The Amphipods Caprella Penantis and Hyale Schmidtii As Biomonitors of Trace Metal Contamination in Intertidal Ecosystems of Algeciras Bay, Southern Spain

Baseline / Marine Pollution Bulletin 58 (2009) 765–786 783 The amphipods Caprella penantis and Hyale schmidtii as biomonitors of trace metal contamination in intertidal ecosystems of Algeciras Bay, Southern Spain

J.M. Guerra-García *, E. Baeza-Rojano, M.P. Cabezas, J.J. Díaz-Pavón, I. Pacios, J.C. García-Gómez

Laboratorio de Biología Marina, Departamento de Fisiología y Zoología, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes 6, 41012 Sevilla, Spain

Nowadays, quick and effective environmental studies in coastal individuals/m2 in Japanese habitats of Sargassum yezoense (Takeu- areas are increasingly being demanded as useful tools for monitor- chi, 1998). Moreover, Caprella penantis has been considered a sen- ing programmes. There is currently a great deal of interest in using sitive species, mainly living in unperturbed areas with high living organisms as pollution biomonitors in aquatic ecosystems, hydrodynamics and low values of organic matter and suspended given that the method used traditionally – chemical analysis of solids (Guerra-García and García-Gómez, 2001). Nevertheless, the the water – does not provide information on the bioavailability potential value of these species as heavy metal biomonitor had of metals present in the environment (Morillo et al., 2005). Fur- not been explored yet in previous studies. thermore, the metal concentrations in water often lie near or be- We selected Algeciras Bay, Southern Spain, as the area to be low the detection limit of instruments and they fluctuate studied for several reasons: (1) it is located in the Strait of Gibral- drastically, depending on water flow and intermittence of dis- tar, with an intense shipping traffic; (2) it is an important indus- charge (Rainbow, 1995). trial area, with chemical factories, refineries, thermal power Trace metals are taken up and subsequently accumulated by plants, iron works, paper mills and ship yards; (3) several previous organisms from both natural sources and from contaminated efflu- ecological studies based on spatial distribution of species and ents which enter estuarine and marine ecosystems through direct physicochemical data have demonstrated a clear gradient of pollu- discharges from coastal communities, ships, rivers and atmo- tion from internal to external stations (sponges: Carballo et al., spheric deposition, and through land run-off (Marsden and Rain- 1996; ascidians, Naranjo et al., 1996; epifauna of sublittoral algae bow, 2004). The term biomonitors denotes those species which and briozoa: Conradi et al., 1997; Sánchez-Moyano and García- accumulate trace metals in their tissues and may therefore be ana- Gómez, 1998; Sánchez-Moyano et al., 2000a,b, 2002; intertidal lysed to monitor the bioavailability of such contaminants in the communities: Fa et al., 2002; Guerra-García et al., 2006). ecosystems (Rainbow and Phillips, 1993). The organisms most Amphipods were sampled in the intertidal area at eight stations commonly used for biomonitoring are bivalve molluscs, especially around Algeciras Bay (Fig. 1) during June 2008. Caprella penantis mussels and oyster of the genera Mytilus, Perna and Crassostrea was collected from the seaweed Asparagopsis armata and Hyale smi- (Rainbow and Phillips, 1993; Morillo et al., 2005). Among crusta- chdtii from Corallina elongata. The animals were transported to the ceans, decapods (crabs, lobsters, prawns, etc.) are large and may laboratory in plastic containers, sorted by species and then killed by generally be easily identified, but they are usually mobile and often freezing. At each station, several environmental parameters were regulate their tissue concentrations of particular trace elements measured ‘‘in situ”: water temperature and oxygen concentration such as copper, manganese and zinc (Rainbow, 1998). However, were measured with an oxymeter Crison Oxi 45 P, and turbidity barnacles and talitridean amphipods appear to have particular was measured in nephelometric turbidity units (ntu) using a porta- promise as cosmopolitan biomonitors (Rainbow and Moore, ble turbidimeter WTW Turb 355 IR. Additionally, water samples for 1986; Rainbow and Phillips, 1993; Ugolini et al., 2004, 2005). metal analyses were taken from the same sampling sites using Amphipod crustaceans have been proposed as model biomonitor polyethylene bottles. Amphipod samples were dried at 30 °C until organisms since they are extremely widespread, occur in high den- constant weight and then finely ground. The dry, powdered solid sities in some communities, are often the principal food for preda- tissue was accurately weighed in a dry, pre-cleaned Teflon diges- tory fishes and birds, and there is considerable potential for metal tion vessel. 2 ml of HNO3, 1 ml of HCl and 3 ml of H2O2 were added accumulated by amphipods to be transferred along marine food to each vessel. The vessels were sealed and place in the microwave chains (Marsden and Rainbow, 2004). Although amphipods are chamber Anton Paar, Multiwave 3000 with a rotating 16-position usually proposed as marine bioindicators based on ecological stud- sample carousel, at 240 °C for 20 min with a maximum pressure ies of species distribution (see Conradi et al., 1997; Guerra-García of 40 bar. After digestion, the solution was brought to 25 ml volume and García-Gómez, 2001), the number of studies quantifying the with deionised waters. Analytical determinations were performed trace metal concentrations in amphipods to tests its validity as bio- by using Inductively Coupled Plasma-Optical Emission Spectropho- monitors is rather low. In fact, most of the studies only provide tometer (ICP-OES Horiba Jobin-Yvon, Ultima 2). Patterns used for measures of heavy metal concentrations in talitridean amphipods ICP measures included Merck ICP multielement calibration stan- (Rainbow and Moore, 1986), and caprellids, an important amphi- dard solution IV (HC612727) and XII (OC461429). The following pod group have not been explored in this sense yet. trace metals were analysed both, in water and amphipod samples: For this study, we selected two amphipods, the gammarid Hyale Ag, As, Cr, Cu, Fe, Hg, Mn, Ni, Pb, V and Zn. The degree of human schmidtii and the caprellid Caprella penantis. These species are pressures at the stations was estimated with the index of general among the dominant taxa in intertidal communities and shallow anthropogenicP stress (Fa, 1998). The index was calculated by the waters. Hyale schmidtii is the most common Hyale in the Mediter- equation S =( Pa/Dt) Â A, where S is the general anthropogenic ranean (Krapp-Schickel, 1993) reaching densities of more than stress, Pa is the total area of each of the main population centres 2 25,000 individuals/m (Bellan-Santini, 1969). Caprella penantis is in a 5 km radius from the site, Dt is the distance of the focus of each a cosmopolitan species which reaches densities higher than population centre to the site, and A is a measure of disturbance 20,000 individuals/m2 in intertidal seaweeds of temperate ecosys- based on a subjective assessment of both ease of access and levels tems (pers. observ.; Guerra-García, 2001) and can exceed 50,000 of human activity, ranked in a scale 1–5 (see Fa, 1998; Guerra-Gar- cía et al., 2004, in press). * Corresponding author. Tel.: +34 954556229; fax: +34 954233480. Water temperature and turbidity values increased in internal E-mail address: [email protected] (J.M. Guerra-García). stations while oxygen concentration was higher in external sites 784 Baseline / Marine Pollution Bulletin 58 (2009) 765–786

8 5 4 6 Iberian Peninsula

Algeciras Bay

2 7

Fig. 1. Map of the study area showing the sampling stations.

(Fig. 2). Internal stations 3, 4, 5 and 6 showed values of anthropo- penantis was more sensitive to stressful conditions, and only could genic stress clearly higher than external stations 1, 2, 7 and 8. In be found in the external stations 1, 2 and 3. Actually, Caprella fact, observations by Fa (1998) indicated that sites under high lev- penantis showed higher concentrations for Cu, Fe, Mn, Ni and Zn els of human pressure are characterised by values of the index of than Hyale schmidtii. Both species, showed a very clear pattern of anthropogenic stress >20, as those registered in internal sites. Con- higher accumulation in internal versus external stations, becoming sequently, environmental measures of this and previous studies interesting taxa to be use as trace metal biomonitors. (see Guerra-García et al., 2006) conﬁrm the pollution gradient The capacity of Caprella penantis in accumulating trace metals along Algeciras Bay stations. In spite of these evidences, measures was higher than Hyale schmidtii, and the range of values is among of heavy metal concentrations in water samples were all below the highest when compared with other amphipods, mostly talitr- detection limit, as in majority of studies (Morillo et al., 2005). How- ids, used as biomonitors in previous studies (Table 2). Caprella ever, most of metals (except for Ag, Cr, Hg and Pb) were accumu- penantis is the ﬁrst caprellid investigated regarding trace metals lated and concentrated by Caprella penantis and Hyale schmidtii accumulation; future studies should be conducted to explore the (Table 1). While Hyale schmidtii was present in all stations, Caprella better potential capacity of these amphipods as biomonitors in comparison with the gammarideans. Recently, several studies have demonstrated that caprellids are extremely sensitive to TBT (Aono TURBIDITY (ntu) OXYGEN (mg/l) and Takeuchi, 2008); they may have lower metabolic capacity to 4 12 degradate TBT than gammarids and, consequently, accumulate it 11 in higher concentrations and have been proposed as excellent 3 10 organisms to monitor temporal and spatial changes in baseline concentrations of butyltins (Takeuchi et al., 2001; Ohji et al., 2 9 2002). Similar reasons could be involved in the higher rate of metal 8 accumulation in Caprella penantis in comparison with gammari- 1 7 dean species. Crustacean employ different physiological strategies

0 6 to avoid toxic effects when accumulating trace metals (see Rain- 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 bow, 2002). For example, decapods regulate body concentrations of essential metals like cooper and zinc; barnacles on the other TEMPERATURE (ºC) ANTHROPOGENIC STRESS hand accumulate high concentrations of these metals with associ- 22 150 ated detoxiﬁcation processes (Rainbow, 2002). In amphipods, accumulated body metal concentrations are the best biomarkers for environmental metal availabilities (Marsden and Rainbow, 20 100 2004). Amphipods appear to store accumulated trace metals from solution and food storing the metal accumulated in the ventral cae- ca of the midgut. So, trace metals accumulated from the diet are 18 50 detoxiﬁed and stored in granules of ventral caecal cells, with more granules present in amphipods from contaminated sites. These 16 0 granules are eventually (up to 30 days) discharged into the alimen- 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 tary tract whence they are excreted (see Rainbow, 2002; Marsden and Rainbow, 2004). Metal accumulation affects the ecology of ani- Fig. 2. Values of turbidity, oxygen and temperature measured in water of the eight stations (Mean ± SD, n = 6). Values of the index of anthropogenic stress (see Fa, mals as a consequence of the energy costs associated with excret- 1998) are also included. ing the incoming metals. Maybe, the situation in caprellidean Baseline / Marine Pollution Bulletin 58 (2009) 765–786 785

Table 1 Metal concentrations (ppm, dry weight) in Caprella penantis and Hyale schmidtii. Mean ± SD. Caprella penantis was absent in stations 4–8.

As Cu Fe Mn Ni Zn Caprella penantis Leach, 1814 St. 1 <0.008 7.0 ± 1.3 117.1 ± 0.5 21.4 ± 1.6 <0.002 81.2 ± 1.0 St. 2 <0.008 <0.008 217.5 ± 0.9 43.9 ± 0.8 <0.002 116.4 ± 0.9 St. 3 <0.008 173.0 ± 1.7 1886.1 ± 1.8 122.4 ± 1.6 21.1 ± 7.8 544.3 ± 0.5 Hyale schmidtii (Heller, 1866) St. 1 <0.008 <0.008 28.3 ± 1.9 67.8 ± 2.1 <0.002 21.6 ± 1.0 St. 2 <0.008 <0.008 55.5 ± 2.6 7.9 ± 0.7 <0.002 24.5 ± 1.5 St. 3 5.7 ± 5.4 85.8 ± 0.9 82.9 ± 1.2 9.9 ± 1.6 4.2 ± 2.7 41.5 ± 0.4 St. 4 7.8 ± 7.1 42.5 ± 1.3 218.7 ± 0.9 27.9 ± 0.6 2.8 ± 2.7 95.6 ± 0.7 St. 5 7.1 ± 3.1 33.7 ± 0.9 339.8 ± 0.7 17.3 ± 0.8 <0.002 65.9 ± 1.4 St. 6 <0.008 <0.008 255.1 ± 2.2 18.2 ± 0.4 <0.002 69.9 ± 0.9 St. 7 <0.008 <0.008 155.1 ± 0.7 6.4 ± 1.7 <0.002 48.0 ± 1.6 St. 8 <0.008 <0.008 30.8 ± 4.4 16.2 ± 1.9 <0.002 32.5 ± 2.6

Table 2 Metal concentrations (ppm, dry weight) in a range of amphipods in previous studies.

Species Location Cu Fe Zn Reference Caprella penantis Spain <0.008–173.0 117.1–1886.1 81.2–544.3 Present study Corophium volutator Sweeden 85.8–115.0 – – Eriksson and Weeks (1994) Gammarus lacusta Scotland 35.2–200.0 71.3–1290.0 81.3–562.0 Rainbow and Moore (1986) Hyale nilsooni Scotland 18.6–53.3 – 15.9–102.0 Moore and Rainbow (1987) Hyale perieri Azores 37.3–92.8 – – Moore and Rainbow (1987) Hyale schmidtii Spain <0.008–85.8 28.3–339.8 21.6–95.6 Present study Orchestia gammarellus Scotland 55.5–123.0 68.9–1800.0 132.0–361.0 Rainbow and Moore (1986) Scotland 46.4–87.9 – 127.0–271.0 Rainbow et al. (1999) England 99.1–185.0 – 143.0–195.0 Moore et al. (1995) France 49.8–70.5 – 103.0–224.0 Rainbow et al. (1999) France 45.1–65.1 – 112.0–136.0 Rainbow et al. (1999) Orchestia mediterranea Scotland 23.6–62.9 – – Marsden and Rainbow (2004) Orchestia tenuis New Zealand 30.5–48.8 – – Rainbow et al. (1993a,b) Paracorophium excavatum New Zealand 90.0–150.0 – – Eriksson and Weeks (1994) Platorchestia platensis Hong Kong 61.4–130.0 – – Marsden and Rainbow (2004) Talitrus saltator Poland 49.7–70.4 161.0–492.0 162.0–262.0 Rainbow et al. (1998a,b) Poland 39.2–57.4 137.0–554.0 94.2–148.0 Fialkowski et al. (2000) Scotland 16.2–40.6 – – Rainbow and Moore (1990) Italy 32.6–87.7 108.7–540.0 112.0–236.0 Ugolini et al. (2004) Talorchestia quoyana New Zealand 15.6–48.2 – 481 Rainbow et al. (1993a)

amphipods is not the same than in gammarids, the excretion Fa. D.A., 1998. The influence of pattern and scale on the rocky-shore macrobenthic capacity could be more limited explaining the higher concentra- communities through the Strait of Gibraltar. Ph.D. Thesis, University of Southampton, UK. tions accumulated. Further research with more caprellid species Fa, D.A., Finlayson, C., García-Adiego, E., Sánchez-Moyano, J.E., García-Gómez, J.C., is necessary to better understand the significance of accumulated 2002. Influence of some environmental factors on the structure and distribution metal concentrations in amphipods. of the rocky shore macrobenthic communities in the Bay of Gibraltar: preliminary results. Almoraima 28, 73–88. Fialkowski, W., Rainbow, P.S., Fialkowska, E., Smith, B.D., 2000. Biomonitoring of Acknowledgements trace metals along the Baltic Coast of Poland using the sandhopper Talitrus saltator (Montagu) (Crustacea: Amphipoda). Ophelia 52, 183–192. Financial support of this work was provided by the Ministerio de Guerra-García, J.M., 2001. Habitat use of the Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa. Ophelia 55, 27–38. Educación y Ciencia (Project CGL2007-60044/BOS) co-financed by Guerra-García, J.M., García-Gómez, J.C., 2001. The spatial distribution of Caprellidea FEDER funds, and by the Consejería de Innovación, Ciencia y Empresa, (Crustacea: Amphipoda): a stress bioindicator in Ceuta (North Africa, Gibraltar Junta de Andalucía (Project P07-RNM-02524). area). PSZNI Marine Ecology 22, 357–367. Guerra-García, J.M., Corzo, J., Espinosa, F., García-Gómez, J.C., 2004. Assessing habitat use of the endangered marine mollusc Patella ferruginea (Gastropoda, Patellidae) in northern Africa: preliminary results and implication for References conservation. Biological Conservation 116, 319–326. Guerra-García, J.M., Maestre, M., González, A.R., García-Gómez, J.C., 2006. Assessing Aono, A., Takeuchi, I., 2008. Effects of tributyltin at concentrations below ambient a quick monitoring method using rocky intertidal communities as bioindicator: levels in seawater on Caprella danilevskii (Crustacea: Amphipoda: Caprellidae). a multivariate approach in Algeciras Bay. Environmental Monitoring and Marine Pollution Bulletin 57, 515–523. Assessment 116, 345–361. Bellan-Santini, D., 1969. Contribution à l’étude des peuplements infralittoraux sur Guerra-García, J.M., Cabezas, P., Baeza-Rojano, E., Espinosa, F., García-Gómez, J.C., in substrat rocheux (étude qualitative et quantitative de la frange supérieure). press. Is the north side of the Strait of Gibraltar more diverse than the south Recueil des travaux de la Station marine d’Endoume 63, 1–294. side? A case study using the intertidal peracarids (Crustacea: Malacostraca) Carballo, J.L., Naranjo, S.A., García-Gómez, J.C., 1996. Use of marine sponges as stress associated to the seaweed Corallina elongata. Journal of the Marine Biological indicators in marine ecosystems at Algeciras Bay (southern Iberian Peninsula). Association of the United Kingdom, doi:10.1017/s0025315409002938. Marine Ecology Progress Series 135, 109–122. Krapp-Schickel, T., 1993. Genus Hyale. In: Ruffo, S. (Ed.), The Amphipoda of the Conradi, M., López-González, P.J., García-Gómez, C., 1997. The amphipod Mediterranean. Part 3. Mémoires d l’Institut océanographique, Monaco, pp. community as a bioindicator in Algeciras Bay (Southern Iberian Peninsula) 728–738. based on a spatio-temporal distribution. PSZNI Marine Ecology 18, 97–111. Marsden, I.D., Rainbow, P.S., 2004. Does the accumulation of trace metals in Eriksson, S.P., Weeks, J.M., 1994. Effects of copper on hypoxia on two populations of the crustaceans affect their ecology-the amphipod example? Journal of benthic amphipod Corophium volutator (Pallas). Aquatic Toxicology 29, 73–81. Experimental Marine Biology and Ecology 300, 373–408. 786 Baseline / Marine Pollution Bulletin 58 (2009) 765–786

Moore, P.G., Rainbow, P.S., 1987. Copper and zinc in an ecological series of Rainbow, P.S., Fialkowski, W., Smith, B.D., 1998b. The sandhopper Talitrus saltator as talitroidean Amphipoda (Crustacea). Oecologia 73, 120–126. a trace metal biomonitor in the Gulf of Gdansk, Poland. Marine Pollution Moore, P.G., Rainbow, P.S., Weeks, J.M., Smith, B., 1995. Observations on copper and Bulletin 36, 193–200. zinc in an ecological series of talitroidean amphipods (Crustacea: Amphipoda) Rainbow, P.S., Amiard-Triquet, C., Amiard, J.C., Smith, B.D., Best, S.L., Nassiri, Y., from the Azores. Acoreana Supplement, 93–102. Langston, W.J., 1999. Trace metal uptake rates in crustaceans (amphipods and Morillo, J., Usero, J., Gracia, I., 2005. Biomonitoring of trace metals in a mine- crabas) from coastal sites in NW Europe differentially enriched with trace polluted estuarine system (Spain). Chemosphere 58, 1421–1430. metals. Marine Ecology Progress Series 183, 189–203. Naranjo, S.A., Carballo, J.L., García-Gómez, J.C., 1996. Effects of environmental stress Sánchez-Moyano, J.E., García-Gómez, J.C., 1998. The arthropod community, on ascidian populations in Algeciras Bay (Southern Spain). Possible marine especially Crustacea, as a bioindicator in Algeciras Bay (Southern Spain) based bioindicators? Marine Ecology Progress Series 144, 119–131. on a spatial distribution. Journal of Coastal Research 14, 1119–1133. Ohji, M., Takeuchi, I., Takahashi, S., Tanabe, S., Miyazaki, N., 2002. Differences in the Sánchez-Moyano, J.E., Estacio, F., García-Adiego, E.M., García-Gómez, J.C., 2000a. The acute toxicities of tributyltin between the Caprellidea and the Gammaridea molluscan epifauna of the alga Halopteris scoparia in Southern Spain as a (Crustacea: Amphipoda). Marine Pollution Bulletin 44, 16–24. bioindicator of coastal environmental conditions. Journal of Molluscan Studies Rainbow, P.S., 1995. Biomonitoring of heavy metal availability in the marine 66, 431–448. environment. Marine Pollution Bulletin 31, 183–192. Sánchez-Moyano, J.E., García-Adiego, E.M., Estacio, F., García-Gómez, J.C., 2000b. Rainbow, P.S., 1998. Phylogeny of trace metal accumulation in crustaceans. In: Effect of environmental factors on the spatial distribution of the epifauna of the Langston, W.J., Bebianno, M. (Eds.), Metal Metabolism in Aquatic Environments. alga Halopteris scoparia in Algeciras Bay, Southern Spain. Aquatic Ecology 34, Chapman and Hall, London, pp. 285–319. 355–367. Rainbow, P.S., 2002. Trace metal concentrations in aquatic invertebrates: why and Sánchez-Moyano, J.E., García-Adiego, E.M., Estacio, F., García-Gómez, J.C., 2002. so what? Environmental Pollution 120, 497–507. Effect of environmental factors on the spatial variation of the epifaunal Rainbow, P.S., Moore, P.G., 1986. Comparative metal analyses in amphipod polychaetes of the alga Halopteris scoparia in Algeciras Bay (Strait of crustaceans. Hydrobiologia 141, 273–289. Gibraltar). Hydrobiologia 470, 133–148. Rainbow, P.S., Moore, P.G., 1990. Seasonal variation in copper and zinc concentrations Takeuchi, I., 1998. Dry weight, carbon and nitrogen components of caprellid in three talitrid amphipods (Crustacea). Hydrobiologia 196, 65–72. amphipods (Crustacea) inhabiting the Sargassum yezoense community of Rainbow, P.S., Phillips, D.J.H., 1993. Cosmopolitan biomonitors of trace metals. Otsuchi Bay, northeastern Japan. Marine Biology 130, 417–423. Marine Pollution Bulletin 26, 593–601. Takeuchi, I., Takahashi, S., Tanabe, S., Miyazaki, N., 2001. Caprella watch: a new Rainbow, P.S., Emson, R.H., Smith, B.D., Moore, P.G., Mladenov, P.V., 1993a. Talitrid approach for monitoring butyltin residues in the ocean. Marine Environmental amphipods as biomonitors of trace metals near Dunedin, New Zealand. Journal Research 52, 97–113. of Marine and Freshwater Research 27, 201–207. Ugolini, A., Borghini, F., Calosi, P., Bazzicalupo, M., Chelazzi, G., Focardi, S., 2004. Rainbow, P.S., Malik, I., O’Brien, P., 1993b. Physicochemical and physiological effects Mediterranean Talitrus saltator (Crustacea, Amphipoda) as a biomonitor of on the uptake of dissolved zinc and cadmium by the amphipod crustacean heavy metals contamination. Marine Pollution Bulletin 48, 526–532. Orchestia gammarellus. Aquatic Toxicology 25, 15–30. Ugolini, A., Borghini, F., Focardi, S., Chelazzi, G., 2005. Heavy metals accumulation in Rainbow, P.S., Moore, P.G., Watson, D., 1998a. Talitrid amphipods (Crustacea) as bio- two syntopic sandhopper species: Talitrus saltator (Montagu) and Talorchestia monitors for copper and zinc. Estuarine, Coastal and Shelf Science 28, 567–582. ugolinii Bellan Santini and Ruffo. Marine Pollution Bulletin 50, 1328–1334.