Marine Pollution Bulletin 60 (2010) 509–514

Contents lists available at ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier .com/locate /marpolbul

Metal concentration and structural changes in Corallina elongata(Corallinales, Rhodophyta) from hydrothermal vents

Ruben P. Couto a,b,*, Ana I. Neto b,c, Armindo S. Rodrigues a,b

a CIRN – Centro de Investigação de Recursos Naturais, Universidade dos Açores, 9501-801 Ponta Delgada, Apartado 1422, Portugalb Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Apartado 1422, Portugalc CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas 289, 4050-123 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Keywords:Calcifying organismsVolcanic activityElement concentrationBioindicatorsOcean acidificationPortugal

0025-326X/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.marpolbul.2009.11.014

* Corresponding author. Address: Departamento dAçores, 9501-801 Ponta Delgada, Apartado 1422, Portfax: +351 296 650 100.

E-mail address: coutoruben@uac.pt (R.P. Couto).

Shallow-water hydrothermal activity is widely present at Azores archipelago. Organisms in such environ-ments present great potential as sentinels of the effects derived from chronically exposure to increasedtemperature, metal concentrations and reduced pH. This study aimed to evaluate metal concentration inCorallina elongata collected at locations exposed and not exposed to shallow-water hydrothermal activityand evaluate changes in its calcareous structure. Elemental concentration was determined and morpho-metric analysis was performed by scanning electron microscopy. Thicker cell walls and a bleachedappearance were observed on C. elongata specimens from the hydrothermally active location, as wellas increased concentrations of elements associated to volcanic activity.

This study reports on metal accumulation and morphometric changes in the calcareous structure of C.elongata from a hydrothermally active location, adding new data for further research on such habitats andcommunities, providing an insight on how coralline algae might be affected by ocean acidification.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Volcanic activity is one of the most powerful geological phe-nomena and can be expressed in range of ways such as lava emis-sions, diffuse degassing from soils, and hydrothermal activity(Cruz, 2003; Ferreira et al., 2005).

The Azores islands (Fig. 1) are located in the northern Atlanticand are of volcanic origin. Volcanic activity responsible for islandformation varied from effusive eruptions, characterized by a steadyoutpouring of lava, to explosive events, with violent fragmentationof lava (Cruz and França, 2006).

The Azores is also characterized by the presence of active deep-sea and shallow-water hydrothermal vents. Organisms that live insuch environments are chronically exposed to ‘‘natural thermalpollution”, high metal concentration, either in the form of particlesor associated with gases from volcanic emissions (Hansell et al.,2006), as well as to acidified seawater adjacent to hydrothermallyactive areas due to the diffusion of acidic volcanic gases (mainlyCO2) (Cruz and França, 2006).

Macrophytic algae play an important role in functioning of mar-ine ecosystems (Gattuso et al., 1998). Algae interact with the envi-

ll rights reserved.

e Biologia, Universidade dosugal. Tel.: +351 296 650 000;

ronment through various processes that include chemicalbioconcentration, excretion, organic matter production anddecomposition. They are often used as indicators for the healthof marine ecosystems and can be valuable bioindicators for metalpollution due to their longevity, metal accumulation capacity andsessile nature (Abdallah et al., 2005; Chaudhuri et al., 2007; Mish-eer et al., 2006; Mohamed and Khaled, 2005).

In temperate regions, such as the Azores, calcareous macroalgaecan thrive and dominate in a large range of habitats from the eulit-toral to the deepest part of the photic layer (Canals and Ballesteros,1997). Articulated coralline algae also support a diverse assem-blage of macrofauna (Kelaher et al., 2001). The cell walls of coral-line algae are heavily impregnated with calcium carbonate, in theform of calcite. This makes calcareous algae very tough and resis-tant to abrasion, making coralline algae one of the most importantstructural elements in many coastal zones. Calcareous algae havebeen studied as potential sentinels for metal pollution (Mohamedand Khaled, 2005; Stengel et al., 2004; Wallenstein et al., 2009)and seawater acidification effects (Hall-Spencer et al., 2008; Martinet al., 2008). They have also been used as ‘‘global palaeothermom-eters” through measuring the percentage of magnesium and cal-cium in skeletal calcite (Mg/Ca ratio), (Halfar et al., 2008;Kamenos et al., 2008; Ries, 2006; Stanley et al., 2002).

Studies on shallow-water hydrothermal vents have been made(Hall-Spencer et al., 2008; Martin et al., 2008), reporting to thoseareas reduced coverage or even total absence of coralline algae,

Fig. 1. Azores archipelago; São Miguel island, showing the hydrothermally active (H) and control (Cs) locations where samples were collected.

510 R.P. Couto et al. / Marine Pollution Bulletin 60 (2010) 509–514

bleached appearance of plants and dissolution of the calcifiedcell walls of coralline algae and invertebrates with carbonatedstructures.

In the Azores, a few studies have examined the effects of shal-low-water hydrothermal activity on marine organisms (Cardigoset al., 2005; Cunha et al., 2008) including algae (Colaço et al.,2006; Wallenstein et al., 2009), but none has examine its effectsin the algae structure. The present study aimed to examine theeffects of shallow-water hydrothermal activity on the chemicalcomposition, anatomy and calcareous structures of the benthiccoralline alga Corallina elongata J. Ellis and Solander. C. elongatawas chosen as it is abundant throughout the Azores (Alvaroet al., 2008; Martins et al., 2008; Tittley and Neto, 2000; Wallen-stein et al., 2006, 2008, 2009), can be easily identified andcollected.

2. Materials and methods

Plants of C. elongata were collected from February to April 2007at four sampling locations around São Miguel island (see Fig. 1):Porto Formoso, characterized by the presence of shallow-waterhydrothermal vents (37�49018.800N, 025�27025.200W, hereafter re-ferred to as H), Lagoa (37�44031.800N, 025�35019.300W), Feteiras(37�46054.600N, 025�46024.700W) and Ferraria (37�51026.800N,025�51010.300W), selected as control locations (hereafter referredto as C1, C2 and C3, respectively). It is worth mentioning that atFerraria there is shallow-water hydrothermal activity, however re-stricted to a small enclosed bay away from the location wherespecimens were collected.

C. elongata was randomly collected throughout a wide intertidalarea at each sampling location on one occasion during low tide. Ateach location temperature and pH were measured using an HI98127 pH meter and thermometer from HANNA Instruments�.

In the laboratory, samples were washed with seawater collectedin the respective sampling location to remove excessive particulatematter, scrubbed with a soft nylon brush to remove epiphytes and

epifaunal species and rinsed very briefly with distilled water to re-move traces of salt. Plants were then dried at 37 �C and kept in adry environment until analysis.

Concentration of Ca – Calcium, Cd – Cadmium, Mg – Magne-sium, Mn – Manganese, Rb – Rubidium and Zn – Zinc was deter-mined by high resolution inductively coupled plasma massspectrometry (HR-ICP-MS) at Activation Laboratories Ltd.” (Ancast-er, Canada) (see Wallenstein et al., 2009).

Analysis of the calcium carbonated structure was made byexamination of plants of C. elongata in the scanning electron micro-scope (SEM, JEOL JSM-5410). Eight specimens from each samplinglocation were mounted onto aluminium stubs with double-sidedtape, coated with carbon and then with gold/palladium 40/60 ina vacuum evaporator (JEOL JEE-400) to ensure a high quality mor-phological image. The aluminium stubs with the algal sampleswere kept in a desiccator at all times. Images from the exterior sur-face of the apex of C. elongata specimens were taken with the soft-ware JEOL (SemAfore�) at 1000� magnification with a workingdistance of 20 mm and 15 keV accelerating voltage, and morpho-metric analysis was performed with the software AxioVision(Zeiss). Cell density was measured in the entire image field and cellwall thickness was measured on 40 randomly selected cells fromeach sample. And the cell area/cell wall thickness ratio wascalculated.

One-way asymmetrical analysis of variance (Table 1) was usedto test for differences in: (1) cell wall thickness, (2) cell density, (3)cell area/cell wall thickness ratio, (4) Ca, Cd, Mg, Mn, Rb and Znconcentrations and the (5) Mg/Ca ratio between the hydrother-mally active location and the control locations.

Generally, the factor location was partitioned into two compo-nents: one contrasting the hydrothermally active and control loca-tions (H vs. Cs) and the other contrasting control locations amongeach other (Cs). Prior to analysis, Cochran’s test was used to checkfor problems of heteroscedascity and data were transformed whenappropriate. Where heterogeneity of variances persisted, untrans-formed data were analysed (Underwood, 1997).

Table 2Temperature and pH values recorded at low tide in the hydrothermally active (H) andcontrol (Cs) locations.

Stations Temperature (�C) pH

H 17.1 6.65C1 16.4 8.27C2 16.1 8.42C3 16.3 8.15

Table 1Asymmetrical analysis of variance comparing the hydrothermally active (H) andcontrol (Cs) locations, where: a = all locations, b = control locations and n = number ofreplicates. Adapted from Terlizzi et al. (2005). These tests assume equivalence amongr2

H vs: Cs , r2Cs and r2

L (see Glasby, 1997).

Source of variation df Expected mean square

Location a � 1 r2e þ nr2

L þ anr2L

H vs. Csa 1 r2e H þ nr2

H vs: Cs þ bnr2L

Csb a � 2 r2e Cs þ nr2

Cs

Residual a(n � 1) r2e

Res H n � 1 r2e H

Res Cs (a � 1)(n � 1) r2e Cs

a Tested over the residual if Cs can be eliminated from the model (not significantat a = 0.25, Underwood, 1997); tested over Cs otherwise.

b Tested over Res Cs.

R.P. Couto et al. / Marine Pollution Bulletin 60 (2010) 509–514 511

3. Results

Higher temperature and lower pH were recorded for the seawa-ter at the hydrothermally active location when compared with thatof control locations (Table 2).

Personal observation revealed that C. elongata is more abundantand homogeneously distributed at the control locations, and thatspecimens occurring at the hydrothermally active location werebleached.

There are significant differences between locations in cell wallthickness among locations (Fig. 2), with higher values recordedfor specimens from the hydrothermally active location (Fig. 3,Table 3). Although not statistically significant but close to it

Fig. 2. SEM photomicrographs of Corallina elongata collected at the control locations (A)plants from the hydrothermally active location.

(p = 0.057), cell density follows the same pattern. The cell area/cellwall thickness ratio was lower in specimens from the hydrother-mally active location, though not statistically significant.

The only element for which there were statistically significantresults was Cadmium with lower values associated with the hydro-thermally active location. Nevertheless, the concentrations of Zn,Rb and Mn were higher at the hydrothermally active location, aswas also the case of Ca and a consequently lower Mg/Ca ratio(Fig. 4, Table 4), even not statistically significant. Mg concentrationdidn’t reveal any pattern between locations.

4. Discussion

The higher temperature and low pH recorded at the hydrother-mally active location is a natural result of the volcanic activity. Infact, unpublished temperature and pH data collected hourly atthe hydrothermally active location during a 12 h period in July2008 when compared to high tide and to average surface seawatertemperature for the same time of the year – 21.1 �C (Instituto Hid-rográfico, 2000) and to average surface ocean pH – 8.2 ± 0.3 units(Royal Society, 2005) show that during low tide there is nearly a3 �C rise in temperature and a decrease of 2 units in pH.

In this location, plants of C. elongata had a lower density (per-sonal observation), a fact that may be related to the lower pH re-corded there (6.6 vs. 8.4). Gao et al. (1993) referred that adecrease in pH inhibits calcification and a reduced coverage oreven total absence of coralline algae has been reported for placeswhere the mean pH was bellow 7.7 (Hall-Spencer et al., 2008; Mar-tin et al., 2008). These studies, however, were developed withplants growing subtidally in the vent area while the present oneis intertidal thus specimens are exposed to hydrothermal activitymainly during periods of flooding tide. Lowest pH conditions occurat low tide (personal observation) when dilution of hydrothermalinputs is lower, thus reaching intertidal organisms mainly due tothe influence of wave action.

The bleached appearance of plants from hydrothermally activelocations is probably related to the lower pH that is probably lead-ing to the dissolution of the calcified cell walls, as reported by Hall-Spencer et al. (2008) for algae and invertebrates with carbonatedstructures.

and at the hydrothermally active location (B), showing (arrows) thicker cell walls in

Fig. 3. Mean (+SE) of: Cell wall thickness, cell density and cell area/cell wallthickness ratio in the hydrothermally active (H) and control (Cs) locations.

Table 3Asymmetrical analysis of variance comparing cell wall thickness, cell density and the cell arSignificant terms in bold.

Source df Cell wall thickness Cell

MS F p MS

L 3 0.79 4.29H vs. Cs 1 1.68 18.52 0.004 9.52Cs 2 0.34 1.47 0.252 1.68Residual 28 0.19 1.97Res H 7 0.09 1.83Res Cs 21 0.23 2.02Transformation None x � 1Cochran’s C = 0.34 C = 0

512 R.P. Couto et al. / Marine Pollution Bulletin 60 (2010) 509–514

The significantly greater cell wall thickness and cell density(which was not statistically significantly) observed in specimensfrom the hydrothermally active location is likely the result of in-creased seawater temperatures. In fact, Lobban et al. (1985) refersto increased growth rates and primary production associated topunctual increases in temperature in hydrodynamic temperatecoastal areas activity. Additionally, the increase in the content ofCa in specimens collected at the hydrothermally active location ob-served in the present study (see Fig. 4, Table 4) suggests a higherrate of algal calcification, likely to be related to the increased tem-perature as reported by Mohamed and Khaled (2005) and to thehigher inorganic carbon concentrations in sea water (due to thedissolution of CO2) which promotes calcification and photosynthe-sis (Gao et al., 1993).

The Mg/Ca ratio did not revealed significant variations betweenhydrothermally active and control locations, similar to that re-ported for coralline red algae by Stanley et al. (2002).

The higher concentrations of Zn, Rb, and Mn in specimens col-lected at the hydrothermally active location are in accordance withthe results of Colaço et al. (2006) for algae at hydrothermally activelocations and by Cunha et al. (2008) in the digestive gland of mar-ine limpets from shallow-water hydrothermal vents. This suggeststhat C. elongata assimilates these elements, which are associatedwith the volcanic activity (Amaral et al., 2006; Cunha et al.,2008; Rodrigues et al., 2008; Zaldibar et al., 2006). Additionally,according to Kamenos et al. (2008) increasing sea water tempera-ture promotes the incorporation of several elements into the cal-cium carbonate structure of several calcareous marine organisms.

Mohamed and Khaled (2005) also report higher cadmium con-centration (11.081 ppm) in C. mediterranea from warmer areas,which they attribute to a faster rate of calcification in those loca-tions resulting in less strict regulation of minor and trace elementuptake. In the present study, the results obtained for Cadmiumwere lower at the hydrothermally active location, suggesting thatthere is a lower input of this element in the seawater at that loca-tion. The higher values recorded at the control locations are likelyrelated to the input of large amounts of artificial fertilizers fromsurrounding pastures with elements becoming bioavailable toorganisms as was previously reported by Rodrigues et al. (2008)for the arthropod Pseudaletia unipuncta (Haworth).

Moreover, a competitive mechanism between H+ and metal ionsthat result in a lower adsorption with decreasing pH (Papageorgiouet al., 2006) might also be influencing the accumulation of someelements at the hydrothermally active location. In fact, due to highpercentage of calcium carbonate, coralline algae have low meta-bolic rates and a lower proportion of metabolic tissue (Littleret al., 1983), and Corallina spp. have been reported to show lowervalues of metal accumulation when compared with non calcifiedalgae (Jordanova et al., 1999; Kut et al., 2000; Stengel et al.,2004; Wallenstein et al., 2009).

ea/cell wall thickness ratio in the hydrothermally active (H) and control (Cs) locations.

density Cell area/cell wall thickness

F p MS F p

293.745.20 0.057 586.87 3.99 0.1840.83 0.448 147.17 2.46 0.109

47.8812.2459.76

000 None.33 C = 0.39

Fig. 4. Element concentration (Mean + SE) in Corallina elongata collected at the hydrothermally active (H) and control (Cs) locations.

Table 4Asymmetrical analysis of variance comparing element concentration in Corallina elongata in the hydrothermally active (H) and control locations (Cs). Significant terms in bold.E = � 10.

Source df Ca Mg Zn Rb Mn Cd Mg/Ca

MS F MS F MS F MS F MS F MS F MS F

L 3 2.68 0.06 448.49 0.04 805.34 0.04 1.48H vs. Cs 1 4.95 3.21 88E�4 0.11 577.22 5.73 0.03 0.64 2079.30 12.70 0.11 218272.88** 1.42 0.94Cs 2 1.54 3.02 0.08 44.83** 384.12 0.71 0.05 2.29 168.37 0.48 43 E�4 1.50 1.51 14.81*

Residual 4 0.38 16E�4 429.47 0.04 302.80 22 E�4 0.08Res H 1 5E�3 12E�4 100.82 0.08 163.81 5E�7 0.02Res Cs 3 0.51 18E�4 539.02 0.02 349.13 29E�4 0.10

Transformation None None None None None None v � 100Cochran’s test C = 0.39 C = 0.49 C = 0.93* C = 0.54 C = 0.83 C = 0.99** C = 0.46

* p < 0.05.** p < 0.01.

R.P. Couto et al. / Marine Pollution Bulletin 60 (2010) 509–514 513

514 R.P. Couto et al. / Marine Pollution Bulletin 60 (2010) 509–514

Shallow-water hydrothermal vents are potential laboratoriesfor studying the effects of global warming and ocean acidificationon marine organisms, such as calcifying organisms (e.g. corallinealgae, Kleypas et al., 2006). Coralline algae, such is C. elongata,are important structuring elements in many coastal zones, andthey are known to have resisted to past fluctuations in pH. It is,however, unknown how they adapt to rapid seawater acidification,as predicted by the increased release of CO2 due to anthropogenicactivities (Pearson and Palmer, 2000). In such a context, andaccording to Haugan et al. (2006), it is fundamental to considerCorallina spp. in key type ecosystems investigations.

Studies examining the element concentration in the sedimentsand seawater as well as studies examining the geochemistry of flu-ids from active shallow-water hydrothermal systems are needed tohelp explaining the relationship between algal metabolism andmetal accumulation.

Acknowledgements

The authors wish to thank Francisco Wallenstein and GustavoMartins for helpful discussions and comments. We are grateful tothe Scanning Electron Microscopic (SEM) Service of Departmentof Biology, University of Azores. Jorge Medeiros provided facilitiesand technical help in the technology of SEM. This work was fundedby Centro de Investigação de Recursos Naturais da Universidadedos Açores (CIRN) and Direcção Regional da Ciência e Tecnologia(DRCT – Regional Government of Azores). Ruben Couto was sup-ported by a PhD grant from DRCT (M3.1.1/I/014A/2005).

References

Abdallah, A.M.A., Abdallah, M.A., Beltagy, A.I., 2005. Contents of heavy metals inmarine seaweeds from the Egyptian coast of the Red Sea. Chem. Ecol. 21, 399–411.

Alvaro, N.V., Wallenstein, F.F.M.M., Neto, A.I., Nogueira, E.M., Ferreira, J., Santos, C.I.,Amaral, A.F., 2008. The use of digital photography for the definition of coastalbiotopes in Azores. Hydrobiologia 596, 143–152.

Amaral, A., Soto, M., Cunha, R., Marigómez, I., Rodrigues, A., 2006. Bioavailabilityand cellular affects of metals on Lumbricus terrestris inhabiting volcanise soils.Environ. Pollut. 142, 103–108.

Canals, M., Ballesteros, E., 1997. Production of carbonate particles by phytobenthiccommunities on the Mallorca-Menorca shelf, northwestern Mediterranean Sea.Deep-Sea Res. Part II 44, 611–629.

Cardigos, F., Colaço, A., Dando, P.R., Ávila, S.P., Sarradim, P.M., Tempera, F.,Conceição, P., Pascoal, A., Santos, R.S., 2005. Shallow water hydrothermal ventfield fluids and communities of the D. João de Castro Seamount (Azores). Chem.Geol. 224, 153–168.

Chaudhuri, A., Mitra, M., Havrilla, C., Waguespack, Y., Schwarz, J., 2007. Heavy metalbiomonitoring by seaweeds on the Delmarva Peninsula, east coast of the USA.Bot. Mar. 50, 151–158.

Colaço, A., Raghukumar, C., Mohandass, C., Cardigos, F., Santos, R.S., 2006. Effect ofshallow-water venting in Azores on a few marine biota. Cah. Biol. Mar. 47, 359–364.

Cruz, J.V., 2003. Groundwater and volcanoes: examples from the Azoresarchipelago. Environ. Geol. 44, 343–355.

Cruz, J.V., França, Z., 2006. Hydrogeochemistry of thermal and mineral watersprings of the Azores archipelago (Portugal). J. Volcanol. Geotherm. Res. 151,382–398.

Cunha, L., Amaral, A., Medeiros, V., Martins, G.M., Wallenstein, F.F.M.M., Couto, R.P.,Neto, A.I., Rodrigues, A., 2008. Bioavailable metals and cellular effects in thedigestive gland of marine limpets living close to shallow water hydrothermalvents. Chemosphere 71, 1356–1362.

Ferreira, T., Gaspar, J.L., Viveiros, F., Marcos, M., Faria, C., Sousa, F., 2005. Monitoringof fumarole discharge and CO2 soil degassing in the Azores: contribution tovolcanic surveillance and public health risk assessment. Ann. Geophys. 48, 787–796.

Gao, K., Aruga, Y., Asada, K., Ishihara, T., Akano, T., Kiyohara, M., 1993. Calcificationin the articulated coralline alga Corallina pilulifera, with special reference to theeffect of elevated CO2 concentration. Mar. Biol. 117, 129–132.

Gattuso, J.P., Frankignoulle, M., Wollast, R., 1998. Carbon and carbonate metabolismin coastal aquatic ecosystems. Annu. Rev. Ecol. Syst. 29, 405–434.

Glasby, T.M., 1997. Analysing data from post-impact studies using asymmetricalanalyses of variance. A case study of epibiota on marinas. Aust. J. Ecol. 22, 448–459.

Halfar, J., Steneck, R.S., Joachimski, M., Kronz, A., Wanamaker Jr., A.D., 2008.Coralline red algae as high-resolution climate recorders. Geology 6, 463–466.

Hall-Spencer, J.M., Rodolfo-Metalpa, R., Martin, S., Ransome, E., Fine, M., Turner,S.M., Rowley, S.J., Tedesco, D., Buia, M.C., 2008. Volcanic carbon dioxide ventsshow ecosystem effects of ocean acidification. Nature 454, 96–99.

Hansell, A.L., Horwell, C.J., Oppenheimer, C., 2006. The health hazards of volcanoesand geothermal areas. Occup. Environ. Med. 63, 149–156.

Haugan, P.M., Turley, C., Pörtner, H.O., 2006. Effects on the Marine Environment ofOcean Acidification Resulting from Elevated Levels of CO2 in the Atmosphere.Report on Ocean Acidification OSPAR.

Instituto Hidrográfico, 2000. Roteiro da Costa de Portugal – Arquipélago dos Açores,2. Edição (PUB. IH-128-NV), Lisboa.

Jordanova, A., Strezov, A., Ayranov, M., Petkov, N., Stoilova, L., 1999. Heavy metalassessment in algae, sediments and water from the Bulgarian black sea coast.Water Sci. Technol. 39, 207–212.

Kamenos, N.A., Cusack, M., Moore, P.G., 2008. Coralline algae are globalpalaeothermometers with bi-weekly resolution. Geochim. Cosmochim. Acta72, 771–779.

Kelaher, B.P., Chapman, M.G., Underwood, A.J., 2001. Spatial patterns of diversemacrofaunal assemblages in coralline turf and their associations withenvironmental variables. J. Mar. Biol. Ass. UK 81, 917–930.

Kleypas, J.A., Feely, R.A., Fabry, V.J., Langdon, C., Sabine, C.L., Robbins, L.L., 2006.Impacts of Ocean Acidification on Coral Reefs and Other Marine Calcifiers: AGuide for Future Research. Report of a Workshop held 18–20 April 2005, St.Petersburg, FL.

Kut, D., Topcuoglu, S., Esen, N., Küçükcezzar, R., Güven, K.C., 2000. Trace metals inmarine algae and sediment samples from the Bosporus. Water Air Soil Pollut.118, 27–33.

Littler, M.M., Littler, D.S., Taylor, P.R., 1983. Evolutionary stages in a tropical barrierreef system: functional-form groups of marine macroalgae. J. Phycol. 19, 229–237.

Lobban, C.S., Harrisson, P.J., Duncan, M.J., 1985. The Physiological Ecology ofSeaweeds. Cambridge University Press, Cambridge.

Martin, S., Rodolfo-Metalpa, R., Ransome, E., Rowley, S.J., Buia, M.C., Gattuso, J.P.,Hall-Spencer, J.M., 2008. Effects of naturally acidified seawater on seagrasscalcareous epibionts. Biol. Lett. 4, 693–695.

Martins, G.M., Thompson, R.C., Hawkins, S.J., Neto, A.I., Jenkins, S.R., 2008. Rockyintertidal community structure in oceanic islands: scales of spatial variability.Mar. Ecol. Prog. Ser. 356, 15–24.

Misheer, N., Kindness, A., Jonnalagadda, S., 2006. Elemental uptake by seaweed,Plocamium corallorhirza along the KwaZulu-Natal Coast of Indian Ocean, SouthAfrica. J. Environ. Sci. Health B 41, 1037–1048.

Mohamed, L., Khaled, A., 2005. Comparative study of heavy metal distribution insome coastal seaweeds of Alexandria. Egypt. Chem. Ecol. 21, 181–189.

Papageorgiou, S.K., Katsaros, F.K., Kouvelos, E.P., Nolan, J.W., Deit, H.L.,Kanellopoulos, N.K., 2006. Heavy metal sorption by calcium alginate beadsfrom Laminaria digitata. J. Hazard. Mater. 137, 1765–1772.

Pearson, P.N., Palmer, M.R., 2000. Atmospheric carbon dioxide concentrations overthe past 60 million years. Nature 406, 695–699.

Ries, J.B., 2006. Mg fractionation in crustose coralline algae: Geochemical, biological,and sedimentological implications of secular variation in the Mg/Ca ratio ofseawater. Geochim. Cosmochim. Acta 70, 891–900.

Rodrigues, A., Cunha, L., Amaral, A., Medeiros, J., Garcia, P., 2008. Bioavailability ofheavy metals and their effects on the midgut cells of a phytophagous insectinhabiting volcanic environments. Sci. Total. Environ. 406, 116–122.

Royal Society, 2005. Ocean Acidification due to Increasing Atmospheric Carbondioxide. Policy Document 12/05. Royal Society, London.

Stanley, S.M., Ries, J.B., Hardie, L.A., 2002. Low-magnesium calcite produced bycoralline algae in seawater of late Creataceous composition. PNAS 99, 15323–15326.

Stengel, D.B., Macken, A., Morrison, L., Morley, N., 2004. Zinc concentrations inmarine macroalgae and a lichen from western Ireland in relation tophylogenetic grouping, habitat and morphology. Mar. Pollut. Bull. 48, 902–909.

Terlizzi, A., Benedetti-Cecchi, L., Bevilacqua, S., Fraschetti, S., Guidetti, P., Anderson,M.J., 2005. Multivariate and univariate asymmetrical analyses in environmentalimpact assessment: a case study of Mediterranean subtidal sessile assemblages.Mar. Ecol. Prog. Ser. 289, 27–42.

Tittley, I., Neto, A.I., 2000. A provisional classification of algal-characterised rockyshore biotopes in the azores. Hydrobiologia 440, 19–25.

Underwood, A.J., 1997. Experiments in Ecology: Their Logical Design andInterpretation Using Analysis of Variance. Cambridge University Press,Cambridge.

Wallenstein, F.F.M.M., Neto, A.I., 2006. Intertidal rocky shore biotopes of the Azores:a quantitative approach. Helgol. Mar. Res. 60, 196–206.

Wallenstein, F.M., Neto, A.I., Alvaro, N.V., Santos, C.I., 2008. Algae-based biotopes ofthe Azores (Portugal): spatial and seasonal variation. Aquat. Ecol. 42, 547–559.

Wallenstein, F.M., Couto, R.P., Amaral, A.S., Wilkinson, M., Neto, A.I., Rodrigues, A.S.,2009. Baseline metal concentrations in marine algae from São Miguel (Azores)under different ecological conditions–Urban proximity and shallow waterhydrothermal activity. Mar. Pollut. Bull. 58, 424–455.

Zaldibar, B., Rodrigues, A., Lopes, M., Amaral, A., Marigomez, I., Soto, M., 2006.Freshwater molluscs from volcanic areas as model organisms to assessadaptation to metal chronic pollution. Sci. Total. Environ. 371, 168–175.