TX, USA.

With observed results of increase in Cd and Zn concentration in H. wrightii from Sepetiba

Bay at 1997, it was shown that the metals mobilization from contaminated sediments

through dredging activities were at least in part, transferred to the biotic compartment via

accumulation by the seagrass population.

Acknowledgements

This work was financially supported by the Brazilian program PRONEX/MCT and by

research grants of CNPq (521688/96-5) and FAPERJ (E-26/170336/98) to G.M. Amado

Filho.

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