The concentrations of heavy metals and the incidence of micronucleated erythrocytes and liver EROD activity in two edible fish from the Paraíba do Sul river basin in Brazil

the Paraíba do Sul river (PSr) basin crosses one of the most populated and industrialized regions of Brazil and receives effluent from the metallurgic, paper mill and agricultural industries. PSr water has multiple uses, including being a supply of potable water and riverine populations which consume some species of its fish. In the present study, we investigated the responses of two biomarkers of aquatic pollution, EroD activity in the liver and micronuclei frequency in peripheral blood, as well as the concentrations of seven heavy metals in the muscle tissue of two species of fish native to the PSR: Geophagus brasiliensis and Hypostomus sp. Sampling was performed in both the wet and dry seasons of 2003, with a total of 339 fish collected from 16 sites along the PSR basin. Fish collected at Levy gasparian, Barra Mansa and três rios both had higher EroD activity and micronuclei frequency. The results also suggested a moderate induction of EROD activity in fish collected from S.L. Paraitinga and Sapucaia. Micronuclei frequency was elevated in fish from S.J. Campos, Além Paraíba, Campos and S.J. Barra. in all cases, heavy metal concentrations did not exceed the maximum considered safe for fish meat consumption.


INTRoDuCTIoN
the Paraíba do Sul river (PSr) drainage basin extends over 55,400 km 2 crossing three of the most industrialized states of Brazil: São Paulo, rio de Janeiro and Minas gerais.the PSr can be divided into three stretches according to the effects of the prevailing economical activities on drainage.the upper stretch, closest to the source of the PSr, is characterized by small cities and localized agricultural activities. in the middle stretch the PSr receives untreated sewage from larger cities and discharge from various industries, including chemical, metallurgic and paper mill plants.the mouth of the Paraibuna river is one of the largest PSr tributaries, which has a record of contamination with polychlorinated biphenyls (PCB), [1] and is located in the middle stretch.The lower stretch is influenced by intensive agricultural practice (sugarcane plantations) and has increased salinity due to the surrounding river mouth delta.the water from the PSr is used for agricultural irrigation, by industry and for domestic purposes (including as a supply for drinking water).Fishing also occurs along the basin.owing to the pollution of the PSr, consumption of its fish might pose health risks to the associated riverine population.
the present study was undertaken in order to evaluate the impact of contamination of the PSr basin by CYP1A inducers (Ethoxyresorufin-O-deethylase (EROD) activity) and genotoxic agents (micronuclei frequency in peripheral blood) on two edible fish.

Concentrations of heavy metals in muscle tissue in conjunction
with genotoxicity and Ahr-ligand (CYP1A, EroD) biomarkers not only indicate the extent of any contamination of edible fish meat by a diversity of industrial contaminants, which may pose risks to human health (such as polycyclic aromatic hydrocarbons (PAHs), polyhalogenated dibenzo-p-dioxins and furans (PCDD/ Fs), polychlorinated biphenyls (PCBs) and some highly toxic metals (Hg, Cd, Pb and others)), but also reveals the bioavailability of these pollutants to vertebrate species living in the PSr basin.

Fish capture and handling
A total of 202 Geophagus brasiliensis and 137 Hypostomus sp., adults of both genders were collected from 16 sites along the PSr and its major tributaries during the wet (January-April) and dry (September-November) seasons of 2003.table 1 shows the names of the closest cities and the river, and geographical coordinates of each sampling site.After capture, fish were immediately measured, weighed and decapitated.

Measurement of heavy metal concentrations
Fish muscle was dissected, as recommended by FAo [2].For quantification of lead (Pb), copper (Cu), chromium (Cr), magnesium (Mg) and nickel (Ni), muscle samples were dried at 80°C for 24 hours and subsequently incubated at 450°C for at least 48 hours, or until calcination was complete.Samples were treated with nitric:chloridric acid solution (3:1), followed by concentrated chloridric acid and re-suspended in 10 mL 0.1N chloridric acid.For analysis of mercury (Hg) concentrations, muscle was chemically digested (hydrogen peroxide, sulfuric:nitric acid solution, 1:1, potassium permanganate and hydroxylamine chlorhydrate).Metal concentrations were determined using atomic absorption spectroscopy, with appropriate standard curves and reference samples [1].

Assessment of the frequency of micronuclei
Peripheral blood was collected and smeared on glass slides for micronuclei determination.Slides were fixed in absolute ethanol and stained with Giemsa.For each fish sample, a total of 1,000 erythrocytes were analyzed for nuclei abnormalities, according to the classification of Da Silva & Fontanetti [3].

EROD assay
Ethoxyresorufin-O-deethylase (EROD) activity was measured in hepatic S9 fractions.Immediately after fishing, fish livers were removed, weighed, wrapped in aluminum foil, and frozen in liquid nitrogen until further use.Liver S9 fractions were prepared by tissue homogenization and centrifugation at 9,000 g for 30 minutes [4].the supernatant was used for total protein and EroD determination.total protein concentrations were quantified using the Bradford assay [5] and EROD activity was determined spectrofluorometrically [6].

ReSuLTS heavy metal concentrations
In any muscle sample obtained from either species of fish, the concentrations of mercury and lead did not exceed the maximum allowable level established by the Brazilian Sanitary Agency (ANViSA) regulations.the concentrations of the other four metals analyzed in the present study were also within the range of values generally found in the literature for non-contaminated fish meat [7,8,9] (Table 2, shows some of the data collected).

Frequency of erythrocytes with micronuclei
the frequency of micronucleated erythrocytes found in the pe-

DISCuSSIoN
The Data in this study were derived from samples of edible fish collected in the dry and wet seasons of 2003.therefore, they are a snapshot of the contamination of PSR fish meat by a diverse range of industrial pollutants (genotoxic and Ahr ligand agents, and toxic metals), which are relevant to human health at the present time.Although these two species and other fish can be used as "sentinel" species for monitoring PSr basin water pollution, it should be borne in mind that the biomarkers respond to the bioavailability of contaminants to target organisms.Persistent compounds with poor solubility in water are deposited at the bottom of the river over time.Contaminants remaining trapped in the sedimentary strata are not bioavailable to fish living in the water column and, thus, these pollutants are not detected by these "sentinel" species.However, if a river sediment is disturbed by natural phenomena or human activity, contaminants deposited on river beds may become re-suspended and ingested (with particulate matter) by fish.Aquatic pollution and contamination of the biota are dynamic phenomena and historical data records are needed to evaluate the impact of anthropological activities and the effectiveness of interventions to control pollution.retrospective epidemiological studies on the impact of pollutants on human health, for instance, would require historical data records in order to estimate past exposure.Some current diseases (e.g.cancer) can be influenced by exposure to contaminants in the environment that took place years or even over a decade ago.
taken together, the concentrations of heavy metals in muscle tissue, liver EroD activities, and the frequency of erythrocytes with micronuclei all indicate that the contamination of edible fish from the PSR basin by metals, PAHs, PCDD/Fs PCBs and related compounds, was not a threat to human health at the time of sampling (2003).Some pollution hotspots, however, were detected.the highest values of micronuclei frequency and EroD activity were found in fish collected in the middle stretch of the river basin between the cities of São José dos Campos and Sapucaia.this region is the most industrialized part of the basin, where the Paraibuna river, one of the largest tributaries, flows into the PSR.The elevated biomarker values in fish from the

Table 1 . Location of Paraiba do Sul river (PSR) sample sites and the number of fish captured at each site in the wet and dry seasons. # City River GPS co-ordinates Fish (N) Latitude (South) Longitude (west) Geophagus brasiliensis (wet/dry) Hypostomus spp. (wet/dry)
(-) no fish caught.http://www.visaemdebate.incqs.fiocruz.br/Parente TEM et al.Biomarkers in fish from Paraíba do Sul river basin, Brazil Vig Sanit Debate 2015;3(1):88-92 | 90 blood of both the species of fish examined in this study (0-0.67‰ in G. brasiliensis and 0-2‰ in Hypostomus sp.) was within the range of values generally reported for other species of fish caught in areas that are unpolluted.thehighestincidence of micronucleated erythrocytes was recorded in fish captured in São José dos Campos, Barra Mansa, três rios, Campos and São João da Barra for G. Brasiliensis, and in São José dos Campos, Levy gasparian, Além Paraíba and São João da Barra for Hypostomus sp.(table3).Hypostomus sp.exhibited a marked difference in their liver EroD activity.While for g. brasiliensis EroD activity ranged from 16 to 260 pmol resorufin.min -miligram of protein -1 (Figure1), in most Hypostomus sp.samples EroD activity was undetectable (data not shown). A far as G. brasiliensis samples were concerned, sampling sites were ranked according to a four-category classification scheme, based on the average level of EROD activity.
Categories of this ranking scheme were defined, taking into account the statistical distribution of the individual values within the whole sample.Locations with a fish liver mean EROD activity value lower than that of the lowest quartile were placed into category 1. Locations where the mean EroD activity values were in between the lower quartile and the median were allocated to category 2, while category 3 comprised locations where the mean EroD activity values of fish were in between the median and the upper quartile, and locations for which mean liver EroD activity values were higher than that of the upper quartile were allocated to category 4 (table4).in both seasons, G. brasiliensis collected at italva were consistently classified into category 1 (lowest activity), while samples from Levy Gasparian were classified into category 4 (highest activity).Fish from Barra Mansa collected during the wet season were classified as category 2, while those caught during the dry season were classified as category 4. Fish from São Luiz do Paraitinga, Três Rios and Sapucaia collected in the dry season were classified as category 4 (the category with the highest activity).During the wet season fish were not sampled at these locations.

Table 2 .
the concentration of heavy metals in the muscle tissue of Geophagus brasiliensis ("acará") and Hypostomus spp.("cascudo") caught during the dry season.