Biotests in Cyanobacterial Toxicity Assessment-Efficient Enough or Not?

Cyanobacteria are a diverse group of organisms known for producing highly potent cyanotoxins that pose a threat to human, animal, and environmental health. These toxins have varying chemical structures and toxicity mechanisms and several toxin classes can be present simultaneously, making it difficult to assess their toxic effects using physico-chemical methods, even when the producing organism and its abundance are identified. To address these challenges, alternative organisms among aquatic vertebrates and invertebrates are being explored as more assays evolve and diverge from the initially established and routinely used mouse bioassay. However, detecting cyanotoxins in complex environmental samples and characterizing their toxic modes of action remain major challenges. This review provides a systematic overview of the use of some of these alternative models and their responses to harmful cyanobacterial metabolites. It also assesses the general usefulness, sensitivity, and efficiency of these models in investigating the mechanisms of cyanotoxicity expressed at different levels of biological organization. From the reported findings, it is clear that cyanotoxin testing requires a multi-level approach. While studying changes at the whole-organism level is essential, as the complexities of whole organisms are still beyond the reach of in vitro methodologies, understanding cyanotoxicity at the molecular and biochemical levels is necessary for meaningful toxicity evaluations. Further research is needed to refine and optimize bioassays for cyanotoxicity testing, which includes developing standardized protocols and identifying novel model organisms for improved understanding of the mechanisms with fewer ethical concerns. In vitro models and computational modeling can complement vertebrate bioassays and reduce animal use, leading to better risk assessment and characterization of cyanotoxins.

Toxic effects of a cyanobacterial strain on Chironomus riparius larvae in a multistress environment.

Cyanobacteria and their toxic metabolites present a global threat to water habitats, but their impact on aquatic organisms in a multistress environment has been poorly investigated. Here we present the results of a survey on the effects of the toxic cyanobacterial strain Trichormus variabilis (heterotypic synonym Anabaena variabilis), and its toxic metabolite, cyanotoxin microcystin-LR, on Chironomus riparius larvae in a multistress environment. An environmentally relevant concentration of microcystin-LR (0.01 mg/L) caused an increase in larvae mortality in an acute toxicity test, which became greater in the presence of environmental stressors (NO3-, NH4+, PO43- and Cd2+), pointing to an additive effect of these agents. Chronic exposure of C. riparius larvae to the microcystin-LR producing strain of T. variabilis in a multistress environment led to a reduction in the larval mass and hemoglobin concentration, and it induced DNA damage in larval somatic cells. The results revealed the additive effect of microcystin-LR in combination with all three tested stressors (NO3-, NH4+, PO43-), and the deleterious effect of chronic exposure of C. riparius larvae to the microcystin-LR producing T. variabilis in a multistress environment. However, the present study further emphasizes the importance of investigating interactions between stressors and cyanotoxins, and their effect on aquatic organisms.

Gene expression changes in Daphnia magna following waterborne exposure to cyanobacterial strains from the genus Nostoc.

Cyanobacteria can produce highly potent cyanotoxins, however, limited information is provided about their toxicity mechanisms in exposed aquatic invertebrates at the molecular level. In the present study, the effects of cyanobacterial strains from the genus Nostoc (Nostoc Z1 and Nostoc 2S3B) in Daphnia magna after waterborne exposure were investigated. Examined endpoints included immobilization (survival) in acute toxicity tests and selected gene expression changes (cyp314, cyp360A8, gst, p-gp, vtg) analyzed by the quantitative real-time polymerase chain reaction (RT-PCR). In addition, enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the observed changes could be due to the presence of microcystins, the most widespread group of cyanotoxins. The results of acute toxicity tests have shown only minor changes in survival rates, which have not exceeded 20% after 48 h of exposure to either strain. On the other hand, significant changes were recorded in molecular responses of Daphnia to tested strains. Treatment with the aquatic strain Nostoc Z1 altered the expression levels of all analyzed genes. Both strains caused a significant p-glycoprotein (p-gp) induction at 75 µg ml-1 which suggests the involvement of p-gp mediated multixenobiotic resistance mechanism (MXR) in facilitating excretion of toxic cyanobacterial compounds in daphnids. Additionally, these strains caused an increase in the expression levels of cyp360A8, indicating that genes related to detoxification processes could be sensitive indicators of cyanobacterial toxicity. Statistically significant induction of cyp314, as well as increases in expression of gst and vtg, were observed only after exposure to Nostoc Z1. This study indicates the potential of certain cyanobacterial metabolites to modify the expression of toxicant responsive genes involved in phase I and phase III of the xenobiotic metabolism, as well as possible interference with growth and reproduction in D. magna. Low microcystin concentrations found in both samples suggest that these cyanotoxins were not responsible for the detected toxic effects.

Evaluation of cyanobacterial toxicity using different biotests and protein phosphatase inhibition assay.

Cyanobacteria are prolific producers of numerous toxic compounds, among which microcystins (hepatotoxins) are the most frequently found. Cyanobacterial bloom in freshwaters is an increasing problem, and there is still a need for rapid and reliable methods for the detection of toxic cyanobacterial samples. In the present study, the toxicity of crude extracts of 11 cyanobacterial strains from different genera has been assessed on two cell lines (human hepatocellular carcinoma HepG2 and rainbow trout (Oncorhynchus mykiss) liver-derived RTL-W1 cells), crustaceans (Daphnia magna and Artemia salina), and zebrafish (Danio rerio) embryos, as well as by protein phosphatase 1 (PP1) inhibition assay and ELISA test to determine whether the toxicity could be due to the presence of hepatotoxins/microcystins. All the tested strains exhibited toxicity on HepG2 cell line (IC50 from 35 to 702 µg mL-1), including Arthrospira (Spirulina) strains, while toxicity against the RTL-W1 cells was detected only in the positive reference Microcystis PCC 7806 and Nostoc 2S9B. Tested strains expressed higher toxicity to D. magna and zebrafish embryos in comparison to A. salina, whereby Nostoc LC1B and Nostoc S8 belonged to the most toxic strains. The PP1-inhibiting compounds have been detected by PP1 assay only in four strains (Microcystis PCC 7806, Oscillatoria K3, Nostoc LC1B, and Nostoc S8), indicating that their toxic potency can be attributed to these compounds. On the other hand, very low levels of microcystins, as confirmed by ELISA, were insufficient to explain toxicity and different toxic potencies of tested cyanobacteria. Results presented in this study suggested HepG2 cell line as a particularly suitable model for cyanobacterial toxicity assessment. In addition, they highlight terrestrial cyanobacterial strains as potent producers of toxic compounds.

Can phytoplankton blooming be harmful to benthic organisms? The toxic influence of Anabaena sp. and Chlorella sp. on Chironomus riparius larvae.

Cyanobacteria and microalgae are abundant biota groups in eutrophic freshwater ecosystems, serving as a food source for many aquatic organisms, including the larvae of non-biting midges (Chironomidae). Many species of cyanobacteria are toxin producers, which can act as stressors to other organisms. The present study aimed to analyze and compare the effects of dietary exposure to the common toxic cyanobacteria Anabaena sp. and non-toxic microalgae Chlorella sp. in Chironomus riparius larvae. Microcystin was detected and quantified in the methanolic extract of Anabaena sp. using the HPLC-DAD technique, and it was identified as microcystin-LR. Both Anabaena sp. and Chlorella sp. were suitable food sources to enable the survival of C. riparius larvae in laboratory conditions, causing negligible mortality and significant differences in the larval mass (ANOVA and Post hoc LSD test; p < 0.05) and hemoglobin concentration (Student's t-test; p < 0.05). Oxidative stress parameters such as advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD) activity, and DNA damage, were also investigated. One-way ANOVA, followed by the Post hoc LSD test, showed a significant increase in AOPP and CAT for the group of larvae fed with Chlorella sp. The same test showed moderate DNA damage in both groups of larvae, with greater damage in the group fed with Anabaena sp. Thus, Chlorella sp. and microcystin-LR producing Anabaena sp. are food sources that did not result in any drastic acute effect on the population level of C. riparius larvae. However, sub-individual-level endpoints revealed significant effects of the treatments, since they caused oxidative stress and DNA damage that may pose a danger to successive generations of test organisms.

Cyanobacterial effects in Lake Ludos, Serbia - Is preservation of a degraded aquatic ecosystem justified?

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludos, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludos, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

A chemical and microbiological characterization and toxicity assessment of the Pancevo industrial complex wastewater canal sediments, Serbia.

The wastewater canal Vojlovica of the Pancevo industrial area, Serbia, is the main collector of the effluents from the local industrial complex. The canal is directly connected to the Europe's second largest river, the Danube. Here, we present a chemical and microbiological analysis of the sediment in order to determine the fate of pollutants over the years, as well as its current condition. Dry matter, clay and organic matter content, a Kjeldahl ammonia, phosphorus, metals, and polychlorinated biphenyls as well as polycyclic aromatic hydrocarbons concentrations were measured. Microbiological analysis included heterotrophic and oil-degrading bacterial counts, isolation of the phenanthrene-degrading bacteria, and identification of cyanobacteria. Generally, in comparison to the results from previous studies, concentrations of the measured pollutants have been in a decline. Specifically, the metal and polycyclic aromatic hydrocarbon concentrations were reduced whereas microbial counts and toxicity tests did not indicate significant pollution. The obtained results are probably a consequence of an improved wastewater treatment and microbial degradation of pollutants.

Massive fish mortality and Cylindrospermopsis raciborskii bloom in Aleksandrovac Lake.

This paper presents a case study of a massive fish mortality during a Cylindrospermopsis raciborskii bloom in Aleksandrovac Lake, Serbia in mid-December 2012. According to a preliminary investigation of the samples taken on November 6 before the fish mortalities and to extended analyses of samples taken on November 15, no values of significant physicochemical parameters emerged to explain the cause(s) of the fish mortality. No industrial pollutants were apparent at this location, and results excluded the likelihood of bacterial infections. Even after freezing, the dissolved oxygen concentration in the water was sufficient for fish survival. High concentrations of chlorophyll a and phaeophytin occurred in the lake, and phytoplankton bloom samples were lethal in Artemia salina bioassays. A bloom of the cyanobacterium C. raciborskii was recorded during November. Although the A. salina bioassays indicated the presence of toxic compounds in the cyanobacterial cells, the cyanotoxins, microcystins, cylindrospermopsin and saxitoxin were not detected.

Epidemiology of primary liver cancer in Serbia and possible connection with cyanobacterial blooms.

Today, the occurrence of harmful cyanobacterial blooms is a common phenomenon and a potential global health problem. Cyanobacteria can produce metabolites highly toxic to humans. More than 80% of reservoirs used for water supply in Central Serbia have bloomed over the past 80 years. A 10-year epidemiological study showed a significant increase in the incidence of primary liver cancer (PLC) in the regions where water from the blooming reservoirs was used for human consumption. At the same time, no correlation was found between the incidence of PLC and other risk factors, such as cirrhosis and hepatitis viruses. Given the strong association with PLC induction and various known possible mechanisms of carcinogenic action, it is highly possible that, cyanotoxins--acting as initiator and promoter--may be the major risk factor that acts synergistically with other risk factors to cause increased incidence of PLC. However, at present, it is still not certain whether cyanotoxins alone were sufficient to induce PLC. Therefore, additional assessment of the health risks that may arise from human exposure to cyanotoxins is advisable.

Human exposure to cyanotoxins and their effects on health.

Cyanotoxins are secondary metabolites produced by cyanobacteria. They pose a threat to human health and the environment. This review summarises the existing data on human exposure to cyanotoxins through drinking water, recreational activities (e.g., swimming, canoeing or bathing), the aquatic food web, terrestrial plants, food supplements, and haemodialysis. Furthermore, it discusses the tolerable daily intake and guideline values for cyanotoxins (especially microcystins) as well as the need to implement risk management measures via national and international legislation.

Cyanotoxins: characteristics, production and degradation routes in drinking water treatment with reference to the situation in Serbia.

Cyanobacteria are members of phytoplankton of the surface freshwaters. The accelerated eutrophication of freshwaters, especially reservoirs for drinking water, by human activity has increased the occurrence and intensity of cyanobacterial blooms. They are of concern due to their ability to produce taste and odors compounds, a wide range of toxins, which have a hepatotoxic, neurotoxic, cytotoxic and dermatotoxic behavior, being dangerous to animal and human health. Therefore, the removal of cyanobacteria, without cell lysis, and releasing of intracellular metabolites, would significantly reduce the concentration of these metabolites in the finished drinking water, as a specific aim of the water treatment processes. This review summarizes the existing data on characteristics of the cyanotoxins, their productions in environment and effective treatment processes to remove these toxins from drinking water.

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia.

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to Østensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the further study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Anabaena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.