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ISSN : 2288-1115(Print)
ISSN : 2288-1123(Online)
Korean Journal of Ecology and Environment Vol.49 No.2 pp.67-79
DOI : https://doi.org/10.11614/KSL.2016.49.2.067

Dynamics and Control Methods of Cyanotoxins in Aquatic Ecosystem

Ho-Dong Park*, Jisun Han, Bong-seok Jeon
Department of Environmental Sciences, Faculty of Science, Shinshu University, Matsumoto 390-8621, Japan
Corresponding author: +81-263-37-2490, +81-263-37-2560, pparkhd@shinshu-u.ac.jp
June 6, 2016 June 15, 2016 June 29, 2016

Abstract

Cyanotoxins in aquatic ecosystems have been investigated by many researchers worldwide. Cyanotoxins can be classified according to toxicity as neurotoxins (anatoxin-a, anatoxin-a(s), saxitoxins) or hepatotoxins (microcystins, nodularin, cylindrospermopsin). Microcystins are generally present within cyanobacterial cells and are released by damage to the cell membrane. Cyanotoxins have been reported to cause adverse effects and to accumulate in aquatic organisms in lakes, rivers and oceans. Possible pathways of microcystins in Lake Suwa, Japan, have been investigated from five perspectives: production, adsorption, physiochemical decomposition, bioaccumulation and biodegradation. In this study, temporal variability in microcystins in Lake Suwa were investigated over 25 years (1991~2015). In nature, microcystins are removed by biodegradation of microorganisms and/or feeding of predators. However, during water treatment, the use of copper sulfate to remove algal cells causes extraction of a mess of microcystins. Cyanotoxins are removed by physical, chemical and biological methods, and the reduction of nutrients inflow is a basic method to prevent cyanobacterial bloom formation. However, this method is not effective for eutrophic lakes because nutrients are already present. The presence of a cyanotoxins can be a potential threat and therefore must be considered during water treatment. A complete understanding of the mechanism of cyanotoxins degradation in the ecosystem requires more intensive study, including a quantitative enumeration of cyanotoxin degrading microbes. This should be done in conjunction with an investigation of the microbial ecological mechanism of cyanobacteria degradation.


초록


    Introduction

    Science and social issues related to cyanobacteria and cyanotoxin problems first emerged in Korea in the 1990s in response to problems with the mariculture of freshwater fish in a dam reservoir. Specifically, a cyanobacterial bloom occurred in the reservoir, after which a musty/earthy taste and odor coupled with algal metabolites, geosmin and 2- methylisoborneol were detected in drinking water. As a result, a cyanobacterial investigation was conducted in dam reservoirs located in four major rivers from 1992 to 1995, and the results revealed widespread occurrence of microcystin and/or anatoxin-a producing cyanobacteria, with cyanotoxin concentrations as high as several hundred μg L-1 (Park et al., 1998). Based on these findings, cyanobacterial problems were reduced by prohibition of mariculture in the dam reservoir to prevent phosphorus loading and subsequent eutrophication. However, during the four major rivers restoration project from 2008 to 2012 in Korea, weirs including estuary dykes were built in several places, which reduced the flow rate and prolonged the residence time. As a result, there have been large cyanobacterial blooms since summer of 2013. In the summer of 2015, dense cyanobacterial blooms and high concentrations of cyanotoxins were observed throughout four major rivers in Korea, leading to concern regarding their potential impact on drinking water and aquatic organisms. Moreover, there is the potential for marine benthic organisms to be exposed to high levels of cyanotoxins when cyanobacteria are discharged into the sea through the estuary dyke.

    Use of tap water for cooking, bathing and drinking was prohibited in the summer of 2014 in Toledo, Ohio, USA, because of the occurrence of cyanobacterial bacterial blooms in adjacent Lake Erie (Jetoo et al., 2015). Indeed, there has been a constant cyanobacterial bloom in the lake since 2011, which has been attributed to climate change, population growth and agriculture.

    In Japan, although microcystin (hepatotoxin) is the main toxin that has been detected in aquatic systems, anatoxin-a (neurotoxin) and cylindrospermopsin (cell death in the liver, kidney, spleen) have also been detected on occasion (Fig. 1). Approximately 20 spot-billed ducks died from August to September in 1995 in a pond located in Hyogo Prefecture, Japan (Matsunaga et al., 1999; Li et al., 2016), as a result of an influx of untreated sewage from treatment facilities in the upper stream destroyed by the Great Hanshin Earthquake in January of that year. The mass mortality was attributed to microcystin produced by Microcystis aeruginosa (Fig. 2). Additionally, 22 domestic ducks died in Lake Biwa in the summer of 2007, and microcystin was detected in their livers (Nakamura et al., 2013) (Table 1). There have also been reports of bird mortality that might have been caused by cyanotoxins, but few studies have investigated these cases thoroughly.

    Bloom-forming cyanobacteria have been observed in eutrophic lakes worldwide for many years. The chemical structure of cyanotoxins has already been elucidated, but their dynamics in water columns are not yet known. In this study, we summarize and discuss the dynamics of cyanotoxins in lake ecosystems and consider methods for their management, including their production, accumulation (by fish and shellfish) and decomposition (by bacteria).

    1.Cyanotoxins

    Cyanotoxins produced by cyanobacterial blooms are classified as neurotoxins (anatoxin-a, anatoxin-a(s), saxitoxin) or hepatotoxins (microcystin, nodularin, cylindrospermop- sin) (Carmichael, 1992). Hepatotoxins such as anatoxin-a and neurotoxins such as cylindrospermopsin, which are known to cause necrosis in the liver, kidney and spleen, have been detected in lakes in Japan (Fig. 1). At least 10 species of cyanobacteria have been reported to produce microcystin, including M. aeruginosa. Microcystin is a cyclic peptide consisting of seven amino acids, and over 90 microcystin variants (MW 909-1115) have been shown to undergo variations in amino acids.

    Fig. 3 shows the toxic effects and metabolism of microcystins. Microcystins exist intracellularly, and are released into extracellular areas following cell damage. Microcystins found in drinking water have liver-specific toxicity. Organic anion transporting polypeptides mediate hepatocellular uptake of microcystins, which then bind and inhibit protein phosphatases 1 and 2A. Inhibition of protein phosphatases activity leads to enhanced phosphorylation by protein kinase, resulting in excessive phosphorylation of proteins. It has been reported that hyperphosphorylated proteins induce destruction of the cytoskeleton and apoptosis in acute toxicity caused by microcystin, causing damage to keratin and plectin (MacKintosh et al., 1990). Moreover, hyperphosphorylated protein has tumor promoting activity that occurs by affecting tumor suppressor gene p53. In chronic toxicity, microcystin has toxic effects on mitochondria in the cell, leading to generation of reactive oxygen species. Reactive oxygen species have been reported to cause cell membrane denaturation via peroxidation of cell membrane lipids, and subsequent liver cancer associated with apoptosis, liver fibrosis and hepatic cirrhosis. Conversely, microcystin is excreted as cysteine and glutathione conjugate, enhancing water solubility in the liver (Kondo et al., 1992).

    Studies of microcystin toxicity have been based on okadaic acid produced by dinoflagellates, because okadaic acid is a protein phosphatase inhibitor similar to microcystin (Imanishi and Harada, 2004). Since the activity of protein kinase is promoted by the inhibition of protein phosphatase, flavonoids have been shown to inhibit the activity of protein kinase. Naringin is a citrus bioflavonoid from the peel, juice and seeds of citrus fruits, especially grapefruit (Fig. 4). The bitter principle of grapefruit, naringin, has been reported to possess antioxidant, anticancer and cholesterol reducing properties (Rajadurai and Prince, 2007). Naringin has also been reported to prevent disruption of the keratin cytoskeleton in rat hepatocytes and microcystin induced apoptotic cell death (Xie et al., 2014).

    Anatoxin-a, which is an alkaloid neurotoxin that was identified in Anabaena flos-aquae in Canadian lakes, can cause death in a few minutes (4~7 min.) via a depolarizing blockade of synaptic transmission in the neuromuscular junction. Anatoxin-a has high toxicity, with an intraperitoneal LD50 of 200~375 μg kg-1 in mice. Trace amounts of anatoxin-a have been detected in Japanese lakes (Harada et al., 1993), and anatoxin-a was isolated from Anabaena and Microcystis species in Japan (Park et al., 1993). Anatoxin-a has structural similarity to the neurotransmitter acetylcholine and binds to the nicotinic acetylcholine receptor in competition with acetylcholine. However, anatoxin-a cannot be degraded by acetylcholinesterase, which rapidly hydrolyzes acetylcholine to acetate and choline, leading to muscle contractions (Swanson et al., 1986).

    Cylindrospermopsin, which is produced by Umezakia natans in Japan, has been reported to have toxic effects, primarily in the liver, as well as in the kidney, intestine, lungs and thymus (Falconer and Humpage, 2006). A severe poisoning incident occurred on Palm Island, Queensland, Australia, in 1979 in which 150 residents (mainly children) had symptoms of gastroenteritis and kidney dysfunction because of cylindrospermopsin released into the water after a Cylindrospermopsis raciborskii bloom on a reservoir treated with copper sulfate (Table 2) (Hawkins et al., 1985). In a recent study, cylindrospermopsin was found to be produced by other species including Anabaena, Aphanizomenon and Raphidiopsis curvata. Furthermore, there is concern that cylindrospermopsin can be introduced into the water supply during certain growth phases and under specific culture conditions (Harada et al., 1994; Banker et al., 1997; Li et al., 2001; Schembir et al., 2001).

    2.Dynamics and bioaccumulation of cyanotoxins

    Cyanotoxins in Lake Suwa, Japan, were first studied in 1977, when a mouse bioassay was conducted by intraperitoneal injection of the extractions from cyanobacterial blooms. Since then, the molecular structure has been determined and improved analytical methods for evaluation of cyanotoxins in lakes have been developed.

    Fig. 5 shows the annual changes in intracellular microcystin concentrations in Lake Suwa from 1992~2012. The highest concentrations of microcystins in 1992 (mid-June and late September) and 1994 (early October) were 120 and 180 μg L-1, respectively, at which time the cell density of M. viridis and M. aeruginosa were measured simultaneously. The concentration of microcystin-RR was higher than that of microcystin-LR throughout the study period, except in 1994. The microcystins concentration decreased greatly after to 1998 to less than 11 μg L-1. Moreover, the microcystins concentration was below 1 μg L-1 in 2010 and 2011, and was below the detection limit during some of these months. There are several possible reasons for this decrease in microcystins after 1998. One is that it was caused by the reduction of nutrient P inflow because of increased sewerage system coverage in the catchment area (Fig. 6). Additionally, the release of nutrient P from sediments (internal loading) has decreased because of decreased cyanobacteria biomass in the lake. Lake water retention time has also decreased during this period in response to strong winds and high levels of precipitation. Finally, the N/P ratio increased in response to the decreased concentration of dissolved phosphorus (Fig. 6). It was recently reported that the biomass of cyanobacteria decreased and the species composition changed from toxic to non-toxic cyanobacteria in response to these factors.

    Previous studies have found that microcystins released from cyanobacteria accumulated in aquatic organisms. Table 1 shows the maximum microcystin contents in freshwater organisms in laboratory experiments and lakes. The microcystin content of zooplankton and fish ranged from 24.5 to 1387 μg g-1 (DW) and 0.02 to 337 μg g-1 (DW and WW), respectively. Marine bivalves accumulated particularly high levels of microcystins. In addition, it is possible that crab larvae accumulate microcystins and therefore act as vectors of the toxin up the aquatic food web. Fig. 7 shows the production, feeding, accumulation, degradation and discharge of microcystins in Lake Suwa. The microcystin values shown in Fig. 7 were determined from samples collected from June to November in 1992 to 1998. Microcystins were not detected in aquatic organisms, except in bivalves of Lake Suwa. As shown in Table 1, the microcystin concentrations of Anodonta woodiana, Cristaria plicata and Unio douglasiae in the midgut gland were 12.6, 297 and 420 μg g-1 (DW), respectively. In addition, the microcystin content of U. douglasiae was found to be 53%, 34%, 6% and 7% in the midgut gland, gills and muscle, gonad, and digestive gland, respectively. An experiment measuring the acute and chronic toxicity of microcystins toward mammals revealed that most accumulate in the liver, which would explain the large amount of microcystin accumulated in the midgut gland because it performs the same function as the liver. Moreover, U. douglasiae collected in Lake Suwa were cultivated during 3 months without microcystins, after which a 10% decrease in microcystins was observed. However, further work is needed to elucidate the dynamics of microcystins between species. Cyanobacterial blooms in lakes travel downstream, eventually reaching the sea. Cyanobacterial blooms in Lake Suwa are discharged to the Tenryu River (Fig. 8). As shown in Fig. 9, microcystins accumulated in aquatic organisms of Tenryu River (Katagami et al., 2004), indicating the need for ecotoxicological evaluation of aquatic organisms located in areas downstream from areas known to be impacted by microcystins.

    3.Removal methods of microcystins

    It has been reported that anatoxins and cylindrospermopsins are released from algal cells after the middle stages of growth (Fig. 1). However, microcystins are not released; therefore, the algal cells should be removed to remove the microcystins. Although the released microcystins can be removed, the treatment cost is expensive. The microcystins can be released via algal cell lysis by bacteria and consumption by predators, as well as use of copper sulfate in drinking water plants.

    The removal of released microcystins can be accomplished by physical and chemical methods. Activated carbon filtration is one physical method used to remove the dissolved pollutants. It has been reported that 0.7 g microcystins were removed by 1.0 g activated carbon. The removal rate of microcystins by powdered activated carbon and granulated activated carbon were 20%~85% and over 90%, respectively (Lambert et al., 1996). Activated carbon filtration is used in drinking water plants to absorb odor. Another physical method for their removal is reverse osmosis, which employs a semi-permeable membrane to remove organic pollutants with a MW>100 (Lawton and Robertson, 1999). Hypochlorite starts to form at pH 5 and is completely ionized at pH 10 by chlorination. The detoxication rate of microcystins by chlorination at pH 5 in 30 minutes, at pH 7 in 22 hours and at pH 10 was 93%, 88% and below 0.5%, respectively (Tsuji et al., 1997). These results reflect the reduced formation of hypochlorous acid, which has high activity. Therefore, the pH of the target water should be maintained at less than neutral pH. However, the adaptation of chlorination in the field is difficult because most of the cyanobacterial blooms are formed at above neutral pH. Another method of removal is ozone treatment, in which ozone gas is employed to remove organic materials. Ozone can decrease the formation of trihalomethane, but this method is more expensive than chlorination and results in the formation of formaldehyde. The removal rate of microcystins by ozone treatment was 99%, but the oxidants generated by the treatment might be influenced in organisms (Rositano et al., 1998). The removal rate of microcystins by treatment with potassium permanganate was 95%, which was similar to the value for chlorination (Rositano et al., 1998). The removal rate of microcystins by solar light without catalyst was 14% over 26 days but was 95% over 29 days when phycocyanin was present in the treatment water (Tsuji et al., 1994). Electrochemical treatment can remove Microcystis cells and microcystins via the reactive oxygen species and/or reactive chlorine species generated on the anode surface during treatment. Based on the results of laboratory experiments, the removal rate of Microcystis cells and microcystins was over 95%. In addition, phosphorus is deposited onto the cathode surface along with calcium ions as CaHPO4 (monetite) and/or CaHPO4·2H2O (brushite) (Jeon et al., 2014). The ability to remove Microcystis cells and microcystins was better when a ceramic anode such as dimensionally stable anode was applied than a Pt anode. Many other methods including hydrogen peroxide treatment and photocatalyst treatment have also been investigated (Lawton and Robertson, 1999).

    Aluminum hydroxide is an amorphous colloidal precipitate with very low solubility at neutral pH and high coagulation and phosphorus adsorption properties. Therefore, salts such as aluminum sulfate have long been applied to treatment of drinking water, industrial and domestic wastewater, sludge, and lakes and reservoirs (Cooke et al., 2005). Most studies conducted to date have reported that aluminum causes no cell damage or lysis, and no subsequent toxin release during conventional treatment of drinking water or in the laboratory (Peterson et al., 1995; Chow et al., 1999). However, a recent study investigated the effect of long-term and high doses of alum treatment because the environmental conditions in lakes and treatment plants differ widely (Han et al., 2013). Therefore, long-term application of large alum doses is not recommended as an in-lake treatment.

    Biological methods or the removal of microcystins employing bacteria, protozoa and rotifers have been investigated. Microcystins are relatively stable under various physical and chemical conditions. Temporal variability in the concentration of microcystins was studied during the warm season over four years (1991~1994) in Lake Suwa, during which time the concentrations of extracellular microcystins remained very small (<4 μg L-1) compared with the levels of intracellular microcystins. The relatively higher percentages of microcystins in filtered lake water (>20%) at the end of the blooms suggests that release of microcystins from cells occurs during senescence of Microcystis cells and the decomposition period. Based on these results, a mechanism for removal of microcystins might be present in aquatic ecosystems. Therefore, a bacterium capable of degrading microcystins was isolated from Lake Suwa. Among 15 strains isolated from Lake Suwa during the Microcystis bloom, only one, strain Y2, was shown to degrade microcystins. The bacterium was tentatively identified as Sphingomonas by manual chemotaxonomy, but 16S rRNA sequencing analysis suggests that it is in fact a new species or genus. When the Y2 bacterium was added to microcystins present in culture medium, they were completely degraded in 6 days (Park et al., 2001). The fate of microcystins in the aquatic ecosystem is considered that microcystins were produced by cyanobacteria and released by cell lysis then released microcystins were decomposed, if a microcystin-degrading bacteria, such as Y2, was present, as the observed concentration of microcystin in filtered lake water was not high compared with the amount of intracellular microcystins. Over 12 genera of microcystin- degrading bacteria have been reported, but only five species were identified in the present study (Table 3). The microcystindegrading bacteria were identified by 16S rRNA gene analysis and investigated to determine the presence of mlrA, mlrB, mlrC, and mlrD, which have previously been reported to be involved in the degradation of microcystin-LR by Sphingomonas sp. strain ACM-3962 (Bourne et al., 2001). MlrA, which encodes enzymes via the mlrA gene, hydrolyzes the cyclic structure of microcystin-LR. The mlrD gene encodes a transporter protein of microcystin and its degradation products. MlrA, MlrB, and MlrC probably catalyze the degradation of microcystin-LR, linear microcystin and the tetrapeptide H-AddaGlu-Mdha-Ala-OH, respectively. The microcystins degradation rate of six genera of microcystin- degrading bacteria that identified the presence of the mlrA gene ranged from several dozen to several hundred mg L-1 day-1 (Dziga et al., 2013). Based on these results, the extracellular microcystins in a lake could be removed in several days.

    Fig. 5 shows the annual changes in intracellular microcystin concentrations in Lake Suwa from 1992~2012. Concentrations of extracellular microcystins remained very small compared with the levels of intracellular microcystins. These findings can be explained by the presence of microcystin- degrading bacteria. The contents of microcystins in the sediments of the lake were not determined; however, the adsorption rate of microcystins to the sediments was measured. The maximum adsorption rates of microcystin- RR and microcystin-LR were 1860 μg·g-1 and 640 μg·g-1, respectively. Qualitative and quantitative analysis of microcystins adsorbed on the sediments was difficult because desorption from the sediments led to poor reproducibility. Therefore, a new analysis method should be investigated. Additionally, bivalves can accumulate and concentrate cyanotoxins. Accordingly, it is necessary to investigate the possibility of accumulation and concentration of cyanotoxins in aquatic organisms other than Bivalvia. In addition, the removal of cyanotoxins from water resources is expected based on the dynamics of microcystin-degrading bacteria (Park, 2008).

    The basis of the prevention of cyanobacterial blooms is the reduction of inflowing nutrients (N and P). Treatment of the load source is most important; however, the efficiency is low because aquatic ecosystems accumulate and recycle loaded nutrients. Indeed, the accumulation of nutrients in the sediments has delayed recovery of the lake. Nevertheless, dredging of the sediments is not an appropriate method of treatment.

    Recently, aquatic plant zones in many Japanese lakes are decreasing because of the installation of artificial facilities in coastal areas. In a naturally eutrophic lake, algal blooms do not form because aquatic plants grow in the shallow part of the lake. Rather, algal blooms are formed in lakes with wide surface areas and few aquatic plants. This is likely because the plants absorb inflowing nutrients and release allelopathic substances. Therefore, it is important to conduct plant recovery projects in coastal lake areas to prevent cyanobacterial blooms. Recovery plans for the coastal areas of Lake Suwa have been implemented to prevent the formation of cyanobacterial blooms. Moreover, the inflow of nutrients into water catchment areas is related to the artificial development of mountains. Therefore, it is also necessary to protect the forest around the water sources and rivers to prevent cyanobacterial blooms.

    Conclusions

    The first death of an animal caused by cyanotoxin (from Nodularia spumigena) was reported in Australia in the 1870s. Additionally, large-scale death of wild birds (over 260,000) occurred in Canada between 1995 and 1998. Moreover, 52 dialysis patients died as a result of water contamination with cyanotoxin in Caruaru, Brazil, in February of 1996. Accordingly, toxic cyanobacterial blooms that occur in lake and dam water used as supply sources pose severe health risks to the populations utilizing these water sources (Fig. 10). Microcystin-LR is a well-known cyanotoxin, and a drinking water guideline value for microcystin-LR of 1.0 μg L-1 has been suggested by the World Health Organization (WHO, 1998). Most countries in the EU have established drinking water guidelines similar to that recommended by the WHO or an independent value. However, no such standards have been established in Korea; therefore, it is necessary to establish guidelines to protect the local environment.

    Figure

    KSL-49-67_F1.gif

    Classification of cyanotoxins, structures, toxicity and cyanotoxin producers.

    KSL-49-67_F2.gif

    Photomicrograph of Microcystis aeruginosa.

    KSL-49-67_F3.gif

    Toxic effects and metabolism of microcystins (adapted and modified from Park (2014)).

    KSL-49-67_F4.gif

    Structure of naringin. Naringin occurs naturally in citrus fruits, especially grapefruit.

    KSL-49-67_F5.gif

    Annual changes of intracellular microcystin concentrations in Lake Suwa, Japan.

    KSL-49-67_F6.gif

    Changes of TN and TP loading from 1975 to 2006. a, TN loading, b, TP loading. Natural, urban and farmland showed as aspect source load in 1975. These data were obtained from Nagano Prefecture.

    KSL-49-67_F7.gif

    Dynamics of microcystin in the aquatic ecosystem of Lake Suwa, Japan (production, feeding, accumulation, degradation and discharge of microcystins) (adapted and modified from Park (2014)).

    KSL-49-67_F8.gif

    Discharge of cyanobacterial blooms from Lake Suwa to Tenryu River, Japan (Kamaguchi water gate and Tenryu River).

    KSL-49-67_F9.gif

    Dynamics of the microcystin in aquatic ecosystem of Tenryu River, Japan (adapted and modified from Park (2005)).

    KSL-49-67_F10.gif

    Occurrence of harmful cyanobacterial blooms and associated health risks.

    Table

    Microcystin content in freshwater organisms.

    *: DW; Dry weight,
    **: WW; Wet weight

    Examples of human exposures to cyanobacterial blooms.

    Bacterial degradation rate of microcystin (modified Hu et al. (2009)).

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