Ecotoxicology and Environmental Safety 118 (2015) 90–97

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Mercury in muscle and brain of catfish from the Madeira river, Amazon, Brazil Wanderley R. Bastos a, José G. Dórea b,n, José Vicente E Bernardi c, Leidiane C. Lauthartte a, Marilia H. Mussy a,d, Marília Hauser e, Carolina Rodrigues da C. Dória d, Olaf Malm e a

Laboratório de Biogeoquímica Ambiental, Universidade Federal de Rondônia, Brazil Faculdade de Ciências da Saúde, Universidade de Brasília, Brazil c Instituto de Química, Universidade de Brasília, Brasília, Brazil d Laboratório de Ictiologia e Pesca, Universidade Federal de Rondônia, Brazil e Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 March 2015 Received in revised form 13 April 2015 Accepted 16 April 2015

The central nervous system is a critical target for Hg toxicity in all living organisms. Total Hg (THg) was determined in brain and muscle samples of 165 specimens of eight species of catfish (Brachyplatystoma filamentosum; Brachyplatystoma platynemum; Brachyplatystoma rousseauxii; Brachyplatystoma vaillantii; Phractocephalus hemiliopterus; Pseudoplatystoma punctifer; Pseudoplatystoma tigrinum; Zungaro zungaro) from the Madeira River, Brazilian Amazon. Despite the narrow range of Fishbase trophic level (4.2–4.6) the median THg concentrations ranged from 0.39 to 1.99 mg/kg and from 0.03 to 0.29 mg/kg respectively in muscle and brain from the studied species. Overall, the median concentration for all samples analyzed was 0.93 mg/kg and 0.16 mg/kg respectively in muscle and brain; most samples (76%) showed muscle Hg concentrations 40.5 mg/kg. There were statistically significant THg differences between sex (female 4males). The correlation between THg concentrations in muscle and brain was statistically significant (r¼ 0.9170; po 0.0001). In the studied specimens, fish total length was significantly correlated with muscle (r ¼0.3163; p ¼0.0001) and brain (r ¼0.3039; p ¼0.0003) THg; however, fish age was negatively and significantly correlated (r ¼  0.2991; p¼0.0012) with THg in muscle but not with THg in brain (r ¼  0.0190; p¼0.8492). Amazonian catfish accumulate high levels of Hg in muscle and brain; however, brain-THg concentrations can be predicted from muscle-THg. Muscle-Hg in catfish can be a tool to detect brain-Hg concentrations associated with environmental Hg. & 2015 Elsevier Inc. All rights reserved.

Keywords: Catfish Methylmercury Brain Muscle Age Sex

1. Introduction The tropical rain forest of the Amazon with its abundance of life (terrestrial and aquatic) and regular annual flooding is a facilitator for methylation of naturally occurring mercury, leading to the production of methylmercury (MeHg). As shown in some studies (Achá et al., 2012; Correia et al., 2012) these wetlands are major sites of MeHg production; MeHg accumulates in fish depending on their trophic level. Indeed, fish from the Amazon Basin have a wide range of total-Hg (THg) concentrations in muscle (Barbosa et al., 2003; Bastos et al., 2015). The THg load is distributed differently in organs (liver4 muscle 4gonad4blood) of catfish (Cizdziel et al., 2003); nevertheless, the central nervous system is the critical target for Hg toxicity (Pereira et al., 2014). n

Corresponding author. Fax: þ55 61 81317800. E-mail address: [email protected] (J.G. Dórea).

http://dx.doi.org/10.1016/j.ecoenv.2015.04.015 0147-6513/& 2015 Elsevier Inc. All rights reserved.

There are laboratory studies demonstrating MeHg adverse effects on reproduction, biochemical processes, damage to cells and tissues of fish occurring at muscle-Hg concentrations of about 0.5– 1.0 mg/kg (wet weight); mathematical model studies used to assess fish toxicology have estimated that the lowest observable adverse effects level (LOAEL) is about 0.5 mg Hg/kg (wet weight) in muscle (see references in Scheuhammer et al., 2015). In fish from Amazonian rivers, these LOAEL levels are frequently exceeded in large specimens at the top of the food web including catfish sampled up to 2003 (Barbosa et al., 2003). Potential risks for Amazon fish species have been proposed by others (Silva et al., 2012). Crump and Trudeau (2009) have reviewed the effects of Hg accumulation in the fish brain, describing “reduced neurosecretory material, hypothalamic neuron degeneration, and alterations in parameters of monoaminergic neurotransmission”. In experimental conditions, catfish decreased brain serotonin, and increased dopamine (Crump and Trudeau, 2009).

W.R. Bastos et al. / Ecotoxicology and Environmental Safety 118 (2015) 90–97

The potential neurotoxic effects of Hg can be assessed by comparing brain Hg concentrations of fish to levels that can affect the central nervous system (CNS). Despite that, we do not have studies of fish-brain Hg concentrations. There is a scarcity of knowledge about fish biomarkers of MeHg neurotoxicity for use in aquatic environment studies. Besides needing baseline information to quantify the impact of methylation environments or contaminant sources, we need to learn fish neurotoxic thresholds. Catfish species are abundant in the Amazon Basin; they are essentially piscivorous and with migratory behavior (Barthem and Goulding, 1997; Fabré and Barthem, 2005). Approximately 2000 catfish species are found in South America; Brachyplatystoma rousseauxii and Brachyplatystoma filamentosum are the most traded in the region and Siluriformes species represent 17% of the fishing industry in the Madeira River basin, (Doria et al., 2012). Amazon catfish occupy the top of the aquatic food chain showing the highest Hg concentrations; in the commonly caught Brachyplatystoma genus these concentrations are above those recommended (1.00 mg/kg) in the Brazilian legislation for human consumption (MS-Ministério da Saúde, 1998; Bastos et al., 2008; Hacon. et al., 2014). There is a general paucity of data regarding the retention of Hg in fish brain. Environmental studies of fish-Hg from the Amazon collected samples during specific expeditions. Frequently, sampling sites were established to assess a specific environmental situation (Oliveira et al., 2010) or to maximize the probability of finding statistical differences related to impacted locations (Dórea and Barbosa, 2007). However, fish consumed by riverines tend to be smaller than fish sold at markets (Oliveira et al., 2010). The differences between fish size (and accompanying Hg concentrations) as consumed by anglers and that caught by scientists have also been raised by Burger et al. (2006). Indeed, differences in fish-Hg information derived from fish size collected by scientists and professional fisherman (common size to be marketed) might have implications for exposure assessment in both humans and wild life (Burger et al., 2006). With the largest diversity of fresh-water fish in the planet, there is a lack of data regarding Hg concentrations in brain of fish from Amazonian tropical rivers. The main objective of this study is to generate information to identify physiological factors useful for monitoring wildlife Hg exposure. To this end we measured Hg in brain and muscle tissues of catfish species abundant in the Amazon Basin, aiming (1) to determine THg and MeHg concentrations in the muscle and brain of Amazonian catfish; (2) to relate these concentrations to fish size, sex, and age.

91

2. Materials and methods In order to optimize the size of specimens while reducing the variability of randomly caught fish, and to minimize financial and logistic efforts, we chose our samples from catfish caught (during the year 2010) by professional fisherman before being dispatched to the fish market in the state capital of Porto Velho; specimens are currently considered to be of commercialized length, but they are smaller than commonly attained maximum length and weight (Table 1). With the lowest effort and cost we could study Hg in freshwater fish at the top of trophic levels. These fish were caught along the Madeira River between the cities of Guajará-Mirim-RO (Long 243219.152 – Lat 8805970.844-UTM) and Humaitá-AM (Long 497790.381 – Lat 9170102.251-UTM); these sites represent fisherman preferences. Fish species and feeding habits were established according to taxonomy and identification keys (Santos et al., 2006); for each specimen the total length was measured (cm) from the tip of the snout to the posterior end of the last vertebra, as well as total weight (g), sex, and gonadal maturation stage were recorded. Sex was determined macroscopically after examination of the gonads; based on gonad inspection and secondary sexual characteristics (Vazzoler, 1996), only reproductively mature catfish were used. For the species of Brachyplatystoma genus the age was determined by counting growth marks of the otoliths, according to data from Santo Antonio Energia and Energia Sustentável do Brasil Madeira River Ichthyofauna Conservation Program (Santos et al., 2012). For the species of Pseudoplatystoma genus the age was determined according to the age-length table (Loubens and Panfili, 2000). A sample of muscle (circa 20 g, wet weight) was removed from the side of the head near the dorsal region; otolith and brain samples were taken after sawing the cranial bones with a stainless steel saw. All samples (of muscle, otolith and brain) were individually labeled and stored frozen until analyses. The brain was stored in Eppendorf vials (5.0 mL). The fish otoliths were washed in water, dried and stored in referenced envelopes for later laboratory processing (Alonso, 2002; Santos et al., 2012). Hg (THg and MeHg) concentrations in muscle tissue and THg in brain tissue were determined by in-house developed and validated methods described elsewhere (Malm et al., 1989 and Bastos et al., 1998) for THg; Liang et al. (1994) and Taylor et al. (2011) for MeHg). Briefly, THg determination was done in samples after treatment with H2SO4:HNO3 (1:1, Tédia, Brazil) solution and KMnO4 (5% w/v-Merck) of 200 mg of muscle tissue and 50 mg of brain (wet weight). After weighing samples, 5.0 mL of the acid mixture was added and taken to a digestion block for 60 min

Table 1 Fish size (standard length and total weight) and total mercury (THg) concentrations in muscle and brain tissues (n ¼165) of several species of catfish in relation to Fishbase* trophic level, and maximum attainable weight and length. Scientific name

Brachyplatystoma filamentosum Brachyplatystoma platynemum Brachyplatystoma rousseauxii Brachyplatystoma vaillantii Phractocephalus hemiliopterus Pseudoplatystoma punctifer Pseudoplatystoma tigrinum Zungaro zungaro ND¼ No data. a

Fishbase (2014).

n

Fishbasea trophic level

Max length (cm)

Max weight (kg)

Standard length range (cm)

Total weight (kg)

Muscle tissue

Brain tissue

THg (mg/kg) median (min–max)

THg (mg/kg) median (min–max)

4.5 70.79

360

200

110–126

9.0–42.0

1.647 (0.705–4.640)

0.268 (0.085–1.109)

61 4.5 70.80

360

200

45–79

1.5–7.0

1.986 (0.413–4.891)

0.290 (0.043–1.150)

4.6 70.8 4.5 70.80 4.2 70.73 ND 4.5 70.0 4.5 70.80

192 150 134 ND 130 140

ND 20 44.2 ND 17 NG

70–101 35–51 49–73 35–59 46–91 45–80

4.0–14.0 0.5–2.5 2.5–8.0 0.7–13.3 6.5–9.0 1.3–13.0

0.634 0.661 0.385 0.388 0.486 0.668

0.098 (0.050–0.645) 0.132 (0.037–0.410) 0.031 (0.012–0.073) 0.051 (0.025–0.401) 0.032 (0.013–0.096) 0.088 (0.024–0.283)

16

25 18 10 16 05 14

(0.292–4.139) (0.123–1.076) (0.183–0.472) (0.201–1.743) (0.244–0.661) (0.295–1.333)

W.R. Bastos et al. / Ecotoxicology and Environmental Safety 118 (2015) 90–97

3. Results This study presents total Hg concentrations in muscle and brain tissues of eight species of catfish of the Siluriforme Order and the Pimelodidae Family (Brachyplatystoma filamentosum – filhote/piraíba, n ¼16; Brachyplatystoma platynemum – babão, n¼ 61; Brachyplatystoma rousseauxii – dourada, n ¼25; Brachyplatystoma vaillantii – piramutaba, n¼ 18; Phractocephalus hemiliopterus – pirarara, n ¼10; Pseudoplatystoma punctifer – surubim, n ¼16, Pseudoplatystoma tigrinum – surubim/carapari, n ¼05; Zungaro zungaro – jaú, n ¼14). Table 1 shows tissue THg concentrations, as well as body measurements (total weight and standard length). The studied species had a very close Fishbase (2014) trophic level (4.2–4.6) and a wider range of size. The range of standard length varied among species but represented common size within each species; species-specific maximum attainable size is much larger than the specimens sampled (Table 1). Approximately 76% of

THg

MeHg

MeHg:THg

100 80

3.00

60 2.00 40 1.00

20 0

ux ea ss ou .r )B (5

(1

0)

B.

.f ila m

pl at yn

en

to s

em

um

um

ii

0.00

)B

MeHg:THg (%)

4.00

THg and MeHg (mg/kg)

(Tecnal-Mod.007A, Piracicaba, São Paulo, Brazil). After digestion, 4.0 mL of KMnO4 solution (5% w/v) was added and left for 30 min more in the digestion block. After cooling to room temperature (725 °C), hydroxylamine hydrochloride drops of solution at 12% (w/v) were added and the digested samples were transferred to a volumetric flask and diluted with 10.0 mL of ultra-pure H2O (MilliQ Plus, Millipore, Bedford, MA, USA). THg determinations were carried out by cold vapor atomic absorption spectrometry (Flow Injection Mercury System- FIMS -400- Perkin Elmer, Ueberlingen, Germany) (Malm et al., 1989; Bastos et al., 1998). For the MeHg determination in dorsal muscle samples we used the EPA (2001) and Liang et al. (1994) methods. A weighed (100 mg wet weight) sample, 5.0 mL of 25% (w/v) KOH methanolic solution was added to extract MeHg in an oven (Nova Instruments, Model NI 1512, São Paulo, Brazil) at 70 °C for 6 h; gentle stirring was applied every hour. The samples were then kept in the dark to avoid possible degradation of MeHg. Ethylation was done with 300 μL of 272 g/L sodium acetate buffer (pH 4.5) followed by the addition of 30 μL of sample and 50 μL of 1% (w/v) tetra ethyl sodium borate solution (Brooks Rand Labs, Seattle, USA) after Taylor et al. (2011); the final volume of 40 mL was attained with ultrapure water (milli-Q, Millipore, Cambridge, MA, USA). MeHg was then determined in a MERX-TM automated system from Brooks Rand Labs (Seattle, USA) equipped with an auto-sampler, a purge and trap unit, a packed column GC/pyrolysis unit, and a Model III atomic fluorescence spectrophotometer. Method accuracy of Hg determinations were ensured by the use of certified material (Tuna Fish, BCR-463 – THg ¼ 2.850 70.160 mg/kg and MeHg ¼ 3.040 70.160 mg/kg) which was run with each batch of samples; mean recovery for THg was 99% (2.810 70.022 mg/kg – n ¼ 07) and for MeHg was 97% (2.980 70.028 mg/kg – n¼ 06). All analysis of samples and certified reference materials were run in triplicate. Limits of detection (LD) and quantification (LOQ) were 0.016 mg/ kg and 0.058 mg/kg and for THg and 0.003 mg/kg and 0.009 mg/kg for MeHg (CITAC, 2002). All glassware was washed clean in 10% HNO3 and rinsed with ultra-pure H2O. With this dataset (n ¼165 samples) of catfish we estimated predictive equations for THg in brain from THg in muscle. The statistical analyses were done with packages contained in XLSTAT (Adinsoft, version 1.01, 2013, Paris, France) and PRISM (version 4.0; San Diego, CA) software, which was also used for data summarization (in figures) and correlation analysis. We used the nonparametric Mann–Whitney U-test (alternative to the t-test for independent samples) in a multiple linear regression analysis to determine the predictive equations of brain-Hg concentrations from muscle-Hg concentrations. We accepted a risk level of o0.05 as statistically significant.

(5

92

Fig. 1. Whisker-box plot representing median and standard deviation of THg and MeHg concentrations in muscle as well as MeHg:THg ratio of three species of catfish.

catfish (126 specimens) examined contained 40.50 mg/kg THg (WHO, 1990). The dominant chemical form of Hg in muscle was MeHg (mean 63%; range 43–88%; Fig. 1). We measured MeHg in muscle tissue samples of 12% (n ¼20) of only three species with sufficient material to determine both THg and MeHg-B. filamentosum; B. platynemum; B. rousseauxii; correlation between MeHg:THg ratio and fish age was negative but statistically nonsignificant (r¼  2769; p¼ 0.2820). Fig. 2 integrates variations in THg concentrations in muscle and brain tissues with brain:muscle ratio (Fig. 2a) and with standard length (3b); a different pattern of tissue Hg concentration is seen between brain:muscle ratio of THg concentrations (Fig. 2a) and fish standard length (Fig. 2b); coincidentally, the largest THg concentrations (in both muscle and brain) was disproportionally higher in B. platynemum than in the other studied species (Table 1). The relationships between THg concentrations in muscle and brain for all samples, and as a function of sex (for a representative number of specimens) are shown in Fig. 3. There was a strong and significant correlation (r ¼ 0.9145; p o0.0001) between muscle and brain THg concentrations (Fig. 3a); regression of mean THg concentration in brain muscle was positive and highly significant. Regression lines for males and females are illustrated in Fig. 3b; the slope was slightly higher for males than for females and statistically significant (p ¼0.001). A boxplot summarizes differences between sexes for both muscle and brain mercury concentrations; males had significantly lower THg concentrations in muscle and brain (Fig. 3c). Most of the fish sampled represented a common length for each species. Fish length and age in relation to THg concentrations are illustrated in Fig. 4. Furthermore, there was a statistically significant correlation between total length and Hg concentrations in muscle. A significant positive linear relationship was seen between total length and mercury concentration in muscle (r ¼ 0.3162; po 0.0001) and brain (r ¼0.3019; p o0.0003); Therefore, we consider our measurements of THg in muscle tissue to be representative of the THg concentration in brain. Fig. 4 shows scatterplots illustrating the relationship between THg in muscle and brain as a function of fish total length and age. Correlation was positive and statistically significant between total length and THg in muscle (r ¼0.3163; p¼ 0.0001) and brain (r ¼0.3039; p ¼0.0003). However when plotted against fish age, THg was negatively correlated in muscle (r ¼  0.2991; p ¼0.0012) and in brain (r ¼  0.0190; p ¼0.8492). Table 2 summarizes studies reporting both muscle and brain THg of different fish trophic levels from several countries in natural environments. Even when comparing similar trophic levels

W.R. Bastos et al. / Ecotoxicology and Environmental Safety 118 (2015) 90–97

4.00

100 75

3.00 50 2.00 25

1.00

0.50

0

(6 1) B. pl at yn (1 6) em B. um fil am en (2 to 5) su m B. ro us se au (1 xi 4) i Z. zu ng ar (1 o 8) B. va illa (0 nt 5) ii P. tig r in (1 um 6) P. pu (1 0) nc P. tif er he m ilio pt er us

0.00

Brain [THg] mg/kg

Brain:Muscle

Brain:Muscle (%)

2.00

40

1.00

20

0.00

0

0.60

0.80

1.00

1.20

Male r=0.9104

0.60 0.40

tif

er

0.20

P. 8) (0

2.00

(0

Fig. 2. Whisker-box plot representing median and quartiles of THg concentrations in muscle, brain, brain:muscle ratio (3a, n ¼165), and bar graph (3b; n¼ 84) showing muscle and brain THg in relation to standard length of eight species of catfish.

6.00

Total [Hg], mg/kg

4. Discussion Our results show that brain-Hg concentrations are strongly correlated with muscle-Hg concentrations; actually muscle-THg is a robust predictor of brain Hg concentrations. Regarding the high correlation between muscle and brain Hg concentrations, our results are in agreement with that of Adams and Sonne (2013) reported in the goliath grouper of US waters. In catfish, brain-Hg concentrations represent 10–25% of that found in muscle; these findings, with the largest number of samples representing fish of high trophic levels, are within a range reported by others for a variety of species (0.3–59%; Table 2); in waters polluted by phenylmercury from the pulp and paper industry, brain-Hg was higher than muscle-Hg (Steinnes et al., 1976). Because of concerns about Hg effects on neurological functions, crucial for survival, brain-mercury can be monitored through muscle-Hg concentrations. Despite that, it is surprising how few studies there are (Table 2) addressing muscle and brain-Hg concentrations and how wide the range of brain:muscle Hg ratio is in

6.00

Male n=27

Female n=15

4.00

2.00

) (M

)

ai Br

cl M

us

n

(M e

n ai

us

cl

e

(F

(F

)

)

0.00

M

(4.1–4.7) the brain:muscle ratio was wider (1.1–58.7) than in our study. In our study, with the largest number of specimens (165 samples from eight species), the results showed a variation of brain:muscle ratio between 8.3% and 25% while in other studies this variation was wider (0.3–59%). This pattern of Hg distribution is confirmed by the experimental study of Kennedy (2003) exposing goldfish (Carassius auratus) to inorganic Hg at levels found in dental amalgam discharges. Indeed, except for the report of Coulibaly et al. (2012), in all studies muscle-Hg was higher than brain-Hg.

4.00

Muscle [THg] mg/kg

9)

P.

0.40

Female r=0.8468

0.00 0.00

pu

pt ilio he

m

P. 6) (0

0.20

0.80

nc

er

um tig

ng zu Z. 4)

(0

6) (1

rin

ar

nt illa va B.

us ro B. 0)

(1

o

ii

i xi au se

em yn at pl

0.10

1.00

Brain [THg] mg/kg

60

us

3.00

um

Total [Hg], mg/kg

100 80

B.

0.20

Muscle [THg] mg/kg

4.00

1) (3

Standard length

Brain

0.30

0.00 0.00

Standard length, cm

Muscle

5.00

0.40

Br

Total [Hg], mg/kg

Brain

Muscle

5.00

93

Fig. 3. Scatter-plots of (a) muscle and brain THg concentrations in all samples (n ¼165), and (b) as a function of sex (n¼ 42) in species with representative number of both sexes; (c) whisker-box plot representing differences in THg concentrations in brain and muscle of selected species with samples of both sexes (n¼ 42, the mean is identified by þ ).

the few existing studies (0.3–59%). It is worth noting that for marine birds feeding exclusively on fish the muscle:brain ratio has been reported as 42.8% (Burger et al., 2014). Krey et al. (2015) show that MeHg:THg ratio in brain of whales varied from 17% to 20% depending on the brain region. Therefore, with this level of predictability, muscle-Hg concentrations could be used as a proxy to study brain-Hg related fish toxicology. In the current study, catfish showed a higher THg concentration than the threshold for neurobehavioral and/or neurochemical effects (Depew et al., 2012). However, large differences in muscleTHg concentrations (circa 5-fold) contrast with narrow differences in trophic levels (Table 2). Trophic position varied between catfish species, ranging from 4.2 to 4.6; variation in THg concentrations and maximum attainable size, however, was wider. Generalizing

94

W.R. Bastos et al. / Ecotoxicology and Environmental Safety 118 (2015) 90–97

1.5

Brain [Hg], ug/g

Muscle [Hg], ug/g

6

4

2

1.0

0.5

0.0

0 0

50

100

0

150

25

Total [Hg], mg/kg

Total [Hg], mg/kg

1.50

S r= -0.4349; p=0.0001 n=115

4.00

2.00

0.00 0.00

2.00

75

100

125

150

Total length, cm

Total length, cm 6.00

50

4.00

6.00

r= -0.01906 n=102

1.00

0.50

0.00 0.00

2.00

4.00

6.00

Age (years)

Age (years)

Fig. 4. Correlation between total Hg concentration in muscle (a) and brain (b) fish total length; correlation age and THg in muscle (c) and brain (d).

trophic level for THg concentrations within top-predator fish is not sufficient. This asymmetry underlies considerable variations related to both constitutional and environmental factors; they may reflect differences in forage items and fish-related constitutional factors (sex, age, and maximum attainable size). The difference in ranges of maximum attainable size (3-fold) and maximum attainable weight (10-fold) among species are biological characteristics controlling Hg metabolism. Therefore, it is challenging to interpret

THg concentrations and their functional significance in wild fish. As discussed by Romanuk et al. (2011) fish body size shapes the demand for food and nutrition; thus, for fish at the top of the aquatic food web, body size should have a positive relationship with mercury concentrations in tissues. THg accumulation in fish also depends on the species trophic level. In the studied catfish (Fishbase, 2014 level between 4.2 and 4.6), we found that THg concentrations in muscle are better correlated with fish length than with fish age. Indeed we showed a significant correlation of

Table 2 Summary of mercury concentrations (mg/kg wet weight) in samples of brain and muscle of fish from different studies. Species

Tr.Levela

n

Muscle (M)

Brain (B)

B:Mb%

Country

Reference

Brachyplatystoma filamentosum Brachyplatystoma platynemum Brachyplatystoma rousseauxii Brachyplatystoma vaillantii Phractocephalus hemiliopterus Pseudoplatystoma punctifer Pseudoplatystoma tigrinum Zungaro zungaro Perca fluviatilis Somniosus microcephalus Epinephelus itajara Pomatomus saltatrix Mugil cephalus Dicentrarchus labrax Liza aurata Sarotherodon melanotheron Mustelus mustelus Trichiurus lepturus Carcharhinus limbatus Rhizoprionodon terraenovae Roccus saxatilis

4.5 70.79 4.5 70.80 4.6 70.8 4.5 70.80 4.2 70.73 NG 4.5 70.0 4.5 70.80 4.4 70.0 4.2 70.6 4.17 0.0 4.5 70.3 2.5 70.17 3.5 70.50 2.3 70.16 2.5 70.19 3.8 70.3 4.4 70.4 4.4 70.4 4.4 70.5 4.7 7 0.2

24 61 25 18 10 16 05 14 43 3 56 40 21 30 40 60 22 31 ND ND 50

1.97 2.11 0.98 0.64 0.35 0.52 0.48 0.73 1.05 4.10 0.63 0.32 0.27 96.2 93.2 0.17 1.77 0.05 3.33 0.76 0.31

0.37 0.35 0.15 0.16 0.03 0.07 0.04 0.10 0.404 0.62 0.37 0.09 0.09 0.3 0.3 0.25 0.16 0.02 1.33 0.45 0.08

18.8 16.6 15.3 25 8.6 13.5 8.3 13.7 38.5 15.1 58.7 28.1 33.3 0.3 0.3 147 9.0 40.0 39.9 59.2 25.8

Amazon, Brazil Amazon, Brazil Amazon, Brazil Amazon, Brazil Amazon, Brazil Amazon, Brazil Amazon, Brazil Amazon, Brazil Slovakia Greenland USA USA India Portugal Portugal Ivory Coast Italy Brazil NG NG USA

This work This work This work This work This work This work This work This work Brázová et al. (2012) Corsolini et al. (2014) Adams and Sonne (2013) Burger et al. (2012) Menon and Mahajan (2013) Mieiro et al. (2012) Mieiro et al. (2012) Coulibaly et al. (2012) Storelli et al. (2011) Cardoso et al. (2009) Núnez-Nogueira (2005) Núnez-Nogueira (2005) Cizdziel et al. (2003)

ND¼ No data given. a b

Fishbase trophic level (Fishbase, 2014). Estimated from the reported means.

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body length with both brain and muscle mercury concentrations. However, the correlation between THg and fish age was weak and statistically non-significant. Catfish has a broad lifespan, and, in this case, showed an age variation between one and six years, therefore with both fast and slower growth rate intervals. Furthermore, it is known that fish of the same cohort or age may exhibit different growth rates. Such evidence of slow and fast growth rate within the same age group has already been reported for B. rousseauxii in the Amazon system (Alonso, 2002). This may explain the lack of statistically significant association between age and THg concentrations. Johnson et al. (2015) reviewed the role of prey in Hg bioaccumulation suggesting that “when preferred prey are rare, predators may need to expend more energy in foraging for less energetically profitably prey”; this may result in reduced growth efficiency and/or age-related Hg elimination, key drivers of bioaccumulation of mercury (Dang and Wang, 2012). Squadrone et al. (2015) recently reported that European catfish (Silurus glanis) exceeded the maximum level ( 40.5 mg/kg) in 37% of analyzed samples in fish muscle; this is a much lower proportion than what we found (77%) in Amazonian catfish. Squadrone et al. (2015) also observed that females displaced higher THg concentrations than males. This is in agreement with our results; we also found statistically significant differences between males and females. Fish eliminate MeHg slowly (Trudel and Rasmussen, 1997) and Hg levels in eggs are affected by MeHg content of adult captive fish (Hammerschmidtt and Sandheinrich, 2005). Therefore it is reasonable to assume that a difference in THg could occur between sexes; there were by far less studies related to sex differences in pollutant levels (including Hg) in fish than other types of wildlife (Burger, 2007). Indeed, in some species there is size related sexual dimorphism (Burger, 2007) with a predominance of females in larger size classes; a common reproductive tactic aimed at increasing fertility (Nikolsky, 1963; Lowe-McConnell, 1999). Therefore, when taking into consideration size variation in catfish our results show a slightly higher Hg concentration in females than in males. Recently, however, Madenjian et al. (2011) reported sex difference in lake trout; male with larger body weight also showed higher Hg concentrations than females. These researchers have studied the rate of feeding and rate of Hg elimination, which may be species-specific. This same group reported that the elimination rate in males was higher than in females in northern pikes (Madenjian et al., 2014); these observations were confirmed for sharks (Pethybridge et al., 2010). Contrary to this, others have reported (Willacker et al., 2013) that certain female-fish had lower THg concentrations than males (Stacy and Lepak, 2012; Gewurtz et al., 2011; Madenjian et al., 2015). Sex difference (males 4 females) in Hg concentrations for walleye (Gewurtz et al., 2011) and for other species have been reported (Monteiro and Lopes, 1990; Penedo de Pinho et al., 2002; Willacker et al., 2013). However, most studies reported non-statistically significant sex difference in fish-Hg concentrations (Magalhaes et al., 2007; Weis and Ashley, 2007; Harmelin-Vivien et al., 2009; Adams, 2009; Martinez-Gomez et al., 2012; Burger et al., 2007; Endo et al., 2008; Mela et al., 2014). Inconsistencies of reported differences in THg concentrations between male and females may relate species (sexual dimorphism included), trophic levels, fish size, and environment. Marketed fish in the Amazon are usually larger in size than corresponding species regularly caught and consumed by subsistence riverine populations (Oliveira et al., 2010). Variations in THg concentrations in muscle samples, both inter- and intra-species, have been extensively studied in fish from the Amazon Basin (Barbosa et al., 2003); the need to consider biological characteristics regarding Hg bioaccumulation is amply recognized. This study adds new features related to brain THg; besides Hg chemical

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speciation, our study includes a large number of catfish species, exploring influences of sex and age. This study also adds information crucial to monitoring fish-Hg contamination and potential toxic effects in the tropical-forest environment of the Amazon. With regard to fish from tropical forests, there is no other study addressing simultaneously Hg in muscle and brain. MuscleHg in catfish can be a tool to detect brain-Hg concentrations associated with environmental Hg contamination. Given the frequency of high Hg concentrations found in species at the top of the Amazonian food web, extrapolation of neurobehavioral findings from rodent models to fish may be an unsustainable assumption. Neural equivalence or parallelism, regarding Hg concentrations in the brain of fish and that of rodent models, has to take into consideration the respective toxicokinetics and toxicodynamics of Hg in fish. Basu (2015) has discussed the role of neurotoxicants in molecular and functional endpoints of aquatic life. Although undoubtedly Hg can affect neurotransmitters in fish, threshold levels of functional relevance remain to be established; in catfish as in other high trophic level fish, the threshold of muscle Hg concentration of 0.50 mg/kg needs more studies to establish its functional relevance in the wild. Trophic level in the studied Amazonian catfish species showed relative small variation (4.2–4.6); however, variations in THg concentrations and maximum attainable size were wider (Table 1). Therefore, generalizing trophic level for THg concentrations within top-predator species is not sufficient. Indeed, a comparison of our results with published catfish studies (Table 2) from different environments (fresh water, estuarine, and marine areas) shows also a wider variation in trophic levels (2.3–4.5). Furthermore, the data on brain:muscle ratio are fragmented and were estimated from mean values (Table 2). Nevertheless, in all studies, regardless of trophic levels, brain-Hg concentrations were always lower than in muscle. The biological traits such as maximum attainable weight of catfish indicate that some of them can indeed have longer lives and perhaps slow growth rates, which may blur the THg accumulation in comparative studies of these high trophic species. Furthermore, bigger catfish tend to eat bigger fish (or aquatic fauna) that likely have more Hg than smaller ones. Hg mean levels in muscle were higher than those in brains; however the ratio varied from 0.3% to 59%. This variation cannot be explained by the species' trophic levels or differences physicochemical conditions of waters (freshwater, estuarine, or marine environments) seen in the literature (Table 2).

5. Conclusions Amazonian catfish are high accumulators of Hg in muscle and brain; catfish Hg concentrations vary due to tissue type (muscle and brain), species, age, sex, biometrics (weight and length), and trophic level. Brain-Hg concentrations can be predicted from muscle-Hg concentrations, thus facilitating assessment of Hg dynamics in catfish.

Capsule abstract Brain-THg concentrations are lower but highly correlated with muscle-THg; thus, muscle-Hg in catfish can be a tool to detect brain-Hg concentrations associated with environmental Hg.

Acknowledgements This work was partly supported by the National Research

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Council of Brazil-CNPq (project-552331/2011-2). The authors also gratefully acknowledge the support of the laboratory of Ichthyology and Fishing of Rondônia Federal University, Santo Antonio Energia for the Grant 21/2012 (to M. H.), and Program Pro-Amazon RO (CAPES).

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