Determination of heavy metals by inductively coupled plasma-optical emission spectrometry in fish from the Piracicaba River in Southern Brazil

A total of 202 fish, representing 16 species, were collected during 2008 (March–October) in the Tanquan region of the Piracicaba River using nets. Flesh samples were collected and analyzed, using inductively coupled plasma-optical emission spectroscopy for Al, As, Cd, Co Cr, Cu, Mn, Mo, Ni, Pb, Se, Sn, Sr, and Zn. The results showed that the flesh of these fish all contained extremely high levels of Al and Sr, and moderately high levels of Cr, As, Zn, Ni, Mn and Pb. The metals were higher in these fish during rainy season, with fish collected during the months of March and October being the highest. In addition, the accumulation of metals was species-dependent. Cascudos (Hypostomus punctatus) and piranhas (Serrasalmus spilopleura) exhibited high levels of almost all of the metals, while curimbata (Prochilodus lineatus) had moderate levels. A few species, including pacu (Piaractus mesopotamicus) and dourado (Salminus maxillosus), had very low levels of most metals. The results show that the Piracicaba River Basin is widely contaminated with high levels of many toxic heavy metals, and that human consumption of some fish species is a human health concern.     

Evaluation of element availability in bottom sediments by synchrotron total reflection X-ray fluorescence analysis (SR-TXRF)

Summary Total reflection X-ray fluorescence (TXRF) was used to evaluate the availability and contamination of inorganic elements in sediment samples from Atibaia River, located at Piracicaba Basin, near the Campinas region, S?o Paulo State, Brazil. The total contents (geological matrix) and the concentration of elements weakly linked to the sediment (available fraction) were determined. The availability of these elements was evaluated through the ratios between available fractions and total contents. The results showed that the elements Mn, Ni, Cu and Zn are easily available in the aquatic environment.     

Time still to restore the polluted Piracicaba river basin

Over the last decades the acceleration of the industrialization and urbanization processes together with the intensive agricultural practices have resulted in an impact on the Piracicaba river basin, state of São Paulo, Brazil. The source rivers drain from an area of low population density, absence of heavy industries, non-significant agriculture, native forest and reforestation, the opposite is found in the middle part of the basin. Samples of riverbed sediments were collected along the basin for chemical analysis. Results showed that the source rivers still preserve their natural characteristics, while the Atibaia river in the middle part shows signs of pollution from the agricultural activity, industrial effluents and urban sewage.     

Concentrations of Fe, Cu and Zn in rum by EDXRF using APDC preconcentration

Ten rum (aguardente) samples commercialized in Piracicaba region, São Paulo State, Brazil Southeast, were analysed by energy-dispersive X-ray fluorescence (EDXRF) using ammonium pyrrolidinedithiocarbamate (APDC) preconcentration in order to determine Fe, Cu and Zn concentrations. The effect of sucrose (2%) was also studied in this methodology, using a multielemental standard solution (40% ethanol). Copper concentrations in two samples were higher than the value allowed by Brazilian law. Limits of detection for these elements were in the ng^.ml^-1 range.     

Arsenic in terrestrial invertebrates from riparian areas of the Piracicaba River Basin,São Paulo State,Brazil

There is no information on arsenic distribution in terrestrial invertebrates from riparian forests of urban and rural areas in Brazil. The objective of this study was to evaluate the As levels in invertebrates from riverine forests of the Piracicaba River Basin, São Paulo, Brazil, using the instrumental neutron activation analysis, k ₀-comparator method. After correction of mass fractions, values higher than 0.10 mg kg⁻¹ were quantified in invertebrates from both urban and agricultural areas. An unexpected As mass fraction of 13 mg kg⁻¹ obtained in the Coleopteran pest Macrodactylus pumilio indicated resistance to As-containing-pesticides.

     

Monitoring of the environmental pollution by trace element analysis in tree-rings using synchrotron radiation total reflection X-ray fluorescence

This paper aims to study the environmental pollution in the tree development, in order to evaluate its use as bioindicator in urban and country sides. The sample collection was carried out in Piracicaba city, São Paulo State, which presents high level of environmental contamination in water, soil and air, due to industrial activities, vehicles combustion, sugar-cane leaves burning in the harvesting, etc. The species Caesalpinia peltophoroides (“Sibipiruna”) was selected because it is widely used in urban forestation. Synchrotron Radiation Total Reflection X-ray Fluorescence technique (SR-TXRF) was employed to identify and quantify the elements and metals of nutritional and toxicological importance in the wood samples. The analysis was performed in the Brazilian Synchrotron Light Source Laboratory, using a white beam for excitation and a Si(Li) detector for X-ray detection. In several samples, P, K, Ca, Ti, Fe, Sr, Ba and Pb were quantified. The K/Ca, K/P and Pb/Ca ratios were found to decrease towards the bark.     

Characterization of Brazilian commercial milks by instrumental neutron activation analysis

Aiming at the determination of toxic and essential elements in Brazilian commercial bovine milk, 25 ultra high temperature (UHT) milk samples were acquired in the local market of Piracicaba, SP. The samples were freeze-dried and analyzed by instrumental neutron activation analysis (INAA) allowing the determination of Br, Ca, Co, Cs, Fe, K, Na, Rb and Zn. When the results were expressed as concentration (mg·l⁻¹) no significant differences were found. However, considering the dry matter, results showed a clear difference between the mass fractions (mg·kg⁻¹ d.w.) of skim milk and whole milk for the elements Br, Ca, K, Na, Rb and Zn, indicating that the removal of fat caused a concentration effect in the dry matter of skim milks. Discrepancies were found between the concentrations of Ca and Na measured by INAA and the values informed in the labels. Ca showed variations within 30% for most samples, while concentrations of Na were up to 190% higher than informed values. The sample preparation and the INAA procedure were appropriate for the determination of Br, Ca, Co, Cs, Fe, K, Na, Rb and Zn in milk samples.     

Neutron activation analysis for assessing chemical composition of dry dog foods

Brazil holds the second largest population of domestic dogs in the world, with 33 million dogs, only behind the United States. The annual consumption of dog food in the country is 1.75 million tons, corresponding to the World’s sixth in trade turnover. Dog food is supposed to be a complete and balanced diet, formulated with high quality ingredients. All nutrients and minerals required for an adequate nutrition of dogs are added to the formulation to ensure longevity and welfare. In this context, the present study aimed at assessing the chemical composition of dry dog foods commercialized in Brazil. Thirty-four samples were acquired in the local market of Piracicaba and analyzed by instrumental neutron activation analysis (INAA) to determine the elements As, Br, Ca, Co, Cr, Cs, Fe, K, La, Na, Rb, Sb, Sc, Se, U, and Zn. In general, the concentrations of Ca, Fe, K, Na, and Zn complied with the values required by the Association of American Feed Control Officials (AAFCO). To evaluate the safety of dog food commercialized in Brazil, further investigation is necessary to better understand the presence of toxic elements found in this study, i.e. Sb and U. INAA was useful for the screening analysis of different types and brands of dry dog foods for the determination of both essential and toxic elements.     

Presence of rhodopsin and porphyropsin in the eyes of 164 fishes, representing marine, diadromous, coastal and freshwater species--a qualitative and comparative study

There are two types of visual pigments in fish eyes; most marine fishes have rhodopsin, while most freshwater fishes have porphyropsin. The biochemical basis for this dichotomy is the nature of the chromophores, retinal (A1) and 3-dehydroretinal (A2), each of which is bound by an opsin. In order to study the regional distribution of these visual pigments, we performed a new survey of the visual pigment chromophores in the eyes of many species of fish. Fish eyes from 164 species were used to examine their chromophores by high-performance liquid chromatography--44 species of freshwater fish, 20 of peripheral freshwater fish (coastal species), 10 of diadromous fish and 90 of seawater fish (marine species) were studied. The eyes of freshwater fish, limb freshwater fish and diadromous fish had both A1 and A2 chromophores, whereas those of marine fish possessed only A1 chromophores. Our results are similar to those of previous studies; however, we made a new finding that fish which live in freshwater possessed A1 if living near the sea and A2 if living far from the sea if they possessed only one type of chromophore.     

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.     

The relationship between the mercury concentration in fish muscles and scales/fins and its significance

The determination of mercury in fish typically involves analysis of muscles. For predicting the concentration of mercury in fish muscle on the basis of the analysis of fish scales or fins, the relationship between total mercury concentrations in fish muscles and in fish scales and fins was studied. Mercury content in fish muscles, scales and fins was determined by atomic absorption spectrometry with thermal decomposition of the sample in a flow of oxygen. A number of scale treatments were applied in order to remove impurities and to enhance the prediction quality. For scale treatment, 40 min of washing with DI water in an ultrasonic bath is recommended. A coefficient of determination r²= 0.93 for the relationship between Hg concentrations in muscles and scales was achieved for 40 fish among the different fish species tested (European bream, perch, roach) from the Hamry Reservoir, Czech Republic. With respect to fin sampling, the coefficient of determination r² for these fish was 0.86. The analysis of fish scales and caudal fins is a useful screening tool for assessing the relative mercury contamination of monitored fish. The method of sampling scales is not suitable for fish species with small scales such as brown trout.      

Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish

Dioxin-like polychlorinated biphenyls (DL-PCBs) often make up the majority of the toxic equivalent (TEQ) contribution of dioxins found in fish samples. For the purpose of making risk assessments, it is therefore important to develop screening methods for determining TEQ concentrations of DL-PCBs in retail fish. We have developed a rapid biosensor immunoassay (BIA) for DL-PCBs that uses a surface plasmon resonance sensor (Biacore 3000). The BIA is highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118) that is generally the most abundant DL-PCB isomer found in fish. The fish extracts were first cleaned up on a multilayer silica gel column followed by an alumina column, then subjected to the assay. The quantitative limit of the assay was 1 ng PCB 118 per gram of tested sample. Dilution and recovery tests using purified fish extracts suggested that the matrix effect was minimized in the assay by diluting the analyzed samples. The assay results for retail fish samples (n=7) agreed well with those obtained by an enzyme-linked immunoassay (ELISA) using the same monoclonal antibody: ELISA has been already validated for determining DL-PCBs in fish samples, so BIA performs well in this analysis. Finally, BIA results for the TEQ concentrations of DL-PCBs in retail fish samples (n=10) correlated well with those obtained by high-resolution gas chromatography coupled to high-resolution mass spectrometry (r=0.89). Our method is therefore useful for screening retail fish to determine the TEQ concentrations of DL-PCBs.     

Microencapsulation of Fish Oil by Simple Coacervation of Hydroxypropyl Methylcellulose

To improve the oxidative stability and application of fish oil, it was microencapsulated by simple coacervation followed by spray drying. Simple coacervation took place by adding malt dextrin into the emulsion of fish oil and hydroxypropyl methylcellulose （HPMC） solution. Influences of several process parameters on the microencapsulation were evaluated and the oxidative stability and microstructure of microcapsules were analyzed. Results showed that the coacervation could be observed only when dextrose equivalent value （DE value） of malt dextrin, concentration of HPMC solution and fish oil percentage in microcapsules were no more than 20. 5% and 40%, respectively. Moreover, microencapsulation efficiency was higher at HPMC solution concentration of 4% and fish oil percentage of less than 30%. The oxidative stability of fish oil was improved by the microencapsulation and done best in the ease of replacing malt dextrin by 40% with acacia. Scanning electronic microscopic photographs showed that the microcapsule obtained was a round, smooth and hollow microcapsule with its wall made up of innumerable small and solid submicrocapsules with the core of fish oil.     

Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography–tandem mass spectrometry

Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74–103 %) in fish blood. Matrix effects (6–26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34–75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP.

Methylmercury in marine fish from Malaysian waters and its relationship to total mercury content

The study evaluated methylmercury concentrations, the methylmercury to total mercury ratio (%MeHg) and their correlations in ten fish species from different trophic levels. Methylmercury levels in fish studied were in the range of 0.007 to 0.914 µg g^?1 wet wt. Muscle tissue of predatory fish contained significantly (p < 0.05) higher content of methylmercury than non-predatory fish. The methylmercury to total mercury ratio ranged from 49.1% to 87.5%, with the highest ratio in predatory fish. This ratio was always higher in muscle tissue compared to the liver tissues, indicating tissue-specific binding and accumulation of methylmercury in the muscle. All the fish species showed strong positive correlation between methylmercury and total mercury levels (R ²> 0.86). Except for long tail tuna and short-bodied mackerel, all fish species showed lower methylmercury levels and estimated weekly intake as compared to the maximum values established by US FDA (of 0.5 µg g^?1) and by FAO/WHO (1.5 µg kg^?1 bodyweight), respectively. This study showed that the percentage of methylmercury is rather high in fish and fish represents the major source of this toxic mercury form to the local population.     

Multielemental evaluation of garbage bags by EDXRF

Garbage bags have been broadly used for collecting household waste in Brazil and worldwide. Many of these bags are made from recycled polymers, and the presence of harmful elements is a matter of concern. In this study, an energy dispersive X-ray fluorescence (EDXRF) approach for direct analysis of garbage bags is proposed. The concentrations of K, Ca, Ti, Cr, Mn, Fe, Ni, Cu, Zn, Br, Rb, Sr and Pb were determined in 14 garbage bags of different brands purchased from local markets in Piracicaba, São Paulo State, Brazil. The EDXRF technique proved to be an effective technique for multielemental, environmentally friendly and direct garbage bag monitoring. For most samples, Ca and Ti presented at minor concentration ranges and K, Cr, Mn, Fe, Ni, Cu, Zn and Pb at minor and trace levels. The EDXRF limits of quantification were K: 220; Ca: 112; Ti: 42; Cr: 19; Mn: 13; Fe: 9.2; Ni: 5.7; Cu: 5.3; Zn: 5.2; Br: 12; Rb: 20; Sr: 28 and Pb:17 μg g^–1. The samples had variable thicknesses, with a superficial density variation of 1.5 to 23% among the triplicates. The trueness of the method was checked by analysing the National Institute of Standards and Technology standard reference material SRM2783.     

Determination of the metal balance shift induced in small fresh water fish by X-ray irradiation using PIXE analysis

The balance of essential elements in organisms can be changed by environmental stresses. A small fresh water fish, the medaka, was irradiated with X-rays (total dose: 17 Gy, which is not a lethal dose for this fish). Essential elements in the liver, gall bladder, kidney, spleen, heart and brain of the fish were measured by the particle induced X-ray emission (PIXE) method and compared with those of a control fish. Various changes in the elemental balance shift were observed. The PIXE method can analyze many elements in a small sample simultaneously, and so the changes in elemental content induced by irradiation were readily determined.     

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.