Alga Pseudokirchneriella Subcapitata Research Articles

Evaluation of acute and chronic ecotoxicity of cyclophosphamide, ifosfamide, their metabolites/transformation products and UV treated samples

Cyclophosphamide (CP) and Ifosfamide (IF) are two nitrogen mustard drugs widely prescribed in cancer therapy. They are continuously released via excreta into hospital and urban wastewaters reaching wastewater treatment plants. Although CP and IF, their metabolites and transformation products (TPs) residues have been found in the aquatic environment from few ng L-1 to tens of μg L-1, their environmental toxic effects are still not well known. The present study aimed to investigate the acute and chronic ecotoxicity of CP and IF and their commercially available human metabolites/TPs, i.e. carboxy-CP, Keto-CP and N-dechloroethyl-CP on different organisms of the aquatic trophic chain. The experiments were performed using the green alga Pseudokirchneriella subcapitata, the rotifer Brachionus calyciflorus and the crustaceans Thamnocephalus platyurus and Ceriodaphnia dubia. Moreover, to assess the treatment conditions in regards to parent compound removal and formation of new TPs, CP and IF were UV- irradiated for 6h, 12h, 24h, 36h and 48h, followed by toxicity evaluation of treated samples by algae, rotifers and crustaceans. Between the parent compounds, IF resulted as more toxic drug under tested conditions, exerting both acute and chronic effects especially on C.dubia (LC50:196.4mgL-1, EC50:15.84mgL-1). Among the tested metabolites/TPs, only carboxy-CP inhibited the reproduction in the rotifer. However, LOEC and NOEC values were calculated for CP and IF for all organisms. In addition, despite a low degradation of CP (28%) and IF (36%) after 48h UV-irradiation, statistically significant effect differences (p<0.05) from not-irradiated and irradiated samples were observed in both acute and chronic assays, starting from 6h UV-irradiation. Our results suggest that the toxic effects found in the aquatic organisms may be attributable to interactions between the parent compounds and their metabolites/TPs.

Investigation of nanotoxicological effects of nanostructured hydroxyapatite to microalgae Pseudokirchneriella subcapitata

The advance of nanotechnology has enabled the development of materials with optimized properties for applications in agriculture and environment. For instance, nanotechnology-based fertilizers, such as the candidate hydroxyapatite (HAp) nanoparticles (Ca10(PO4)6(OH)2), can potentially increase the food production by rationally supplying phosphorous to crops, although with inferior mobility in the environment (when compared to the soluble counterparts), avoiding eutrophication. Nonetheless, the widespread consumption of nanofertilizers also raises concern about feasible deleterious effects caused by their release in the environment, which ultimately imposes risks to aquatic biota and human health. Nanoparticles characteristics such as size, shape, surface charge and chemical functionality strongly alter how they interact with the surrounding environment, leading to distinct levels of toxicity. This investigation aimed to compare the toxicity of different HAp nanoparticles, obtained by two distinct chemical routes, against algae Pseudokirchneriella subcapitata, which composes the base of the aquatic trophic chain. The as synthesized HAp nanoparticles obtained by co-precipitation and co-precipitation followed by hydrothermal method were fully characterized regarding structure and morphology. Toxicity tests against the microalgae were carried out to evaluate the growth inhibition and the morphological changes experienced by the exposition to HAp nanoparticles. The results showed that high concentrations of coprecipitated HAp samples significantly decreased cell density and caused morphological changes on the algal cells surface when compared to HAp obtained by hydrothermal method. HAp nanoparticles obtained with dispersing agent ammonium polymethacrylate (APMA) indicated negligible toxic effects for algae, due to the higher dispersion of HAp in the culture medium as well as a reduced shading effect. Therefore, HAp nanoparticles obtained by the latter route can be considered a potential source of phosphorous for agricultural crops in addition to reduce eutrophication.

Cell reproductive patterns in the green alga Pseudokirchneriella subcapitata (=Selenastrum capricornutum) and their variations under exposure to the typical toxicants potassium dichromate and 3,5-DCP.

Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. Currently, it is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. However, despite this organism’s, our knowledge of its cell biology—for example, the patterns of nuclear and cytoplasmic division in the mitotic stage—is limited. Although it has been reported that P. subcapitata proliferates by popularity forming four daughter cells (autospores) through multiple fission after two nuclear divisions, here, we report two additional reproductive patterns by which two autospores are formed by binary fission (“two-autospore type”) and eight autospores are formed by multiple fission (“eight-autospore type”). Moreover, we found that cell reproductive patterns differed markedly with the culture conditions or with exposure to either of two typical toxicants, potassium dichromate (K2Cr2O7) and 3,5-dichlorophenol (3,5-DCP). The eight-autospore type occurred at the highest frequency in the early phase of culture, but it disappeared under 3,5-DCP at 2.0 mg/L. Under 0.3 mg/L K2CrO7 (Cr(VI)) the eight-autospore type took substantially longer to appear than in control culture. The two-autospore type occurred only in the late phase of culture. To our knowledge, this is the first detailed evaluation of the reproductive patterns of P. subcapitata, which changed dramatically in the presence of toxicants. These findings suggest that observation of the reproductive patterns of P. subcapitata will help to elucidate different cell reactions to toxicants.

Short‐ and Long‐Term Exposure to Heavy Metals Induced Oxidative Stress Response in Pseudokirchneriella subcapitata

Algal cells can be exposed to toxicants for a short‐term due to accidental discharges or, more commonly, for a long‐term. The present work aimed to assess the ability of Cd, Cr, Cu, and Zn to induce accumulation of reactive oxygen species (ROS) in the alga Pseudokirchneriella subcapitata after a short (6 h) or a long (72 h) exposure time. The relationship between the ROS induction and the content of reduced glutathione (GSH) was also examined. For this purpose, three nominal concentrations of each metal were used corresponding approximately to 72 h‐EC10 and 72 h‐EC50 values and a high concentration (&gt;72 h‐EC90 values). Intracellular ROS accumulation and GSH content were evaluated using a fluorescent‐based approach. A long‐term (chronic) exposure of algal cells to Cd, Cu, and Zn, at the highest concentrations tested, induced an increase of intracellular ROS and GSH content. The increase of GSH content might be a form of algal cells to redress the imbalance caused by the oxidative stress. However, the increase of GSH was not enough to protect the algal cells against the long‐term exposure to oxidative stress. The exposure of algal cells to low or intermediate metals concentrations induced a modification of GSH content; however, no increase of ROS production was detected, which indicates that the toxic symptoms exhibited by algal cells, under these conditions, cannot be attributed to intracellular ROS accumulation.

Zinc toxicity to the alga Pseudokirchneriella subcapitata decreases under phosphate limiting growth conditions

Previous studies have suggested that phosphorus (P) deficiency can increase the sensitivity of microalgae to toxic trace metals, potentially due to reduced metal detoxification at low cell P quota. The existing evidence is, however, inconsistent. This study was set up to determine the combined effects of zinc (Zn) and P supplies on Zn and P bioaccumulation and growth of the green microalgae Pseudokirchneriella subcapitata. Zinc toxicity was investigated in (i) a 24h growth rate assay with cells varying in initial cell P quota (0.5–1.7% P on cell dry weight) with no supplemental P during Zn exposure (Expt. 1) and in (ii) a 48h growth assay initiated with cells at the end of a 14-days steady state culture at three P addition rates (RARs) between 0.8 and 1.6day−1 (Expt.2). The solution Zn concentrations required to reduce final cell density by 10% relative to control (EbC10) were 5-fold (Expt.1) or 2-fold (Expt.2) lower at the highest P supply than at the lowest P supply, i.e. Zn was more toxic at higher P supply, in contrast with the suggestions from previous studies. Cell P quota increased with increasing Zn in the exposure solution (Expt.2), thereby partially overcoming P deficiency under moderate Zn toxicity compared to low Zn exposure. Similarly, cell Zn increased with increasing P supply, potentially induced by Zn–P complexation or precipitation inside the cell. A dynamic growth model accounting for effects of external Zn and internal P on the specific growth rate was calibrated to all data. This model shows that the effect of solution Zn on specific growth rate (ErC50) was statistically unaffected by cell P quota. In contrast, this model predicts that the EbC10 (i.e. EC10 based on cell numbers) varies with P supply because cell P depends on external P and Zn. Moreover, scenario analysis predicts even contrasting trends of the EbC10 with increasing P supply depending on the duration of the growth assay and the P supply scenario. Our data at two experimental scenarios and the prediction under various relevant scenarios suggest a weaker effect of secondary stress factor (Zn) when nutrient deficiency (first stress factor) is prevailing.

Chronic toxicity of an environmentally relevant mixture of pharmaceuticals to three aquatic organisms (alga, daphnid, and fish).

Principles of concentration addition and independent action have been used as effective tools to predict mixture toxicity based on individual component toxicity. The authors investigated the toxicity of a pharmaceutical mixture composed of the top 10 detected active pharmaceutical ingredients (APIs) in the Tama River (Tokyo, Japan) in a relevant concentration ratio. Both individual and mixture toxicities of the 10 APIs were evaluated by 3 short-term chronic toxicity tests using the alga Pseudokirchneriella subcapitata, the daphnid Ceriodaphnia dubia, and the zebrafish Danio rerio. With the exception of clarithromycin toxicity to alga, the no-observed-effect concentration of individual APIs for each test species was dramatically higher than the highest concentration of APIs found in the environment. The mixture of 10 APIs resulted in toxicity to alga, daphnid, and fish at 6.25 times, 100 times, and 15,000 times higher concentrations, respectively, than the environmental concentrations of individual APIs. Predictions by concentration addition and independent action were nearly identical for alga, as clarithromycin was the predominant toxicant in the mixture. Both predictions described the observed mixture toxicity to alga fairly well, whereas they slightly underestimated the observed mixture toxicity in the daphnid test. In the fish embryo test, the observed toxicity fell between the predicted toxicity by concentration addition and independent action. These results suggested that the toxicity of environmentally relevant pharmaceutical mixtures could be predicted by individual toxicity using either concentration addition or independent action.

Ecotoxicity and genotoxicity of cyclophosphamide, ifosfamide, their metabolites/transformation products and their mixtures

Cyclophosphamide (CP) and ifosfamide (IF) are commonly used cytostatic drugs that repress cell division by interaction with DNA. The present study investigates the ecotoxicity and genotoxicity of CP, IF, their human metabolites/transformation products (TPs) carboxy-cyclophosphamide (CPCOOH), keto-cyclophosphamide (ketoCP) and N-dechloroethyl-cyclophosphamide (NdCP) as individual compounds and as mixture. The two parent compounds (CP and IF), at concentrations up to 320 mg L−1, were non-toxic towards the alga Pseudokirchneriella subcapitata and cyanobacterium Synecococcus leopoliensis. Further ecotoxicity studies of metabolites/TPs and a mixture of parent compounds and metabolites/TPs performed in cyanobacteria S. leopoliensis, showed that only CPCOOH (EC50 = 17.1 mg L−1) was toxic. The measured toxicity (EC50 = 11.5 mg L−1) of the mixture was lower from the toxicity predicted by concentration addition model (EC50 = 21.1 mg L−1) indicating potentiating effects of the CPCOOH toxicity. The SOS/umuC assay with Salmonella typhimurium revealed genotoxic activity of CP, CPCOOH and the mixture in the presence of S9 metabolic activation. Only CPCOOH was genotoxic also in the absence of metabolic activation indicating that this compound is a direct acting genotoxin. This finding is of particular importance as in the environment such compounds can directly affect DNA of non-target organisms and also explains toxicity of CPCOOH against cyanobacteria S. leopoliensis. The degradation study with UV irradiation of samples containing CP and IF showed efficient degradation of both compounds and remained non-toxic towards S. leopoliensis, suggesting that no stable TPs with adverse effects were formed. To our knowledge, this is the first study describing the ecotoxicity and genotoxicity of the commonly used cytostatics CP and IF, their known metabolites/TPs and their mixture. The results indicate the importance of toxicological evaluation and monitoring of drug metabolites as they may be for certain aquatic species more hazardous than parent compounds.

An interlaboratory comparison of nanosilver characterisation and hazard identification: Harmonising techniques for high quality data

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag+-species (the concentration of Ag+-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4±6.8nm primary size, mean total Ag concentration 41.14mg/L, 46–68% of soluble Ag+-species in stock, 123.8±12.2nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01–1mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1–10mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10–100mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag+-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag+-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

Acute and chronic ecotoxicity of carbaryl with a battery of aquatic bioassays

ABSTRACTThe ecotoxic effects of carbaryl (carbamate insecticide) were investigated with a battery of four aquatic bioassays. The nominal effective concentrations immobilizing 50% of Daphnia magna (EC50) after 24 and 48 h were 12.76 and 7.47 µg L−1, respectively. After 21 days of exposure of D. magna, LOECs (lowest observed effect concentrations) for cumulative molts and the number of neonates per surviving adult were observed at carbaryl concentration of 0.4 µg L−1. An increase of embryo deformities (curved or unextended shell spines) was observed at 1.8 and 3.7 µg L−1, revealing that carbaryl could act as an endocrine disruptor in D. magna. Other bioassays of the tested battery were less sensitive: the IC50-72h and IC10-72h of the algae Pseudokirchneriella subcapitata were 5.96 and 2.87 mg L−1, respectively. The LC50-6d of the ostracod Heterocypris incongruens was 4.84 mg L−1. A growth inhibition of H. incongruens was registered after carbaryl exposure and the IC20-6d was 1.29 mg L−1. Our results suggest that the daphnid test sensitivity was better than other used tests. Moreover, carbaryl has harmful and toxic effects on tested species because it acts at low concentrations on diverse life history traits of species and induce embryo deformities in crustaceans.

Phytotoxicity and genotoxicity assessment of imazethapyr herbicide using a battery of bioassays.

The imazethapyr herbicide (formulation Verosil(®)) was evaluated for phytotoxicity and genotoxicity using a battery of bioassays: (1) the growth inhibition of the green alga Pseudokirchneriella subcapitata, (2) the root growth and germination of the higher plant Lactuca sativa, (3) the genetic damage using the Salmonella/microsome test, and (4) the aneugenic and clastogenic effects on Allium cepa. The Verosil(®) formulation was highly toxic to the non-target green alga (median effective concentration (EC50) = 1.05 ± 0.05 mg active ingredient (a.i.) L(-1)), and concentrations above 10 mg a.i. L(-1) inhibited root elongation in lettuce: relative growth index (RGI) between 0.28 ± 0.01 and 0.66 ± 0.10. No genotoxic effect was observed in S almonella typhimurium at 100 mg a.i. L(-1), either with or without the microsomal fraction. However, significant differences in the frequency of chromosomal aberrations in anaphases and telophases (bridges, chromosome fragments, and vagrants) were observed in A. cepa at concentrations between 0.01 and 1 mg a.i. L(-1) with respect to the control. The frequencies of micronuclei showed significant differences with respect to the control at concentrations between 0.001 and 0.1 mg a.i. L(-1). A very high mitotic index (MI = 93.8 ± 5.8) was observed associated with a high number of cells in the prophase stage at 100 mg a.i. L(-1), indicating cytotoxicity. These results showed that imazethapyr is toxic to the non-target populations in both aquatic and terrestrial ecosystems. This herbicide might also exert clastogenic and aneugenic mitotic damage in higher plants. Therefore, the imazethapyr formulation may constitute an environmental risk to plants.

Modelling the effects of pulse exposure of several PSII inhibitors on two algae

Subsequent to crop application and during precipitation events, herbicides can reach surface waters in pulses of high concentrations. These pulses can exceed the Annual Average Environmental Quality Standards (AA-EQS), defined in the EU Water Framework Directive, which aims to protect the aquatic environment. A model was developed in a previous study to evaluate the effects of pulse exposure for the herbicide isoproturon on the alga Scenedesmus vacuolatus. In this study, the model was extended to other substances acting as photosystem II inhibitors and to other algae. The measured and predicted effects were equivalent when pulse exposure of atrazine and diuron were tested on S. vacuolatus. The results were consistent for isoproturon on the alga Pseudokirchneriella subcapitata. The model is thus suitable for the effect prediction of phenylureas and triazines and for the algae used: S. vacuolatus and P. subcapitata. The toxicity classification obtained from the dose–response curves (diuron>atrazine>isoproturon) was conserved for the pulse exposure scenarios modelled for S. vacuolatus. Toxicity was identical for isoproturon on the two algae when the dose–response curves were compared and also for the pulse exposure scenarios. Modelling the effects of any pulse scenario of photosystem II inhibitors on algae is therefore feasible and only requires the determination of the dose–response curves of the substance and growth rate of unexposed algae. It is crucial to detect the longest pulses when measurements of herbicide concentrations are performed in streams because the model showed that they principally affect the cell density inhibition of algae.

Dynamics of Metal Partitioning at the Cell–Solution Interface: Implications for Toxicity Assessment under Growth-Inhibiting Conditions

Metal toxicity toward microorganisms is usually evaluated by determining growth inhibition. To achieve a mechanistic interpretation of such toxic effects, the intricate coupling between cell growth kinetics and metal partitioning dynamics at the cell-solution interface over time must be considered on a quantitative level. A formalism is elaborated to evaluate cell-surface-bound, internalized, and extracellular metal fractions in the limit where metal uptake kinetics is controlled by internalization under noncomplexing medium conditions. Cell growth kinetics is tackled using the continuous logistic equation modified to include growth inhibition by metal accumulation to intracellular or cell surface sites. The theory further includes metal-proton competition for adsorption at cell-surface binding sites, as well as possible variation of cell size during exposure to metal ions. The formalism elucidates the dramatic impacts of initial cell concentration on metal bioavailability and toxicity over time, in agreement with reported algae bioassays. It further highlights that appropriate definition of toxicity endpoints requires careful inspection of the ratio between exposure time scale and time scale of metal depletion from bulk solution. The latter depends on metal internalization-excretion rate constants, microorganism growth, and the extent of metal adsorption on nonspecific, transporter, and growth inhibitory sites. As an application of the theory, Cd toxicity in the algae Pseudokirchneriella subcapitata is interpreted from constrained modeling of cell growth kinetics and of interfacial Cd-partitioning dynamics measured under various exposure conditions.

Cadmium accumulation and toxicity in the unicellular alga Pseudokirchneriella subcapitata: Influence of metal-binding exudates and exposure time.

Predicting metal availability and toxicity for chronic (several hours or days) metal exposure scenarios, even for unicellular algae, is a major challenge to existing toxicity models. This is because several factors affecting metal uptake and toxicity, such as the release of metal-binding exudates, changes in the kinetics of metal uptake and toxicity over time, and algal physiological acclimation to internalized metals, are still poorly understood. The present study assessed the influence of these factors on Cd uptake and toxicity in laboratory batch cultures of the freshwater alga Pseudokirchneriella subcapitata. To do so, changes in the free Cd(2+) concentrations caused by the release of metal-binding algal exudates were monitored, (109)Cd accumulation in algal cells was measured, and Cd-induced inhibition of algal growth as a function of exposure time (from 12 h to 96 h) was followed. Results indicate that metal-binding exudates may decrease the proportion of the free Cd(2+) ion in solution up to 2-fold, a decrease that affects Cd uptake and toxicity. Pseudokirchneriella subcapitata has the capacity to decrease net Cd uptake rate on short time scales (<24 h), but this reduction in the Cd uptake rate disappeared after 24 h, and Cd toxicity occurred at relatively high Cd concentrations in solution. These data illustrate some of the pitfalls of standard algal toxicity assays, which were designed for acute exposures, and suggest how robust chronic bioassays might be developed.

Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress

The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72h-EC10 and 72h-EC50 values and a high concentration (above 72h-EC90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of ~16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals.

Elimination and detoxification of fungicide miconazole and antidepressant sertraline by manganese peroxidase-dependent lipid peroxidation system

The imidazole fungicide miconazole (MCZ) and the antidepressant sertraline (SER) have the potential to adversely affect aquatic organisms when they are released into the environment. To assess if they could be broken down, MCZ and SER were treated with laccase and manganese peroxidase (MnP) from lignin-degrading white-rot fungi. The MnP-dependent lipid peroxidation system with Tween 80 containing unsaturated fatty acid was effective in eliminating MCZ and SER; the MnP-Tween 80 system helped eliminate 88% of MCZ and 85% of SER after 24 h of treatment. Furthermore, this system eliminated a metabolite of SER (desmethylsertraline or DSER), and no differences between the elimination rates of DSER and SER were observed throughout the treatment period. It was also confirmed that the MnP-Tween 80 system caused a complete loss of growth inhibition by MCZ of the gram-positive bacterium Bacillus subtilis and reduced the growth inhibition by SER of the green alga Pseudokirchneriella subcapitata by 78% after 24 h of treatment. On the other hand, no appreciable elimination of MCZ and SER was obtained by treatment with laccase, MnP, or the laccase-mediator system with 1-hydroxybenzotriazole.

Chitosan nanoparticles loaded the herbicide paraquat: The influence of the aquatic humic substances on the colloidal stability and toxicity

Polymeric nanoparticles have been developed for several applications, among them as carrier system of pesticides. However, few studies have investigated the fate of these materials in the environment in relation to colloidal stability and toxicity. In nature, humic substances are the main agents responsible for complexation with metals and organic compounds, as well as responsible for the dynamics of these nanoparticles in aquatic and terrestrial environments. In this context, the evaluation of the influence of aquatic humic substances (AHS) on the colloidal stability and toxicity of polymeric nanoparticles of chitosan/tripolyphosphate with or without paraquat was performed. In this study, the nanoparticles were prepared by the ionic gelation method and characterized by size distribution measurements (DLS and NTA), zeta potential, infrared and fluorescence spectroscopy. Allium cepa genotoxicity studies and ecotoxicity assays with the alga Pseudokirchneriella subcapitata were used to investigate the effect of aquatic humic substances (AHS) on the toxicity of this delivery system. No changes were observed in the physical–chemical stability of the nanoparticles due to the presence of AHS using DLS and NTA techniques. However some evidence of interaction between the nanoparticles and AHS was observed by infrared and fluorescence spectroscopies. The ecotoxicity and genotoxicity assays showed that humic substances can decrease the toxic effects of nanoparticles containing paraquat. These results are interesting because they are important for understanding the interaction of these nanostructured carrier systems with species present in aquatic ecosystems such as humic substances, and in this way, opening new perspectives for studies on the dynamics of these carrier systems in the ecosystem.

Selecting a sensitive battery of bioassays to detect toxic effects of metals in effluents

The use of bioassay batteries is necessary to evaluate toxic effects at various biological levels. The selection of bioassays without prior testing and determination of the most sensitive/suitable groups for each impact may allow the discharge of effluents that pose a threat to the environment. The present study tested and selected a battery of sensitive ecotoxicological bioassays for detecting toxic effects of metals. The sensitivities of six organisms were evaluated (algae Pseudokirchneriella subcapitata and Chlorella vulgaris, Cladocera Daphnia similis and Ceriodaphnia dubia, and fish Poecilia reticulata and Danio rerio) after exposure to 10 individual metal species deemed toxic to the aquatic environment (Ag+, Cd2+, Cu+, Cu2+, Cr3+, Cr6+, Pb2+, Ni2+, Zn2+, and Hg2+) and to real (steel-mill) and laboratory simulated effluents. In the bioassays, fish were the least sensitive; D. rerio showed no sensitivity to any of the effluents tested. P. subcapitata was a good bioindicator of Cr3+ toxicity, and D. similis was the most sensitive organism to Hg2+; but the toxic effect of effluents with higher levels of Hg2+ was better detected by C. dubia. The most sensitive battery of bioassays to detect low concentrations of dissolved metals in effluents was the 72-h chronic test with C. vulgaris and the 48-h acute test with C. dubia.

Ecotoxicological evaluation of poly(epsilon-caprolactone) nanocapsules containing triazine herbicides.

The triazine class of herbicides includes the compounds ametryn, atrazine, and simazine, which are used to control weeds in plantations of crops such as maize, sorghum, and sugar cane. Despite their acceptance in agriculture, these herbicides can be dangerous to the environment, depending on their toxicity, the degree of contamination, and the duration of exposure. Controlled release systems are increasingly used to mitigate problems of toxicity and minimize environmental impacts, and can also increase herbicide efficiency. The objective of this work was to prepare poly(epsilon-caprolactone) nanocapsules containing ametryn and atrazine, and evaluate their toxicity to aquatic organisms as well as in cytogenetic tests employing human lymphocyte cultures. The PCL nanocapsules were prepared according to the interfacial deposition of pre-formed polymer method. Ecotoxicological assays were performed with the alga Pseudokirchneriella subcapitata and the microcrustacean Daphnia similis. The cytogenetic tests consisted of observing mitotic index alterations after exposing lymphocyte cell cultures to different formulations. Encapsulation of the herbicides in the nanocapsules resulted in lower toxicity to the alga and higher toxicity to the microcrustacean, compared to the herbicides alone. The cytogenetic tests showed that formulations of nanocapsules containing the herbicides were less toxic than the herbicides alone. The findings indicate the potential of the nanocapsule formulations in agricultural applications, where they could help to reduce the quantities of herbicides used as well as impacts on the environment and human health.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Natural dissolved organic matter mobilizes Cd but does not affect the Cd uptake by the green algae Pseudokirchneriella subcapitata (Korschikov) in resin buffered solutions

Natural dissolved organic matter (DOM) can have contrasting effects on metal bioaccumulation in algae because of complexation reactions that reduce free metal ion concentrations and because of DOM adsorption to algal surfaces which promote metal adsorption. This study was set up to reveal the role of different natural DOM samples on cadmium (Cd) uptake by the green algae Pseudokirchneriella subcapitata (Korschikov). Six different DOM samples were collected from natural freshwater systems and isolated by reverse osmosis. In addition, one 13C enriched DOM sample was isolated from soil to trace DOM adsorption to algae. Algae were exposed to standardized solutions with or without these DOM samples, each exposed at equal DOM concentrations and at equal non-toxic Cd2+ activity (∼4nM) that was buffered with a resin. The DOM increased total dissolved Cd by factors 3–16 due to complexation reactions at equal Cd2+ activity. In contrast, the Cd uptake was unaffected by DOM or increased maximally 1.6 fold (13C enriched DOM). The 13C analysis revealed that maximally 6% of algal C was derived from DOM and that this can explain the small increase in biomass Cd. It is concluded that free Cd2+ and not DOM-complexed Cd is the main bioavailable form of Cd when solution Cd2+ is well buffered.