Nitrogen and phosphorus limitation of oceanic microbial growth during spring in the Gulf of Aqaba

Bioassay experiments were performed to identify how growth of key groups within the microbial community was simultaneously limited by nutrient (nitrogen and phosphorus) availability during spring in the Gulf of Aqaba's oceanic waters. Measurements of chlorophyll a (chl a) concentration and fast repetition rate (FRR) fluorescence generally demonstrated that growth of obligate phototrophic phytoplankton was co-limited by N and P and growth of facultative aerobic anoxygenic photoheterotropic (AAP) bacteria was limited by N. Phytoplankton exhibited an increase in chl a biomass over 24 to 48 h upon relief of nutrient limitation. This response coincided with an increase in photosystem II (PSII) photochemical efficiency (F v /F m), but was preceded (within 24 h) by a decrease in effective absorption crosssection (σPSII) and electron turnover time (τ). A similar response for τ and bacterio-chl a was observed for the AAPs. Consistent with the up-regulation of PSII activity with FRR fluorescence were observations of newly synthesized PSII reaction centers via low temperature (77K) fluorescence spectroscopy for addition of N (and N + P). Flow cytometry revealed that the chl a and thus FRR fluorescence responses were partly driven by the picophytoplankton (æ10 μm) community, and in particular Synechococcus. Productivity of obligate heterotrophic bacteria exhibited the greatest increase in response to a natural (deep water) treatment, but only a small increase in response to N and P addition, demonstrating the importance of additional substrates (most likely dissolved organic carbon) in moderating the heterotrophs. These data support previous observations that the microbial community response (autotrophy relative to heterotrophy) is critically dependent upon the nature of transient nutrient enrichment. © Inter-Research 2009

UPTAKE AND TOXIC EFFECTS OF SURFACE MODIFIED NANOMATERIALS IN FRESHWATER AQUATIC ORGANISMS

Nanomaterials are a class of materials with unique properties due to their size, and the association of these properties with the toxicity of nanomaterials is poorly understood. The present study assessed the toxic effects of stable aqueous colloidal suspensions of three distinctly different classes of nanomaterials in aquatic organisms. The fullerene, C70, was stabilized through non-covalent surface modification with gallic acid. Toxicity of C70-gallic acid was confirmed to exhibit similar toxic effects as C60-fullerene, including changes in antioxidative processes in Daphnia magna. Daphnia magna fecundity was significantly reduced in 21d bioassays at C70-gallic concentrations below quantifiable limits (0.03 mg/L C70). Antioxidant enzyme activities of glutathione peroxidase and superoxide dismutase as well as lipid peroxidation suggested that exposed organisms experienced oxidative stress. Carbon dots are a class of nanomaterials proposed for use as nontoxic alternatives to semiconductor quantum dots for photoluminescent applications, because of the difference in toxicity of their core components: carbon as opposed to heavy metals. In vivo analysis of treated organisms by confocal fluorescence microscopy revealed carbon dots were absorbed and systemically distributed regardless of particle size. The present study did not find any evidence of acute toxicity at concentrations up to 10mg/L carbon dots. These concentrations also failed to produce negative effects in Ceriodaphnia dubia bioassays to predict chronic toxicity. Carbon dots also failed to elicit developmental toxic effects in zebrafish. The toxic effects of semiconductor quantum dots have been partially attributed to the release of heavy metals with their degradation, particularly cadmium. Laser ablation inductively coupled mass spectrometry was used to compare the uptake of cadmium, selenium and zinc in Daphnia magna treated to CdSe/ZnS quantum dots or CdCl2. These quantum dots were observed to accumulate primarily in the gut lumen and no evidence of uptake of intact quantum dots was observed. Evidence suggests degradation of the quantum dots release of component ions with accumulation of Cd and Zn in the gut epithelia. Quantum dots elicited acute toxicity at 0.66 mg/L Cd but promoted increased reproduction at 40 µg/L

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations

Ferramentas integrativas para avaliação dos efeitos do biochar na biota do solo

Biochar application to soil as an agricultural amendment, as well as a carbon sink, is a focus of increasing interest, despite the underlying factors determining its behaviour, toxicity and fate in soil remaining poorly understood. The main aim of this study was to integratively evaluate the ecotoxicological potential of a wood chip biochar in soil at representative application rates, through combining the responses of multiple soil organisms, and structural and functional parameters, at relevant spatial and temporal scales. To achieve this, the specific objectives were defined and addressed within four experimental chapters. The effects on soil biota of biochar alone and a biochar-compost mixture from a commercial vineyard in Central Portugal, were monitored with laboratory bioassays. Both fresh and field-aged biochar and biochar-compost were tested by evaluating the endpoints survival and reproduction of the collembolan Folsomia candida and food consumption and biomass change of terrestrial isopod Porcellionides pruinosus. Freshly-amended soil did not induce significant changes on organisms’ performance, while the organisms’ fitness was reduced when exposed to the field-aged soil and amended-soil, which was subjected to various climatic factors and conventional pesticides. The results suggested that bioavailability of potentially toxic compounds, like pesticides, might not decrease over time by the presence of biochar and biochar-compost in vineyards that receive conventional plant protection products. Subsequently, research was conducted on the potential inherent toxicity of biochar on biota, as influenced by particle size and application rates, where the experimental design was based on a preliminary earthworm (Eisenia andrei) avoidance behaviour assay. The main experiment was conducted over 28 days in greenhouse microcosms in which survival, weight losses and vertical distribution of E. andrei and bait-lamina consumption were assessed, and combined the evaluation of leachates toxicity looking into endpoints luminescence inhibition of bacterium Vibrio fischeri and immobilisation of the cladoceran Daphnia magna. In addition, a laboratory feeding experiment was performed to address the weight change and the possible link with polyaromatic hydrocarbons (PAH)-type metabolites in the earthworms’ tissues. The results showed that smaller particles (<0.5 mm) of woodchip biochar might pose sub-lethal toxicity to soil biota, suggesting that there is a connection in behavioural (avoidance), individual (weight changes, naphthalene-type metabolites in earthworms’ tissue) and functional (bait-lamina consumption) endpoints. Next, the link was explored between the interaction of soil invertebrates from different functional groups, such as earthworms (E. andrei) and isopods (P. pruinosus), and activity of soil microbial enzymes in biochar-amended soil, alongside the main mechanisms of earthworm’ responses. The latter was investigated with biomarkers of effect. Microbial response was sampling time-, invertebrate presence-, and enzyme-dependent. Reproduction of E. andrei was not affected by the exposure to the woodchip biochar. Biomarkers responded as early warning tools, by showing an increase in lipid peroxidation and cellular energy allocation decrease in exposed earthworms. At last, higher tier testing was conducted in indoor small-scale terrestrial ecosystem models over 42 days, by assessing the effects of biochar, biochar-compost and inorganic fertilizer (NPK) and their combinations, on the earthworm E. andrei survival and weight loss, bait-lamina consumption and a morphological and production traits of rapid cycling plant Brassica rapa, as well as of their leachates on growth inhibition of aquatic macrophyte Lemna minor. The results revealed low-to-no effect on earthworms, and slight stimulations in production parameters in plants, namely in the treatment of combined biochar-compost with mineral fertilizer. L. minor growth was a sensitive endpoint. The study indicated that possibility of nutrients leaching stimulation might not be excluded, which could pose a hazard to aquatic systems. Together, the results demonstrate that biological responses to woodchip biochar varied from sub-lethal to neutral and/or stimulatory, depending on the test organism and endpoint, biochar treatment and application rate. Further, they highlight that for a comprehensive understanding of biochar effects on biota and associated mechanisms, it is paramount to evaluate various indicator species and endpoints, that include different exposure routes and levels of biological organisation and interactions, under representative exposure scenariosA aplicação de biochar no solo como aditivo agrícola, bem como fonte de carbono, é um foco de crescente interesse, apesar de vários fatores subjacentes determinarem o seu comportamento, toxicidade e destino no solo, apesar de pouco compreendidos. O principal objetivo deste estudo foi avaliar de forma integrada o potencial ecotoxicológico de aplicações representativas de um biochar produzido de raspas e resíduos de madeira no solo, combinando respostas de vários organismos edáficos e parâmetros estruturais e funcionais, em escalas espaciais e temporais relevantes. Para isso, os objetivos específicos foram definidos e abordados em quatro capítulos experimentais. Os efeitos sobre a biota do solo deste biochar e de uma mistura de biochar com compostagem (biochar-composto) em vinhas com fins comerciais no centro de Portugal foram monitorizados em bioensaios de laboratório. O biochar e o biochar-composto foram testados através da avaliação da sobrevivência e reprodução do colêmbolo Folsomia candida e do consumo de alimento e biomassa do isópode terrestre Porcellionides pruinosus. O solo imediatamente modificado com a adição do biochar e biochar-composto não induziu mudanças significativas no desempenho dos organismos, enquanto a aptidão dos organismos foi reduzida quando expostos ao esse solo envelhecido em campo e ao solo retificado, que foi submetido a vários fatores climáticos e pesticidas convencionais. Os resultados sugerem que a biodisponibilidade de compostos potencialmente tóxicos, como pesticidas, pode não diminuir em termos temporais pela presença de biochar e biochar-composto em vinhas que recebem este tipo de produtos fitofarmacêuticos convencionais. Posteriormente, a toxicidade inerente do biochar foi avaliada na biota, tendo em conta a influência do tamanho das partículas e taxas de aplicação, onde o delineamento experimental foi baseado num ensaio preliminar de comportamento (evitamento) no lumbricídeo Eisenia andrei. A experiência principal foi conduzida durante 28 dias em microcosmos de estufas onde foram avaliadas a sobrevivência, perda de peso e distribuição vertical de E. andrei e o consumo de "bait-lamina", combinando a avaliação datoxicidade dos lixiviados com o objetivo de determinar a inibição de luminescência da bactéria Vibrio fischeri e a imobilização do cladócero Daphnia magna. Além disso, foi realizada uma experiência de alimentação em laboratório para abordar a alteração de peso e a possível ligação com m etabólitos de hidrocarbonetos poliaromáticos (HPAs) nos tecidos do s lumbricídeos. Os resultados mostraram que partículas pequenas (< 0.5 mm) de biochar de madeira podem causar toxicidade sub-letal no biota do solo, sugerindo que há uma relação com o comportamento (evitamento), ao nível individual (alterações de peso, metabólitos tipo naftaleno em tecido de lumbricídeos) e parâmetros funcionais (consumo de "bait-lamina"). Em seguida, explorou-se a interação entre invertebrados de solo de diferentes grupos funcionais, os lumbricídeos (E. andrei) e os isópodes (P pruinosus), e a sua relação com a atividade enzimática do solo, em solo biologicamente alterado, juntamente com os principais mecanismos de respostas dos lumbricídeos. Este último foi avaliado com biomarcadores de efeito. A resposta microbiana mostrou ser dependente do tempo de amostragem, da presença de invertebrados e da enzima em causa. A reprodução de E. andrei não foi afetada pela exposição ao biochar de madeira. Os biomarcadores responderam como ferramentas de alerta precoce, mostrando um aumento na peroxidação lipídica e diminuição da alocação de energia celular em lumbricídeos expostas. Finalmente, testes de complexidade mais elevada foram conduzidos em modelos de ecossistemas terrestres de pequena escala em 42 dias, avaliando os efeitos de biochar, biochar-composto e fertilizante inorgânico (NPK) e as suas combinações, na sobrevivência e perda de peso de E. andrei, consumo de "bait-lamina", assim como a componente morfológica e de produção da planta Brassica rapa (de ciclo de vida rápido), bem como a inibição do crescimento da macrófita aquática Lemna minor exposta aos respetivos lixiviados. Os resultados revelaram poucos ou nenhuns efeitos nos lumbricídeos e pequenas estimulações nos parâmetros de produção nas plantas, nomeadamente no tratamento de biochar-composto combinado com fertilizante mineral. O crescimento de L. minor foi um dos parâmetros sensível. O estudo indicou que a possibilidade de estímulo de lixiviação de nutrientes pode não ser excluída, o que pode representar um risco para os sistemas aquáticos. Assim sendo, os resultados demonstram que as respostas biológicas ao biochar de resíduos de madeira variaram de efeitos subletais a neutros e / ou de estímulo, dependendo do organismo e parâmetro do teste, do tratamento com biochar e da taxa de aplicação. Além disso, é de destacar que, para uma compreensão abrangente dos efeitos de biochar na biota e nos mecanismos associados, é fundamental avaliar várias espécies e parâmetros indicadores, que incluam diferentes vias de exposição e níveis de organização biológica e interações, sob cenários de exposição representativosPrograma Doutoral em Biologi

Microbial detoxification of deoxynivalenol (DON), assessed via a Lemna minor L. bioassay, through biotransformation to 3-epi-DON and 3-epi-DOM-1

Mycotoxins are toxic metabolites produced by fungi. To mitigate mycotoxins in food or feed, biotransformation is an emerging technology in which microorganisms degrade toxins into non-toxic metabolites. To monitor deoxynivalenol (DON) biotransformation, analytical tools such as ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) are typically used. However, these techniques do not give a decisive answer about the remaining toxicity of possible biotransformation products. Hence, a bioassay using Lemna minor L. was developed. A dose-response analysis revealed significant inhibition in the growth of L. minor exposed to DON concentrations of 0.25 mg/L and higher. Concentrations above 1 mg/L were lethal for the plant. This bioassay is far more sensitive than previously described systems. The bioassay was implemented to screen microbial enrichment cultures, originating from rumen fluid, soil, digestate and activated sludge, on their biotransformation and detoxification capability of DON. The enrichment cultures originating from soil and activated sludge were capable of detoxifying and degrading 5 and 50 mg/L DON. In addition, the metabolites 3-epi-DON and the epimer of de-epoxy-DON (3-epi-DOM-1) were found as biotransformation products of both consortia. Our work provides a new valuable tool to screen microbial cultures for their detoxification capacity

Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni

Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies

Evaluation of a Microalgal Bioassay Based on Community Level Physiological Profiling (CLPP)

This thesis examined the use of commercially available Biolog 96-well plates containing different carbon sources for a microalgal bioassay. Quaternary ammonium compounds (QACs) were used as potentially toxic model compounds to demonstrate the applicability of the assay. Toxicity of dodecyl trimethyl ammonium chloride (DTAC) and didecyl dimethyl ammonium bromide (DDAB) on the growth of Scenedesmus obliquus varied significantly under autotrophic, heterotrophic and mixotrophic growth conditions. The concentration for 50% growth inhibition effect (EC50) of DTAC followed the order: autotrophic (0.48 ± 0.03 mg of DTAC/L) \u3e heterotrophic (1.46 ± 0.04 mg of DTAC/L) \u3e mixotrophic (2.11 ± 0.06 mg of DTAC/L), whereas for DDAB, the order of inhibition was different as: heterotrophic (0.52 ± 0.02 mg of DDAB/L) \u3e autotrophic (1.18 ± 0.08 mg of DDAB/L) \u3e mixotrophic (1.35 ± 0.02 mg of DDAB/L). Moreover, EC50 values were a function of carbon source and growth regime, showing that only EC­50 values do not fully capture the toxic effects of a potentially toxic compound might have on algal communities. Therefore, a new assay was developed based on community level physiological profiling (CLPP). Five different mixtures of artificially defined microalgal communities were employed and the changes in substrate utilization patterns by the treatment of hexadecyl trimethyl ammonium chloride (CTAC) were quantified using principal component analysis (PCA). The toxic effect of CTAC was significant (P \u3c 0.05), showing 58% inhibition compared to the control and the effect was more pronounced for the treatment than that obtained by varying the initial composition of the defined algal communities. The newly developed assay was further applied on wetland water samples, wastewaters (i.e. primary and secondary), river water, and activated carbon treated and untreated oil sand process waters (OSPWs). The assay was able to generate a distinguishable response among these samples, measuring small differences within the respective water groups and larger differences between them

Progress in fluorescence biosensing and food safety towards point-of-detection (PoD) system

The detection of pathogens in food substances is of crucial concern for public health and for the safety of the natural environment. Nanomaterials, with their high sensitivity and selectivity have an edge over conventional organic dyes in fluorescent-based detection methods. Advances in microfluidic technology in biosensors have taken place to meet the user criteria of sensitive, inexpensive, user-friendly, and quick detection. In this review, we have summarized the use of fluorescence-based nanomaterials and the latest research approaches towards integrated biosensors, including microsystems containing fluorescence-based detection, various model systems with nano materials, DNA probes, and antibodies. Paper-based lateral-flow test strips and microchips as well as the most-used trapping components are also reviewed, and the possibility of their performance in portable devices evaluated. We also present a current market-available portable system which was developed for food screening and highlight the future direction for the development of fluorescence-based systems for on-site detection and stratification of common foodborne pathogens