Changes in the morphology and cell ultrastructure of a microalgal community exposed to a commercial glyphosate formulation and a toxigenic cyanobacterium.

Human activities significantly influence the health of aquatic ecosystems because many noxious chemical wastes are discharged into freshwater bodies. Intensive agriculture contributes to the deterioration by providing indirectly fertilizers, pesticides, and other agrochemicals that affect the aquatic biota. Glyphosate is one of the most used herbicides worldwide, and microalgae are particularly sensitive to its formulation, inducing displacement of some green microalgae from the phytoplankton that leads to alterations in the floristic composition, which fosters the abundance of cyanobacteria, some of which can be toxigenic. The combination of chemical stressors such as glyphosate and biological ones, like cyanotoxins and other secondary metabolites of cyanobacteria, could induce a combined effect potentially more noxious to microalgae, affecting not only their growth but also their physiology and morphology. In this study, we evaluated the combined effect of glyphosate (Faena®) and a toxigenic cyanobacterium on the morphology and ultrastructure of microalgae in an experimental phytoplankton community. For this purpose, Microcystis aeruginosa (a cosmopolitan cyanobacterium that forms harmful blooms) and the microalgae Ankistrodesmus falcatus, Chlorella vulgaris, Pseudokirchneriella subcapitata, and Scenedesmus incrassatulus were cultivated, individually and jointly, exposing them to sub-inhibitory concentrations of glyphosate (IC10, IC20, and IC40). Effects were evaluated through scanning electron (SEM) and transmission electron (TEM) microscopy. Exposure to Faena® produced alterations in the external morphology and ultrastructure of microalgae both individually and in combined cultures. SEM evidenced the loss of the typical shape and integrity of the cell wall and an increase in the biovolume. TEM revealed reduction and disorganization of the chloroplast, variation in starch and polyphosphate granules, formation of vesicles and vacuoles, cytoplasm degradation, and cell wall continuity loss. The presence of M. aeruginosa was, for microalgae, an additional stress factor adding to the chemical stress produced by Faena®, increasing the damage in their morphology and ultrastructure. These results alert to the effects that can be caused by glyphosate and the presence of toxigenic bacteria on the algal phytoplankton in contaminated and anthropic and eutrophic freshwater ecosystems.

Commercial pesticides for urban applications induced population growth and sub-cellular alterations in Raphidocelis subcapitata (Chlorophyceae) at concerning environmental concentrations.

Information regarding the safety and environmental risks of pesticides intended for urban use remains limited. This study aimed to assess the effects of four common pesticides on the microalga Raphidocelis subcapitata: DIAZINON® 25% C. E., Roundup®, URBACIN® 20C. E., and VAPODEL® 20% C. E., which are commercial formulations of diazinon, glyphosate, dichlorvos, and cypermethrin, respectively. According to 96-h inhibition of population growth bioassays, the four pesticide toxicities exemplified the following order: DIAZINON® (diazinon) > Roundup® (glyphosate) > VAPODEL® (dichlorvos) > URBACIN® (cypermethrin). Increasing pesticide concentrations elicited alterations in the specific growth rates (µmax). The macromolecule contents and photosynthetic pigments increased in groups exposed to the highest concentrations of DIAZINON® 25%, Roundup®, and URBACIN® 20 compared to the control group, despite these treatments inducing lower population growth rates. VAPODEL® 20% induced higher growth rates and lower macromolecule content compared to the control. Since active ingredients were not quantified, certain comparisons may prove limiting, but it is important to assess the effects of the whole mixtures in the form that they enter the environment, especially for urban-intended applications or generic formulations with higher additive contents. Finally, this study demonstrated that commercial pesticide formulations designed for urban applications might pose a threat to freshwater microalgae due to their underestimated toxic potential, but further studies are required.

Toxigenic Microcystis aeruginosa (Cyanobacteria) affects the population growth of two common green microalgae: Evidence of other allelopathic metabolites different to cyanotoxins.

Agriculture runoffs and discharge of wastewaters are the major causes of eutrophication. Although eutrophication could promote the thriving of any phytoplankter, harmful algal blooms (HABs) are dominated frequently by cyanobacteria. Currently, HABs dominated by the toxigenic cyanobacterium Microcystis aeruginosa in lakes and reservoirs are the main environmental concerns worldwide. This study aimed to determine how M. aeruginosa (Ma) modifies the population growth of Pseudokirchneriella subcapitata (Ps) and Ankistrodesmus falcatus (Af). Growth kinetics were determined for each species and in the combinations: Ps-Ma, Af-Ma, Af-Ps, and Ps-Af-Ma. At the end of experiments, photosynthetic pigments, phycobiliproteins, and microcystins were quantified. A logistic equation significantly described the growth trend for all of the tested species, enabling the identification of negative effects on early stages in the population growth of co-cultures with the cyanobacterium; in addition, the interaction effects on the growth rate and in the maximum attainable population density were determined. The biomasses of A. falcatus and P. subcapitata were significantly higher when cultured individually than in all of the combinations with the cyanobacterium. The concentrations of chlorophyll a and b, as well as carotenoids, were lower in combined cultures, but phycobiliprotein content in the cultures with M. aeruginosa was not significantly affected. Microcystis aeruginosa negatively affected the growth of the microalgae, but A. falcatus was significantly more inhibited than P. subcapitata; however, microcystin concentrations were significantly reduced in the co-cultures with microalgae. These results could help to explain the displacements of microalgae when cyanobacteria are present, giving rise to cyanobacterial blooms in eutrophic freshwaters.

Exposure to sublethal concentrations of the glyphosate-based herbicide Faena® increases sensitivity in the progeny of the American cladoceran Daphnia exilis (Herrick, 1895).

The use of herbicides has increased over the last decades. Glyphosate is the most widely used herbicide commercialized in more than 750 formulations. While information about glyphosate's toxicity on different non-target aquatic organisms has been vastly documented, we know little about the transgenerational effects in aquatic biota. This study determined the cross-generation effects produced by the glyphosate-based herbicide Faena® on the American cladoceran Daphnia exilis. Measured endpoints were survival, reproductive responses, metabolic biomarkers, and the size of neonates. D. exilis was exposed to glyphosate concentrations of 2.09, 2.49, and 3.15 (mg L-1) (as content in Faena®) during 21 days starting from neonates, at 25°C, 16:8 photoperiod, fed with 8 × 105 cells mL-1 of Pseudokirchneriella subcapitata. The LC50 was 4.22 mg L-1. Survival, accumulated progeny, and the number of clutches in the parental generation (P1) were significantly higher than those observed in the first generation (F1). Exposure to the herbicide completely inhibited reproduction in the F1. The size of the neonates varied among treatments and broods in P1; nevertheless, neonate size (body and total lengths, as well as body width) was significantly affected in F1. Toxic effects on the survival and reproduction of D. exilis were significantly increased in the F1 exposed to Faena®. Results warn about the augmented effect on progeny where parents were exposed to this herbicide. Multigenerational adverse effects could be expected in freshwater zooplankton exposed to Faena®. The frequently claimed low toxicity of glyphosate must be revised to control the indiscriminate use of this herbicide.

A high-throughput assay for screening environmental pollutants and drugs impairing predator avoidance in Daphnia magna.

This study addresses short-term habituation of the escape response in the aquatic crustacean Daphnia magna evoked by sudden changes in light intensity, using a high-throughput system. Daphnia magna exhibits a marked phototactic behaviour and swim away from light to avoid predation by fish. Currently, there is no information available on the habituation of this phototactic response. The Daphnia photomotor response assay (DPRA) measures the distance moved after a sudden increase in light intensity. Using DPRA, it is possible to determine not only the magnitude of the phototactic response, but also its habituation after repetitive cycles of light and darkness. The progressive reduction observed in response to a series of light stimuli in the proposed assay meet the criteria for habituation. Most cholinergic and serotonergic modulators enhanced photomotor responses and reduced habituation. Dopaminergic and histaminergic modulators also reduced habituation, whereas diazepam was the only compound that increased habituation. Imidacloprid, apomorphine, diphenhydramine, diazepam, and memantine decreased photomotor responses. Thus, the DPRA was also predictive in assessing the effects of neuroactive and neurotoxic environmental contaminants such as selective serotonin reuptake inhibitors, diazepam, organophosphorous, and neonicotinoid pesticides. We conclude that the proposed DPRA may be an effective screening tool for compounds that can impair predation avoidance behaviour in aquatic organisms.

Seasonal changes in the bacterial community structure of three eutrophicated urban lakes in Mexico city, with emphasis on Microcystis spp.

Artificial urban lakes commonly have physicochemical conditions that contribute to rapid anthropogenic eutrophication and development of cyanobacterial blooms. Microcystis is the dominat genus in most freshwater bodies and is one of the main producter of microcystins. Using 454-pyrosequencing we characterized the bacterial community, with special emphasis on Microcystis, in three recreational urban lakes from Mexico City in both wet and dry seasons. We also evaluated some physicochemical parameters that might influence the presence of Microcystis blooms, and we associated the relative abundance of heterotrophic and autotrophic bacterial communities with their possible metabolic capacities. A total of 14 phyla, 18 classes, 39 orders, 53 families and 48 bacterial genera were identified in both seasons in the three urban lakes. Cyanobacteria had the highest relative abundance followed by Proteobacteria and Actinobacteria. Microcystis was the dominant taxon followed by Arthrospira, Planktothrix and Synechococcus. We also found heterotrophic bacteria associated with the blooms, such as Rhodobacter, Pseudomonas, Sphingomonas and, Porphyrobacter. The highest richness, diversity and dominance were registered in the bacterial community of the Virgilio Uribe Olympic Rowing-Canoeing Track in both seasons, and the lowest values were found in the Chapultepec Lake. The canonical correspondence analysis showed that dissolved oxygen and NO3-N concentrations might explain the presence of Microcystis blooms. The metabolic prediction indicated that these communities are involved in photosynthesis, oxidative phosphorylation, methane metabolism, carbon fixation, and nitrogen and sulfur metabolism. The lakes studied had a high prevalence of Microcystis, but average values of microcystins did not exceed the maximum permissible level established by the United States Environmental Protection Agency for recreational and cultural activities. The presence of cyanobacteria and microcystins at low to moderate concentrations in the three lakes could result in ecosystem disruption and increase animal and human health risks.

Multistressor negative effects on an experimental phytoplankton community. The case of glyphosate and one toxigenic cyanobacterium on Chlorophycean microalgae.

Aquatic ecosystems face serious pollution issues. Discharges of toxic substances and eutrophication may lead to changes in the phytoplankton community and foster cyanobacterial blooms. Glyphosate-based herbicides are chemical stressors of microalgae that may affect the structure of phytoplankton communities, and also stimulate the synthesis of cyanotoxins by cyanobacteria. The simultaneous presence of glyphosate and toxigenic cyanobacteria increases the stress on microalgae, jointly affecting their growth and development. This study evaluated the combined effect of a toxigenic cyanobacterium and glyphosate in the development of an experimental microalgal community. We studied the effect of Microcystis aeruginosa on the population growth of the microalgae Ankistrodesmus falcatus, Chlorella vulgaris, Pseudokirchneriella subcapitata, and Scenedesmus incrassatulus. We also evaluated the combined effect of sub-inhibitory glyphosate (Faena®) concentrations on the content of macromolecules and the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as on the concentration of TBARS. These effects were evaluated through the integrated biomarker response (IBR). In individual experiments, microalgae showed lower growth rates versus M. aeruginosa. In the mixed bioassays, both M. aeruginosa and microalgae showed reduced growth. IC50 values for Faena® ranged from 1.022 to 2.702 mg L-1. In the microalgae + cyanobacteria bioassays, the herbicide lowered the growth rates of microalgae but stimulated the proliferation of M. aeruginosa. The joint action of both stressors affected growth rate and population dynamics, macromolecule content, and led to increased CAT and GPx levels. Faena® influenced growth rate and caused oxidative stress. On the other hand, the herbicide stimulated the synthesis of cyanotoxins, which further affected microalgal development. The experimental community was not only affected by the herbicide, but the mixed culture with cyanobacteria magnified the effects of chemical stress. These results illustrate the potential damage to phytoplankton expected in anthropically eutrophic water bodies that are also polluted by glyphosate.

Exposure to the azo dye Direct blue 15 produces toxic effects on microalgae, cladocerans, and zebrafish embryos.

Aquatic pollution caused by dyes has increased together with the growth of activities using colorants such as the textile, leather, food, and agrochemicals industries. Because most popular azo dyes are synthesized from benzidine, a carcinogenic compound, a threat to aquatic biota could be expected. The use of single species for toxicity assessment provides limited data, so a battery of test organisms, including representatives of different trophic levels such as algae, zooplankters, and fish, could undoubtedly provide more information. Therefore, our study was aimed at evaluating the toxic effect of the azo dye Direct blue 15 (DB15) on a battery of bioassays using a primary producer (Pseudokirchneriella subcapitata), a primary consumer (Ceriodaphnia dubia), and a secondary consumer (Danio rerio). P. subcapitata was more sensitive to DB15 (IC50 = 15.99 mg L-1) than C. dubia (LC50: 450 mg L-1). In the algae exposed to DB15, chlorophyll-a and -b were significantly increased, and carotenoids were reduced. The concentrations of protein, carbohydrates, and lipids per cell in P. subcapitata exposed to all DB15 concentrations were significantly higher than that measured in control. At 25 mg L-1 of DB15, survival, total progeny, and the number of released clutches were significantly decreased, and the start of reproduction was delayed in C. dubia. DB15 did not induce lethal or sublethal effects in D. rerio embryos at any of the tested concentrations from 24 to 72 h post-fertilization (hpf), but from 96 to 144 hpf, the larvae exposed to 100 and 500 mg L-1 developed yolk sac edema, curved tail, and skeletal deformations. After 144 hpf, DB15 produced a significant increase in embryos without a heartbeat, as the concentration of dye raised. The textile-used, azo dye DB15, caused toxic effects of different magnitude on microalgae, cladocerans, and zebrafish embryos; for this reason, the discharge of this colorant into waterbodies should be regulated to prevent environmental impacts.

Analysis of neurotransmitters in Daphnia magna affected by neuroactive pharmaceuticals using liquid chromatography-high resolution mass spectrometry.

Neurotransmission plays an essential role during the central nervous system (CNS) development. During the last years, several studies based on the changes produced in neurotransmitters of aquatic organisms caused by pharmaceuticals have been reported. Daphnia magna, the aquatic ecotoxicological model organism, shares several of the neurotransmitters targeted by antidepressant and other neuro-active drugs with vertebrates. Therefore, a method based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) has been applied for the first time to study the levels of 41 neurotransmitters in Daphnia magna under the effect of four different neuro-active pharmaceuticals (sertraline, venlafaxine, duloxetine and fluoxetine). In addition, the performance of LC-HRMS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method using LC-HRMS is a new tool for neurotoxicology research using the Daphnia magna model. As a result, general differences on the concentrations of those neurotransmitters exposed to the mentioned pharmaceuticals were observed.

Congo red dye diversely affects organisms of different trophic levels: a comparative study with microalgae, cladocerans, and zebrafish embryos.

Global consumption of synthetic dyes is roughly 7 × 105 tons per year, of which the textile industry expends about two-thirds. Consumption of synthetic dyes produces large volumes of wastewater discharged into aquatic ecosystems. Colored effluents produce toxic effects in the hydrobionts, reduce light penetration, and alter the photosynthetic activity, causing oxygen depletion, among other effects. Some dyes, such as Congo red (CR), are elaborated with benzidine, a known carcinogenic compound. Information regarding dye toxicity in aquatic ecosystems is scarce; therefore, our study was aimed at evaluating the toxicity of CR on a battery of bioassays: the microalga Pseudokirchneriella subcapitata, the cladocerans Daphnia magna and Ceriodaphnia rigaudi, and the zebrafish Danio rerio. P. subcapitata was the most sensitive species to CR (IC50, 3.11 mg L-1); in exposed individuals, population growth was inhibited, but photosynthetic pigments and macromolecule concentrations were stimulated. D. magna was tolerant to high dye concentrations, the determined LC50 (322.9 mg L-1) is not an environmentally relevant value, but for C. rigaudi, LC50 was significantly lower (62.92 mg L-1). In zebrafish embryos, exposure to CR produced yolk sac edema, skeletal deformities, and stopped larvae hatching; lack of heart beating was the only observed lethal effect. CR affected organisms of different trophic levels diversely. Particularly, the effects observed in microalgae confirm the vulnerability of primary producers to dye-polluted wastewaters, because dyes produced toxic effects and interfered with photosynthesis. Different cladoceran species displayed different acute effects; thus, species sensitivity must also be considered when toxicity of dyes is assessed. Inhibition of fish larvae hatching is a significant effect not previously reported that warns about the toxicity of dyes in fish population dynamics. Synthetic azo colorants should be considered as emerging pollutants because they are discharged into the aquatic environment and are not currently included in the environmental regulation of several countries.

Using a new high-throughput video-tracking platform to assess behavioural changes in Daphnia magna exposed to neuro-active drugs.

Recent advances in imaging allow to monitor in real time the behaviour of individuals under a given stress. Light is a common stressor that alters the behaviour of fish larvae and many aquatic invertebrate species. The water flea Daphnia magna exhibits a vertical negative phototaxis, swimming against light trying to avoid fish predation. The aim of this study was to develop a high-throughput image analysis system to study changes in the vertical negative phototaxis of D. magna first reproductive adult females exposed to 0.1 and 1 µg/L of four neuro-active drugs: diazepam, fluoxetine, propranolol and carbamazepine. Experiments were conducted using a custom designed experimental chamber containing four independent arenas and infrared illumination. The apical-located visible light and the GigE camera located in front of the arenas were controlled by the Ethovision XT 11.5 sofware (Noldus Information Technology, Leesburg, VA). Total distance moved, time spent per zone (bottom vs upper zones) and distance among individuals were analyzed in dark and light conditions, and the effect of different intensities of the apical-located visible light was also investigated. Results indicated that light intensity increased the locomotor activity and low light intensities allowed to better discriminate individual responses to the studied drugs. The four tested drugs decreased the response of exposed organisms to light: individuals moved less, were closer to the bottom and at low light intensities were closer each other. At high light intensities, however, exposed individuals were less aggregated. Propranolol, carbamazepine and fluoxetine induced the most severe behavioural effects. The tested drugs at environmental relevant concentrations altered locomotor activity, geotaxis, phototaxis and aggregation in D. magna individuals in the lab. Therefore the new image analysis system presented here was proven to be sensitive and versatile enough to detect changes in diel vertical migration across light intensities and low concentration levels of neuro-active drugs.

Exposure to the herbicide 2,4-D produces different toxic effects in two different phytoplankters: A green microalga (Ankistrodesmus falcatus) and a toxigenic cyanobacterium (Microcystis aeruginosa).

The extensive use of 2,4-dichlorophenoxiacetic acid (2,4-D) in agriculture is an important source of pollution to water and soil. Toxicity of commonly used herbicides to non-target, planktonic photosynthetic organisms has not been described completely yet. Therefore, we determined the effect of subinhibitory 2,4-D concentrations on the Chlorophycean alga Ankistrodesmus falcatus and on a toxigenic strain of the cyanobacterium Microcystis aeruginosa. Population growth, photosynthetic pigments, macromolecular biomarkers (carbohydrates, lipids, and protein), and antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPx], and superoxide dismutase [SOD]) were quantified, and the integrated biomarker response (IBR) was calculated. Scanning electron microscope (SEM) and transmission electron microscope (TEM) observations were also performed. The 96-h median inhibitory concentration (IC50) for 2,4-D was 1353.80 and 71.20mgL-1 for the alga and the cyanobacterium, respectively. Under 2,4-D stress, both organisms increased pigments and macromolecules concentration, modified the activity of all the evaluated enzymes, and exhibited ultrastructural alterations. M. aeruginosa also increased microcystins production, and A. falcatus showed external morphological alterations. The green alga was tolerant to high concentrations of the herbicide, whereas the cyanobacterium exhibited sensitivity comparable to other phytoplankters. Both organisms were tolerant to comparatively high concentrations of the herbicide; however, negative effects on the assessed biomarkers and cell morphology were significant. Moreover, stimulation of the production of cyanotoxins under chemical stress could increase the risk for the biota in aquatic environments, related to herbicides pollution in eutrophic freshwater ecosystems.

Two stage heterotrophy/photoinduction culture of Scenedesmus incrassatulus: potential for lutein production.

A biomass production process including two stages, heterotrophy/photoinduction (TSHP), was developed to improve biomass and lutein production by the green microalgae Scenedesmus incrassatulus. To determine the effects of different nitrogen sources (yeast extract and urea) and temperature in the heterotrophic stage, experiments using shake flask cultures with glucose as the carbon source were carried out. The highest biomass productivity and specific pigment concentrations were reached using urea+vitamins (U+V) at 30°C. The first stage of the TSHP process was done in a 6L bioreactor, and the inductions in a 3L airlift photobioreactor. At the end of the heterotrophic stage, S. incrassatulus achieved the maximal biomass concentration, increasing from 7.22gL-1 to 17.98gL-1 with an increase in initial glucose concentration from 10.6gL-1 to 30.3gL-1. However, the higher initial glucose concentration resulted in a lower specific growth rate (µ) and lower cell yield (Yx/s), possibly due to substrate inhibition. After 24h of photoinduction, lutein content in S. incrassatulus biomass was 7 times higher than that obtained at the end of heterotrophic cultivation, and the lutein productivity was 1.6 times higher compared with autotrophic culture of this microalga. Hence, the two-stage heterotrophy/photoinduction culture is an effective strategy for high cell density and lutein production in S. incrassatulus.

Maternal-embryonic metabolic and antioxidant response of Chapalichthys pardalis (Teleostei: Goodeidae) induced by exposure to 3,4-dichloroaniline.

Chapalichthys pardalis is a viviparous fish, microendemic to the Tocumbo Region in the state of Michoacán, Mexico. Despite the peculiar type of reproduction of goodeid fish and their mother-embryo interaction, the effects on embryos induced by maternal exposure to aquatic xenobiotics are still unknown. The objective of the present work was to determine the maternal-embryonic metabolic and antioxidant response of C. pardalis exposed to 3,4-dichloroaniline (3,4-DCA), a compound considered highly noxious to the environment because of its high toxicity and persistence, which has been used as reference toxicant in toxicological bioassays. We determined the median lethal concentration (LC50, 96 h) and then exposed pregnant females to 3.3, 2.5, and 0.5 mg L-1 of 3,4-DCA (equivalent to LC1, LC0.01, and LC50/10, respectively) during 21 days. We assessed the activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), macromolecules content (proteins, lipids, carbohydrates), glucose, and lactate concentration, as well as the oxidative damage, by measuring thiobarbituric acid reactive substances, and protein oxidation. To interpret results, we used the integrated biomarker response (IBRv2). The average LC50 was of 5.18 mg L-1 (4.8-5.5 mg L-1; p = 0.05). All females exposed to concentrations of 3.3 and 2.5 mg L-1 lost 100% of the embryos during the bioassay, whereas those exposed to 0.5 mg L-1 showed alterations in the antioxidant activity and oxidative damage, being the embryos and the maternal liver the most affected, with IBRv2 values of 10.09 and 9.21, respectively. Damage to macromolecules was greater in embryos and the maternal liver, with IBRv2 of 16.14 and 8.40, respectively. We conclude that exposure to xenobiotics, like 3,4-DCA, in species with a marked maternal-embryonic interaction represents a potential risk for the development and survival of the descendants, thereby, potentially affecting the future of the population.

Exposure to silver nanoparticles produces oxidative stress and affects macromolecular and metabolic biomarkers in the goodeid fish Chapalichthys pardalis.

Silver nanoparticles (AgNPs) are the most commercialized nanomaterial worldwide, mainly due to their microbicidal activity. Although, AgNPs have been shown to be toxic to aquatic species, their effect on endemic fish, like Goodeidae, has not been demonstrated. Endemic species are under strong pressures by anthropogenic contamination and destruction of their habitat; therefore, we studied adult Chapalichthys pardalis, an endemic fish of Mexico. We evaluated the toxic effect of AgNPs through oxidative stress, macromolecular and metabolic biomarkers. We determined the LC50 (96h) and performed subchronic tests (21days) using sublethal AgNPs concentrations (equivalent to CL1 and CL10). At the end of the bioassay, we quantified 10 stress biomarkers in the liver, gills, and muscle, including the antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione [GPx]), thiobarbituric acid reactive species (TBARS), protein oxidation (CO), macromolecules (proteins, lipids, and carbohydrates), and metabolites (glucose and lactate). In addition, we determined the integrated biomarkers response (IBR). LC50 was of 10.32mgL-1. Results of subchronic exposure (21days) revealed that AgNPs produce oxidative stress in C. pardalis adults, as evidenced by a diminution in antioxidant enzymes activity and an increase in TBARS and oxidized proteins. AgNPs also diminished levels of macromolecules and generated a high-energy consumption, reflected in the reduction of glucose levels, although lactate levels were not altered. The IBR analysis evidenced that the largest effect was produced in organisms exposed to LC10, being the liver and gills the organs with the greatest damage. Results demonstrated that exposure to AgNPs induces acute and chronic toxic effects on C. pardalis and forewarns about the impact that these nanomaterials can exert on these ecologically relevant aquatic organisms.

Biodegradation of a commercial mixture of the herbicides atrazine and S-metolachlor in a multi-channel packed biofilm reactor.

Atrazine and S-metolachlor are two of the most widely used herbicides for agricultural purposes; consequently, residues of both compounds and their metabolites had been detected in ground and superficial waters. Unlike atrazine, the complete degradation of metolachlor has not been achieved. Hence, the purpose of this research is to study the biodegradation of a commercial mixture of atrazine and S-metolachlor in a prototype of a multi-channel packed-bed-biofilm reactor (MC-PBR) designed with the aim of solving the problems of pressure drop and oxygen transfer, typically found on this type of bioreactors.Because the removal efficiency of the herbicides was increased when Candida tropicalis was added to the original microbial community isolated, the reactor was inoculated with this enriched community. The operational conditions tested in batch and continuous mode did not affect the removal efficiency of atrazine; however, this was not the case for S-metolachlor. The removal rates and efficiencies showed a notable variation along the MC-PBR operation.

Validation of a two-generational reproduction test in Daphnia magna: An interlaboratory exercise.

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.

Congo red dye affects survival and reproduction in the cladoceran Ceriodaphnia dubia. Effects of direct and dietary exposure.

Nearly 7 00000 tons of dyes are produced annually throughout the world. Azo dyes are widely used in the textile and paper industries due to their low cost and ease of application. Their extensive use results in large volumes of wastewater being discharged into aquatic ecosystems. Large volume discharges constitute a health risk since many of these dyes, such as Congo Red, are elaborated with benzidine, a known carcinogenic compound. Information regarding dye toxicity in aquatic ecosystems is limited. Therefore, the aim of the present study was to evaluate the effect of Congo Red on survival and reproduction of Ceriodaphnia dubia. We determined the 48 h median lethal concentration (LC50) and evaluated the effects of sublethal concentrations in subchronic exposures by using as food either fresh algae or algae previously exposed to the dye. LC50 was 13.58 mg L-1. In subchronic assays, survival was reduced to 80 and 55 %, and fertility to 40 and 70 %, as compared to the control, in C. dubia fed with intoxicated cells or with the mix of intoxicated + fresh algae, respectively, so the quantity and type of food had a significant effect. We determined that Congo Red is highly toxic to C. dubia since it inhibits survival and fertility in concentrations exceeding 3 mg L-1. Our results show that this dye produces negative effects at very low concentrations. Furthermore, our findings warn of the risk associated with discharging dyes into aquatic environments. Lastly, the results emphasize the need to regulate the discharge of effluents containing azo dyes.

How do toxic metals affect harmful cyanobacteria? An integrative study with a toxigenic strain of Microcystis aeruginosa exposed to nickel stress.

Nickel (Ni) is an essential metal for some organisms, but also a common toxic pollutant released into the water. Toxicity of Ni has not been completely established for cyanobacteria; for this reason, we evaluated the effect of sub-inhibitory Ni concentrations on a toxigenic strain of Microcystis aeruginosa and on microcystins production. Population growth, photosynthetic pigments concentration, biomarkers, including antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPx], and superoxide dismutase [SOD]), as well as macromolecules (proteins, carbohydrates and lipids) were quantified; SEM and TEM observations were also performed. Population growth was affected starting at 3µgL(-1), and at 24µgL(-1) growth was completely inhibited; the 96-h Ni(2+) IC50 was 3.7µgL(-1). Ni exposure increased pigments concentration, augmented all the macromolecules, and increased activities of CAT and GPx; alterations on the internal cell structure were also observed. The integrated biomarker response revealed that Ni(2+) augmented the antioxidant response and the macromolecules content. Ni stress also increased microcystins production. M. aeruginosa was affected by Ni at very low concentrations, even lower than those established as safe limit to protect aquatic biota. Aside from the toxic effects produced in this cyanobacterium, stimulation to produce toxins could potentiate the environmental risks associated with water pollution and eutrophication.