Impacts of nanoparticles to microbes and invertebrates: from community responses to cellular targets

Tese de doutoramento in Sciences Specialization in BiologyThe incredible development in nanotechnology since the last decade has brought the “nanoworld” to our regular life. However, the extensive global growth in commercial production and usage of nanomaterial-based products raised the question whether nanomaterials when released to the environment can constitute a potential risk to biota and ecosystem processes. Being large reservoirs, natural waters are likely to be the ultimate sink of nanomaterials. In forested streams, microbes, predominantly fungi, decompose plant litter from riparian vegetation and transfer carbon and energy via invertebrate shredders to higher trophic levels. Freshwater decomposers are sensitive to changes in water quality with implications to ecosystem functioning. Considering the recent development of nanotechnology, assessing the potential toxicity of nanomaterials against freshwater decomposers and examining their ecological and physiological responses to nanoparticle exposure will contribute to a safer use of nanomaterials. In this study, by using a microcosm approach, we found that nanocopper oxide (nanoCuO), nanosilver, and their ionic precursors severely affected leaf litter decomposition by stream-dwelling microbes, as indicated by a decrease in microbial biomass, fungal sporulation and species richness. Moreover, the analysis of fungal and bacterial communities, based on DNA fingerprints from denaturing gradient gel electrophoresis and fungal sporulating species, revealed shifts in species composition and changes towards a better adapted community under the stress induced by nano and ionic metals. Moreover, the negative effects of metal nanoparticles were less pronounced than those of their ionic forms. Nanoparticle size (12, 50 and 80 nm) and the presence of humic acid (HA) influenced the toxicity of nanoCuO against stream-dwelling microbial decomposers. The toxicity of nanoCuO increased in a dose-dependent manner and with the decrease in nanoparticle size. Bacteria were more sensitive than fungi to nanoCuO, because EC50 values for biomass of bacteria were much lower than those of fungi. Fungal reproduction was more sensitive to nanoCuO than leaf decomposition or microbial biomass. HA alone also had negative effects on microbial diversity and activity, but the presence of HA alleviated the negative effects of smaller size nanoCuO (12 or 50 nm). Alterations in leaf surface morphology further supported the impacts of nanoparticles and HA on microbial activity on decomposing leaves, as shown by scanning electron microscopy. We also showed that nanoCuO had lethal and sublethal effects on Allogamus ligonifer, a common invertebrate shredder in Southwest European streams that prefers high quality stream water. The feeding behaviour and growth of the invertebrate were affected in a dose-dependent manner. Effects were due to both nanoCuO and ionic copper leached from nanoCuO that adsorbed or accumulated in the shredder body. The feeding behaviour of the invertebrate shredder was more inhibited as nanoparticle size decreased. The toxicity of smaller size nanoCuO to the shredder was alleviated by the presence of HA. A postexposure feeding experiment showed a very low recovery of the invertebrate feeding behaviour after stress removal. The exposure of aquatic fungal populations to nanoCuO led to a decrease in biomass production, alterations in cell-wall morphology, increased biosorption of nanoCuO and induction of extracellular laccase activity in a time and dose-dependent manner. Fungal populations from metal-polluted streams were more resistant/tolerant to the stress induced by nanoCuO than those from non-polluted streams. Differences in laccase activity among fungi appeared to be related to the presence of laccase-like genes in the copper-binding domain. Exposure to nanoCuO or ionic copper led to lower intracellular accumulation of reactive oxygen species (ROS), plasma membrane disruption, and DNA-strand breaks in fungal populations isolated from metal-polluted streams than in those from non-polluted streams. The activities of glutathione reductase and superoxide dismutase were higher in fungi from metal-polluted than from non-polluted streams, but the opposite was found for glutathione peroxidase activity. Results suggested that fungi from metal-polluted streams have higher capacity to deal with the oxidative stress induced by nanoCuO, probably due to their ability to maintain a high ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). In contrast to metal nanoparticles, polyhydroxy fullerene (PHF) nanoparticles stimulated the growth of the yeast Saccharomyces cerevisiae, which was used as model of eukaryotic organism. Moreover, the oxidative stress induced by cadmium ions to yeast cells was mitigated by the presence of PHF. A maximum growth recovery was obtained after 26h of exposure to 500 ppm PHF at pH 6.8. Results suggested that PHF nanoparticles have antioxidant and free-radical scavenging properties.Na última década ocorreu um desenvolvimento exponencial da nanotecnologia o que trouxe o "nanomundo" à nossa vida do dia a dia. No entanto, o crescimento a nível global da produção e do uso de produtos com base em nanomateriais levanta a questão de saber se os nanomateriais, quando libertados para o meio ambiente, podem constituir um risco potencial para as comunidades biológicas e para os processos dos ecossistemas a elas associados. Os ecossistemas de água doce são susceptíveis de constituir o reservatório final dos nanomateriais. Nos rios e ribeiros florestados, os microrganismos, principalmente os fungos, decompõem o material vegetal proveniente da vegetação ribeirinha e promovem a transferência do carbono e da energia para os níveis tróficos superiores através da actividade dos invertebrados trituradores. Os decompositores de água doce são sensíveis a alterações na qualidade da água, com implicações para o funcionamento do ecossistema. Assim, a avaliação da potencial toxicidade dos nanomateriais para os decompositores de água doce e a análise das suas respostas ecológicas e fisiológicas à exposição a nanopartículas contribuirá para uma utilização mais segura dos nanomateriais. Neste estudo, usando uma abordagem em microcosmos, mostrámos que as nanopartículas de óxido de cobre, as nanopartículas de prata, e os seus precursores iónicos afectavam negativamente a decomposição da folhada por comunidades de microrganismos aquáticos, como indicado por uma diminuição da biomassa microbiana (fungos e bactérias), da esporulação dos fungos e da riqueza em espécies de fungos. A análise da comunidades de fungos, por electroforese em gradiente desnaturante do DNA microbiano e com base na morfologia das conídias libertadas da folhada em decomposição, revelou alterações na estrutura das comunidades no sentido de uma comunidade melhor adaptada ao stress induzido pelos metais quer nas formas nano quer iónicas. Além disso, os efeitos negativos das nanopartículas metálicas foram menos pronunciados do que os das suas formas iónicas. O tamanho das nanopartículas de óxido de cobre (12, 50 e 80 nm) e a presença do ácido húmico (HA) influenciou a toxicidade das nanopartículas para os microrganismos decompositores. A toxicidade das nanopartículas de óxido de cobre aumentou com a dose e com a diminuição do tamanho das partículas. As bactérias foram mais sensíveis do que os fungos às nanopartículas de óxido de cobre, porque os valores de EC50 para a biomassa de bactérias foram muito mais baixos do que os dos fungos. A reprodução dos fungos foi mais sensível à exposição às nanopartículas de óxido de cobre do que a decomposição da folhada ou a biomassa microbiana. O HA sozinho também teve efeitos negativos sobre a diversidade e a actividade dos microrganismos. Contudo, a presença de HA mitigou os efeitos negativos das nanopartículas de óxido de cobre de menor tamanho (12 ou 50 nm). As alterações na morfologia da superfície da folhada, reveladas por microscopia electrónica de varrimento, corroboraram os efeitos das nanopartículas e do HA na actividade microbiana nas folhas em decomposição. Os nossos resultados também mostraram que as nanopartículas de óxido de cobre tiveram efeitos letais e subletais em Allogamus ligonifer, um invertebrado detritívoro comum em rios do Sudoeste Europeu com elevada qualidade ecológica. A presença de nanopartículas de óxido de cobre afectou o comportamento alimentar e o crescimento do invertebrado de uma forma dependente da dose. Os efeitos negativos no animal pareceram ser devidos à adsorção ou acumulação no corpo do invertebrado de nanopartículas e de cobre iónico libertado das nanopartículas. O comportamento alimentar dos invertebrados foi mais inibido na presença de nanopartículas de menor tamanho comparativamente às de maior tamanho. A toxicidade das nanopartículas de óxido de cobre de menor tamanho para o invertebrado foi atenuada pela presença de HA. Uma experiência de alimentação de pósexposição mostrou uma baixa recuperação do comportamento alimentar dos invertebrados após a remoção do stress imposto pelas nanopartículas. A exposição de populações de fungos aquáticos às nanopartículas de óxido de cobre levou a uma diminuição da biomassa produzida, a alterações na morfologia da parede celular, ao aumento da bioadsorção das nanopartículas de óxido de cobre e à indução da actividade de lacases extracelulares de uma forma dependente da dose e do tempo. As populações de fungos isoladas de rios poluídos com metais foram mais resistentes/tolerantes ao stress induzido pelas nanopartículas metálicas do que as isoladas de rios não poluídos. As diferenças observadas na actividade das lacases entre os fungos pareceram estar associadas à presença ou ausência de genes do tipo das lacases. A exposição a nanopartículas de óxido de cobre ou a cobre iónico induziu menor acumulação intracelular de espécies reactivas de oxigénio e menos danos na membrana plasmática e no DNA de fungos isolados de rios poluídos com metais do que em fungos isolados de rios não poluídos. As actividades da glutationa reductase e da superóxido dismutase foram mais elevadas em fungos isolados de rios poluídos com metais do que em fungos isolados de rios não poluídos. Contudo, o oposto foi observado para a actividade da glutationa peroxidase. Os resultados sugerem que os fungos de rios poluídos com metais têm maior capacidade para lidar com o stress oxidativo induzido pelas nanopartículas de óxido de cobre provavelmente devido à sua capacidade de manter uma razão elevada de glutationa reduzida (GSH) em relação à glutationa oxidada (GSSG) nas células. Em contraste com o observado para as nanopartículas metálicas, as nanopartículas de poli-hidroxi-fulereno (PHF) estimularam o crescimento da levedura Saccharomyces cerevisiae, a qual foi utilizada neste trabalho como modelo de organismo eucariota. Por outro lado, o stress oxidativo induzido por iões de cádmio na levedura foi atenuado pela presença de PHF. A recuperação máxima do crescimento da levedura foi obtida após 26 horas de exposição a 500 mg L-1 de PHF e a pH 6,8. Os resultados sugerem que as nanopartículas de PHF têm propriedades antioxidantes

Plastic interactions with pollutants and consequences to aquatic ecosystems: what we know and what we do not know

Plastics are a group of synthetic materials made of organic polymers and some additives with special characteristics. Plastics have become part of our daily life due to their many applications and uses. However, inappropriately managed plastic waste has raised concern regarding their ecotoxicological and human health risks in the long term. Due to the non-biodegradable nature of plastics, their waste may take several thousands of years to partially degrade in natural environments. Plastic fragments/particles can be very minute in size and are mistaken easily for prey or food by aquatic organisms (e.g., invertebrates, fishes). The surface properties of plastic particles, including large surface area, functional groups, surface topography, point zero charge, influence the sorption of various contaminants, including heavy metals, oil spills, PAHs, PCBs and DDT. Despite the fact that the number of studies on the biological effects of plastic particles on biota and humans has been increasing in recent years, studies on mixtures of plastics and other chemical contaminants in the aquatic environment are still limited. This review aims to gather information about the main characteristics of plastic particles that allow different types of contaminants to adsorb on their surfaces, the consequences of this adsorption, and the interactions of plastic particles with aquatic biota. Additionally, some missing links and potential solutions are presented to boost more research on this topic and achieve a holistic view on the effects of micro- and nanoplastics to biological systems in aquatic environments. It is urgent to implement measures to deal with plastic pollution that include improving waste management, monitoring key plastic particles, their hotspots, and developing their assessment techniques, using alternative products, determining concentrations of micro- and nanoplastics and the contaminants in freshwater and marine food-species consumed by humans, applying clean-up and remediation strategies, and biodegradation strategies.This study was supported by the strategic programme UID/BIA/04050/2019 and the Emergemix project (PTDC/BIABMA/30922/2017) funded by national funds through the Portuguese Foundation for Science and Technology (FCT) I.P. and by the ERDF through the COMPETE2020ProgramaOperacionalCompetitividade e Internacionalizacao

Impacts of CuO nanoparticles on aquatic detritus foodwebs

FEDER-POFC-COMPETE and the Portuguese Foundation for Science and Technology supported this study (PEst-C/BIA/UI4050/2011, NANOECOTOX-PTDC/AAC-AMB/121650/2010) and A. Pradhan was supported by FCT (SFRH/BD/45614/2008)

Polyhydroxyl fullerene can mitigate toxicity effects of cadmium on yeasts

With inception of nanotechnology, nanomaterials are currently in the prime-line of research. Polyhydroxyl fullerene is one of the widely commercialized nanomaterials and applied in electronics and biomedical fields. Unlike fullerene, polyhydroxyl fullerene is known to be biocompatible, and recent studies reported its antioxidant properties. On the other hand, cadmium toxicity has been often attributed to the ability of this metal to induce oxidative stress. To assess antioxidant properties of polyhydroxyl fullerene, the model yeast Saccharomyces cerevisiae was exposed to cadmium (≤ 5 ppm, 3 levels) in the presence or absence of polyhydroxyl fullerene (≤ 500 ppm, 3 levels) at different pH values (5.8, 6.3 and 6.8). The size distribution and dispersion of polyhydroxyl fullerene in the stock suspension were measured with DLS (z-average 143 nm, PdI 0.365). Yeast growth, plasma membrane integrity and accumulation of reactive oxygen species (ROS) were investigated in the presence or absence of Cd2+ and/or polyhydroxyl fullerene. Yeast growth was inhibited up to 34% by exposure to Cd2+, but was not affected by polyhydroxyl fullerene. At pH 5.8, the exposure to the highest concentration of Cd2+ (5 ppm) and polyhydroxyl fullerene (500 ppm) stimulated yeast growth by 59 and 61% at the exponential and late exponential growth phases, respectively. Growth increased even more when pH increased to 6.8. Severe plasma membrane disruption and ROS accumulation were observed after exposure to the highest concentration of Cd2+ in the absence of polyhydroxyl fullerene. Membrane disruption and ROS accumulation decreased with increasing concentration of polyhydroxyl fullerene and with the increase in pH to 6.8. Overall, results indicated that polyhydroxyl fullerene is not toxic to yeasts and may potentially trigger antioxidant defense mechanisms to mitigate metal-induced toxicity.Funding: FEDER-POFC-COMPETE and FCT supported this study (PEst-C/BIA/UI4050/2011, NANOECOTOX- PTDC/AAC-AMB/121650/2010) and A. Pradhan (SFRH/BD/45614/2008)

Nano copper oxide is a threat to an endemic shredder of the Iberian Peninsula

FEDER-POFC-COMPETE and the Portuguese Foundation for Science and Technology supported this study (PEst-C/BIA/UI4050/2011, NANOECOTOX-PTDC/AAC-AMB/121650/2010) and A. Pradhan was supported by the Portuguese Foundation for Science and Technology (SFRH/BD/45614/2008)

Humic acids and nanoparticle size change the toxicity of nano CuO to freshwater microbes and invertebrates

Increased commercial application of metal oxide nanoparticles increases the chance of their exposure to surface waters, generating a potential risk to biota and associated ecological processes. The probable threat however may depend on nanoparticle size and also the interactions with natural organic matter present in water, such as humic substances. In streams, microbes and invertebrate shredders are key players in detritus foodwebs to transfer energy from plant-litter to higher trophic levels. We investigated the impacts of nano CuO size (12, 50 and 80 nm powder) and concentration (up to 400 ppm; 5 levels) and the influence of humic acid (HA ≤100 ppm; 3 levels) on stream-dwelling microbial decomposers and the invertebrate shredder Allogamus ligonifer. In the absence of HA, the exposure of microbially-colonised leaves to different sizes of nano CuO reduced leaf decomposition. The effects became more severe as nano CuO concentration increased and nanoparticle size decreased. The exposure of shredders to sublethal concentrations of nano CuO decreased leaf consumption rate and the effects were stronger for nanoparticles with lower size. The exposure to higher concentrations of HA alone reduced leaf decomposition by microbes and leaf consumption by the invertebrate. Conversely, the exposure to HA led to a decrease in nano CuO toxicity, particularly at lower nanoparticle sizes.Acknowledgement: FEDER-POFC-COMPETE and FCT supported this study (PEst-C/BIA/UI4050/2011 and PTDC/AAC-AMB/121650/2010), AP (SFRH/BD/45614/2008) and PG (SFRH/BD/75516/2010)

Effects of metal nanoparticles on freshwater rotifers may persist across generations

Nanotechnology has become one of the fastest growing industries in the current century because nanomaterials (NMs) are present in an ever-expanding range of consumer products increasing the chance of their release into natural environments. In this study, the impacts of two metal nanoparticles (Ag-NPs and CuO-NPs) and their equivalent ionic forms (Ag+ and Cu2+) were assessed on the lentic freshwater rotifer Brachionus calyciflorus and on its ability to adapt and recover through generations. In our study, Ag-NPs and CuO-NPs inhibited the rotifer population growth rate and caused mortality at low concentrations (< 100 μg L-1). Ag-NPs and CuO-NPs decreased in the medium when organisms were present (48 h exposure: 51.1 % and 66.9 %, respectively), similarly Ag+ and Cu2+ also decreased from medium in presence of the organisms (48 h: 35.2 % and 47.3 %, respectively); although the metal concentrations removed from the medium were higher for nanoparticles than metal ions, metal ions showed higher effects then their respective nanoparticle forms. Rotifer populations exposed for 4 generations to the toxicants were able to recover the population growth rate, but some rotifers showed developmental delay and inability to reproduce even after the removal of the toxicants. Intracellular accumulation of reactive oxygen species as well as plasma membrane damage were found in the rotifers at concentrations corresponding to EC10 (Ag-NPs = 1.7 μg L-1, Ag+ = 4.5 μg L-1, CuO-NPs = 46.9 μg L-1, Cu2+ = 35 μg L-1) of the population growth rate. Our results showed, for the first time, that effects of metal nanoparticles and metal ions on rotifer populations may persist along several generations. This should be taken into account when assessing risks of metal nanoparticles in freshwaters.This work was supported by the strategic programme UID/BIA/ 04050/2019 and the Emergemix project (PTDC/BIABMA/30922/2017) funded by national funds through the Portuguese Foundation for Science and Technology (FCT) I.P. and by the European Regional Development Fund (ERDF) through the COMPETE2020 – Programa Operacional Competitividade e Internacionalizaçao. ˜ Nuno Martins (UMINHO/BD/ 24/2016) was also supported by The Doctoral Program in Applied and Environmental Microbiology - NORTE-08-5369-FSE-000060 funded by NORTE2020 through the European Social Fund

Individual and mixed effects of anticancer drugs on freshwater rotifers: a multigenerational approach

Human population growth has led to an increased release of chemical contaminants into aquatic environments. Emerging chemical contaminants (ECCs) are of increasing concern because they can affect non-target organisms in aquatic ecosystems. The application of anticancer drugs is increasing because of enhanced cancer rates and use of chemotherapy. We assessed the impacts of two widely used anticancer drugs known for their distinct modes of action, namely 5-fluorouracil (5-FU) and doxorubicin (DOX), on the freshwater rotifer Brachionus calyciflorus across generations. Rotifer mortality (24 h) and population growth (48 h) were assessed to determine initial lethal and sub-lethal effects. Exposure of rotifers to 5-FU (up to 200 mg L-1) did not cause mortality, while DOX caused mortality at high concentrations (EC50 = 15.6 mg L-1). Effects of 5-FU on population growth rate was higher than DOX (5-FU EC50 = 10.49 mu g L-1, DOX EC50 = 8.78 mg L-1). The effects of the drugs in binary mixture on population growth rates were dose dependent; significant antagonistic effects were found when 5-FU was present in the mixture at high concentrations. Finally, a transgenerational assay for five generations revealed that rotifers were able to recover their population growth rate after fourth generation when exposed to 5-FU; however, population became non-viable after the second generation of exposure to DOX. At the cellular level, accumulation of reactive oxygen species and plasma membrane damage were observed at EC10 and increased at EC50 for both drugs. After exposure of rotifers to 5-FU across generations, there were signs of oxidative stress recovery, as shown by a decrease in ROS accumulation and plasma membrane damage. Our results showed for the first time that the adverse effects of anticancer drugs on freshwater rotifer populations are drug and dose dependent and can persist or be attenuated along generations.- This work was supported by the strategic programme UID/BIA/04050/2020 and the Emergemix project (PTDC/BIABMA/30922/2017) funded by portuguese funds through the Portuguese Foundation for Science and Technology (FCT) I.P. and by the European Regional Development Fund (ERDF) through the COMPETE2020 -Programa Operacional Competitividade e Internacionalizacao. This work was also supported by NORTE-08-5369-FSE-000060 by NORTE2020 through the European Social Fund and N. Martins (UMINHO/BD/24/2016)

Proteomic responses to nanoparticulate and ionic silver in fungi from metal-polluted and non-polluted streams

Enhanced production and usage of silver nanoparticles (AgNPs) raise concerns about their potential impacts in aquatic ecosystems. Effects of AgNPs and Ag+ were assessed based on the variations in the overall proteome and the activities of selected antioxidant enzymes in two fungal strains of Articulospora tetracladia, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61). For that, fungi were exposed to concentrations of AgNPs and Ag+ affecting 20% of growth (EC20). A total of 432 proteins were identified, of which 172 belonged to At72 and 260 to At61; 71 were expressed in both strains. At72 had 58% and 52% of the proteins induced by AgNPs and Ag+, respectively. For At61, the percentages were higher ( ˜77%). The higher percentages of proteins suppressed by either Ag forms suggest higher stress in At72 than in At61, which is consistent with the background of this fungal strain. Major groups of proteins were related to carbohydrate metabolism, amino-acid and protein biosynthesis. Both Ag forms also induced stress-responsive proteins, including catalase and superoxide dismutase which remained consistent with the profile of their enzymatic activities. These results supported the ability of these fungi in initiating an efficient antioxidant response to cope with Ag-induced toxicity. Overall, the functional proteomic approach can be useful to get a mechanistic insight on the stress induced by AgNPs or Ag+ in aquatic fungi that play a key role in plant litter decomposition in stream

Proteomic responses to nanoparticulate and ionic silver in aquatic fungi

Enhanced use of silver nanoparticles (AgNPs) has inevitably resulted in their release into aquatic environments raising concern about the risk to aquatic biota and related ecological functions. Functional proteomics is an emerging technology that provides high-throughput analyses augmenting measurements of direct and highly sensitive responses at the cellular and sub-cellular levels. The impacts of AgNPs and its ionic precursor (Ag+ in AgNO3) at low exposure concentrations (close to environmental realism) on a fungal strain isolated from a non-polluted stream were assessed based on the variations in the overall proteome as well as in the activity of selected antioxidant enzymes. A total of 352 proteins were identified, but only 151 proteins were responsive (significantly up- or down-regulated relative to control) of which 65% presented matching alterations. Out of these 151 proteins, 62% increased abundance under stress induced by AgNPs and 56% under stress induced by Ag+. Exposure to both forms of silver induced proteins related to stress response, in particular, antioxidant enzymes. The antioxidant enzymatic responses were consistent with the proteomic responses, suggesting that the ability to initiate an efficient antioxidant response is essential for the fungus to cope with Ag-induced toxicity. Moreover, several proteins involved in the metabolism of carbohydrates, amino acids and lipids were altered. This evidence may reflect the need of generating energy to support the cellular defense mechanisms. Some of the significantly altered proteins were associated with the correct folding of nascent and stress accumulated misfolded proteins or degradation of transiently denatured and unfolded proteins preventing their aggregation. Others were related to the regulation of translation suggesting a compromised protein synthesis system. Overall, the functional proteomic approach can be useful to expand the knowledge on silver-induced stress responses in aquatic fung