Assessment of the single and mixture ecotoxicity of pharmaceuticals of environmental concern using aquatic test organisms / Avaliação da ecotoxicidade individual e das misturas de fármacos de preocupação ambiental usando organismos-teste aquáticos

Pharmaceuticals are contaminants of emerging concern which have been a target of increasing attention by the scientific community. Pharmaceuticals presenting high consumption, incomplete metabolism and incomplete removal at wastewater treatment plants have been frequently detected in aquatic ecosystems worldwide. This is the case of the pharmaceuticals metformin (MET), bisoprolol (BIS), sotalol (SOT) and ranitidine (RAN). However, ecotoxicity data for these contaminants are scarce, especially regarding behavior effects and chronic toxicity. In addition, the knowledge regarding the joint toxicity of these pharmaceuticals on non-target organisms is still incipient, which makes their environment risk assessment uncertain. This study aimed to fill these knowledge gaps for these four pharmaceuticals, by carrying out toxicity tests using five test organisms from three trophic levels. Different endpoints were assessed in tests with Raphidocelis subcapitata (algae), Lemna minor (macrophyte), Daphnia similis (crustacean), Hydra attenuate (cnidarian) and Danio rerio (fish). The binary and quaternary mixture acute toxicity for these pharmaceuticals were assessed on D. similis and D. rerio embryo tests, respectively. This study also aimed to evaluate the predictive accuracy of the Concentration addition (CA) and the Independent action (IA) classic models. In addition, the nature of the possible toxicological interactions between the pharmaceuticals in binary mixtures were also evaluated, using the Combination Index-isobologram (CI) method. The modelling of the concentration-response curves and the associated statistical analyses were performed using the automated spreadsheet ToxCalcMix v.1.0 and the software OriginPro 2015. The software CompuSyn was used for performing the mixture analyses with the CI method. The experimental planning of the binary mixture tests was performed using the fractioned factorial design, in order to cover several possible ratio and level-dependent effects with a reduced number of test organisms. The results obtained in this study are shown in four articles. In article 1, we provided a critical review and discussed the misunderstandings, deficiencies and data gaps on the ecotoxicity data of pharmaceuticals and personal care products mixtures published in the literature. In the following articles, the results obtained from the single and mixture toxicity tests performed in this study were presented and discussed. The pharmaceuticals MET (article 2) and BIS (article 3) were classified as hazardous to the aquatic environment, in the acute toxicity category. However, an ecological risk is not expected for the pelagic freshwater species exposed to these two pharmaceuticals, based on the chronic data obtained. The results obtained from the mixture toxicity tests (article 4) showed that most of the observed toxicity effects from the binary mixtures were in the zone between the predicted effects by the CA and IA models. The CI model showed to be an useful tool to describe the possible toxicological interactions occurring between the pharmaceuticals in joint action. Even statistically significant non-effect concentrations of the pharmaceuticals added up to induce significant adverse effects in mixtures (something from nothing). It was concluded that ecological risk assessment based on single toxic effects can underestimate the real impact of environmental contaminants on aquatic ecosystems

A contaminação ambiental por fármacos tem sido alvo de crescente preocupação pela comunidade científica. Fármacos de elevado consumo, incompleto metabolismo e remoção incompleta em estações de tratamento de esgoto, como é o caso da metformina (MET), bisoprolol (BIS), sotalol (SOT) e ranitidina (RAN), têm sido frequentemente detectados em matrizes aquáticas do mundo todo. Apesar disso, dados ecotoxicológicos consistentes para esses contaminantes são escassos, principalmente com relação a efeitos comportamentais e oriundos de estudos crônicos. Além disso, o entendimento dos efeitos de suas ações combinadas em organismos não-alvo é ainda incipiente, o que gera incertezas na avaliação dos seus riscos ambientais. Esta pesquisa teve por objetivo preencher essas lacunas de conhecimentos para esses quatro fármacos, por meio da realização de testes com cinco diferentes organismos-teste de três diferentes níveis tróficos. Foram analisados diferentes parâmetros avaliativos em testes com os organismos aquáticos Raphidocelis subcapitata (alga), Lemna minor (macrófita), Daphnia similis (crustáceo), Hydra attenuata (cnidário) e Danio rerio (peixe). As toxicidades agudas das misturas binárias e quaternárias desses quatro fármacos também foram avaliadas em testes com D. similis e embriões de D. rerio, respectivamente. Este trabalho também teve por objetivo avaliar a acurácia preditiva dos modelos de adição de concentração (CA) e ação independente (IA) e analisar a natureza das possíveis interações toxicológicas entre os fármacos, em misturas binárias, usando o modelo do Índice de Combinação (CI). A modelagem das relações concentração-resposta e as análises estatísticas associadas foram realizadas empregando-se a planilha automatizada ToxCalcMix versão 1.0 e o software OriginPro 2015. O software CompuSyn foi utilizado para as análises envolvendo o CI. O planejamento experimental dos testes de misturas binárias foi realizado por meio do design fatorial fracionado, a fim de cobrir diversas possíveis interações em várias proporções e níveis de efeitos, com a redução do número de organismos-teste. Os resultados desta pesquisa estão apresentados em quatro artigos. No artigo 1, realizou-se uma revisão crítica com relação às lacunas de conhecimentos e deficiências identificadas a partir da análise da literatura sobre a ecotoxicologia de misturas de fármacos e de produtos de higiene pessoal. Nos artigos seguintes, foram apresentados e discutidos os resultados oriundos dos testes com os quatro fármacos avaliados neste estudo. Os fármacos MET (artigo 2) e BIS (artigo 3) foram classificados como perigosos para o ambiente aquático, na categoria de toxicidade aguda. Contudo, um risco ecológico não é esperado para as espécies pelágicas de água doce expostas a esses dois fármacos, com base nos dados de toxicidade crônica obtidos. Os resultados dos testes de misturas (artigo 4) permitiram concluir que a maior parte dos efeitos observados das misturas binárias estiveram na zona entre os efeitos preditos pelos modelos clássicos de CA e IA. O modelo do CI mostrou-se uma ferramenta útil para descrever a natureza das possíveis interações toxicológicas que ocorrem entre os fármacos em ações combinadas. Mesmo concentrações de nenhum efeito estatisticamente significativo dos fármacos causaram efeitos adversos significativos quando em misturas (something from nothing). Concluiu-se que avaliações de risco ecológicas baseadas em efeitos tóxicos individuais de contaminantes ambientais podem subestimar o real impacto desses compostos em ecossistemas aquáticos

Biting midges (Diptera: Ceratopogonidae) from an urban forest fragment in Central Amazon (Brazil): Effects of opening areas on abundance, richness, and composition

ABSTRACT We assessed the immature stages of Ceratopogonidae (Diptera) in artificial containers in an urban forest fragment in Manaus (Brazil), including their behavioral, biological and ecological information. In addition, we evaluated the effects of deforestation in an open and forested area on Ceratopogonidae communities. Immatures were sampled between August 2012 and July 2014 in artificial containers installed in both habitat types. We collected 685 immatures of seven morpho-species of Bezzia Kieffer, Culicoides Latreille, Dasyhelea Kieffer, Forcipomyia Meigen, and Palpomyia Meigen. In the open area, we recorded higher temperature and electrical conductivity values than in the forested area; however, these variables did not differ between seasons. Water volume was higher in open area and in rainy season, while pH was similar in both areas and seasons. Species richness was higher in forested area, but did not differ between seasons. We did not record differences in abundance between areas or seasons. Community composition differed between areas, but not between seasons. We provide the first records of Culicoides (Hoffmania) insignis Lutz and C. (Haematomyidium) quasiparaensis Clastrier in artificial containers from the state of Amazonas. Our results suggest that the preservation of forested areas in Amazonas is fundamental for the maintenance of the life cycle of some species of Ceratopogonidae.

Additional list of species of aquatic macrophytes in the lower basin of the Xingu River / Lista adicional de macrófitas aquáticas do baixo Rio Xingu

Abstract In this paper the authors present an additional list of aquatic macrophytes in the lower basin of the Xingu River.

Resumo Neste trabalho os autores apresentam uma lista adicional de macrófitas aquáticas no baixo Rio Xingu.

Additional list of species of aquatic macrophytes in the lower basin of the Xingu River / Lista adicional de macrófitas aquáticas do baixo Rio Xingu

In this paper the authors present an additional list of aquatic macrophytes in the lower basin of the Xingu River.

Neste trabalho os autores apresentam uma lista adicional de macrófitas aquáticas no baixo Rio Xingu.

Epiphytic marine pigmented bacteria: A prospective source of natural antioxidants

Awareness on antioxidants and its significance in human healthcare has increased many folds in recent time. Increased demand requisite on welcoming newer and alternative resources for natural antioxidants. Seaweed associated pigmented bacteria screened for its antioxidant potentials reveals 55.5% of the organisms were able to synthesize antioxidant compounds. DPPH assay showed 20% of the organisms to reach a antioxidant zone of 1 cm and 8.3% of the strains more than 3 cm. Pseudomonas koreensis (JX915782) a Sargassum associated yellowish brown pigmented bacteria have better activity than known commercial antioxidant butylated hydroxytoluene (BHT) against DPPH scavenging. Serratia rubidaea (JX915783), an associate of Ulva sp. and Pseudomonas argentinensis (JX915781) an epiphyte of Chaetomorpha media, were also contributed significantly towards ABTS (7.2% ± 0.03 to 15.2 ± 0.09%; 1.8% ± 0.01 to 15.7 ± 0.22%) and FRAP (1.81 ± 0.01 to 9.35 ± 0.98; 7.97 ± 0.12 to 18.70 ± 1.84 μg/mL of AsA Eq.) respectively. 16S rRNA gene sequence analysis revealed bacteria that have higher antioxidant activity belongs to a bacterial class Gammaproteobacteria. Statistical analysis of phenolic contents in relation with other parameters like DPPH, ABTS, reducing power and FRAP are well correlated (p < 0.05). Results obtained from the current study inferred that the seaweed associated pigmented bacteria have enormous potential on antioxidant compounds and need to be extracted in a larger way for clinical applications.

Antibacterial properties of silver nanoparticles synthesized by marine Ochrobactrum sp.

Metal nanoparticle synthesis is an interesting area in nanotechnology due to their remarkable optical, magnetic, electrical, catalytic and biomedical properties, but there needs to develop clean, non-toxic and environmental friendly methods for the synthesis and assembly of nanoparticles. Biological agents in the form of microbes have emerged up as efficient candidates for nanoparticle synthesis due to their extreme versatility to synthesize diverse nanoparticles with varying size and shape. In the present study, an eco favorable method for the biosynthesis of silver nanoparticles using marine bacterial isolate has been attempted. Very interestingly, molecular identification proved it as a strain of Ochrobactrum anhtropi. In addition, the isolate was found to have the potential to form silver nanoparticles intracellularly at room temperature within 24 h. The biosynthesized silver nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscope (TEM) and scanning electron microscope (SEM). The UV-visible spectrum of the aqueous medium containing silver nanoparticles showed a peak at 450 nm corresponding to the plasmon absorbance of silver nanoparticles. The SEM and TEM micrographs revealed that the synthesized silver nanoparticles were spherical in shape with a size range from 38 nm - 85 nm. The silver nanoparticles synthesized by the isolate were also used to explore its antibacterial potential against pathogens like Salmonella Typhi, Salmonella Paratyphi, Vibrio cholerae and Staphylococcus aureus.

Shredders are abundant and species-rich in tropical continental-island low-order streams: Gorgona Island, Tropical Eastern Pacific, Colombia / Trituradoras son abundantes y ricas en especies en corrientes de bajo orden de islas continentales tropicales: Isla Gorgona, Pacífico Oriental Tropical, Colombia

Macroinvertebrate shredders may have been overlooked in tropical streams due to the geographical bias of early studies, methodological limitations, and the complex influences of local-scale factors. While shredders seem to be scarce in most oceanic island streams, we here test if they are abundant in a continental island. Gut content analyses of benthic macroinvertebrates were used to identify shredding taxa in streams located in different types of forest in Gorgona Island (Tropical Eastern Pacific). General dietary overlap (GO) was quantified and relative biomass, relative frequency and the leaf litter percentage in the guts were used to establish the relative importance of each taxon in the shredding guild. Various indices were used to identify the spatial arrangement (i.e. contagious or random) of each taxon and shredding guild among streams. We identified 31 shredding taxa that were divided into specialist-shredders (14 taxa), generalist-shredders (10), and collector-shredders (7). There was a complete GO (0.75, p<0.001) for the guild. Cockroaches (Epilampra) were the most represented shredders due to the greatest contribution to guild total biomass and to the highest content of leaf litter in their guts. These organisms were more important than shrimps and crabs in terms of abundance and biomass in leaf pack samples. Potimirin shrimps ranked second and Stenochironomus midges ranked third. Among aquatic insects, other secondarily important species were Leptohyphes (Ephemeroptera), Macrelmis, Anchytarsus and Tetraglosa (Coleoptera). Ten taxa exhibited contagious spatial pattern and twenty-one exhibited a random distribution. Resource distribution (i.e., leaf packs) between streams was random too. The guild was contagiously distributed, but this result could be highly influenced by the taxa with contagious distribution. Mean abundance, richness and mean biomass of shredders were not significantly correlated with any of the environmental variables measured. Three factors seemly explain the high richness and abundance of shredders in Gorgona Island: (1) its continental origin, (2) its current proximity to the continent (35 km), and (3) the high diversity and availability of leaf litter and woody debris inputs to the streams. Although crabs, shrimps, aquatic insects and semiaquatic-cockroaches coexisted in Gorgona Island, the latter were the most important leaf litter shredders in terms of biomass. Rev. Biol. Trop. 62 (Suppl. 1): 85-105. Epub 2014 February 01.

Existe la posibilidad de que los macroinvertebrados fragmentadores hayan sido subvalorados en las quebradas tropicales debido al sesgo geográfico de los estudios realizados, a limitaciones metódicas y a la influencia de factores de escala local. A pesar de que los fragmentadores son escasos en las quebradas de islas oceánicas, hipotetizamos que son abundantes en Isla Gorgona. Los análisis de contenidos estomacales de estos organismos se usaron para identificar los taxones fragmentadores colectados en quebradas con diferentes tipos de bosque. Se cuantificaron el índice general de sobreposición dietaria (GO por su siglas en inglés), y el índice de valor de importancia de cada taxón en el gremio (con base en biomasa relativa, frecuencia relativa y proporción de tejido vegetal en los contenidos estomacales). El arreglo espacial de cada taxón y el del gremio entre quebradas (i.e., contagioso o aleatorio) se determinó a partir de varios índices. Se identificaron 31 fragmentadores, los cuales se dividieron en especialistas (14 taxones), generalistas (10) y colectores-fragmentadores (7). Hubo un GO completo del gremio (0.75, p<0.001). Debido a su alta contribución de biomasa y de abundancia al gremio, y a la elevada proporción de tejido vegetal consumido, las cucarachas (Epilampra) fueron los fragmentadores-especialistas más representativos, superiores incluso a camarones y cangrejos. El camarón Potimirin fue el segundo taxón más importante seguido por el díptero Stenochironomus. Entre los insectos acuáticos, otros taxones de importancia secundaria fueron Leptohyphes (Ephemeroptera), Macrelmis, Anchytarsus y Tetraglosa (Coleoptera). Diez taxones mostraron un patrón espacial de contagio, y 21 se organizaron aleatoriamente, al igual que los recursos (i.e., “paquetes” de hojarasca). Aunque el gremio se organizó contagiosamente, estos resultados pudieron estar altamente influenciados por los taxones con distribución del mismo tipo. No se encontraron correlaciones significantes entre la abundancia media, la riqueza y la biomasa media con ninguna de las variables ambientales. Tres factores explicaron la alta riqueza y la abundancia de los fragmentadores en Isla Gorgona: (1) el origen continental, (2) su proximidad al continente (35km), y (3) la alta diversidad y disponibilidad de hojarasca y de desechos de madera dentro de las quebradas. Aunque cangrejos, camarones, insectos acuáticos y cucarachas-semiacuáticas coexistieron en la isla, estas últimas fueron, en términos de biomasa, los fragmentadores más importantes.

Aquatic macroinvertebrates associated with Eichhornia azurea (Swartz) Kunth and relationships with abiotic factors in marginal lentic ecosystems (São Paulo, Brazil) / Macroinvertebrados aquáticos associados à Eichhornia azurea (Swartz) Kunth e suas relações com as variáveis abióticas em ecossistemas lênticos marginais (São Paulo, Brasil)

Marginal lakes are characterised by their having high biological diversity due to the presence of aquatic macrophytes in their coastal zones, providing habitats for refuge and food for animal community members. Among the fauna components associated with macrophytes, aquatic macroinvertebrates are important because they are an energy source for predators and fish. In six lakes and two different seasons (March and August 2009), the ecological attributes of aquatic macroinvertebrate community associated with Eichhornia azurea were compared and the controlling environmental factors were identified. Since the attributes of macroinvertebrate community are strictly associated with abiotic variables of each distinct habitat, our hypothesis was that each site associated with the same floating aquatic macrophyte (E. azurea) should have a typical composition and density of organisms. We identified 50 taxa of macroinvertebrates, with greater taxa richness for aquatic insects (37 taxa) divided into eight orders; the order Diptera being the most abundant in the two study periods. On the other hand, higher values of total taxa richness were recorded in August. Dissolved oxygen and pH presented the greatest number of significant positive correlations with the different taxa. The animals most frequently collected in the six lakes in March and August 2009 were Hirudinea, Oligochaeta, Hydrachnidae, Conchostraca, Ostracoda, Noteridae, Ceratopogonidae, Chironomidae, Culicidae, Caenidae, Pleidae, Aeshnidae, Libellulidae, Coenagrionidae and Nematoda. Only densities of Trichoptera, Ostracoda and Conchostraca presented the highest significant differences between lakes in both study periods and considering the composition of macroinvertebrates no significant differences were registered for macroinvertebrate composition.

Lagoas marginais são caracterizadas por apresentar elevada diversidade biológica, devido à presença de macrófitas aquáticas nas suas zonas litorâneas, constituindo habitats de refúgio e alimento para comunidades associadas. Entre os seus integrantes, destacam-se os macroinvertebrados aquáticos, fonte de energia para predadores e ictiofauna. Em seis lagoas e em duas épocas distintas (março e agosto de 2009), os atributos ecológicos da comunidade de macroinvertebrados aquáticos associados à Eichhornia azurea foram comparados, identificando-se os fatores ambientais controladores. Uma vez que os atributos da comunidade de macroinvertebrados estão estritamente associados às variáveis abióticas de cada habitat distinto, nossa hipótese era de que cada local com a presença da mesma macrófita aquática flutuante (E. azurea) deveria ter uma típica composição e densidade de organismos. Foram identificados 50 taxa de macroinvertebrados, com maior riqueza registrada para os insetos aquáticos (37 taxa), distribuídos em oito ordens; sendo a ordem Diptera a mais abundante nos dois períodos de estudo. Por outro lado, maiores valores de riqueza total de taxa foram registrados em agosto. Oxigênio dissolvido e pH apresentaram o maior número de correlações significativas positivas com os diferentes taxa. Os animais mais frequentes, coletados nas seis lagoas estudadas em março e agosto de 2009, foram Hirudinea, Oligochaeta, Hydrachnidiae, Conchostraca, Ostracoda, Noteridae, Ceratopogonidae, Chironomidae, Culicidae, Caenidae, Pleidae, Aeshnidae, Libellulidae, Coenagrionidae e Nematoda. Apenas as densidades de Trichoptera, Ostracoda e Conchostraca apresentaram maior diferença significativa entre lagoas, em ambos os períodos de estudo. Considerando-se a composição de macroinvertebrados, diferenças significativas não foram registradas.

Genomic Approaches in Marine Biodiversity and Aquaculture

Recent advances in genomic and post-genomic technologies have now established the new standard in medical and biotechnological research. The introduction of next-generation sequencing, NGS,has resulted in the generation of thousands of genomes from all domains of life, including the genomes of complex uncultured microbial communities revealed through metagenomics. Although the application of genomics to marine biodiversity remains poorly developed overall, some noteworthy progress has been made in recent years. The genomes of various model marine organisms have been published and a few more are underway. In addition, the recent large-scale analysis of marine microbes, along with transcriptomic and proteomic approaches to the study of teleost fishes, mollusks and crustaceans, to mention a few, has provided a better understanding of phenotypic variability and functional genomics. The past few years have also seen advances in applications relevant to marine aquaculture and fisheries. In this review we introduce several examples of recent discoveries and progress made towards engendering genomic resources aimed at enhancing our understanding of marine biodiversity and promoting the development of aquaculture. Finally, we discuss the need for auspicious science policies to address challenges confronting smaller nations in the appropriate oversight of this growing domain as they strive to guarantee food security and conservation of their natural resources.