Using single-species and algal communities to determine long-term adverse effects of silver nanoparticles on freshwater phytoplankton.

The physical and chemical properties of silver nanoparticles (AgNPs) have led to their increasing use in various fields such as medicine, food, and industry. Evidence has proven that AgNPs cause adverse effects in aquatic ecosystems, especially when the release of Ag is prolonged in time. Several studies have shown short-term adverse effects of AgNPs on freshwater phytoplankton, but few studies have analysed the impact of long-term exposures on these populations. Our studies were carried out to assess the effects of AgNPs on growth rate, photosynthesis activity, and reactive oxygen species (ROS) generation on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa, and additionally on microcystin (MC-LR) generation from these cyanobacteria. The tests were conducted both in single-species cultures and in phytoplanktonic communities exposed to 1 ngL-1 AgNPs for 28 days. The results showed that cell growth rate of both single-species cultures decreased significantly at the beginning and progressively reached control-like values at 28 days post-exposure. This effect was similar for the community-cultured cyanobacteria, but not for the green algae, which maintained a sustained decrease in growth rate. While gross photosynthesis (Pg) increased in both strains exposed in single cultures, dark respiration (R) and net photosynthesis (Pn) decreased in S. armatus and M. aeruginosa, respectively. These effects were mitigated when both strains were exposed under community culture conditions. Similarly, the ROS generation shown by both strains exposed in single-species cultures was mitigated when exposure occurred in community cultures. MC-LR production and release were significantly decreased in both single-species and community exposures. These results can supply helpful information to further investigate the potential risks of AgNPs and ultimately help policymakers make better-informed decisions about their utilization for environmental restoration.

Bacillus spp. metabolites are effective in eradicating Aedes aegypti (Diptera: Culicidae) larvae with low toxicity to non-target species.

The growing spread of dengue, chikungunya and Zika viruses demand the development of new and environmentally safe control methods for their vector, the mosquito Aedes aegypti. This study aims to find novel larvicidal agents from mutualistic (endophytic and rhizospheric) or edaphic bacteria that have no action against non-target organisms. Eleven out of the 254 bacterial strains tested were able to kill Ae. aegypti larvae. Larvicidal activity did not depend on presence of cells, since culture supernatants or crude lipopeptide extracts (CLEs) killed the larvae. Bacillus safensis BacI67 and Bacillus paranthracis C21 supernatants were the best performing supernatants, displaying the lowest lethal concentrations (LC50 = 31.11 µL/mL and 45.84 µL/mL, respectively). Bacillus velezensis B64a and Bacillus velezensis B15 produced the best performing CLEs (LC50 = 0.11 mg/mL and 0.12 mg/mL, respectively). Mass spectrometry analysis of CLEs detected a mixture of surfactins, iturins, and fengycins. The samples tested were weakly- or non-toxic to mammalian cells (RAW 264.7 macrophages and VERO cells) and non-target organisms (Caenorhabditis elegans, Galleria mellonella, Scenedesmus obliquus, and Tetrahymena pyriformis) - especially B. velezensis B15 CLE. The biosynthetic gene clusters related to secondary metabolism identified by whole genome sequencing of the four best performing bacteria strains revealed clusters for bacteriocin, beta-lactone, lanthipeptide, non-ribosomal peptide synthetases, polyketide synthases (PKS), siderophores, T3PKS, type 1 PKS-like, terpenes, thiopeptides, and trans-AT-PKS. Purification of lipopeptides may clarify the mechanisms by which these extracts kill Ae. aegypti larvae.

Effect of glyphosate on the growth, morphology, ultrastructure and metabolism of Scenedesmus vacuolatus.

The effects of a commercial glyphosate formulation on the oxidative stress parameters and morphology (including the ultrastructure) of the phytoplanktonic green microalga Scenedesmus vacuolatus were evaluated. After 96 h of exposure to increasing herbicide concentrations (0, 4, 6, 8 mg L-1 active ingredient) with the addition of alkyl aryl polyglycol ether surfactant, the growth of the cultures decreased (96 h-IC50- 4.90 mg L-1) and metabolic and morphology alterations were observed. Significant increases in cellular volume (103-353%) and dry weight (105%) and a significant decrease in pigment content (41-48%) were detected. Oxidative stress parameters were significantly affected, showing an increase in the reactive oxygen species (ROS) and reduced glutathione (GSH) contents, oxidative damage to lipids and proteins and a decrease in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and the detoxifying enzyme glutathione-S-transferase (GST). Cells exposed to glyphosate formulation were larger and showed an increase in vacuole size, bleaching, cell wall thickening and alteration of the stacking pattern of thylakoids. The results of this study showed the participation of oxidative stress in the mechanism of toxic action of the commercial glyphosate formulation on S. vacuolatus and the relation between the biochemical, morphological and ultrastructure alterations.

Environmental evaluation of flocculation efficiency in the separation of the microalgal biomass of Scenedesmus sp. cultivated in full-scale photobioreactors.

In this paper the environmental evaluation of the separation process of the microalgal biomass Scenedesmus sp. from full-scale photobioreactors was carried out at the Research and Development Nucleus for Sustainable Energy (NPDEAS), with different flocculants (iron sulfate - FeCl3, sodium hydroxide - NaOH, calcium hydroxide - Ca(OH)2 and aluminum sulphate Al2(SO4)3, by means of the life cycle assessment (LCA) methodology, using the SimaPro 7.3 software. Furthermore, the flocculation efficiency by means of optical density (OD) was also evaluated. The results indicated that FeCl3 and Al2(SO4)3 were highly effective for the recovery of microalgal biomass, greater than 95%. Though, when FeCl3 was used, there was an immediate change in color to the biomass after the orange colored salt was added, typical with the presence of iron, which may compromise the biomass use according to its purpose and Al2(SO4)3 is associated with the occurrence of Alzheimer's disease, restricting the application of biomass recovered through this process for nutritional purposes, for example. Therefore, it was observed that sodium hydroxide is an efficient flocculant, promoting recovery around 93.5% for the ideal concentration of 144 mg per liter. It had the best environmental profile among the compared flocculant agents, since it did not cause visible changes in the biomass or compromise its use and had less impact in relation to acidification, eutrophication, global warming and human toxicity, among others. Thus, the results indicate that it is important to consider both flocculation efficiency aspects and environmental impacts to identify the best flocculants on an industrial scale, to optimize the process, with lower amount of flocculant and obtain the maximum biomass recovery and decrease the impact on the extraction, production, treatment and reuse of these chemical compounds to the environment. However, more studies are needed in order to evaluate energy efficiency of the process coupled with other microalgal biomass recovery technologies. In addition, studies with natural flocculants, other polymers and changes in pH are also needed, as these are produced in a more sustainable way than synthetic organic polymers and have the potential to generate a biomass free of undesirable contaminants.

Growth modeling of the green microalga Scenedesmus obliquus in a hybrid photobioreactor as a practical tool to understand both physical and biochemical phenomena in play during algae cultivation.

In recent years, numerous studies have justified the use of microalgae as a sustainable alternative for the generation of different types of fuels, food supplementation, and cosmetics, as well as bioremediation processes. To improve the cost/benefit ratio of microalgae mass production, many culture systems have been built and upgraded. Mathematical modeling the growth of different species in different systems has become an efficient and practical tool to understand both physical and biochemical phenomena in play during algae cultivation. In addition, growth modeling can guide design changes that lead to process optimization. In the present work, growth of the green microalga Scenedesmus obliquus was modeled in a hybrid photobioreactor that combines the characteristics of tubular photobioreactors (TPB) with thin-layer cascades (TLC). The system showed productivity greater than 8.0 g m-2 day-1 (dry mass) for CO2 -fed cultures, and the model proved to be an accurate representation of experimental data with R2 greater than 0.7 for all cases under variable conditions of temperature and irradiance to determine subsystem efficiency. Growth modeling also allowed growth prediction relative to the operating conditions of TLC, making it useful for estimating the system given other irradiance and temperature conditions, as well as other microalgae species.

Cylindrospermopsin induced changes in growth, toxin production and antioxidant response of Acutodesmus acuminatus and Microcystis aeruginosa under differing light and nitrogen conditions.

Growing evidence suggests that some bioactive metabolites (e.g. cyanotoxins) produced by cyanobacteria have allelopathic potential, due to their inhibitory or stimulatory effects on competing species. Although a number of studies have shown that the cyanotoxin cylindrospermopsin (CYN) has variable effects on phytoplankton species, the impact of changing physicochemical conditions on its allelopathic potential is yet to be investigated. We investigated the physiological response of Microcystis aeruginosa (Cyanobacteria) and Acutodesmus acuminatus (Chlorophyta) to CYN under varying nitrogen and light conditions. At 24h, higher microcystins content of M. aeruginosa was recorded under limited light in the presence of CYN, while at 120h the lower levels of the toxins were observed in the presence of CYN under optimum light. Total MCs concentration was significantly (p<0.05) lowered by CYN after 120h of exposure under limited and optimum nitrogen conditions. On the other hand, there were no significant (p>0.05) changes in total MCs concentrations after exposure to CYN under high nitrogen conditions. As expected, limited light and limited nitrogen conditions resulted in lower cell density of both species, while CYN only significantly (p<0.05) inhibited the growth of M. aeruginosa. Regardless of the light or nitrogen condition, the presence of CYN increased internal H2O2 content of both species, which resulted in significant (p<0.05) changes in antioxidant enzyme (catalase, peroxidase, superoxide dismutase and glutathione S-transferase) activities. The oxidative stress caused by CYN was higher under limited light and limited nitrogen. These results showed that M. aeruginosa and A. acuminatus have variable response to CYN under changing light and nitrogen conditions, and demonstrate that need to consider changes in physicochemical conditions during ecotoxicological and ecophysiological investigations.

Does anatoxin-a influence the physiology of Microcystis aeruginosa and Acutodesmus acuminatus under different light and nitrogen conditions?

Due to changing global climatic conditions, a lot of attention has been given to cyanobacteria and their bioactive secondary metabolites. These conditions are expected to increase the frequency of cyanobacterial blooms, and consequently, the concentrations of cyanotoxins in aquatic ecosystems. Unfortunately, there are very few studies that address the effects of cyanotoxins on the physiology of phytoplankton species under different environmental conditions. In the present study, we investigated the effect of the cyanotoxin anatoxin-a (ATX-A) on Microcystis aeruginosa (cyanobacteria) and Acutodesmus acuminatus (chlorophyta) under varying light and nitrogen conditions. Low light (LL) and nitrogen limitation (LN) resulted in significant cell density reduction of the two species, while the effect of ATX-A on M. aeruginosa was not significant. However, under normal (NN) and high nitrogen (HN) concentrations, exposure to ATX-A resulted in significantly (p < 0.05) lower cell density of A. acuminatus. Pigment content of M. aeruginosa significantly (p < 0.05) declined in the presence of ATX-A, regardless of the light condition. Under each light condition, exposure to ATX-A caused a reduction in total microcystin (MC) content of M. aeruginosa. The detected MC levels varied as a function of nitrogen and ATX-A concentrations. The production of reactive oxygen species (H2O2) and antioxidant enzyme activities of both species were significantly altered by ATX-A under different light and nitrogen conditions. Our results revealed that under different light and nitrogen conditions, the response of M. aeruginosa and A. acuminatus to ATX-A was variable, which demonstrated the need for different endpoints of environmental factors during ecotoxicological investigations.

Biodegradation of carbamazepine using freshwater microalgae Chlamydomonas mexicana and Scenedesmus obliquus and the determination of its metabolic fate.

This study evaluated the toxicity and cellular stresses of carbamazepine (CBZ) on Chlamydomonas mexicana and Scenedesmus obliquus, and its biodegradation by both microalgal species. The growth of both microalgal species decreased with increase of CBZ concentration. The growth of S. obliquus was significantly inhibited (97%) at 200 mg CBZ L(-1), as compared to the control after 10days; whereas, C. mexicana showed 30% inhibition at the same experimental conditions. Biochemical characteristics including total chlorophyll, carotenoid contents and enzyme activities (SOD and CAT) for both species were affected by CBZ at relatively high concentration. C. mexicana and S. obliquus could achieve a maximum of 35% and 28% biodegradation of CBZ, respectively. Two metabolites (10,11-dihydro-10,11-expoxycarbamazepine and n-hydroxy-CBZ) were identified by UPLC-MS, as a result of CBZ biodegradation by C. mexicana. This study demonstrated that C. mexicana was more tolerant to CBZ and could be used for treatment of CBZ contaminated wastewater.

Effect of the temperature, pH and irradiance on the photosynthetic activity by Scenedesmus obtusiusculus under nitrogen replete and deplete conditions.

This paper evaluates the effect of the irradiance, pH and temperature on the photosynthetic activity (PA) of Scenedesmus obtusiusculus under N-replete and N-deplete conditions through oxygen measurements. The highest PA values were 160 mgO2 gb(-1) h(-1) at 620 µmol m(-2) s(-1), 35 °C and pH of 8 under N-replete conditions and 3.3 mgO2 gb(-1) h(-1) at 100 µmol m(-2) s(-1), 28.5 °C and pH of 5.5 for N-deplete conditions. Those operation conditions were tested in a flat-panel photobioreactor. The biomass productivity was 0.97 gb L(-1) d(-1) under N-replete conditions with a photosynthetic efficiency (PE) of 4.4% yielding 0.85 gb mol photon(-1). Similar biomass productivity was obtained under N-deplete condition; and the lipid productivity was 0.34 gL L(-1) d(-1) with a PE of 7.8% yielding 0.39 gL mol photon(-1). The apparent activation and deactivation energies were 16.1 and 30 kcal mol(-1), and 11.9 and 15.3 kcal mol(-1), for N-replete and N-deplete conditions, respectively.

Effect of indigo dye effluent on the growth, biomass production and phenotypic plasticity of Scenedesmus quadricauda (Chlorococcales).

The effect of indigo dye effluent on the freshwater microalga Scenedesmus quadricauda ABU12 was investigated under controlled laboratory conditions. The microalga was exposed to different concentrations of the effluent obtained by diluting the dye effluent from 100 to 175 times in bold basal medium (BBM). The growth rate of the microalga decreased as indigo dye effluent concentration increased (p <0.05). The EC50 was found to be 166 dilution factor of the effluent. Chlorophyll a, cell density and dry weight production as biomarkers were negatively affected by high indigo dye effluent concentration, their levels were higher at low effluent concentrations (p <0.05). Changes in coenobia size significantly correlated with the dye effluent concentration. A shift from large to small coenobia with increasing indigo dye effluent concentration was obtained. We conclude that even at low concentrations; effluents from textile industrial processes that use indigo dye are capable of significantly reducing the growth and biomass production, in addition to altering the morphological characteristics of the freshwater microalga S. quadricauda. The systematic reduction in the number of cells per coenobium observed in this study further confirms that environmental stress affects coenobium structure in the genus Scenedesmus, which means it can be considered an important biomarker for toxicity testing.

The growth of Monoraphidium sp. and Scenedesmus sp. cells in the presence of thorium.

Toxicity of thorium by Monoraphidium sp. and Scenedesmus sp. was studied. Microalgal cultures were inoculated in ASM-1 medium in presence and absence of thorium. Its effect was monitored by direct counting on Fuchs-Rosenthal chamber and with software. The toxicity of thorium over the species was observed for concentrations over 50.0 mg/L. After 30 days, Monoraphidium cells decreased their concentration from 4.23 × 10(6) to 4.27 × 10(5) and 8.57 × 10(5) cells/mL, in the presence of 50.0 and 100.0 mg/L of thorium, respectively. Scenedesmus sp. cells were more resistant to thorium: for an initial cell concentration of 7.65 × 10(4) cells/mL it was observed a change to 5.25 × 10(5) and 5.12 × 10(5) cells/mL, in the presence of thorium at 50.0 and 100.0 mg/L, respectively. This is an indication that low concentrations of the radionuclide favored the growth, and that Scenedesmus cells are more resistant to thorium than Monoraphidium cells. The software used for comparison with direct count method proved to be useful for the improvement of accuracy of the results obtained, a decrease in the uncertainty and allowed recording of the data. The presence of thorium suggests that low concentrations have a positive effect on the growth, due to the presence of the nitrate, indicating its potential for ecotoxicological studies.

An in situ test to explore the responses of Scenedesmus acutus and Lepocinclis acus as indicators of the changes in water quality in lowland streams.

This bioassay was designed with the aim of exploring the responses of two wild planktonic microalgae exposed in situ (72 h) as indicators of the changes in water quality. Monocultures of both strains within dialysis membrane bags were placed at two sites in a small lowland stream. Site 1 is located at a suburban area with low horticultural activity and Site 2 is impacted by toxic industrial discharges and urban land use. There was a decrease in population growth of both species at Site 2 compared with Site 1. The comparison of the algae exposed in situ with the normal specimens cultured at the laboratory indicated a significant increase in the cellular volume for both species at both sites. Abnormal shape was recorded at both sites, the percentage being significantly greater for Scenedesmus acutus at Site 2. Significant changes in pyrenoids size were observed in S. acutus and in the percentage of fragmented nuclei in Lepocinclis acus. Also in the latter abnormal paramylon grains were observed. These responses were accentuated at Site 2. This bioassay was sensitive, short term, low cost, and therefore is a suitable tool to contribute with the monitoring and ecological risk assessment of lowland streams.

Adaptation of microalgae to a gradient of continuous petroleum contamination.

In order to study adaptation of microalgae to petroleum contamination, we have examined an environmental stress gradient by crude oil contamination in the Arroyo Minero River (AMR), Argentina. Underground crude oil has constantly leaked out since 1915 as a consequence of test drilling for possible petroleum exploitation. Numerous microalgae species proliferated in AMR upstream of the crude oil spill. In contrast, only four microalgal species were detected in the crude oil spill area. Species richness increases again downstream. Microalgae biomass in the crude oil spill area is dominated by a mesophile species, Scenedesmus sp. Effects of oil samples from AMR spill on photosynthetic performance and growth were studied using laboratory cultures of two Scenedesmus sp. strains. One strain (Se-co) was isolated from the crude oil spill area. The other strain (Se-pr) was isolated from a pristine area without petroleum contamination. Crude oil has undetectable effects on Se-co strain. In contrast crude oil rapidly destroys Se-pr strain. However, Se-pr strain can adapt to low doses of petroleum (≤ 3% v/v total hydrocarbons/water) by means of physiological acclimatization. In contrast, only rare crude oil-resistant mutants are able to grow under high levels of crude oil (≥ 10% v/v total hydrocarbons/water). These crude oil-resistant mutants have arisen through rare spontaneous mutations that occur prior to crude oil exposure. Species richness in different areas of AMR is closely connected to the kind of mechanism (genetic adaptation vs. physiological acclimatization) that allows adaptation. Resistant-mutants are enough to assure the survival of microalgal species under catastrophic crude oil spill.

Oxidative stress and antioxidant defenses in two green microalgae exposed to copper.

The aim of this work was to assess the effects of 1 week copper exposure (6.2, 108, 210 and 414microM) on Scenedesmus vacuolatus and Chlorella kessleri. The strains showed different susceptibility to copper. Copper content was determined in both strains by total X-ray reflection fluorescence analysis (TXRF). In S. vacuolatus, the increase of medium copper concentration induced an augmentation of protein and MDA content, and a significant decrease in the chlorophyll a/chlorophyll b ratio. S. vacuolatus showed a significant increase of catalase activity in 210 and 414microM of copper, and a significant increment of SOD activity and GSH content only in 414microM of copper. On the contrary, C. kessleri did not show significant differences in these parameters between 6.2 and 108microM of copper. Increased copper in the environment evokes oxidative stress and an increase in the antioxidant defenses of S. vacuolatus.

Effect of cadmium on cellular viability in two species of microalgae (Scenedesmus sp. and Dunaliella viridis).

We determined the effect of several concentrations of cadmium (0, 5, 10, and 20 microg/l) on cellular viability in the microalgae Scenedesmus sp. and Dunaliella viridis, by measuring growth at 0, 24, 48, 72, and 96 h and pigment production at 10 days. Algae were obtained from the Nonvascular Plant Laboratory collection, in the Facultad Experimental de Ciencias, Universidad del Zulia, Venezuela. Growth was measured by cellular counting, while pigment content was evaluated using conventional spectrophotometric techniques. Growth of both species decreased in the exposed cultures comparing with the control, but its behavior was similar, because in both control and exposed cultures, its was observed an adaptive phase in the first hours, as well as a growth phase after 72 h. Cadmium concentrations above 10 microg/l produced an adverse effect on pigment production, depending on the concentration and/or exhibition time. However, even though cadmium inhibited growth and pigment production, levels of both parameters indicated cellular viability, demonstrating the adaptability of the algae cultures when they were exposed to the metal.

Growth, photosynthetic and respiratory responses to sub-lethal copper concentrations in Scenedesmus incrassatulus (Chlorophyceae).

In the present paper we investigated the effects of sub-lethal concentrations of Cu2+ in the growth and metabolism of Scenedesmus incrassatulus. We found that the effect of Cu2+ on growth, photosynthetic pigments (chlorophylls and carotenoids) and metabolism do not follow the same pattern. Photosynthesis was more sensitive than respiration. The analysis of chlorophyll a fluorescence transient shows that the effect of sub-lethal Cu2+ concentration in vivo, causes a reduction of the active PSII reaction centers and the primary charge separation, decreasing the quantum yield of PSII, the electron transport rate and the photosynthetic O2 evolution. The order of sensitivity found was: Growth>photosynthetic pigments content=photosynthetic O2 evolution>photosynthetic electron transport>respiration. The uncoupled relationship between growth and metabolism is discussed.

QSARs for aromatic hydrocarbons at several trophic levels.

Quantitative structure-activity relationships (QSARs) with aromatic hydrocarbons were obtained. Biological response was measured by acute toxicity of several aquatic trophic levels. The chemicals assayed were benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, isopropylbenzene, n-propylbenzene, and butylbenzene. Acute toxicity tests were carried out with Scenedesmus quadricauda, as representative of primary producers; Daphnia spinulata, a zooplanctonic cladoceran; Hyalella curvispina, a benthic macroinvertebrate; and Bryconamericus iheringii, an omnivorous native fish. The EC50 or LC50 was calculated from analytical determinations of aromatic hydrocarbons. Nonlinear regression analysis between the logarithm of the octanol-water partition coefficient (log Kow) of each compounds and the toxicity end points was performed. QSARs were positively related to increases in log Kow at all trophic levels. Intertaxonomic differences were found in comparisons of algae with animals and of invertebrates with vertebrates. We observed that these differences were not significant with a log Kow higher than 3 for all organisms. Aromatic hydrocarbons with log Kow values of less than 3 showed different toxicity responses, with algae more resistant than fish and invertebrates. We concluded that this was a result of the narcotic mode of action related to liposolubility and the ability of the compound to reach its target site in the cell. The bioconcentration factor (BCF) achieved to start nonpolar narcosis fell almost 1 order of magnitude below the BCF expected from the log Kow. Predicted critical body residues for nonpolar narcosis ranged between 2 and 1 mM.

Phenotypic plasticity in Scenedesmus incrassatulus (Chlorophyceae) in response to heavy metals stress.

The microalgae genus Scenedesmus is commonly found in freshwater bodies, wastewater facilities and water polluted with heavy metals. Phenotypic plasticity in Scenedesmus has been documented in response to a wide variety of conditions; however, heavy metals have not been comprehensively documented as phenotypic plasticity inducers. In this study, we report the phenotypic plasticity of Scenedesmus incrassatulus (a non-spiny, four-cell coenobium forming species) in response to EC(50) value of copper, cadmium and hexavalent chromium. S. incrassatulus was grown in batch cultures in the presence of each metal. Chlorophyll-a content, cell size, parameters derived from the schematic energy-flux model for photosystem II, and morphotype expressions were recorded. Divalent cation metals induced unicellular forms, and hexavalent chromium produced out-of-shape coenobia corresponding to various stages of autospore formation. The changes induced by divalent metals were interpreted as phenotypic plasticity, because they were always associated to population doublings and were reversible when toxicant pressure was removed (only for Cu). Copper was the best inductor of unicellular forms and also affected significantly all the photosynthetic parameters measured. The developed morphotypes could confer ecological advantages to S. incrassatulus in metal stressed environments.