Polishing entire stems and roots using sandpaper under water: An alternative method for macroscopic analyses.

PREMISE: Polishing entire stem and root samples is an effective method for studying their anatomy; however, polishing fresh samples to preserve woods with soft tissues or barks is challenging given that soft tissues shrink when dried. We propose sanding fresh or liquid-preserved samples under water as an alternative, given that it preserves all tissues in an intact and clear state. METHODS AND RESULTS: By manually grinding the surface of the samples under water using three ascending grits of waterproof sandpapers, an excellent polished sanded surface is obtained. The wood swarf goes into the water without clogging the cell lumina, rendering the surfaces adequate for cell visualization and description. We show results in palms, liana stems, roots, and wood blocks. CONCLUSIONS: Using this simple, inexpensive, rapid technique, it is possible to polish either fresh, dry, or liquid-preserved woody plant samples, preserving the integrity of both the soft and hard tissues and allowing for detailed observations of the stems and roots.

Embryo stage of development is not decisive for reproductive outcomes in frozen-thawed embryo transfer cycles.

OBJECTIVE: To evaluate if the outcomes of IVF/ICSI in frozen-thawed embryo transfer and fresh embryo transfer cycles differ in relation to cleavage and blastocyst stages. METHODS: Retrospective cohort study to compare IVF/ICSI outcomes between fresh embryo transfer and frozen-thawed embryo transfer cycles, according to the stage of embryo development. Analysis was carried out on 443 consecutive embryo transfer cycles performed between January 1st and December 31st, 2014. Women aged up to 38 and submitted to embryo transfer cycles with fresh (n = 309) or frozen-thawed (n = 134) embryos at a private center for assistance in human reproduction were considered for analysis. Results in each group were stratified according to the stage of embryo development: cleavage stage and blastocyst stage. Main outcome measures were implantation rate, clinical pregnancy rate, ongoing pregnancy rate and live birth rate per cycle. RESULTS: In the fresh embryo transfer group, for cleavage stage versus blastocyst stage, respectively, implantation rates were 22% and 47% (p = 0.0005); clinical pregnancy rates were 34% and 64% (p = 0.0057); the ongoing pregnancy rates were 30% and 61% (p = 0.0046) and live birth rates were 28% and 55% (p = 0.0148). There were no significant differences in the rates between cleavage and blastocyst stages in the frozen-thawed group, neither between fresh and frozen-thawed cleavage embryo transfers nor between fresh and frozen-thawed blastocyst transfers. CONCLUSION: Our results confirm that blastocyst transfer is better than cleavage stage in fresh embryo transfer cycles. In frozen-thawed cycles, cleavage or blastocyst stages seem to offer similar reproductive outcomes.

Sequential hair mercury in mothers and children from a traditional riverine population of the Rio Tapajós, Amazonia: seasonal changes.

Riverine populations of the Brazilian Amazon depend on fish for their principal source of protein, which changes in availability with seasonal fluctuations in the aquatic ecosystem. We report seasonal (high and low waters) and interannual changes in total mercury and methylmercury concentrations in hair of subsistence fish-eaters from a traditional riverine village on the banks of the Rio Tapajós in the Brazilian Amazonia. Retrospective exposure assessment was conducted from hair concentrations in segments representative of 2 years. A total of 32 mothers and their 51 children were studied. They reported high fish consumption (4-14 times a week). Ranges of total mercury and methylmercury concentrations in hair were 1.0-51.0 and 0.5-41.4microgg(-1), with means of 12.4 and 10.2microgg(-1), respectively. The relative mean value for methylmercury was 80%. There was a significant correlation between mothers and children for both total-Hg (r=0.4826; P=0.003) and methyl-Hg (r=0.5214; P=0.004). Mercury concentrations along hair strands of individuals showed significant variation coinciding with seasonal high and low waters. The changes in the aquatic environment impacted fish species availability, which is reflected in the total and MeHg concentrations in hair.

Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake.

Lake Paranoá is a tropical reservoir for the City of Brasilia, which became eutrophic due to inadequate sewage treatment associated with intensive population growth. At present, two wastewater treatment plants are capable of processing up to 95% of the domestic sewage, thereby successfully reducing eutrophization. We evaluated both genotoxic and cytotoxic parameters in several fish species (Geophagus brasiliensis, Cichla temensis, Hoplias malabaricus, Astyanax bimaculatus lacustres, Oreochromis niloticus, Cyprinus carpio and Steindachnerina insculpita) by using the micronucleus (MN) test, the comet assay and nuclear abnormality assessment in peripheral erythrocytes. The highest frequencies of MN were found in Cichla temensis and Hoplias malabaricus, which were statistically significant when compared to the other species. However, Steindachnerina insculpita (a detritivorous and lake-floor feeder species) showed the highest index of DNA damage in the comet assay, followed by C. temensis (piscivorous). Nuclear abnormalities, such as binucleated, blebbed, lobed and notched cells, were used as evidence of cytotoxicity. Oreochromis niloticus followed by Hoplias malaricus, ominivorous/detritivotous and piscivorous species, respectively, presented the highest frequency of nuclear abnormalities, especially notched cells, while the herbivorous Astyanax bimaculatus lacustres showed the lowest frequency compared to the other species studied. Thus, for biomonitoring aquatic genotoxins under field conditions, the food web should also be considered.

Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake

Lake Paranoá is a tropical reservoir for the City of Brasilia, which became eutrophic due to inadequate sewage treatment associated with intensive population growth. At present, two wastewater treatment plants are capable of processing up to 95% of the domestic sewage, thereby successfully reducing eutrophization. We evaluated both genotoxic and cytotoxic parameters in several fish species (Geophagus brasiliensis, Cichla temensis, Hoplias malabaricus, Astyanax bimaculatus lacustres, Oreochromis niloticus, Cyprinus carpio and Steindachnerina insculpita) by using the micronucleus (MN) test, the comet assay and nuclear abnormality assessment in peripheral erythrocytes. The highest frequencies of MN were found in Cichla temensis and Hoplias malabaricus, which were statistically significant when compared to the other species. However, Steindachnerina insculpita (a detritivorous and lake-floor feeder species) showed the highest index of DNA damage in the comet assay, followed by C. temensis (piscivorous). Nuclear abnormalities, such as binucleated, blebbed, lobed and notched cells, were used as evidence of cytotoxicity. Oreochromis niloticus followed by Hoplias malaricus, ominivorous/detritivotous and piscivorous species, respectively, presented the highest frequency of nuclear abnormalities, especially notched cells, while the herbivorous Astyanax bimaculatus lacustres showed the lowest frequency compared to the other species studied. Thus, for biomonitoring aquatic genotoxins under field conditions, the food web should also be considered.

Evaluation of genotoxicity and effects on reproduction of nonylphenol in Oreochromis niloticus (Pisces: Cichlidae).

Nonylphenol ethoxylate (NPE) is widely used as a component of detergents, paints, pesticides, and many other products. In the aquatic environment NPE breakdown to 4-nonylphenol (NP), which is more stable and persistent. NP is estrogenic in fish, avian, and mammals and is described as an environmental pollutant with endocrine disruptor characteristics. The genotoxicity of NP was evaluated through micronuclei assay and single cell gel electrophoresis (Comet assay) in peripheral erythrocytes of Oreochromis niloticus exposed in vivo. The study on reproductive development was also carried out in male and female gonads of O. niloticus. Lethal concentration (LC 50%) of 0.032 ml l(-1) was previously determined. We ran assays with O. niloticus exposed to concentrations of 1.0, 10.0, and 16.0 microl l(-1) of NP diluted in water. Our results showed that NP was not genotoxic. However, 3-day exposure to NP in concentrations of 1.0, 10.0, and 16.0 microl l(-1) of water increased the frequency of reproductive stages in males and females. The histology of the reproductive tract of the treated fish was significantly altered in females treated with 16.0 microl l(-1) of water when compared to controls. Analogous estrogenic effects were observed, such as accelerated maturation of oocytes and spermatogenesis. These results showed that the O. niloticus reproductive system is sensitive to NP estrogenicity.

Anthropogenic impact of mercury accumulation in fish from the Rio Madeira and Rio Negro rivers (Amazonia).

Fish is an important concentrator of mono-methyl mercury and the main route to human contamination. We compared fish Hg bioaccumulation (within similar weight ranges) in two Amazonian river habitats during high-water seasons. The Rio Madeira has been greatly impacted by agriculture, alluvial gold extraction, and a hydroelectric reservoir, whereas the Rio Negro is much less affected by these human activities. The species at the top of the food web, Hoplias malabaricus (piscivorous; 80-668 ng Hg/g) and Cichla spp. (piscivorous; 42-747 ng Hg/g) showed the highest range of Hg concentrations. Nonpiscivorous species with comparable weight range, such as Potamorhina latior (detritivorous; 20-157 ng Hg/g) and Myleus torquatus (herbivorous; 2-182 ng Hg/g), had lower Hg concentrations. Triportheus elongatus (omnivorous; 5-350 ng Hg/g), with the lowest weight range, also showed a low range of Hg concentrations. Despite the Rio Madeira's higher sediment load as well as environmental impacts (deforestation, agriculture, hydroelectric reservoir, and alluvial gold mining) on natural Hg release, fish Hg bioaccumulation was no different between the two river habitats for nonpiscivorous species. In this small observational study only the species at the top of the food web (M. torquatus, Cichla spp, T. elongatus) showed higher mean Hg concentrations in the Rio Madeira than the dominantly wilderness habitat of the Rio Negro.

Annual flooding and fish-mercury bioaccumulation in the environmentally impacted Rio Madeira (Amazon).

Regular annual flooding of the Amazonian rivers changes the aquatic environment affecting fish feeding strategies. The Rio Madeira has been greatly impacted by deforestation for agricultural projects, damming for a hydroelectric power plant, and alluvial gold extraction. We studied fish-Hg concentrations within defined weight ranges of representative species at the top of the food web, comparing high and low water seasons. Selected piscivorous species (Cichla spp, Hoplias malabaricus, Pinirampus pirinampu, Serrasalmus spp) showed a large variation of Hg concentrations but only "traíra" (Hoplias malabaricus) showed a statistically significant difference between seasons. However, the bioaccumulation trends during high and low waters were similar for "tucunaré" (Cichla spp) and "traíra" (Hoplias malabaricus) but different for "piranhas" (Serrasalmus spp), "barba chata" (Pinirampus pirinampu) and the detritivorous Prochilodus nigricans. Fish-Hg bioaccumulation is species specific; changes in feeding strategies brought by flooding seasons can change the bioaccumulation pattern without systematically affecting the overall accrual of methylmercury in tertiary consumer species. It appears that naturally occurring Hg and the high sediment load of the Rio Madeira are secondary factors in the Hg bioaccumulation pattern of fish species at the top of the food chain.

Fish consumption, mercury exposure and serum antinuclear antibody in Amazonians.

Exposure to intrinsic Hg in fish was studied in Amazon populations with high prevalence of malaria disease. High fish-eater riverines were compared to urban (Manaus residents) low fish-eater riverines in regards to Hg exposure (hair-Hg) and serum antinuclear antibodies (ANA). Most riverines (99.0%) ate fish daily compared to only 3% of controls. Fish species high in MeHg was consumed more frequently (45.5%) by riverines than controls (18.8%). Mean hair-Hg (34.5 ppm) of riverines was significantly higher than controls (1.0 ppm). Although positive serum ANA was more frequently observed in riverine fish-eaters (12.4%) than controls (2.9%), there was no significant association between hair-Hg and ANA. High prevalence of malaria reporting among riverines was neither associated with Hg exposure nor with serum ANA. An autoimmune dysfunction is unlikely to occur as a result of MMHg exposure due to fish consumption.

Fish mercury bioaccumulation as a function of feeding behavior and hydrological cycles of the Rio Negro, Amazon.

The acidic black waters of the Rio Negro are unimpacted by anthropogenic sources and yet, are characterized by fish containing relatively high Hg concentrations. Regular annual flooding alters the aquatic environment, thereby affecting fish feeding strategies. We studied the impact of annual flooding on fish-Hg bioaccumulation. Tucunarés (Cychla spp, carnivorous), Peixe-cachorro (Hydrolycus scomberoides, carnivorous), Traíra (Hoplias malabaricus, carnivorous), Piranha-branca (Serrasalmus aff. eigenamanni, carnivorous), Piranha-preta (Serrasalmus rhombeus, carnivorous), Acará (Acarichthys heckellii, omnivorous), Aracú (Leporinus friderici, omnivorous), Orana-preta (Hemiodus unimaculatus, omnivorous), Sardinha (Triportheus elongatus, omnivorous), Branquinha (Potamorhina latior, detritivorous), Jaraqui-escama-fina (Semaprochilodus taeniurus, detritivorous), and Pacú-branco (Myleus torquatus, herbivorous) were studied during high (July) and low waters (February) and categorized by body mass. Regardless of hydrological period, ranges of total-Hg concentrations were higher for carnivorous than for omnivorous, detritivorous and herbivorous species. Some species (Traíra, Sardinha, Peixe-cachorro, Piranha branca, Piranha preta) showed changes in feeding patterns as indicated by an inverse trend of Hg bioaccumulation with season. Species with similar trends of Hg bioaccumulation showed higher Hg concentrations during the flooded season (Aracú, Pacú branco and Orana preta) and some had lower Hg (Acará, Branquinha and Tucunarés). Fish Hg concentration is species specific and reflects changes in feeding-behavior dominance brought by annual inundation.

Analysis of genetic susceptibility to mercury contamination evaluated through molecular biomarkers in at-risk Amazon Amerindian populations

We investigated Individual differences in susceptibility to methylmercury (MeHg) contamination and its relationship with polymorphisms of the detoxifying enzyme glutathione S-transferase (GST). In Brazil, some Amerindian tribes from the Amazon region have an increased level of mercury in their hair. Samples of hair and blood were taken from inhabitants of two villages in the Kayabi and Munduruku Amerindian communities to investigate mercury levels in association with genetic polymorphism of GSTs. Other molecular biological markers were also studied, such as hemoglobin, haptoglobin and glucose 6-phosphate dehydrogenase (G-6-PDH). Higher levels of mercury contamination were found in the Kayabi villagers, who had a null genotype (GSTM1 0/0, also denominated GSTM1 null) frequency of 26%, than in the Munduruku villagers, for which the null genotype frequency was 0%. Individuals with the GSTM1 null phenotype had higher concentrations of mercury in their hair than individuals with GSTM1+/+ phenotypes (F = 21.51, p < 0.0001). No association with other markers studied was observed. This study suggests that GSTM1 may be involved in the biotransformation of mercury in humans

Fish consumption (hair mercury) and nutritional status of Amazonian Amer-Indian children.

Fish are abundant and important dietary items for the Amer-Indians, and total hair-Hg (HHg) concentration is a reliable marker of fish consumption. We studied the impact of fish consumption (HHg) on the nutritional status of Indian children of Eastern Amazonia. Weight-for-height Z score (WHZ) was measured, and HHg was determined in 203 children younger than 10 years of age in three villages. There was significantly higher fish consumption in Kayabi children (16.55 microg Hg/g; SD, 11.44) than in children of the Munduruku villages of Missão-Cururu (4.76 microg Hg/g; SD, 2.09) and Kaburua (2.87 microg Hg/g; SD, 2.13). Anthropometric indices showed WHZ means of -0.27, -0.22, and 0.40, respectively, for Kayabi, Missão-Cururu and Kaburua villages. Despite a different pattern of fish-protein consumption between tribes, there was no significant correlation between WHZ and HHg concentrations (r2 = 0.0079; P < 0.2021). Dietary differences among Amazonian tribes can be traced and used in measuring health outcomes. Higher fish consumption, although important for Kayabis, was compensated by other protein sources by the Kaburua villagers.

Hair mercury (signature of fish consumption) and cardiovascular risk in Munduruku and Kayabi Indians of Amazonia.

Fish is an important natural resource in the diet of inhabitants of the Amazon rain forest and a marker of its consumption (hair Hg) was used to compare selected cardiovascular risk parameters between tribes of Eastern Amazonia. Three Munduruku (Terra Preta, Kaburua, Cururu) villages and one Kayabi village at the banks of head rivers (Tapajos, Tropas, Kabitutu, Cururu, Curuzinho, Teles Pires) of the Tapajos Basin were studied in relation to fish Hg concentrations, mercury in hair (fish consumption) and erythrocytes, body mass index (height/weight, kg/cm2), and blood pressure. The mean fish Hg concentrations were higher in predatory (578.6 ng/g) than in nonpredatory species (52.8 ng/g). Overall only 26% of fish Hg concentrations were above 500 ng/g, and only 11% were above 1000 ng/g. There was no systematic trend in fish Hg concentrations from rivers with a history of gold-mining activities. The biomarker of fish consumption (hair Hg) was significantly associated with erythrocyte-Hg (r=0.5181; P=0.0001) and was significantly higher in Kayabi (12.7 microg/g) than in the Munduruku (3.4 microg/g). Biomarker-assessed fish consumption rate was higher in the Kayabi (110 g/day) than in the Munduruku villages (30 g/day). Although no significant differences in body mass index (BMI) were observed between tribes, there was a trend of lower increase in blood pressure with age among the higher fish consumers (Kayabi). Summary clinical evaluation did not detect neurologic complaints compatible with Hg intoxication (paraparesis, numbness, tremor, balancing failure), but endemic tropical diseases such as clinical history of malaria showed a high prevalence (55.4%). Fish is an abundant natural resource, important in the Indian diet, that has been historically consumed without perceived problems and can easily be traced through hair Hg. The exposure to freshwater fish monomethyl mercury is less of an issue than endemic infectious diseases such as malaria and lack of basic medical services.

Piranhas (Serrasalmus spp.) as markers of mercury bioaccumulation in Amazonian ecosystems.

Mercury (Hg) concentration in fish depends on feeding strategies and age/size within the species as well as on water parameters related to acidity and Hg speciation. We chose two species of piranhas (Serrasalmus aff. eigenmanni and Serrasalmus rhombeus) to test their suitability as markers of Hg bioaccumulation. The results of Hg concentrations in piranhas of the Rio Negro, with no history of gold-mining activity, are similar to other Amazonian rivers with intensive gold mining. An increase in water Hg associated with low pH favored higher mean Hg concentrations in the two species. S. rhombeus in this habitat showed a consistently higher Hg concentration than S. eigenmanni. This result was attributed to fish size because of differences in correlation coefficient between S. rhombeus (r = 0.4443; P < 0.0001) and S. eigenmanni (r = 0.1520; P = 0.0135). Matching for fish weight resulted in comparable ranges of Hg concentrations between the two species.

Mercury in hair and in fish consumed by Riparian women of the Rio Negro, Amazon, Brazil.

This work puts the risks and benefits of a mercury acquisition through a fish-dominated diet in the context of the other endemic health problems in the Amazon. Fish consumption rate was assessed after determining mercury concentrations in the most consumed fish and in hair of women at fertile age (15 to 45 years of age), living in remote localities of the Rio Negro basin (not impacted by gold mining). Fish-meals were consumed once a day (7.1%) but in most cases (78.6 %) at least twice a day with an estimated mean fish consumption rate of 170.5 g (range 23 to 293 g). The fish-species most preferred were Tucunarés (Cichla spp), Pacus (Mylossoma aureum and Myleus micans), Piranhas (Sarrasalmus spp), Cará (Satanoperca jurupari), Jaraquí (Semaprochilodus taeniurus). The mean Hg concentration in these species varies widely (38 to 592 ng g(- 1)) but 27% of samples had Hg concentrations above 500 ng g(- 1), and only 7% were above 1000 ng g(- 1). The hair-Hg concentrations ranged from 6.5 to 32.6 microg g(- 1) with 82% of them having concentrations above 10 microg g(- 1) (w.w.) hair. Hair-Hg concentrations were positively correlated with women's age (r=0.4500; P=0.0111). Clinical evaluation did not detected symptoms (paraparesis, tremor, numbness of limbs, sensory disturbances) compatible with Minamata disease, but there was a high (71%) incidence of clinical history of malaria. Fish is an abundant natural resource, rich in essential nutrients and important to the diet of 'ribeirinhos' of Brazilian Amazonia. We hypothesize that lack of basic medical care in these communities outweighs risk of naturally occurring fish-MMHg. Therefore food habits based on fish consumption is part of a successful strategy for survival and should be considered as a health asset.