Widely used herbicide metolachlor can promote harmful bloom formation by stimulating cyanobacterial growth and driving detrimental effects on their chytrid parasites.

Metolachlor (MET) is a widely used herbicide that can adversely affect phytoplanktonic non-target organisms, such as cyanobacteria. Chytrids are zoosporic fungi ubiquitous in aquatic environments that parasitize cyanobacteria and can keep their proliferation in check. However, the influence of organic pollutants on the interaction between species, including parasitism, and the associated ecological processes remain poorly understood. Using the host-parasite system consisting of the toxigenic cyanobacterium Planktothrix agardhii and its chytrid parasite Rhizophydium megarrhizum, we investigated the effects of environmentally relevant concentrations of MET on host-parasite interactions under i) continuous exposure of chytrids and cyanobacteria, and ii) pre-exposure of chytrids. During a continuous exposure, the infection prevalence and intensity were not affected, but chytrid reproductive structures were smaller at the highest tested MET concentration. In the parasite's absence, MET promoted cyanobacteria growth possibly due to a hormesis effect. In the pre-exposure assay, MET caused multi- and transgenerational detrimental effects on parasite fitness. Chytrids pre-exposed to MET showed reduced infectivity, intensity, and prevalence of the infection, and their sporangia size was reduced. Thus, pre-exposure of the parasite to MET resulted in a delayed decline of the cyanobacterial cultures upon infection. After several parasite generations without MET exposure, the parasite recovered its initial fitness, indicating that detrimental effects are transient. This study demonstrates that widely used herbicides, such as MET, could favor cyanobacterial bloom formation both directly, by promoting cyanobacteria growth, and indirectly, by inhibiting their chytrid parasites, which are known to play a key role as top-down regulators of cyanobacteria. In addition, we evidence the relevance of addressing multi-organism systems, such as host-parasite interactions, in toxicity assays. This approach offers a more comprehensive understanding of the effects of pollutants on aquatic ecosystems.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Manganese-oxidizing bacteria form multiple cylindrospermopsin transformation products with reduced human liver cell toxicity.

Cylindrospermopsin (CYN) is a toxic alkaloid highly persistent in aquatic environments. Biological removal of CYN was described previously. However, no transformation products formed by biological processes could be identified so far. Here, we describe that various manganese-oxidizing bacteria (MOB) transform CYN completely at an initial mean concentration of 7 mg L-1 (17 µM) within 3 to 34 days. Regardless of the strain, and transformation rate, transformation of CYN by MOB led to the same seven transformation products identified by mass spectrometry, which suggests that the removal of CYN by MOB follows a similar mechanism. Oxidation was the main transformation process, and the uracil moiety was the most susceptible part of the CYN molecule. In vitro cytotoxicity tests with the transformation products of CYN formed by one of the tested strains against the two human liver cell lines HepG2 and HepaRG, revealed that the transformation products were substantially less toxic than pure CYN for both cell lines. The results suggest that incubation with MOB might be an option for water treatment to remove CYN and may allow more detailed studies on the fate of CYN in the environment.

Manganese-oxidizing bacteria isolated from natural and technical systems remove cylindrospermopsin.

The cyanotoxin cylindrospermopsin was discovered during a drinking water-related outbreak of human poisoning in 1979. Knowledge about the degradation of cylindrospermopsin in waterbodies is limited. So far, only few cylindrospermopsin-removing bacteria have been described. Manganese-oxidizing bacteria remove a variety of organic compounds. However, this has not been assessed for cyanotoxins yet. We investigated cylindrospermopsin removal by manganese-oxidizing bacteria, isolated from natural and technical systems. Cylindrospermopsin removal was evaluated under different conditions. We analysed the correlation between the amount of oxidized manganese and the cylindrospermopsin removal, as well as the removal of cylindrospermopsin by sterile biogenic oxides. Removal rates in the range of 0.4-37.0 µg L-1 day-1 were observed. When MnCO3 was in the media Pseudomonas sp. OF001 removed ∼100% of cylindrospermopsin in 3 days, Comamonadaceae bacterium A210 removed ∼100% within 14 days, and Ideonella sp. A288 and A226 removed 65% and 80% within 28 days, respectively. In the absence of Mn2+, strain A288 did not remove cylindrospermopsin, while the other strains removed 5-16%. The amount of manganese oxidized by the strains during the experiment did not correlate with the amount of cylindrospermopsin removed. However, the mere oxidation of Mn2+ was indispensable for cylindrospermopsin removal. Cylindrospermopsin removal ranging from 0 to 24% by sterile biogenic oxides was observed. Considering the efficient removal of cylindrospermopsin by the tested strains, manganese-oxidizing bacteria might play an important role in cylindrospermopsin removal in the environment. Besides, manganese-oxidizing bacteria could be promising candidates for biotechnological applications for cylindrospermopsin removal in water treatment plants.

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.

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.

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.

Nickel has biochemical, physiological, and structural effects on the green microalga Ankistrodesmus falcatus: An integrative study.

In recent years, the release of chemical pollutants to water bodies has increased due to anthropogenic activities. Ni(2+) is an essential metal that causes damage to aquatic biota at high concentrations. Phytoplankton are photosynthesizing microscopic organisms that constitute a fundamental community in aquatic environments because they are primary producers that sustain the aquatic food web. Nickel toxicity has not been characterized in all of the affected levels of biological organization. For this reason, the present study evaluated the toxic effects of nickel on the growth of a primary producer, the green microalga Ankistrodesmus falcatus, and on its biochemical, enzymatic, and structural levels. The IC50 (96h) was determined for Ni(2+). Based on this result, five concentrations were determined for additional tests, in which cell density was evaluated daily. At the end of the assay, pigments and six biomarkers, including antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD]), and macromolecules (proteins, carbohydrates and lipids), were quantified; the integrated biomarker response (IBR) was determined also. The microalgae were observed by SEM and TEM. Population growth was affected starting at 7.5 µg L(-1) (0.028 µM), and at 120 µg L(-1) (0.450 µM), growth was inhibited completely; the determined IC50 was 17 µg L(-1). Exposure to nickel reduced the concentration of pigments, decreased the content of all of the macromolecules, inhibited of SOD activity, and increased CAT and GPx activities. The IBR revealed that Ni(2+) increased the antioxidant response and diminished the macromolecules concentration. A. falcatus was affected by nickel at very low concentrations; negative effects were observed at the macromolecular, enzymatic, cytoplasmic, and morphological levels, as well as in population growth. Ni(2+) toxicity could result in environmental impacts with consequences on the entire aquatic community. Current regulations should be revised to protect primary producers.