Organotropism of methylmercury in fish of the southeastern of Brazil

Accepted Manuscript Organotropism of methylmercury in fish of the southeastern of Brazil L.S. Azevedo, M.G. Almeida, W.R. Bastos, M.S. Suzuki, M.C.N.N. Recktenvald, M.T.S. Bastos, C.S. Vergílio, C.M.M. de Souza PII:

S0045-6535(17)31133-5

DOI:

10.1016/j.chemosphere.2017.07.081

Reference:

CHEM 19614

To appear in:

ECSN

Received Date: 2 December 2016 Revised Date:

11 July 2017

Accepted Date: 15 July 2017

Please cite this article as: Azevedo, L.S., Almeida, M.G., Bastos, W.R., Suzuki, M.S., Recktenvald, M.C.N.N., Bastos, M.T.S., Vergílio, C.S., de Souza, C.M.M., Organotropism of methylmercury in fish of the southeastern of Brazil, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.07.081. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Organotropism of methylmercury in fish of the southeastern of Brazil L.S. Azevedo a,*, M.G. Almeida a, W.R. Bastos b, M.S. Suzuki a, M.C.N.N. Recktenvald b

a

, M.T.S. Bastos b, C.S. Vergílio c, C.M.M. de Souza a Laboratório de Ciências Ambientais, Centro de Biociências e Biotecnologia,

Rio de Janeiro, RJ, CEP: 28013-602, Brazil. b

Laboratório de Biogeoquímica Ambiental, Universidade Federal de Rondônia, Porto

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Velho, Rondônia, RO, CEP: 76815-800, Brazil. c

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Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes,

Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do

*

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Espírito Santo, Alegre, Espírito Santo, ES, CEP: 29500-000, Brazil. Corresponding author: [email protected]

ABSTRACT

This is one of the first studies to evaluate the effect of biometric variables (total length

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and weight), diet, and abiotic matrices (sediment and water column) on the bioaccumulation of methylmercury in tissues (muscle, liver, and gills) of four fish (two carnivore-invertivores, Pimelodus fur and Pachyurus adspersus; one carnivore-

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piscivore, Oligosarcus hepsetus; and one omnivore, Pimelodella lateristriga) in the lower section of a river in southeastern Brazil. Samples of fish (n=120), water (n=5) and

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sediment (n=5) were collected at five sites characterized by pollution with mercury due to the use of organomercury fungicides and stream bed gold mining, commonly carried out in that section of the river in the 1980s. The results show that biometric variables are strongly correlated with methylmercury levels in muscle (r= 0.61, p<0.0005) of P. fur. As a rule, concentrations of total mercury and methylmercury did not vary considerably between the organs of the species of different food habits, because of the environmental conditions in the study area. Despite the low concentrations of mercury in sediments

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ACCEPTED MANUSCRIPT (<0.05 mg.kg-1 wet.wt), this compartment is a representative source of this pollutant for the organisms investigated, due to the close contact these animals keep with it in view of the low water columns in that section of the river. Keywords:

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Bioaccumulation; Fish; Mercury; Methylmercury; Paraíba do Sul river; Sediment 1. Introduction

The various uses of soils in southeastern Brazil, a region characterized by large

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urban and industrial centers, place intense anthropic pressure on its main drainage basin, where the Paraíba do Sul river (PSR) is one of the main water courses. These waters are

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exposed to the discharge of municipal sewage and industrial effluents as well as to the effects of mining and construction of dams. Several authors have taken biogeochemical approaches to investigate the effects of pollution in various sites in the region (Araújo et al., 2010; Rocha et al., 2015), recording the circulation of mercury (Hg) discharged by

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activities such as the use of organomercury fungicides in sugar cane plantations and gold mining (aluvionar sediments) in the lower section of the river (Câmara, 1990; Lima, 1990). However, these studies did not take into account the potential

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transportation of Hg and the association of the methylmercury (CH3Hg+) with the region’s fish species.

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Binding covalently with the sulfhydryl group of proteins, CH3Hg+ is one of the

most toxic forms of Hg. The compound is characterized by long residence times in tissues of living organisms, which promote its bioaccumulation and biomagnification (Trudel and Rasmussen, 1997) Diet is the main pathway of Hg and CH3Hg+ into fish (Hall et al., 1997), while absorption through gills comes second (Jaeger et al., 2009). Importantly, the species at the top of the trophic chain normally present the highest levels of the organic form of

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ACCEPTED MANUSCRIPT Hg (Clarkson et al., 2003). In this regard, retention of Hg in different fish tissues may induce the emergence of adverse toxic effects, causing histological damage like hepatic lesions and reducing the exchange area of gill lamellae (Mela et al., 2007; Fernandes et al., 2013). Furthermore, ecological factors like balance, posture, as well as performance

ability to escape from predators may also be affected.

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in swimming (Garcia et al., 2000; Berntssen et al., 2003) and in both predation and the

In this scenario, the present study evaluated bioaccumulation of total Hg (THg)

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and CH3Hg+ in tissues (muscle, liver, and gills) of four fish species of different food habits living in the lower section of PSR, southeastern Brazil, a region with a record of

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exposure to Hg due to the use of organomercury fungicides and gold mining activities. To find the factors that influence the bioaccumulation of THg and CH3Hg+, we looked into the relationship between bioaccumulation of these Hg chemical species and biometric variables, food habit, and abiotic matrices (sediment and water column). We

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put forward the hypothesis that biometric characteristics, the ecology of fish, and the environmental conditions in the study area influence the accumulation of contaminants

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in muscle, liver, and gills.

2. Materials and methods

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2.1 Study area

Covering 62,074 km², the drainage basin of PSR is used as water source in three

highly urbanized Brazilian states, namely Rio de Janeiro, São Paulo, and Minas Gerais. In total, 6.7 million people live in the area (IBGE, 2010). Discharge reaches 4,384 m3 s1

in the rainy season, from December to February, and drops to a minimum in the dry

season (between June and August), with 181 m3 s-1 (ANA, 2006). More specifically, the study area is located in the northwest portion of the PSR

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ACCEPTED MANUSCRIPT drainage basin. Samples were collected in five sites along approximately 120 km: (1) São Sebastião do Paraíba (SSP), (2) Itaocara (ITA), (3) São Fidelis (SFI), (4) Baltazar (RPO), and (5) Guarani (RDR). Sites RPO and RDR are located in the sub-basins of rivers Pomba and Dois Rios, both of which are tributaries to PSR (Fig. 1). The study

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área and the collection sites are shown in the map published by Rocha et al. (2015).

The fish examined and water samples were collected in May and July 2014. A prolonged drought affected the lower section of PSR in 2014 and 2015 (AGEVAP,

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2016), when no significant statistical differences were observed (Tukey test, α=0.05; p>0.05) in total rainfall between the dry season (April to September) and the rainy

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season (October to March) (INMET, 2017). Therefore, it was assumed that the influence of seasonality on the distribution of THg and CH3Hg+ in fish, particulate matter, and sediment was negligible.

2.2.1 Fish

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2.2 Sampling

In total, 120 adult individuals of four fish species (N = 30 for each species) from

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three groups of food habits (two carnivore-invertivores, Pimelodus fur and Pachyurus adspersus; one carnivore-piscivore, Oligosarcus hepsetus; and one omnivore,

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Pimelodella lateristriga) were captured with the assistance of Projeto Piabanha, an initiative set up by local stakeholders interested in preserving the PSR (with permission given by Brazil’s Ministry for the Environment, authorization 36260-0). Net mesh sizes used were 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, and 70 mm, which afforded to sort the adult individuals of each species. The fish captured were identified according to site and biometric parameters (total length, TL e total weight, TW). TL and TW of species were obtained measuring with an ichthyometer and weighing in analytical balance. Gender was determined by 4

ACCEPTED MANUSCRIPT gross inspection of gonads (Vazzoler, 1996). Muscle, liver, and gill tissues were excised, lyophilized (dry wt.) and used in the chemical analysis to quantify THg and CH3Hg+. 2.2.2 Fish ecology

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Pimelodus fur is a catfish of the Siluriformes order of benthopelagic habits at trophic level 3.3 ± 0.5 (Fishbase, 2017). The species feeds mainly on small invertebrates (insects, mollusks, and others). Pimellodela lateristriga is another siluriform catfish,

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with a trophic level of 3.6 ± 0.5 (Fishbase, 2017). This species’ diet is more generalist, thus differing form that of P. fur. Neither of the two species has considerable

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commercial value, although the demersal habit of these animals means that they touch the sediment directly, which is the main THg and CH3Hg+ in aquatic ecosystems. Therefore, both are appropriate models to study the influence of abiotic matrices on Hg bioaccumulation. Oligosarcus hepsetus is a fish of the Charachiformes order, with

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pelagic habits and at trophic level 4.2 ± 0.73. This piscivorous species is at the highest trophic level, compared with the others included in this study. Pachyurus adspersus (“brazilian croaker”) belongs to the Perciformes order,

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Sicaenidae family, of benthopelagic habits that may be considered similar to those of P. fur (Fishbase, 2017) and it is at trophic level 3.6 ± 0.4. This is the most commercially

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interesting species examined in this study, and plays an important role in the region’s fishing industry (Vieira, 2010) 2.2.3 Suspended particulate matter and bottom sediment Five liters of the river water were collected in polyethylene flasks at each site. Suspended particulate matter (SPM) was collected by saturation of 0.7-µm of porosity, ϕ 47 mm GF/F filters and lyophilized (L10, Liotop). Limnologic variables like temperature (Thermo Scientific Orion 3 STAR), pH, electric conductivity, O2 saturation

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ACCEPTED MANUSCRIPT (Thermo Scientific Orion STAR A221), and transparency (Secchi disk) were measured at every site during field excursions. Bottom sediment samples were collected from the top 10-cm layer in the five sites in October 2013. Samples were sieved and the passing fraction (< 63 µm) was

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lyophilized and macerated in a ball mill. 2.3 Chemical analyses 2.3.1 Total mercury in fish tissues

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The chemical digestion of muscle (0.1 g dry wt.), liver (0.01 g dry wt.), and gill (0.1 g dry wt.) to determine THg was carried out in five steps (adapted from Bastos et

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al., 1998): (i) extraction with 1 mL H2O2 30% and 3 mL H2SO4 97%:HNO3 65% (1:1); (ii) digestion in block digester system at 60ºC upon total solubilization of the sample; (iii) addition of 5 mL KMnO4 5%; (iv) titration with NH2OH.HCl 12%; (v) filtration through Whatman 40 paper and completion to a 20-mL volume with ultra-pure water

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(milli-Q, Millipore, Cambridge, MA, USA). THg was detected using a mercury analyzer (Quick trace M-7500, CETAC) with a detection limit of 0.001 mg kg-1. The accuracy of the method was evaluated using the dogfish muscle reference material

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(DORM-3, NRC, concentration 0.382 ± 0.060 mg.kg-1 dry wt) with recovery of > 90.0%. Reproducibility was assessed using triplicates for every 80 samples (coefficient

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of variation < 10.0%). The results were expressed as wet wt using the equation: concentration in wet wt= concentration dry wt x (100 - water percent level). 2.3.2 Total mercury in suspended particulate matter and sediment Digestion of the sediment matrix (0.5 g dry wt.) and of filters with SPM (the samples comprised two filters per site) was carried out in five steps: (i) extraction with 1 to 2 mL Milli-Q H2O and 8 mL 3 HCl 37%: 1HNO3 65%; (ii) digestion in a block digester system at 60ºC for 2 h; (iii) addition of 5 mL Milli-Q H2O and 10 mL KMnO4

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ACCEPTED MANUSCRIPT 5% after cooling; (iv) digestion in a block digester system at 60ºC for 30 min; and (v) filtration through Whatman paper 40 and completion to a 25-mL volume with ultra-pure water (milli-Q, Millipore, Cambridge, MA, USA) (Bastos et al., 1998). The results of THg in sediment were kindly provided by Rocha et al. (2015).

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THg in SPM was quantified using a mercury analyzer (Quick trace M-7500, CETAC) with detection limit of 6x10-8 mg L-1.In sediment, THg was analyzed using a mercury analyzer (Mercury Analyses System, FIMS-400 – Perkin-Elmer) with

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detection limit of 0.0005 mg kg-1. The accuracy of the method for sediment was evaluated using the estuarine sediment sample reference material (IAEA-405, Mel,

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concentration 0.81 mg.kg-1 dry wt) with recovery of 93.8%. Reproducibility was assessed using triplicates in all samples (coefficient of variation < 8%). The accuracy of the method for SPM was evaluated using the marine sediment reference material (MESS-3, NRC, concentration 0.091 ± 0.009 mg.kg-1 dry wt) with recovery of >94.0%

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and reproducibility was <10.0%. 2.3.3 Methylmercury in fish tissues

Quantification of CH3Hg+ was carried out as described in EPA Method 1630

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(2001) and Liang et al. (1994), in four steps: (i) addition of 50 mL of KOH in methanol 25% w/v; (ii) heating in stove (NI 1512, Nova Instruments) to 70ºC for 6 h; (iii)

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addition of 300 µL sodium acetate buffer 272 g L-1 (pH 4.5) followed by 3 µL of sample and 50 µL of a solution of sodium tetraethylborate 1% w/v (Taylor et al, 2011); (iv) completion to 40 mL with ultra-pure water (milli-Q, Millipore, Cambridge, MA, USA). The analysis was carried out in gas chromatography-cold vapor-atomic fluorescence spectroscopy (GC-CV-AFS, MERX-TM automated system from Brooks Rand Labs Seattle, USA). Accuracy was assessed using the fish protein reference samples (DORM2, NRC, concentration 4.47 ± 0.032 mg.kg-1 dry wt) with a recovery rate of 93% and

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ACCEPTED MANUSCRIPT detection limit of 0.0014 mg kg-1. Reproducibility was assessed using duplicates for every samples (coefficient of variation < 10%). The results were expressed as wet wt using the equation: concentration in wet wt = concentration dry wt x (100 - water percent level).

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2.4 Total lipid extraction

The extraction of lipids from the muscle of the fish species (five lyophilized samples per species) was carried out according to Folch et al. (1957).

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2.5. Statistical analysis

Normality of data was tested using the Shapiro-Wilk test. In order to meet

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ANOVA requirements, data were transformed using a maximum likelihood function (Box Cox). The differences in concentrations of THg, CH3Hg+, and the percent CH3Hg+ levels in each tissue were analyzed using ANOVA followed by the Tukey test (p < 0.05). The Pearson correlation (α = 0.05) was used to correlate the concentrations of

Rstudio 3.2.2.

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CH3Hg+ with biometric data. All statistical tests were conducted using the software

3. Results and discussion

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3.1. Bioaccumulation of THg and CH3Hg+

Since there are no significant differences among males and females in terms of

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bioaccumulation of mercury (p > 0.05) we used the whole data set without discriminate the number of female and males individuals. Being liposoluble, Hg tends to accumulate in liver tissue and muscle (McIntyre and Beauchamp, 2007). In the present study, the concentrations of THg were highest in liver, followed by muscle and gills (Table 1, p < 0.05), as observed in previous research (Halvelková et al., 2008; Vieira et al., 2011; Bergés-Tiznado et al., 2015). The exception was observed for O. hepsetus, when THg levels were high in liver and muscle samples (with no statistically significant

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ACCEPTED MANUSCRIPT difference), followed by gills (p < 0.05).

A different pattern was observed for

CH3Hg+. In O. hepsetus, P. lateristriga and P.fur, CH3Hg+ concentrations were highest in muscle (p < 0.05), followed by liver and gill samples (with no statistically significant difference between the last two tissues), while in P. adspersus CH3Hg+concentration

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was highest in muscle, followed by liver and then gills (p < 0.05) (Table 1). High levels of CH3Hg+ in fish muscle are expected due to the high affinity of the pollutant for the thiol groups constituting amino acids like cysteine, for instance (Ruelas-Inzunza et al.,

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2003; Leaner and Mason, 2004). This relationship was confirmed by the high percent values of CH3Hg+ in muscle of O. Hepsetus (73.63 ± 20.53%), P. adspersus (78.74 ±

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22.89%), P. lateristriga (85.00 ± 29.67%), and P. fur (56.99 ± 29.44%) (Fig. 2). The CH3Hg+ concentrations in the liver of the fish species analyzed were statistically lower than those observed in muscle (p < 0.05), and the percent values in the organ were the lowest, differing statistically from muscle and gills (p < 0.05). The

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liver plays a key role in the detoxification of CH3Hg+, converting the compound into less bioaccumulative inorganic forms of the metal, thus reducing its toxicity (Suda and Hirayma, 1992; Wiener and Spry, 1995; Watras et al., 1998; Gonzalez et al., 2005;

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Branco et al., 2007; Branco et al., 2011).

The calculated levels of lipids (%) in the muscle of species (P. lateristriga:

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26.85 ± 21.34; P. adspersus: 31.53 ± 18.02; P. fur: 30.61 ± 18.45; O. Hepsetus: 5.60 ± 2.66) did not correlate significantly with the levels of THg and CH3Hg+. Non-significant correlations between these variables have been observed by other authors (Pethybridge et al., 2010). The mean lipid level in O. hepsetus was low, compared with the other species. Classified as a pelagic piscivore, its diet may be presumed to contain fattier items, considering the prey consumed by P. fur, P. adspersus, and P. lateristriga.

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ACCEPTED MANUSCRIPT Nevertheless, the prolonged drought in 2014 and 2015 (AGEVAP, 2016) may have reduced the availability of the prey consumed by O. hepsetus, forcing it to explore other trophic resources in a restricted food scenario. In a study about the trophic ecology of fish species in the lower section of the PSR, Rocha et al. (2015) observed that the

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isotopic niche of O. hepsetus overlapped significantly with that of P. fur, P. adspersus, P. lateristriga, and other demersal fish, and suggested that these species compete for trophic resources.

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A correlation between CH3Hg+ concentration in muscle and weight was also expected, since this chemical species is slowly excreted from the fish muscle (Peterson

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and Sicke, 2007; Storelli et al., 2007; Carrasco et al., 2011; Polak-Juszczak, 2012 ; Burger et al., 2013; Storelli et al., 2005; Quiu et al., 2011; Maceda-Veiga et al., 2012). Nevertheless, P. fur was the only species in which the levels of CH3Hg+ in muscle correlated positively and significantly with TW (p < 0.0005) (Table 2 and Fig. 3). The

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consumption of preys from higher trophic levels by an individual of a given fish species as it grows in size may explains the positive correlation between these variables (Trudel and Rasmussen, 2006).

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The non-significant correlations between the levels of CH3Hg+ in the muscle of the other three species may be explained by the poor distribution of individuals along

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the weight intervals considered (< 20 g, 20 – 40 g, 41 – 60 g, 61 – 80 g, 81 – 100 g, 101 -120 g, 121 – 140 g, 141 – 160 g, and >160 g). A large number of the individuals of O. hepsetus (63%), P. adserpsus (36%), and P. lateristriga (70%) were in the 20 - 40-g range. For P. fur, the highest percent number of individuals in the same weight range (which was also 20 - 40 g) was 23%. Except for the < 20% and 141 - 160-g weight intervals, all other weight classes represented between 10% and 16% of individuals. This indicates a more even distribution of P. fur samples between the weight intervals.

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ACCEPTED MANUSCRIPT Also, the non-significant correlations between TW and the CH3Hg+ levels in liver and gills suggest that these tissues are only lightly influenced by weight gain (Table 2). Even though the liver is a tissue that can accumulate Hg levels that exceed those stored in muscle (Table 1), the organ plays an important role in the detoxification

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and excretion of CH3Hg+ through demethylation reactions that convert the organic form of Hg into less toxic and bioaccumulative species. Such metabolic function prevents the accumulation of CH3Hg+ in the liver in view of weight gain. Though the correlation

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coefficients for L and TW in the four species (Table 2) were not significant, they are negative, which agrees with the liver’s demethylation and excretion ability.

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3.2 Influence of diet

The invertivore species (P. fur) presented the highest THg levels in muscle, while the piscivorous one (O. hepsetus) had the lowest THg in liver (p < 0.05). The levels of THg in the gills of the four species did not differ significantly (p > 0.05).

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Concerning CH3Hg+, the concentrations in muscle did not differ across species (p > 0.05), and the levels in the liver of P. fur, O. Hepsetus, and P.lateristriga were lower than those recorded in the liver of P. adspersus (p < 0.05). The comparisons between

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the levels of contaminant in gills were not carried out, because of the small number of samples from P. lateristriga and O. herpsetus.

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These results do not support the expected hierarchy of food habits in terms of the

bioaccumulation of THg and CH3Hg+ (piscivores > invertivores > omnivores). However, the distribution of these contaminants in the tissues of the species studied may indicate that these organisms are exploring other, similar food resources. The main reasons backing this statement are the environmental conditions in the study area during sample collection and the literature data about isotopic analyses (C and N) in the muscle of the four species examined.

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ACCEPTED MANUSCRIPT During droughts, the diversity of the prey consumed by fish may decline (Abelha et al., 2001). In this scenario, fish may have to explore alternative trophic resources as a survival strategy (Begon et al., 2005). The determination of the isotopic signatures of δ13C and δ15N conducted by

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Rocha et al. (2015) for the four species included in the present study indicate that the niche of the pelagic carnivore species living in the lower section of PSR (represented mainly by O. hepsetus) overlaps significantly with the niches taken by P. fur, P.

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lateristriga, and P. adspersus, and that these animals share trophic resources. Therefore, the intake of THg and CH3Hg+ from nutrition may not have varied considerably

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between the fish examined, explaining the statistically similar concentrations of CH3Hg+ in the muscle of these species. It should be emphasized, however, that the differences observed in the concentrations of THg and CH3Hg+ in the liver of these species are not good indicators of the influence of food habits, since this tissue is able to

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demethylate the organic form of Hg, promoting the excretion of the metal.

3.3 Influence of abiotic matrices

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In the present study, the levels of THg measured in sediment were as many as 1,000 times higher than in the water column (the mean values of physicochemical

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parameters observed in the two collection periods were: transparency (m) = 2.77 ± 0.71; T (°C) = 23.35 ± 1.61; pH= 7.4 ± 0.1; O2 saturation (%) = 99.62; electric conductivity (µS cm-1) = 46.96 ± 6.04), considering the overall extension of the sampling sites (approximately 120 km) and the fact that all species were captured in all collection sites (Table 3). The effect of exposure to sediment, concerning protection of aquatic life, is represented by two parameters, the threshold effect level (TEL = 0.147 mg kg-1) and the probable effect level (PEL = 0.486 mg kg-1) (MacDonald et al., 1996), which indicate

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ACCEPTED MANUSCRIPT the risk of deleterious effects to the aquatic biota (Table 3). The comparison of sediment variables observed in the present study and TEL and PEL values indicate that the sediment in all collection sites poses only a very low hazard of adverse effects to the local biota.

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However, even under such conditions, we observed high CH3Hg+ percent values in the gills of all fish species analyzed, especially the demersal species P. lateristriga (Fig. 2). These results may be associated with the closer contact the species has with

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sediment, as discussed in previous studies about the relationship between the extent of contamination in this compartment and THg and CH3Hg+ bioaccumulation in fish

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(Kasper et al., 2009; Hosseini et al., 2013; Rocha et al., 2015). Abdolvand et al. (2013) also attributed the influence of sediment on the high percent levels (> 90%) and high concentrations of CH3Hg+ observed in gills of the benthonic specie Epinephelus diacanthus (1.025 ± 0.05 mg kg-1, dry wt.) in river Arvand, Iran.

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The effect of sediment on bioaccumulation in gills may be potentiated in shallow aquatic ecosystems, as in the PSR, during low rainfall periods (considering the transparency of the water column, which was as low as 2.77 ± 0.71 m). These

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conditions prompt the need for a foraging strategy characterized by incursions along the entire water column (Rocha et al., 2015), when the bottom sediment is stirred,

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increasing the system’s turbid zone and therefore the amounts of available suspended particulate matter, mainly at greater depths. Therefore, physical characteristics of the system like water column height as well as ecologic factors such as a species’ habitat and foraging strategy may either increase or decrease the influence of abiotic matrices on the bioaccumulation of Hg and CH3Hg+ by fish.

4. Conclusion

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ACCEPTED MANUSCRIPT The levels of CH3Hg+ in muscle were influenced by weight gain of the fish analyzed. Nevertheless, the number of individuals should distribute the most homogeneous way possible across the weight intervals for each species. Also, TW did not affect THg and CH3Hg+ levels in liver and gills considerably.

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It was not possible to define the order of the concentrations of THg and CH3Hg+ in muscle, liver, and gills of the four species investigated based on the food habits of each, due to the prolonged drought in the PSR during the collection period and the

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overlapping of trophic niches of the species studied. The high percent values of CH3Hg+ in the gills of these fish, especially in the demersal P. lateristriga indicate the

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importance of sediment as a representative source of Hg and CH3Hg+ in PSR.

Acknowledgements

The authors are grateful to Projeto Piabanha (permission given by Brazil’s

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Ministry for the Environment, 36260-2) for capturing fish, to Prof Carlos E. V. de Carvalho and Prof Marcos S. M. de B. Salomão (Universidade Estadual do Norte Fluminense Darcy Ribeiro) for providing physico-chemical data, to Laboratório de

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Radioisótopos Eduardo Penna Franca (LREPF) of Universidade Federal do Rio de Janeiro for permission to use the mercury analyses system (FIMS-400). Cristina M

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Souza received a grant from Fundação de Amparo à Pesquisa do RJ (FAPERJ - E26/111.790/2013). References

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ACCEPTED MANUSCRIPT Fig. 1. Lower section of the drainage basin of river Paraíba do Sul and sampling sites. Fig. 2. Percent CH3Hg+ values in three tissues of four fish species captured in Paraíba do Sul river. Different letters indicate statistically significant difference (p < 0.05). Fig. 3. Pearson correlation between concentrations of CH3Hg+ (wet wt.) and total weight of P. fur captured in Paraíba

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+ -1 Table 1. Biometric data and THg and CH3Hg concentration in four fish species captured in Paraíba do Sul river (mg kg wet wt.).

Biometric data (mean±SD)

THg (mean±SD)

Min-Max

Min-Max

Min-Max

Species

TL (cm)

TW (g)

Muscle (N)

Pimelodella lateristriga

16.48 ± 1.74

30.09 ± 11.93

(Omnivorous)

12.4 - 20.0

14.8 - 71.7

0.085 ± 0.054 (30) 0.015-0.207

0.189 ± 0.106 (30) 0.032 –0.4683

0.032 ± 0.021 (30) 0.003 – 0.099

0.079 ±0.053 (20) 0.019 – 0.236

0.014 ±0.008 (18) 0.001 – 0.040

0.016 ±0.008 (3) 0.007 – 0.021

Pachyurus adspersus

19.51 ± 2.81

57.87 ± 29.0

(Invertivore)

15.3 - 25.7

25.62 - 161.38

0.061 ± 0.042aA (30) 0.007 – 0.164

0.211± 0.13bA(30) 0.020 – 0.458

0.025 ± 0.015cA (30) 0.001 – 0.06

0.065 ±0.039dC (16) 0.011 - 0.163

0.044 ±0.020eD (20) 0.007 - 0.086

0.020 ±0.011f(19) 0.004 - 0.044

Pimelodus fur

19.76 ± 3.89

75.65 ± 45.63

(Invertivore)

13.4 - 27.5

21.14 - 206.2

0.144 ± 0.076 (30) 0.062 – 0.351

0.249 ± 0.128 (30) 0.065 – 0.637

0.030 ± 0.016 (30) 0.009 – 0.076

0.074 ±0.039 (28) 0.023 - 0.227

0.017 ±0.010 (21) 0.003 - 0.046

0.013 ±0.008 (24) 0.003 - 0.033

Oligosarcus hepsetus

16.64 ± 2.52

37.55 ± 22.71

aA 0.077 ± 0.041 (30)

0.106 ± 0.074aB (30)

0.029 ± 0.014bA (30)

0.059 ±0.031dC (30)

0.018 ±0.013eE (16)

0.019 ±0.013e (7)

(Piscivore)

11.3 - 22.7

15.5 -112.5

0.019 – 0.203

0.012 – 0.341

0.007 – 0.062

0.018 - 0.146

0.003 - 0.049

0.005 - 0.041

aB

bA

bA

Different lowercase letters indicate significant statistical difference (p < 0.05) in THg and CH3Hg+ between tissues of the same specie Different uppercase letters indicate significant statistical difference (p < 0.05) in THg and CH3Hg+ between the same tissues of different species N = number of samples TL = total length

cA

cA

Muscle (N) dC

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TW = total weight

Gills (N)

Liver (N) eC

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Liver (N)

CH3Hg+ (mean±SD)

Gill (N) e

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M x TW -0.08 0.61* 0.11 -0.17

L x TW -0.28 -0.012 -0.02 -0.15

G x TW 0.06 0.36 IS -0.37

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ITA

S 21º38'04'' W 42º02'01.9''

1.97 x 10-6 ± 1.3 x 10-6

0.047 ± 0.001

RPO

S 21º34'51.6'' W 42º08'25.1'' S 21º44'14.6'' W 41º055'55.1'' S 21º38'04.9''

1.12 x 10-6 ± 1.38 x 10-6

0.045 ± 0.005

1.57 x 10-6 ± 7.67 x 10-7

0.035 ± 0.002

8.69 x 10-7 ± 6.02 x 10-8

0.062 ± 0.001

RDR SFI

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Table 3. Concentration of THg in the SPM (volumetric basis mg L-1) and sediment (mg kg-1 wet wt.) in five sites in Paraíba do Sul river. Geographical SPM Sediment Coordinates (Mean ± SD) (Mean ± SD) -7 -8 S 21º44'54.7'' 0.041 ± 0.004 SSP 1.36 x 10 ± 2.03 x 10 W 42º20'32.7''

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W 41º45'41.2'' SSP: São Sebastião do Paraíba, ITA: Itaocara, SFI: São Fidelis, POR: Baltazar, RDR: Guarani SPM: Suspended Particulate Matter.

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Concentration of CH3Hg+ did not vary among feed habits.

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Sediment as a representative source of mercury to fish.

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