Effects of metal contamination in situ on osmoregulation and oxygen consumption in the mudflat fiddler crab Uca rapax (Ocypodidae, Brachyura)

    Effects of metal contamination in situ on osmoregulation and oxygen consumption in the mudflat fiddler crab Uca rapax (Ocypodidae, Brachyura) Mariana V. Capparelli, Denis M. Abessa, John C. McNamara PII: DOI: Reference:

S1532-0456(16)30038-2 doi: 10.1016/j.cbpc.2016.03.004 CBC 8200

To appear in:

Comparative Biochemistry and Physiology Part C

Received date: Revised date: Accepted date:

17 December 2015 4 March 2016 9 March 2016

Please cite this article as: Capparelli, Mariana V., Abessa, Denis M., McNamara, John C., Effects of metal contamination in situ on osmoregulation and oxygen consumption in the mudflat fiddler crab Uca rapax (Ocypodidae, Brachyura), Comparative Biochemistry and Physiology Part C (2016), doi: 10.1016/j.cbpc.2016.03.004

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ACCEPTED MANUSCRIPT Effects of metal contamination in situ on osmoregulation and oxygen consumption in

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the mudflat fiddler crab Uca rapax (Ocypodidae, Brachyura)

Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,

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Mariana V. Capparelli1*, Denis M. Abessa2 and John C. McNamara1,3

Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil Universidade Estadual Paulista, Campus de São Vicente, São Vicente 11380-972, SP,

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Brazil

Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião 11600-000, SP,

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Brazil

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Running title: Osmoregulation and O2 consumption in metal-contaminated Uca rapax

Keywords: environmental contamination, metal pollution, osmoregulation, oxygen consumption, gill Na+/K+-ATPase activity, fiddler crab, Uca rapax

*Corresponding author: Dr. Mariana Vellosa Capparelli, Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900 Ribeirão Preto 14040-901 SP, Brasil. Tel. +55 16 33154395. E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract The contamination of estuaries by metals can impose additional stresses on estuarine species, which may exhibit a limited capability to adjust their regulatory processes and

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maintain physiological homeostasis. The mudflat fiddler crab Uca rapax is a typical estuarine crab, abundant in both pristine and contaminated areas along the Atlantic coast

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of Brazil. This study evaluates osmotic and ionic regulatory ability and gill Na+/K+ATPase activity in different salinities (<0.5, 25 and 60 ‰ S) and oxygen consumption

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rates at different temperatures (15, 25 and 35 °C) in U. rapax collected from localities along the coast of São Paulo State showing different histories of metal contamination

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(most contaminated Ilha Diana, Santos > Rio Itapanhaú, Bertioga > Picinguaba, Ubatuba [pristine reference site]). Our findings show that the contamination of Uca

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rapax by metals in situ leads to bioaccumulation and induces biochemical and physiological changes compared to crabs from the pristine locality. Uca rapax from the contaminated sites exhibit stronger hyper- and hypo-osmotic regulatory abilities and

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show greater gill Na+/K+-ATPase activities than crabs from the pristine site, revealing

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that the underlying biochemical machinery can maintain systemic physiological processes functioning well. However, oxygen consumption, particularly at elevated

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temperatures, decreases in crabs showing high bioaccumulation titers but increases in crabs with low/moderate bioaccumulation levels. These data show that Uca rapax chronically contaminated in situ exhibits compensatory biochemical and physiological adjustments, and reveal the importance of studies on organisms exposed to metals in situ, particularly estuarine invertebrates subject to frequent changes in natural environmental parameters like salinity and temperature.

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ACCEPTED MANUSCRIPT Introduction Estuarine environments are subject to widely fluctuating abiotic variables such as salinity, dissolved oxygen, pH and temperature all of which impose severe

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physiological restrictions on their inhabitants (Matthiessen & Law, 2002; Amado et al. 2006a). Despite the physiological stresses arising from this natural physico-chemical

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variation, estuarine organisms often exhibit compensatory mechanisms that maintain their physiological homeostasis (Mauro & Moore, 1987; Romano & Zeng, 2010).

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Importantly, estuaries are often located in areas suitable for human development, leading to multiple sources of environmental contamination (Basnyat et al. 1999,

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Lamparelli et al. 2001; Abreu-Mota et al. 2014).

Pollution in estuaries is a significant environmental issue, not only due to the

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ecological importance of estuaries, but also because contaminants constitute an additional stress on estuarine inhabitants (Depledge et al. 1995). However, since estuaries consist of ecosystems defined by dominant biological communities adapted to

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elevated spatial and temporal variability, the detection of stress of strictly anthropogenic

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origin is complex. This phenomenon has been termed the ‘estuarine quality paradox’ (Elliot & Quintino, 2007), and has lead to the development of strategies to minimize the

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interference of natural factors in stress analyses. These include the use of different ‘lines of evidence’ (Chapman, 2007a, b; Choueri et al. 2009) and the measurement of responses at the individual level as biochemical and physiological biomarkers (Stegeman et al. 1992; Cunha et al. 2007). Assessing and monitoring the environmental health of estuarine ecosystems in situ can be particularly difficult given the effects of multiple sources of contamination whose interactions are poorly known (Chapman et al.1987). Further, adjustments in the physiological and biochemical processes of estuarine species in response to long-term exposure to incipient or moderate levels of pollutants in situ have been little studied (Otitoloju & Don-Pedro, 2004). Most investigations evaluate responses to intense, short-term exposure to xenobiotics in the laboratory (Zanders & Rojas, 1996; Brown et al. 2003). Organisms inhabiting chronically polluted areas tend to exhibit a limited capability to adjust their regulatory processes to maintain physiological homeostasis and to reduce the effects of pollutants and other stressors (Harris & Santos, 2000). However, certain populations may develop tolerance to chronic exposure to moderate pollutant concentrations (Klerks & Weis, 1987). 3

ACCEPTED MANUSCRIPT Chronic exposure to metals affects the mechanisms of hemolymph osmotic and ionic regulation in fish and crustaceans (Zanders & Rojas, 1996, 1997; Lignot et al. 2000; Garcia-Santos et al. 2011; Rainbow and Black, 2005). At low salinity,

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hyperosmoregulating decapod crustaceans gain water by osmosis and lose ions by diffusion (Péqueux, 1995; Freire et al. 2008). To maintain salt balance, ions are actively

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captured by ion transporting pumps like the gill Na+/K+-ATPase (McNamara & Faria, 2012), which also increases the uptake of metal ions of similar size and charge

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(Nugegoda & Rainbow 1989; Rainbow, 1996). Such increased metal uptake can impair hyper-osmotic regulatory ability (Péqueux, 1995; Hebel et al. 1997, Bianchini et al.

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2008; Bȫttcher, 1991; 1993; Grosell & Wood, 2002; Bianchini et al. 2004). Oxygen consumption in decapod crustaceans is affected by factors including life

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cycle stage, sex (DeCoursey & Vernberg, 1972), molting cycle (Bergey & Weis, 2007), temperature (Vernberg & Vernberg, 1974), salinity stress (Depledge, 1987, Péqueux, 1995) and metal exposure. Compensatory changes, whether immediate or long term,

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require physiological and biochemical adjustments (Laird & Haefner, 2002; Bamber &

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Depledge, 1997). Oxygen consumption often decreases during acute exposure to metals, indicative of metal-induced pathological damage, owing to the direct inhibition of

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cellular respiration and interference in various respiratory processes (Depledge, 1984). However, oxygen consumption may remain unchanged or even increase, depending on metal concentration. These effects are not mutually exclusive and may be additive, the final effect depending on metal concentration and duration of exposure. Metal stress when combined with high temperature can result in metabolic deficiency, eventually affecting an organism’s fitness in polluted estuaries (Holmstrup, et al. 2010, Schiedek et al. 2006; Sokolova &Lannig, 2008, Pörtner, H.O., 2010). Fiddler crabs of the genus Uca are scavengers and feed mainly on microscopic algae and protozoa found on sand grains, and on organic matter brought on the high tide (Crane, 1975). The genus is typical of intertidal zones in which the crabs build burrows used as a mating site and as a refuge from potential predators, and from daily and seasonal fluctuations in natural environmental parameters. Most of the ten Uca species found in Brazil are gregarious and sympatric, and exhibit diurnal habits and peak activity during low tides (Powers & Cole, 1976; Masunari, 2006). The species used in this investigation is the semi-terrestrial, estuarine, mudflat fiddler crab Uca rapax (Crustacea, Ocypodidae), distributed from Florida (USA), the Gulf of Mexico, the 4

ACCEPTED MANUSCRIPT Antilles and Venezuela, and from Pará to Santa Catarina states in Brazil (Thurman et al. 2013). This land-mark crab inhabits estuarine areas constantly subjected to ample diurnal and seasonal fluctuations in temperature and salinity and shows great versatility

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in using substrates with different grain size characteristics (Thurman, 1984; 1987). In Brazil, the species is abundant in both pristine and chronically contaminated areas.

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The biological responses of adult Uca rapax to cadmium and salinity have been examined under laboratory conditions (Zanders & Rojas, 1997), as has the effect of

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mercury on larval oxygen consumption (DeCoursey & Vernberg, 1972). However, systemic physiological parameters like osmoregulation and oxygen consumption in Uca

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species subjected to different levels of environmental contamination in situ are entirely lacking, and no investigation has addressed the effect of metals on U. rapax from

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contaminated locations in situ.

The coast of São Paulo state in southeastern Brazil exhibits a clear gradient of environmental contamination that increases from the northern to the central coastal area

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(Figure 1). The Santos Estuarine System (SES), located within the central coastal zone,

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is a critical area that accumulates urban and industrial waste from the petrochemical pole in Cubatão, and is one of the most polluted estuaries on earth (CETESB, 2001).

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Within the SES, metals such as Cd, Zn, Ni and Cu are important contaminants at localities such as Ilha Diana and Rio Itapanhaú (Lamparelli et al., 2001; Abessa et al., 2008; Salaroli, 2013), while Ba is also a relevant contaminant in some areas. The present study aims to investigate adjustments in physiological (osmotic and ionic regulatory ability and oxygen consumption) and biochemical (gill Na+/K+-ATPase activity) biomarkers in an indicator species, the fiddler crab Uca rapax, collected from habitats located in two areas presenting a history of chronic, environmental metal contamination (Ilha Diana and Rio Itapanhaú), and from a pristine control area outside the Santos estuarine system (Picinguaba).

Materials and methods Site locations and characteristics Adult specimens of Uca rapax were collected during the winter of 2012 (June-July) and summer of 2013 (January-February) from three study sites located on the southern Atlantic coast of the State of São Paulo, Brazil (Figure 1). Sites were chosen based on

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ACCEPTED MANUSCRIPT their degree of anthropogenic contamination and on their distance from known sources of relevant pollution.

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Site 1. Ilha Diana, Santos (ID) (23º 55’ 04.5” S; 46° 18’ 31.5”W) is located within the Santos Estuarine System (SES), and is influenced by multiple sources of contamination

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such as heavy industries, sewage from specific and diffuse sources, urban drainage and storm waters, domestic and industrial landfills, and the Port of Santos. The SES is

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considered to be a critical area in terms of pollution, and various contaminants such as metals and aromatic hydrocarbons have been detected in potentially toxic

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concentrations (Lamparelli et al. 2001; Quináglia, 2006; Abessa et al. 2008). During crab collections, summer and winter water temperatures were 24 and 21 °C,

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respectively; air temperatures were 28 and 23 °C. Water salinities near the crabs’ burrows were 10 and 28 ‰ salinity.

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Site 2. The second sampling site was chosen in the lower portion of the Rio Itapanhaú,

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Bertioga (RI) (23º 50’ 0.2” S; 46° 09’ 10.6” W). This site is located within the northern region of the SES, and shows some degree of degradation due to local urban expansion,

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the discharge of domestic effluents, and the development of nautical infrastructure and sport fishing marinas (Zaroni, 2006; Cunha-Lignon et al. 2009c) while still receiving contaminants from the SES (Salaroli, 2013). During crab collections, summer and winter water temperatures were 25 and 21 °C, respectively; air temperatures were 28 and 23 °C. Water salinities near the crabs’ burrows were 13 and 10 ‰ salinity.

Site 3. The third sampling site was located in the estuary of the Rio da Fazenda, Picinguaba (P) (23º 22’ 73.0” S; 44° 50’ 50.0” W), situated within the Serra do Mar State Park at the Picinguaba headquarters, near Ubatuba. The park constitutes an ecological corridor of Atlantic Rainforest that is legally protected. This site is considered to be pristine, since it is distant from relevant sources of anthropogenic contamination. During crab collections, summer and winter water temperatures were 28 and 25 °C, respectively; air temperatures were 27 and 21 °C. Water salinities near the crabs’ burrows were 0 and 29 ‰ salinity.

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ACCEPTED MANUSCRIPT Crab collection and laboratory maintenance About 100 adult crabs were collected manually at each sampling site. Only nonovigerous, intermolt crabs of carapace width greater than 10 mm were used in the

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analyses. Crabs were transported to the laboratory in plastic boxes (15 x 15 x 6 cm) of approximately 1,500 cm3 volume, containing small sponge cubes moistened with water

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from the collection site. Approximately 20 crabs were placed in each box for transport. In the laboratory, the crabs were held in the same boxes containing water and a thin

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layer of sediment from the collection site, maintained on a slightly inclined plane so that the crabs had free access to a dry surface. Water in the boxes was renewed daily with

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water from the same collection site, and the boxes were cleaned. To adjust to laboratory conditions before use in the experiments, after collection the crabs were maintained

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unfed for three days at 25 ºC in an air conditioned room under a natural 12 h light: 12 h dark photoperiod.

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2.1. Sediment sampling and analysis

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A single sample of about ≈570 g of sediment was collected from the crabs’ burrows at each collecting site during the crab collections. For each sample, sediment

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from a circular area of 130 cm2 was sampled to a depth of 5 cm and transported to the laboratory in a sealed plastic container. A 50-g aliquot of each sediment sample was placed in screw top Falcon tube and the samples stored in a freezer at -20 ºC for later analysis of metal content.

Distribution of sediment grain sizes was analyzed based on the protocol proposed by Murdoch & MacKnight (1991), and the grain size distributions were classified using the Wentworth scale (Wentworth, 1922). The calcium carbonate content of each sample was measured using the method described by Hirota & Szyper (1976). The organic matter (OM) content of the sediment samples was estimated using the method described by Loring & Rantala (1992).

2.2 Metal content and concentrations in burrow sediments and crab tissues Analytical measurements of metal concentrations in ≈50-g aliquots of surface sediment (up to 5-cm depth) from U. rapax burrows, and in the crabs’ gills and hepatopancreas were performed employing inductively coupled plasma mass 7

ACCEPTED MANUSCRIPT spectrometry (ICP-MS, ELAN DRC II, Perkin Elmer), following a previously described methodology (Batista et al. 2009). All reagents used were of analytical grade except for HNO3 that was purified

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previously in a quartz sub-boiling distiller (Kürner Analysentechnik). Only high purity deionized water (18.2 Mohm-cm resistivity) obtained using a Milli-Q water purification

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system (Millipore, Bedford, MA, USA) was used. Standard multi-element (10 mg L-1) and rhodium (1000 mg L-1) solutions were obtained from PerkinElmer (Shelton, CT,

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USA).

Five grams of sediment or 0.1 g of each tissue was placed in a metal-free,

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propylene Falcon tube (Becton Dickinson) before analysis. Samples were weighed accurately into a PFA digestion vessel, and 5 mL of 14 mol L-1 nitric acid + 2 mL of

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30% H2O2 (v/v) were added. The vessel was irradiated in a microwave oven and decomposition was performed according to the following heating program: (i) step 1 (power 700 W, 4.5 min, 160 °C); (ii) step 2 (power 0 W, 0.5 min, 160 °C); (iii) step 3

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(power 800 W, 5.0 min, 230 °C); (iv) step 4 (power 0 W, 20 min, 35 °C) (adapted from

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Nardi et al., 2009). The samples were left to cool and the volume made up to 50 mL with Milli-Q water. Rhodium was then added as internal standard to a final

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concentration of 10 µg/L. The concentrations of the chemical elements Ba, Cd, Cu, Zn and Ni were then estimated using ICP-MS. These particular elements were chosen as they are important contaminants at the sites located within the Santos Estuarine System, i. e., Ilha Diana and Rio Itapanhaú.

2.3. Sediment metal enrichment factor and bioaccumulation index To quantify metal pollution in the sediment samples, a geo-accumulation index (IGeo) or sediment metal enrichment factor was used: IGeo = log2 [Cn (1.5 Bn)-1] where Cn is the measured concentration of an individual metal in sediment type ‘n’ and Bn is the geochemical background value of the metal. The adjustment factor ‘1.5’ in the equation compensates for possible variation in the background value due to lithogenic effects. The geo-accumulation index is used widely in geochemical evaluations of impacted environments (Förstner, 1983). Using the average composition of shale as a background reference allows comparison of the overall degree of contamination in 8

ACCEPTED MANUSCRIPT different areas. The geo-accumulation index is divided into seven grades: 0 (IGeo <0), 1 (IGeo >0 to 1), 2 (IGeo >1 to 2), 3 (IGeo >2 to 3), 4 (IGeo >3 or 4), 5 (IGeo >4 to 5) and 6 (IGeo >5). Grade 6 corresponds to an enrichment of 100 times greater than the average

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sediment composition. Bioaccumulation of each metal in the crab tissues is expressed as a metal

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bioaccumulation index and was estimated from the ratio of the concentration of each

sediment (Bechtel Jacobs Company, 1998).

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2.2. Osmotic and ionic regulatory ability

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metal in the gills or hepatopancreas to the corresponding metal concentration in the

Groups of 10 adult, male or female crabs chosen at random from each locality

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were placed individually into plastic boxes containing 50 mL of one of three experimental media to which they were then exposed for 5 days: (i) distilled water (<0.5 ‰ S, ≈6 mOsm/kg H2O, 2 mmol L-1 Cl-, a hypo-osmotic medium); (ii) an isosmotic

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salinity (25 ‰ S, 700 mOsm/kg H2O, 370 mmol L-1 Cl-); or (iii) a hyper-osmotic

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salinity (60 ‰ S, 1800 mOsm/kg H2O, 960 mmol L-1 Cl-). Saline media (25 and 60 ‰ S) were prepared by adding artificial sea salt (Instant

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Ocean) to distilled water (<0.5 ‰ S); salinities were verified using a hand-held optical refractometer (American Optical, Model 10419). Mortality was recorded every 24 h, and after 5 days a 50-µL hemolymph sample was taken from the ventral hemocele of each crab using a syringe and 13 x 0.3-gauge needle inserted through arthrodial membrane at the base of the last pereiopod. Osmolality was measured in 10-µL hemolymph aliquots using a Wescor 5500 (Logan, Utah) vapor pressure micro-osmometer. Cl- concentration was measured by microtitration (Schales & Schales, 1941, adapted by McNamara & Santos, 1996) in 10µL hemolymph aliquots using a microtitrator (Metron Herisou, type E 485), employing mercuric nitrate as the titrant and S-diphenylcarbazone as the indicator. Na+ concentration was measured by emission spectrophotometry using an atomic absorption spectrophotometer (GBC, model 932AA) in 10 µL of hemolymph, diluted 1: 20,000 times in distilled water.

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ACCEPTED MANUSCRIPT 2.3 Gill Na+/K+-ATPase activity Gill Na+/K+-ATPase activity was measured as the K+-phosphatase activity of the Na+/K+-ATPase in posterior gill homogenates prepared from groups of 10 crabs each,

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exposed to salinities of <0.5 (distilled water, hypo-osmotic medium), 25 (isosmotic medium) or 60 ‰ S (hyper-osmotic medium) for 5 days. After chilling the crabs briefly

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on ice, posterior gill pairs 6 to 8 from each crab were pooled (N=10) and homogenized in a dry ice/acetone bath using a 20 mmol L-1 Imidazole buffer, pH 6.8, containing 250

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mmol L-1 sucrose, 6 mmol L-1 EDTA and a protease inhibitor cocktail (homogenization buffer).

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The hydrolysis of p-nitrophenylphosphate ditris salt (pNPP) (K+-phosphatase activity) by each gill homogenate from each crab was assayed spectrophotometrically at

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410 nm and 25 °C, continuously monitoring the release of the p-nitrophenolate ion for 15 min using a microplate reader (Spectromax Plus 348) under standard conditions as described by Furriel et al. (2000). K+-phosphatase activity was assayed by adding an

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aliquot of each gill homogenate to a reaction medium containing KCl 10 mmol L-1

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MgCl2 5 mmol L-1, Hepes buffer 10 mmol L-1 pH 7.5, 10 mmol L-1 pNPP (total K+phosphatase activity) or the same medium containing 3 mmol L-1 ouabain (oubain

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insensitive activity). The difference in activities represents the K+-phosphatase activity.

2.4. Measurement of oxygen consumption rate Ten crabs (carapace width ≈15 mm, wet mass 1.5 ± 0.3 g) from each locality were weighed (Gehaka, BG 400, 1 mg accuracy), placed individually into acrylic respirometer chambers of 150-mL total volume, and held during a 12-h adjustment period at one of three temperatures, 15, 25 or 35 °C, in a constant temperature chamber (Fanem BOD incubator, ±0.5 °C precision). The respirometers were coupled by Crystal plastic tubing to a multiplexer manometer and a gas analyzer (Sable Systems, Las Vegas, NV), and were perfused with a constant flow of air from a small aquarium pump. Each respirometer contained a thin layer (3 mm deep) of brackish water from the collection site (salinity adjusted to 12 ‰ S) to allow gill wetting by the individual crabs. Oxygen

consumption

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measured

employing

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intermittent

respirometric technique (Steffensen, 1989). After the 12-h adjustment period, each respirometer was perfused with air at a standard rate of flow (180 mL min-1) via the multiplexer manometer leading to the O2 sensor and pump in the gas analyzer, 10

ACCEPTED MANUSCRIPT establishing the baseline percentage of atmospheric O2 (20.8%). The respirometers containing the crabs were then sealed by closing the respective manometer stopcocks and remained without air flow for 50 min. Airflow was then resumed and the output

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from each respirometer pumped to the O2 sensor that monitored the respective percentage of atmospheric O2 present in the air stream. The decrease in this percentage

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corresponds to the amount of O2 consumed by each crab in each respirometer during the sealed phase, compared to the baseline reading.

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Two consecutive replicates were performed for each crab at each temperature, the lowest value being used to calculate oxygen consumption rate, more indicative of

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standard metabolic rate. The data were collected using the Sable Systems Expedata application that enables calculation of the percentage decrease in oxygen concentration

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in the air flushed through each respirometer.

After the final reading, the crabs were removed from the respirometers, anesthetized in crushed ice and killed by freezing in a freezer at -20 °C for 10 min. They

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were then transferred to a drying oven (MA 033, Marconi) at 60 °C for 24 h, allowed to

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cool in a desiccator and quickly weighed using an analytical balance (Gehaka, BG 400, 1 mg accuracy) to obtain their dry mass. Oxygen consumption rate was calculated from the formula:

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Oxygen consumption (mLO2. g-1. h-1) = IA x flow rate (mL min-1)/[(100 × dry mass (g) x duration of sealed phase (min)] x 60 min where IA is the integral of the signal (% O2) received from the respirometer provided by the Expedata application; flow rate is flux of air (180 mL min-1) through the respirometer during the open phase; dry mass is the mass (g) of each crab after standard drying for 24 h at 60 °C; duration of sealed phase is the length of time during which each respirometer remained sealed (50 min). The factor ‘100’ in the denominator converts the percentage O2 values obtained from the IA values to volume of O2. The factor ‘60’ transforms the value obtained to an hourly rate.

2.5 Statistical analyses All data are given as the mean ± SEM. After satisfying criteria for normality of distribution and equality of variance, the data sets were analyzed using a three-way analysis of variance (locality, season and salinity) for the principal metal concentrations in the sediments and tissues, hemolymph osmolality, sodium and chloride 11

ACCEPTED MANUSCRIPT concentrations, gill K+-phosphatase activity and oxygen consumption rate. Two-way analyses of variance (locality and temperature or salinity) were also performed independently of season as a factor for the same parameters.

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Differences between means within the same parameter were established using the Student–Newman–Keuls multiple comparisons procedure. A minimum significance

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level of P=0.05 was employed throughout.

3. Results

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3.1. Burrow sediments: grain size, calcium carbonate and organic matter content Particle size analysis was used to characterize the sediment fractions from the

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specific localities where Uca rapax was collected. Sediments from Ilha Diana and Picinguaba consisted mainly of fine and very fine sand, together with silt, constituting ≈90% of the total sediment, in summer and winter. Rio Itapanhaú sediments exhibited

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representative percentages across all size fractions, although sandy fractions

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predominated (80%). Sediments from all localities contained about 60% calcium

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carbonate and around 20% organic matter (Table 1).

3.2. Metal contents and concentrations in burrow sediments and crab tissues Table 2 shows the metal contents and respective concentrations in the burrow sediments and in the gills and hepatopancreas of Uca rapax collected from the Picinguaba, Rio Itapanhaú and Ilha Diana localities during the winter of 2012 (JuneJuly) and summer of 2013 (January-February). The Rio Itapanhaú sediments exhibited higher metal concentrations compared to the other sites in both seasons. Specimens of Uca rapax from Rio Itapanhaú and Ilha Diana tended to show metal concentrations greater than those from Picinguaba. A detailed analysis of the metal distributions and their respective bioaccumulation indexes is given below. Picinguaba is considered to be the least polluted reference locality. Only barium and cadmium concentrations in the tissues were affected by season (3-way ANOVA, P<0.05). Crabs from Picinguaba collected in winter had more Ba and Cd in both tissues than in summer. Those from Rio Itapanhaú showed more Cd in the gills in winter, and more Ba and Cd in the hepatopancreas in summer. In crabs from Ilha

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ACCEPTED MANUSCRIPT Diana, Ba was highest in the hepatopancreas in winter and in the gills in summer. Cadmium was highest in the hepatopancreas in summer. Considering winter and summer seasons separately, Ba concentration was

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affected by both tissue and locality, including an interaction effect (2-way ANOVA, P<0.05). There was a greater accumulation of Ba in the hepatopancreas during winter

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and summer. Using Picinguaba as the least polluted reference locality, crabs collected from Rio Itapanhaú showed a higher Ba concentration in the gills and hepatopancreas

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during winter and summer. The Ba content in the burrow sediments of Uca rapax was 5- to 10-fold greater than in the tissues, and the bioaccumulation index was low (<1.0)

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in crabs from all localities and seasons. The bioaccumulation index for Ba in the hepatopancreas was higher than for the gills.

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Cadmium also was affected by tissue and locality with an interaction effect (twoway ANOVA, P<0.05). During summer, Cd concentration was greater in the hepatopancreas and gills of Rio Itapanhaú crabs. Cadmium concentration in the

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sediments was lower than in the tissues. The bioaccumulation index for Cd was >1 in

winter and summer.

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crabs from all locations, and highest in the hepatopancreas of Picinguaba crabs during

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Locality and tissue both affected Cu concentration (two-way ANOVA, P<0.05). In winter, Rio Itapanhaú crabs showed the highest Cu concentrations in the hepatopancreas, and in summer crabs from Rio Itapanhaú and Ilha Diana had higher Cu concentrations in the gills and hepatopancreas compared to Picinguaba crabs. Cu concentrations in the sediments from all localities were lower than in the crab tissues. The bioaccumulation indexes were >1 at all three localities in winter, and at Rio Itapanhaú and Ilha Diana during summer. The greatest bioaccumulation indexes for Cu were found for the hepatopancreas of crabs from Rio Itapanhaú and Ilha Diana. Zinc concentration was affected only by tissue in crabs collected in winter and summer (two-way ANOVA, P<0.05). In winter, crabs showed higher Zn concentration in the hepatopancreas and in the gills in summer. Zinc concentrations in the sediments were higher at the Rio Itapanhaú site in winter and summer compared to Picinguaba and Rio Itapanhaú. The highest bioaccumulation index for Zn was seen in crabs from Ilha Diana in both seasons. Nickel concentration was affected by locality and tissue with an interaction effect (two-way ANOVA, p<0.05). In winter, crabs showed a higher Ni concentration in 13

ACCEPTED MANUSCRIPT the hepatopancreas than in the gills, with no difference among localities. In summer, Ni concentration was highest in the hepatopancreas except for crabs from Rio Itapanhaú. Nickel concentration in sediments from all localities was 10- to 40-fold higher than in

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the crab tissues, and was highest at Picinguaba in winter and at Rio Itapanhaú in summer. Bioaccumulation indexes for Ni were higher in the hepatopancreas than the

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gills, and >1 in crabs from Ilha Diana in summer.

The enrichment factor (LGeo) was 1 for cadmium alone in sediments from Ilha

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Diana during summer. LGeo values for all other metals were close to zero at all localities

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and in both seasons.

3.3. Hemolymph osmotic and ionic regulatory ability

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A similar pattern of strong hyper-/hypo-osmotic regulatory abilities was disclosed for crabs from Picinguaba, Ilha Diana and Rio Itapanhaú (Figure 2, Winter, Summer). Compared to crabs exposed for 5 days to 25 ‰ S, hemolymph osmolality

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(≈800 mOsm/kg H2O) decreases to around ≈580 mOsm/kg H2O in those held in distilled

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water, and increases to ≈1,100 mOsm/kg H2O in those kept at 60 ‰ S. Crabs that show the highest hemolymph osmolalities in distilled water, and lowest values at 60 ‰ S are

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considered the better osmoregulators, since the osmotic gradients they maintain reflect stronger hyper- and hypo-osmotic regulatory ability, respectively. Season alone (P>0.05) had no effect on hemolymph osmolality in Uca rapax when also considering locality and exposure salinity (3-way ANOVA, P<0.05). However, there was a significant interaction effect between salinity (distilled water, 25 and 60 ‰ S) and locality (Picinguaba, Ilha Diana and Rio Itapanhaú) (P<0.05). Crabs collected in winter (Figure 2, Winter) from Ilha Diana and Rio Itapanhaú showed higher hemolymph osmolalities when in distilled water than did Picinguaba crabs. At 60 ‰ S, crabs from Ilha Diana and Rio Itapanhaú had lower hemolymph osmolalities than those from Picinguaba. In summer, hemolymph osmolality in distilled water was higher in crabs from Rio Itapanhaú compared to Ilha Diana and Picinguaba. There were no differences in hypo-regulatory ability at 60 ‰ S among the crabs from the different localities (Figure 2, Summer). [Na+] in the hemolymph of Uca rapax was unaffected by season (P>0.05) considering all factors together (locality, salinity, season) (3-way ANOVA, P<0.05). However, a significant interaction effect (P<0.05) was seen when assessing locality and 14

ACCEPTED MANUSCRIPT salinity independently of season. In crabs collected in winter (Figure 3, Winter), hemolymph [Na+] was highest in those from Rio Itapanhaú in distilled water and at 25 ‰ S compared to Picinguaba. There were no differences at 60 ‰ S. In crabs collected

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in summer, those from Ilha Diana and Rio Itapanhaú showed higher [Na+] than those from Picinguaba at 60 ‰ S, (Figure 3, Summer).

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[Cl-] in the hemolymph of Uca rapax is dependent on salinity and season (3-way ANOVA, P<0.05). Crabs collected in winter from Ilha Diana and Rio Itapanhaú showed

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higher hemolymph [Cl-] at 60 ‰ S than those from Picinguaba (Figure 4, Winter). In crabs collected in summer, hemolymph [Cl-] was lower in crabs from Rio Itapanhaú

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than from Picinguaba in distilled water, while at 25 ‰ S [Cl-] was higher in crabs from Ilha Diana and Rio Itapanhaú than in those from Picinguaba (Figure 4, Summer).

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3.3. Gill Na+/K+-ATPase (K+-phosphatase) activity

Gill K+-phosphatase activity was affected by locality, salinity and season with an

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interaction effect between locality and season (3-way ANOVA, P<0.05). Crabs from

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Ilha Diana and Rio Itapanhaú showed higher K+-phosphatase activities in distilled water in summer than winter (P<0.05) (Figure 5, Winter, Summer). When evaluating season

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separately, activities in crabs collected in winter and summer were affected by locality and salinity (2-way ANOVA, P<0.05). Crabs collected in winter from Ilha Diana and Rio Itapanhaú showed higher activities in distilled water compared to crabs from Picinguaba, both in winter and in summer (Figure 5, Winter, Summer). At 60 ‰ S, crabs from Ilha Diana and Rio Itapanhaú showed higher activities than the Picinguaba site also both in winter and in summer (Figure 5 Winter, Summer).

3.4. Oxygen consumption rate Locality, temperature and season all affect oxygen consumption rate, with an interaction effect between locality and temperature (3-way ANOVA, P<0.05). Oxygen consumption increased with increasing temperature in crabs from all locations and seasons. Analyzing season separately, oxygen consumption rates in crabs collected in winter and summer were affected by locality and temperature (2-way ANOVA, P<0.05). Crabs in winter showed higher consumption rates at 15 and 25 °C than crabs in summer (Figure 6, Winter, Summer). For crabs collected in winter (Figure 6, Winter), 15

ACCEPTED MANUSCRIPT those from Ilha Diana and Rio Itapanhaú showed higher oxygen consumption rates at 15 °C than those from Picinguaba. At 25 °C, crabs from Rio Itapanhaú had higher consumption rates than those from Picinguaba. At 35 °C, Ilha Diana crabs showed

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lower consumption rates than those from other localities. In summer, there were no significant differences in consumption rates at 15 °C among localities. At 25 and 35 °C,

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consumption rates increased more in crabs from Rio Itapanhaú than in crabs from other localities (Figure 6, Summer). The Q10 values for O2 consumption rates by crabs from

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the different localities in summer and winter are given in the insets to Figure 6, Winter and Summer. Overall, consumption rates were more sensitive to temperature increase

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between 15 and 25 °C than between 25 and 35 °C in both summer and winter.

4. Discussion

Metal contents in burrow sediments and tissues of Uca rapax

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We evaluated metal concentrations in burrow sediments using the sediment

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quality values proposed by Choueri et al., (2009) for the Santos Estuarine System (SES). Only the summer Ni concentration in the Rio Itapanhaú sediment can be

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considered moderately high. All other values are low. Copper concentrations in the gills and hepatopancreas were higher in crabs from Rio Itapanhaú and Ilha Diana than in those from Picinguaba, both in winter and in summer. Zinc tissue concentrations were higher in summer.

Among the sites evaluated, Ilha Diana (Santos) can be considered particularly contaminated by metals (Abessa et al 2008; Luiz-Silva, 2002, 2006; Lamparelli et al. 2001). However, the enrichment factor (LGeo), based on the local geochemical background (Luiz-Silva, 2006), did not confirm metal contamination at this locality, at least in sediments collected from the crabs’ burrows. Low burrow-sediment metal concentrations may result from burrow location in supralittoral zone, subject to washing by rain and high tides. Further, such sandy sediments tend to adsorb less metal ions compared to fine sediments like silt and clay, since sand grains exhibit less surface area and fewer active sites that can bind metal ions by ion exchange and complexation (see Ujevic et al., 2000; Lukman et al., 2013; Gupta & Bhattacharyya 2014). Thus, the ecological niche occupied by U. rapax may reduce exposure to sediment metals. This strategy is not entirely effective however, since the Cd, Cu and 16

ACCEPTED MANUSCRIPT Zn concentrations in the gills and hepatopancreas of crabs from Ilha Diana and Picinguaba in winter were greater than in the respective sediments, indicating metal

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uptake from the sediment or water, or complexed with organic matter.

Osmoregulation

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Uca rapax is a strong hyper-/hypo-osmoregulator, able to maintain its hemolymph osmolality fairly constant over a wide range of ambient salinities

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(Thurman, 2002). Uca rapax from all collection sites examined here exhibited this pattern of strong hyper/hypo-osmoregulation. However, crabs from Ilha Diana and Rio

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Itapanhaú, chronically contaminated sites with a history of metal contamination, showed a greater ability to hyper- and hypo-regulate their hemolymph osmolality in winter, and

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similar or lesser Na+ and Cl- hyper-/hypo-regulation in summer than did Picinguaba crabs. These findings suggest that Uca rapax from chronically contaminated localities can adjust its osmoregulatory abilities in winter and inhabit stressful contaminated

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environments characterized by daily changes in salinity. In summer, the crabs appear to

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be unable to provide additional energy for efficient ionic regulation. Such semiterrestrial crabs also exhibit behavioral mechanisms that avoid contact with

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contaminated water. These crabs can spend lengthy periods far from water, storing fluid in their gill chambers, reducing the need for direct contact with water (Hartnoll, 1998). However, they are thus more subject to osmotic and heat stress during the periods when they remain emerged.

Crustaceans occupying contaminated areas do make physiological adjustments (reviewed by Klerks & Weis, 1987). Carcinus maenas shows lower mortality at a Zncontaminated site than at two less contaminated localities, and migrates to the lesscontaminated areas (Bryan & Gibbs, 1983). Uca pugnax from a polluted environment shows greater resistance to methylmercury compared to crabs from a non-polluted area (Callahan & Weis, 1983). Uptake rates for Cu, Zn and Pb are significantly higher in the amphipod Gammarus marinus from a clean river compared to three metal-polluted rivers in south-west England, suggesting that amphipods from the polluted rivers might be more resistant owing to their reduced metal uptake (Wright, 1986). Uca rapax from Ilha Diana and Rio Itapanhaú, showed increased gill Na+/K+-ATPase activities at all salinities and better hemolymph hyper- and hypo- osmoregulatory abilities compared to Picinguaba crabs. Crabs from these sites are exposed to chronic metal contamination 17

ACCEPTED MANUSCRIPT and may generate compensatory enzymatic responses related to ion transport, which underlie systemic responses like osmoregulatory ability. Metals in the environment can compete with other ions such as Na+ (Grossell et al. 2002; Rainbow & Black, 2002) and

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Ca2+ (Paquin et al. 2000; Gensemer et al. 2002) by binding to physiologically active sites in the gill cells, consequently being incorporated by the organism. The Na+/K+-

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ATPase is involved in metal transport across the gill epithelium (Handy & Baines, 2002), and metals can increase enzyme activity by a hormetic effect (Calabrese &

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Baldwin, 2007), resulting in better osmoregulatory capability.

Exposure to sub-lethal metal concentrations in laboratory experiments is well

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known to impair osmoregulatory capacity by inducing regulatory disturbances, e. g., by inhibiting gill Na+/K+-ATPase activity in fish and invertebrates, especially at low

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salinities (Rodriguez Moreno et al.1998, Grosell et al. 2002; Henry et al. 2012). The estuarine crabs Neohelice granulata (Bianchini & Castilho 1999) and Carcinus maenas (Chan & Rainbow et al. 1992) exposed to sub-lethal Zn concentrations, and Eriocheir

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sinensis exposed to metals (Roast et al. 2002), show decreased hyper-osmoregulatory

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ability accompanied by decreased gill Na+/K+-ATPase activity. However, up to a specific metal concentration threshold, during chronic exposure in situ, Na+/K+-ATPase

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activity may increase as seen in carp (Labeo rohita) exposed to iron oxide nanoparticles (Remya et al. 2014), and in Neohelice granulata to Cu (Boyle et al. 2013), considered a compensatory mechanism. In Uca rapax from Ilha Diana and Rio Itapanhaú, increased gill Na+/K+-ATPase activities also appear to be compensatory, underpinning osmoregulatory ability.

Oxygen consumption

Oxygen consumption increased markedly at 35 °C in summer crabs from Rio Itapanhaú compared to Ilha Diana and Picinguaba. In summer, metabolic demands are greater at elevated temperatures and the scope for increased oxygen consumption is higher in crabs from contaminated sites, since they must deal not only with metal contamination but also respond to increased temperature (Vernberg & Vernberg, 1974). While metals are known to decrease O2 consumption (Spicer & Weber, 1992; Depledge, 1984; Bamber & Depledge, 1997), rates increase in the crabs Potamonautes warreni (Vosloo et al., 2002) and Callinectes sapidus (Aagaard & Depledge, 1993). However,

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ACCEPTED MANUSCRIPT these studies used crabs exposed in the laboratory to short term, low to moderate metal concentrations; little is known of the responses of crabs exposed to metals in situ. O2 consumption rates in crabs from Ilha Diana did not increase at 35 °C in

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winter, and increased less than in crabs from the other localities in summer. Ilha Diana crabs may be unable to enhance consumption rates at elevated temperatures, possibly

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owing to the energetic demands of metal depuration and osmotic regulation. Thus, metal contamination appears to limit their ability to deal with additional stressors like

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increased temperature. While Cu and Zn bioaccumulation was detected in the gills and hepatopancreas of Uca rapax from both Ilha Diana and Rio Itapanhaú, those from Ilha

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Diana showed metabolic impairment (O2 consumption increased less) while those from Rio Itapanhaú still exhibited a compensatory response. The effects of chronic

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contamination on O2 consumption appear to be exacerbated at higher experimental temperatures and are also seasonally influenced. Apparently, the potential impact of metals on energy demand by Uca rapax in situ depends on the crab’s metabolic ability

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to increase compensatory energy production. These findings are relevant to global

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climate change scenarios, since under increasingly warmer conditions, U. rapax may be unable to cope with contamination-induced stress.

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Our findings show that chronic exposure of Uca rapax to low to moderate metal concentrations in situ leads to bioaccumulation and induces biochemical and physiological changes compared to crabs from an uncontaminated locality. Uca rapax from contaminated sites maintain stronger hyper- and hypo-osmotic gradients and show greater gill Na+/K+-ATPase activities than do crabs from the pristine reference site, revealing that the underlying biochemical machinery can maintain such physiological processes functioning well. However, oxygen consumption, especially at elevated temperatures, either decreases or increases in crabs from contaminated localities. The energetic costs of these physiological adjustments may influence other processes like fecundity, fertility, egg hatching, larval survival, and molting and mating success, leading to a reduction in population viability. These findings reveal the importance of studies on organisms contaminated by metals in situ, particularly in estuarine invertebrates subject to frequent changes in natural environmental parameters like salinity and temperature.

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ACCEPTED MANUSCRIPT Acknowledgements This investigation received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 2011/22537-0 to JCM) from which

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MVC received a doctoral scholarship (FAPESP 2011/08065-9). JCM (CNPq 300662/2009-2) and DMA (CNPq 308649/2011) received research scholarships from

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the Conselho Nacional de Desenvolvimento Científico e Tecnológico. This study is part of a doctoral dissertation submitted by MVC to the graduate program in Comparative

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Biology, FFCLRP/USP and received additional support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 33002029031P8). We thank

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Susie Keiko for technical assistance, students from the Núcleo de Estudos em Poluição e Ecotoxicologia Aquática (NEPEA/UNESP) for help with crab collections and

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experiments, and Lucas Gonçalves Morais for preparing the site map. Dr. Fernando Barbosa kindly provided access to metal analysis facilities at the Departamento de Análises

Clínicas,

Toxicológicas

e

Bromatológicas,

Faculdade

de

Ciências

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Farmacêuticas at Ribeirão Preto/USP, and Prof. Francisco Leone provided access to

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biochemical assay equipment at the Departamento de Química, Faculdade de Filosofia,

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Ciências e Letras at Ribeirão Preto/USP.

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Matthiessen, P., Law, R.J., 2002. Contaminants and their effects on estuarine and coastal organisms in the United Kingdom in the late twentieth century. Environ. Pollut. 120, 739–757. Mauro, N.A., Moore, G.W., 1987. Effects of environmental pH on ammonia excretion, blood pH, and oxygen uptake in fresh water crustaceans. Comp. Biochem. Physiol. C. 87, 1-3. McNamara, J.C., Faria, SC., 2012. Evolution of osmoregulatory patterns and gill ion transport mechanisms in the decapod Crustacea: a review. J. Comp Physiol. B. 182, 997-1014. Santos, F.H., McNamara, J.C, 1996. Neuroendocrine modulation of osmoregulatory parameters in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, Decapoda). J. Exp. Mar. Biol. Ecol. 206, 109-120. Murdoch, A., MacKnight, S., 1991. Handbook of Techniques for Aquatic Sediments Sampling, CRC Press, Inc., Boca Raton, EL, 1991, 210 pages. Remediation, 3, 135– 136. Nardi, E. P., Evangelista, F. S., Tormen, L., SaintPierre, T. D., Curtius, A. J., de Souza, S. S. 2009. The use of inductively coupled plasma mass spectrometry (ICPMS) for the determination of toxic and essential elements in different types of food samples. Food Chem. 112, 727–732. Nugegoda, D., Rainbow, P.S., 1989. Salinity, osmolality and zinc uptake in Palaemon elegans (Crustacea: Decapoda). Mar. Ecol. Prog. Ser. 55,149-157. Otitoloju, A.A., Don-Pedro, K.N., 2004. Integrated laboratory and field assessments of heavy metals accumulation in edible periwinkle, Tympanotonus fuscatus var. radula (L.). Ecotoxicol. Environ. Safety. 57,354–362. Paquin, P.R., Santore, R.C., Wu, K.B., Anid, P.J., Kavvadas, P.D., Di Toro, D.M., 2000. Revisiting the aquatic impacts of copper discharged by water-cooled copper alloy condensers used by power and desalination plants. Environ. Sci. Policy. 3, 165–174. Péqueux, A., 1995. Osmotic regulation in Crustacea. J. Crust. Biol. 15,1–60. Pörtner, H.O., 2010. Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881-893. Powers, L.W., Cole, J.F., 1976. Temperature variation in fiddler crab microhabitats. J. Exp. Mar. Biol. Ecol. 21, 141–157. Quináglia, G.A., 2006. Caracterização dos níveis basais de concentração de metais nos sedimentos do sistema estuarino da baixada santista. Ph D thesis. Universidade de São Paulo. Instituto de Química. p.239. Rainbow, P. S., 1996. Phylogeny of trace metal accumulation in crustaceans. In Metal Metabolism in Aquatic Environments (Langston, W. J. & Bebianno, M., eds). Chapman and Hall, London. Rainbow, P.S., 1997. Ecophysiology of trace metal uptake in crustaceans. Estuar. Coast. Shelf. Sci. 44,169–175. Rainbow, P.S., Black,W.H., 2002. Effects of changes in salinity and osmolality on the rate of uptake of zinc by three crabs of different ecologies. Mar. Ecol. Prog. Ser. 244, 205–217. Rainbow, P.S., Black, W.H., 2005. Cadmium, zinc and uptake of calcium by two crabs, Carcinus maenas and Eriocheir sinensis. Aquat. Toxicol. 72, 45-65. Remya, M., Ramesh, M., Saravanan, R.K., Poopal, S., Bharathi, Nataraj, D., 2014. Iron oxide nanoparticles to an Indian major carp, Labeo rohita: impacts on hematology, Ion regulation and gill Na+/K+ ATPase activity. J. King. Saud. Univ. Sci. 27, 15-160. 24

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Roast, S.D., Rainbow, P.S., Smith, B.D., Nimmo, M., Jones, M.B., 2002. Trace metal uptake by the Chinese mitten crab Eriocheir sinensis: the role of osmoregulation. Mar. Environ. Res. 53, 453–464. Rodríguez Moreno, P.A., Schwarzbaum, P.J., Rodríguez, E.M., 1998. Effects of cadmium on gill Na,K-ATPase of the estuarine crab Chasmagnathus granulata (Decapoda, Grapsidae) during postmolt: in vivo and in vitro studies. Bull. Environ. Contam. Toxicol. 61, 629–636. Romano, N., Zeng, C., 2010. Survival, osmoregulation and ammonia-N excretion of blue swimmer crab, Portunus pelagicus, juveniles exposed to different ammonia-N and salinity combinations. Comp. Biochem. Physiol. C 151, 222–228. Steffensen, J.J., 1989. Some errors in respirometry of aquatic breathers - how to avoid and correct for them. Fish. Physiol. Biochem. 6,49-59. Salaroli, A.B., 2013. Distribuição de elementos metálicos e As em sedimentos superficiais ao longo do Canal de Bertioga (SP). Ph D thesis. Universidade de São Paulo. Instituto Oceanográfico. Schales, O., Schales, S.S. 1941. A simple and accurate method for the determination of chloride in biological fluids. J. Biol. Chem. 140, 879–883. Schiedek, D., Broeg, K., Barsiene, J., Lehtonen, K., Gercken, J., Pfeifer, S., 2006. Biomarker responses as indication of contaminant effects in blue mussel (Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern Baltic Sea. Mar. Pollut. Bull. 53, 387–405. Sokolova, I. M., Lannig, G., 2008. Interactive effects of metal pollution and temperatures and fluctuating daily temperatures on metabolism in aquatic ectotherms: implications of global climate change. Clim. Res. 37, 181-201. Spicer, J. I. & Weber, R. E. 1992 Respiratory impairment by water-borne copper and zinc in the edible crab Cancer pagurus (L.) (Crustacea: Decapoda) during hypoxic exposure. Mar. Biol. 112, 429–435. Stegeman, J.J., Brouwer, M., Di Giulio, R.T., Förlin, L. Fowler, B.A., Sanders, B.M., Van Veld, P.A. 1992. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In: Huggett, R.J. Thurman, C.L. 2002. Osmoregulation in six sympatric fiddler crabs (genus Uca) from the North western Gulf of Mexico. Mar Ecol. 23,269–284. Thurman, C.L., 1984. Ecological notes on fiddler crabs of south Texas, with special reference to Uca subcylindrica. J. Crustac. Biol. 4, 665–681. Thurman, C.L., 1987. Fiddler crabs (Genus Uca) of eastern Mexico (Decapoda: Brachyura: Ocypodidae). Crustaceana. 53, 95-105. Thurman. C.L., Faria, S.C., McNamara, J.C., 2013.The distribution of fiddler crabs (Uca) along the coast of Brazil: implications for biogeography of the western Atlantic Ocean. Mar. Biodivers. Rec. 6,1-21. Ujevič, I., Odzak, N., Baric, A., 1998. Relationship between Mn, Cr, Pb and Cd concentrations, granulometric composition and organic matter content in the marine sediments from a contaminated coastal area. Fresen. Environ. Bull. 7, 183–189. Vernberg, F.J., Vernberg, W.B., 1974. Pollution and Physiology of Marine Organisms. Academic Press, New York, 492 pp. Vosloo, A., Van Aardt, W.J., Mienie, L.J. 2002. Sublethal effects of copper on the freshwater crab Potamonautes warreni. Comp. Biochem. Physiol. A. 133, 695-702. Wentworth, C. K. 1922. A scale of grade and class terms for clastic sediments. J. Geol. 30, 377-392. 25

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99.6 99.6 78.0 67.1 99.0 97.0

22.0 18.0 24.0 21.0 21.0 26.0

64.0 67.0 62.6 63.9 65.8 62.6

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CaCO 3

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winter summer Rio Itapanhaú winter summer Ilha Diana winter summer

Organic matter

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Picinguaba

Sand

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Table 1. Seasonal variation in the content of sand, organic matter and calcium carbonate (%) in sediment samples (N=1) collected from the Ilha Diana, Rio Itapanhaú and Picinguaba sites during the winter (June and July) of 2012 and summer (January and February) of 2013. See Figure 1 for site locations.

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ACCEPTED MANUSCRIPT Table 2.

Metal contents (µg/gram sediment) in burrow sediments (N=1) and in

homogenates (N=3) of gills and hepatopancreas (µg/g tissue) from Uca rapax collected at the Picinguaba, Rio Itapanhaú and Ilha Diana sites during the summer of 2012 and

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winter of 2013 (see Figure 1 for site localities). Values in parentheses indicate the bioaccumulation factor for each metal (tissue: sediment metal concentration ratio).

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Metal contents are compared between the gill and hepatopancreas homogenates for the same collection site, and among collection sites for each tissue. Data are the mean ± a

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SEM. *Significantly different from respective gill metal concentration (P<0.05). Significantly different from respective value for tissues from crabs collected at

Sediment

Picinguaba Gill

Hepatopancreas

Sediment

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Picinguaba (pristine site, P<0.05).

Rio Itapanhaú Gill Hepatopancreas

Winter < 0.01 (0.02)

0.5 ± 0.0* (0.2)

19.0

0.0

0.1 ± 0.0 (25.0)

0.2 ± 0.0 (50.0)

0.0

Cu Ni Zn

0.9 4.2

43.0 ± 13.0 (48.0) 0.1 ± 0.0 (0.0)

70.0 ± 30.0 (78.0) 0.9 ± 0.0* (0.2)

3.0 3.0

7.0

13.0 ± 0.8 (1.8)

31.0 ± 6.0* (4.5)

25.0

2.0

<0.01 (0.0)

0.1 ± 0.0* (0.0)

30.0

Summer

0.0

0.0 ± 0.0 (0.8)

Cu

1.0

26.0 ± 4.0 (0.0)

Ni

1.0

0.0 ± 0.0 (0.0)

Zn

7.5

11.0 ± 3.0 (1.5)

0.4 ± 0.0* (80.0)

a

0.7 ± 0.1 (28.0)

51.0 ± 3.0 (17.0) 0.1 ± 0.0 (0.0) 10.0 ± 4.0 (0.4)

a

0.5 ± 0.0 (0.0)

a

1.0 ± 0.0* (0.0)

2.5

0.2 ± 0.1* (8.0)

0.0

a

353.0 ± 70.0* (118.0) 0.7 ± 0.0* (0.2) 30.0 ± 8.0* (1.2)

a

4.0 ± 0.0* (0.1) a

Gill

Hepatopancreas

<0.01 (0.0)

0.3 ± 0.0*(0.1)

0.1 ± <0.0 (12.0)

0.1 ± 0.0 (12.0)

a

1.3 1.0

76.0 ± 5.0 (59.0) 0.1 ± 0.0 (0.1)

3.0

20.0 ± 2.0 (6.6)

2.0

0.1 ± 0.0 (0.0)

0.0

0.1 ± 0.0 (25.0)

a

141.0 ± 38.0* (108.0) 0.7 ± 0.0*(0.7) 33.0 ± 3.0 (11.0)

a

0.1 ± 0.0 (0.0)

0.2

0.4 ± 0.1 (2.0)

36.0 ± 5.0* (0.0)

4.0

65.0 ± 3.0 (16.0)

1.6

44.0 ± 3.0 (27.0)

0.0 ± 0.0* (0.0)

5.0

0.7 ± 0.0 (0.1)

0.2 ± 0.0* (0.0)

1.0

0.1 ± 0.0 (0.1)

1.3 ± 0.0* (1.3)

4.0 ± 0.1* (0.5)

30.0

33.0 ± 4.0 (1.0)

12.0 ± 4.0* (0.4)

3.0

12.0 ± 2.0 (4.0)

44.0 ± 0.1*(14.0)

a

a

2.8 ± 0.3* (12.5) a

532.0 ± 40.0* (133.0) a

0.2 ± 0.0* (62.0)

a

a

548.0 ± 207.0* (342.0)

a

a

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Ba

a

0.5 ± 0.1 (0.0)

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2.4

Cd

D

Ba

Ilha Diana Sediment

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Figure 1. Collection sites (1, Ilha Diana; 2, Rio Itapanhaú, both metal contaminated; 3,

SC

RI

southeastern Atlantic coast of the State of São Paulo, Brazil.

PT

Picinguaba, pristine control area) for the mudflat fiddler crab Uca rapax located on the

Figure 2. Hemolymph osmolality in Uca rapax collected from the Picinguaba (P), Ilha

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Diana (ID) and Rio Itapanhaú (RI) sites (see Figure 1 for locations) during the winter of 2012 (June-July) and summer (January-February) of 2013. Crabs were exposed with free access to a dry surface to media of <0.5 ‰ S (distilled water), 25 ‰ S (isosmotic

MA

seawater, 750 mOsm/kg H2O) or 60 ‰ S (hypersaline seawater, 1,800 mOsm/kg H2O) for 5 days. Data are the mean ± SEM (N=7-10). *Significantly different from 25 ‰ S for the same collection site. aSignificantly different from Picinguaba at the same salinity

D

(P<0.05).

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Figure 3. Hemolymph sodium concentrations in Uca rapax collected from the Picinguaba (P), Ilha Diana (ID) and Rio Itapanhaú (RI) sites (see Figure 1 for locations)

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during the winter of 2012 (June-July) and summer (January-February) of 2013. Crabs were exposed with free access to a dry surface to media of <0.5 ‰ S (distilled water, 3 mM Na+), 25 ‰ S (isosmotic seawater, 350 mM Na+) or 60 ‰ S (hypersaline seawater, 840 mM Na+) for 5 days. Data are the mean ± SEM (N=7-10). *Significantly different from 25 ‰ S for the same collection site. aSignificantly different from Picinguaba at the same salinity (P<0.05).

Figure 4. Chloride concentration in the hemolymph of Uca rapax collected from the Picinguaba (P), Ilha Diana (ID) and Rio Itapanhaú (RI) sites (see Figure 1 for locations) during the winter (June-July) and summer (January-February) of 2013. Crabs were exposed with free access to a dry surface to media of <0.5 ‰ S (distilled water, 5 mM Cl-), 25 ‰ S (isosmotic seawater, 400 mM Cl-) or 60 ‰ S (hypersaline seawater, 960 mM Cl-) for 5 days. Data are the mean ± SEM (N=7-10). *Significantly different from 25 ‰S for the same collection site. aSignificantly different from Picinguaba at the same salinity (P<0.05).

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ACCEPTED MANUSCRIPT Figure 5. K+-phosphatase activity of the Na+/K+-ATPase in crude homogenates of the posterior gills of Uca rapax collected from the Picinguaba (P), Ilha Diana (ID) and Rio Itapanhaú (RI) sites during the winter (June-July) and summer (January-February) of

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2013. Crabs were exposed in the laboratory for 5 days to either distilled water (<0.5 ‰ S), isosmotic seawater (25 ‰ S) or hypersaline seawater (60 ‰ S). Data are the mean ±

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SEM (N=7-10). *Significantly different from isosmotic seawater (25 ‰ S) for the same collection site (P<0.05). aSignificantly different from crabs from Picinguaba (pristine

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control site) at the same salinity (P<0.05)

Figure 6. Oxygen consumption rates (mLO2. g-1. h-1) of intact Uca rapax collected from

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the Picinguaba (P), Ilha Diana (ID) and Rio Itapanhaú (RI) sites in the winter (JuneJuly) and summer (January-February) of 2013. After a 12-h adjustment period in the

MA

respirometers at each temperature (15, 25 or 35 °C), oxygen consumption data were obtained during a 50-min sampling period using an intermittent flow-through method (Steffensen, 1989). Data are the mean ± SEM (N=7-10). *Significantly different from

D

25 °C (control temperature) for the same collection site (P<0.05). aSignificantly

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different from Picinguaba (pristine control site) at the same temperature (P<0.05). Insets show the Q10 values for oxygen consumption rates between 15 and 25 °C, and between

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25 and 35 °C, for crabs from the different sites.

30

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FIGURE 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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winter

99.6

summer

99.6

winter

78.0

summer

67.1

winter

99.0

summer

97.0

64.0

18.0

67.0

24.0

62.6

21.0

63.9

21.0

65.8

26.0

62.6

TE AC CE P

Ilha Diana

CaCO3

D

Rio Itapanha ú

RI

Picingua ba

Organic matter 22.0

SC

Sand

NU

Season

MA

Locality

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Table 1. Seasonal variation in the content of sand, organic matter and calcium carbonate (as %) in sediment samples collected from burrows of the fiddler crab Uca rapax at the pristine Picinguaba (control site), and metal-contaminated Rio Itapanhaú and Ilha Diana sites, during the winter (June and July) (N=1) of 2012 and summer (January and February) (N=1) of 2013 (see Figure 1 for data on site locality).

37

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Table 2. Metal contents (µg/g sediment) in burrow sediments and in homogenates (N=3) prepared from gills and hepatopancreas (µg/g

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tissue) of the fiddler crab Uca rapax collected at the pristine Picinguaba (control), and metal-contaminated Rio Itapanhaú and Ilha Diana sites,

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during the winter (January and February) (N=1) of 2012 and summer (June and July) of 2013 (N=1) (see Figure 1 for data on site locality).

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Values in parentheses indicate the bioaccumulation factor for each metal (i. e., ratio of tissue: sediment metal concentration). Data are the mean ±

MA

SEM. *Significantly different from respective gill metal concentration (P<0.05). aSignificantly different from respective value for tissues from crabs collected at the Picinguaba site (P<0.05).

S ediment

Gill

PT ED

Picinguaba Hepato pancreas

S

d

.0

u

.9

i

.2

n

.0

0 0 4 7

<0.01 (0.0)

*0.1 ± 0.0 (0.0)

a

25.0

0.5 ± 0.1 (0.0) a 0.7 ± 0.1 (28.0) 51.0 ± 3.0 (17.0) 0.1 ± 0.0 (0.0) 10.0 ± 4.0 (0.4)

30.0

a 0.5 ± 0.0 (0.0)

CE

.4

*0.5 ± 0.0 (0.2) 0.2 ± 0.0 (50.0) 70.0 ± 30.0 (78.0) *0.9 ± 0.0 (0.2) *31.0 ± 6.0 (4.5)

AC

a

<0.01 (0.02) 0.1 ± 0.0 (25.0) 43.0 ± 13.0 (48.0) 0.1 ± 0.0 (0.0) 13.0 ± 0.8 (1.8)

Gill

ediment

inter 2

Rio Itapanhaú

19.0 0

.0 3 .0 3 .0

Ilha Diana Hepatopan creas

S ediment

a

*1.0 ± 0.0 (0.0) *0.2 ± 0.1 (8.0) a *353.0 ± 70.0 (118.0) *0.7 ± 0.0 (0.2) *30.0 ± 8.0 (1.2)

2 .5 0 .0 1 .3 1 .0 3 .0

Gill

<0.01 (0.0) 0.1 ± <0.0 (12.0) a 76.0 ± 5.0 (59.0) 0.1 ± 0.0 (0.1) 20.0 ± 2.0 (6.6)

Hepatopanc reas

*0.3 ± 0.0 (0.1) 0.1 ± 0.0 (12.0) a *141.0 ± 38.0 (108.0) *0.7 ± 0.0 (0.7) 33.0 ± 3.0 (11.0)

S ummer 2 a

.0

a *4.0 ± 0.0 (0.1)

2 .0

a 0.1 ± 0.0 (0.0)

0.1 ± 0.0 (0.0)

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.5

7

5 .0 30.0

PT

n

1

RI

.0

4 .0

SC

i

1

*2.8 ± 0.3 (12.5) a *532.0 ± 40.0 (133.0) a *0.2 ± 0.0 (0.0) *12.0 ± 4.0 (0.4)

.0 1 .6 1 .0 3 .0

0.1 ± 0.0 (25.0) a 44.0 ± 3.0 (27.0) a 0.1 ± 0.0 (0.1) 12.0 ± 2.0 (4.0)

*0.2 ± 0.0 (62.0) a *548.0 ± 207.0 (342.0) a *1.3 ± 0.0 (1.3) *44.0 ± 0.1 (14.0)

NU

.0

0

a

MA

u

.2

0.4 ± 0.1 (2.0) a 65.0 ± 3.0 (16.0) a 0.7 ± 0.0 (0.1) 33.0 ± 4.0 (1.0)

PT ED

.0

0

*0.4 ± 0.0 (80.0) *36.0 ± 5.0 (0.0) *0.0 ± 0.0 (0.0) *4.0 ± 0.1 (0.5)

CE

d

0.0 ± 0.0 (0.8) 26.0 ± 4.0 (0.0) 0.0 ± 0.0 (0.0) 11.0 ± 3.0 (1.5)

AC

0

39