Microcystins In Water Research Articles

Adsorption of Microcystin LR by Activated Carbon Fibers

Blooms of toxic microorganisms are commonly found in freshwater in several areas of the world. Freshwater poisonings are mainly caused by species of cyanobacteria, also known as blue-green algae. They develop in eutrophic water wherever proper conditions for their growth are found, including a mild wind, a water temperature between 15 and 30 C, a neutral or alkaline pH and a rather high level of mineral nutrients. As a consequence of the extended use of nitrates and phosphates, blooms occur more and more frequently. There is a risk that these blooms contaminate water supplies that are used as recreational areas or as drinking water reservoirs. This is a serious water quality problem because many of the cyanobaterial species are able to produce potent toxins. The most frequently reported toxins are hepatotoxins. They are classified as microcystins and nodularins, the former representing the largest group. All microcystins have a common cyclic heptapeptide structure consisting of (-DAla-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where MeAsp stands for erythro-β-methylaspartic acid, Mdha for Nmethyldehydroalanine, Adda for 3-amino-9-methoxy-2,6,8trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Z for the variable amino acids that give its name to the molecule. For example, microcystin LR has X = Leusine and Z = Arginine, microcystin RR has X = Arginine and Z = Arginine. Microcystins can be synthesized by various genera of cyanobacteria such as Microcystis, Oscillatoria, Anabaena or Nostoc. They have been responsible for the poisoning of fish, birds, wild and domestic animals in many countries. Adverse effects on human health have also been recognized. Recent researches have actually shown that microcystins act as type 1, 2A and 3 protein phosphatase inhibitors, as well as tumor promoters when present in nanomolar concentrations. Nowadays, WHO guidelines have been established for the monitoring of microcystins in water. Maximal values of 0.1 μg/L in drinking water for a long term exposure or 1 μg/L for a short term exposure have been proposed on the basis of laboratory experiments of toxicity on mice and pigs. Blooms of blue-green algae, which have been increasing in recent years, have the potential to cause problems for the water supply industry. Recent occurrences of toxic algal blooms have highlighted the potential risk of toxins entering drinking water supplies, and posing a threat to public health. This necessitates a need for research to determine the effectiveness of different water treatment processes for removing algal toxins. At present, slow sand filtration has shown some potential to remove toxins by biodegradation. However, not all types of toxin were successfully removed. The limited amount of work on ozonation has shown that it is an effective process for removing toxins, even with relatively low ozone doses. Ozonation does produce by-products, but these have been shown to be non-toxic. Activated carbon adsorption has been shown to be capable of effectively removing different toxins. With powdered activated carbon (PAC), high PAC doses were generally required. However, most of the tests were conducted with higher toxin concentrations than would probably be found in raw waters. Granular activated carbon (GAC) can effectively remove toxins, but the studies have not fully assessed the most suitable GAC. In addition, the PAC doses required may be impractical to dose at a treatment works. For GAC adsorption to be effective, contact times of more than 15 minutes should be considered, assuming that microcystin does not biodegrade on the GAC. Activated Carbon Fiber (ACF) is a recently developed adsorbent which has more superior adsorbing capacity to active carbons. Recently, Chungbuk National University (Chungju, South Korea) has developed a manufacturing process of ACF by using various precursors of fabrics, cottons, viscous rayons and mixed fabrics etc. ACF made by fabrics and cottons has never been used to remove microcystins in water sample. There is no published data available that has investigated water treatment processes using ACF made by fabrics and cottons, In this study we aimed to obtain basic data on the adsorption of the toxic peptide, microcystin LR to the ACF that was prepared in Chungbuk National University. Our future goal would be to identify and develop ACF treatment processes suitable for removal of algal toxins and to provide a basis for estimating potential water treatment usage.

Recent advances in the sample preparation, liquid chromatography tandem mass spectrometric analysis and environmental fate of microcystins in water

This review article covers recent developments in the analysis of microcystins (MCs), the natural toxins produced in cyanobacterial blooms that occur in many eutrophic waters. We report applications of new extraction methodologies, such as immunosorbents for sample preparation, and current advances in liquid chromatography with tandem mass spectrometry for detection and identification of new transformation products. Due to the complex nature of MCs, there is a growing interest in analyzing MCs and characterizing their transformation products. The widespread occurrence of toxic cyanobacterial blooms in aquatic resources that are used for drinking water supply has raised public health concern. In addition, the presence of MCs in surface waters has resulted in poisoning animals and killing fish worldwide. For this reason, we review the fate of MCs in surface waters, and include current knowledge about how various environmental conditions, such as pH, temperature and sunlight, influence the biodegradation of these toxins under natural conditions. Lastly, we also discuss studies that investigate the potential removal mechanisms of MCs from drinking water supplies, such as advance oxidation processes.

Elimination of microcystins by water treatment processes—examples from Sulejow Reservoir, Poland

Eutrophication is a serious problem in Polish freshwaters. Mass occurrences of toxic cyanobacteria in reservoirs cause problems in the production of safe drinking water and the diversity of produced toxins complicates monitoring of freshwaters. The aim of this study was to estimate the efficiency of water treatment processes in the removal of microcystins (MCs), cyanobacterial hepatotoxins. Elimination of microcystins was studied at two waterworks, which supply drinking water to the city of Lodz from Sulejow Reservoir. The consecutive steps of pre-oxidation, coagulation, sand filtration, ozonation and chlorination used in the water treatment showed effective elimination of microcystins in water from Sulejow Reservoir in 2002 and 2003. The highest total concentration of microcystin (variants MC-RR, MC-YR, MC-LR) amounted to 6.7 μg l −1 in raw water and was detected on the 13th of August 2002. In 2003 the water utility decided to increase the contribution of ground water in the production of drinking water. This resulted in a decrease of microcystin in water during and after the treatment process. The current management strategy of the waterworks company includes mixing of surface water and ground water, which reduces the hazards caused by toxic cyanobacterial blooms in the reservoir.

Influence of ultrasonic field on microcystins produced by bloom-forming algae

Under the background of algae removal and growth inhibition by ultrasonic irradiation, the effects of ultrasonic irradiation on removal of Microcystis, the concentration variation of microcystins (MC) produced by Microcystis in Microcystis suspension, and sonochemical degradation of microcystins in water, were studied in the paper. The results showed that ultrasonic irradiation could efficiently inhibit the growth of Microcystis, and ultrasonic irradiation shorter than 5 min would not introduce the increase of microcystins dissolved in Microcystis suspension simultaneity. Also, microcystins dissolved in Microcystis suspension would not increase as ultrasonic power increasing. Further research showed that microcystins were effectively degraded in ultrasonic fields. After 20 min ultrasonic irradiation at 150 kHz and 30 W, the removal rate of microcystins reached 70%.

Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?

This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcystins and compares this to the guidance values proposed for microcystins in water by the World Health Organization, and for blue-green algal food supplements by the Oregon State Department of Health. The basis of the risk assessment underlying these guidance values is viewed as being critical due to overt deficiencies in the data used for its generation: (i) use of one microcystin congener only (microcystin-LR), while the other presently known nearly 80 congeners are largely disregarded, (ii) new knowledge regarding potential neuro and renal toxicity of microcystins in humans and (iii) the inadequacies of assessing realistic microcystin exposures in humans and especially in children via blue-green algal food supplements. In reiterating the state-of-the-art toxicology database on microcystins and in the light of new data on the high degree of toxin contamination of algal food supplements, this review clearly demonstrates the need for improved kinetic data of microcystins in humans and for discussion concerning uncertainty factors, which may result in a lowering of the present guidance values and an increased routine control of water bodies and food supplements for toxin contamination. Similar to the approach taken previously by authorities for dioxin or PCB risk assessment, the use of a toxin equivalent approach to the risk assessment of microcystins is proposed.

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.

Enzyme‐Linked Immunosorbent Assay Detection of Microcystins Using New Monoclonal Antibodies

New monoclonal antibodies (mAbs) against the microcystin‐leucine–arginine variant (microcystin‐LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an enzyme‐linked immunosorbent assay (ELISA) experiment was made for the detection of cyanobacterial hepatotoxins, microcystins in water sampled in Soyang Lake, Korea. The performance of the ELISA test with mAbs established in this study was evaluated. The ELISA detection was compared with HPLC detection. Since the detection limit of HPLC is several orders of magnitude higher than with ELISA, attention was also paid to concentration of samples with solid phase extraction cartridges.

Comparison of Fluorescence Immunochromatography and HPLC for the Trace Analysis of Algal Toxins

New monoclonal antibodies (mAbs) against microcystin (MC), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. Using these mAbs, an immunochromatographic assay system was designed for the detection of cyanobacterial hepatotoxins, MCs in water. The immunochromatographic assay system is composed of anti‐MCs‐mAb, fluorescence conjugates in detection solution, an immunochromatographic assay strip, and a laser fluorescence scanner. The performance of the immunochromatographic assay system was compared with high performance liquid chromatography (HPLC) detection. As the detection limit of HPLC is several orders of magnitude higher than with immunochromatographic assay systems, attention was also given to preconcentration of samples with solid phase extraction cartridges.

The use of Lepidium sativum in a plant bioassay system for the detection of microcystin-LR

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Detection of microcystins in water generally relies on specialised equipment and a delay of several days for transport and analysis. Little work has, however, been done on establishing a simple, cost-effective and sensitive plant bioassay for the detection of microcystin-LR (MCLR) in water at the WHO Tolerable Daily Intake guideline level of 1 μg/l. We investigated the effect of a MCLR extract at 1 and 10 μg/l on the growth of Lepidium sativum over 6 days. Exposure to 10 μg/l MCLR resulted in a significant decrease in root and leaf lengths and fresh weights of seedlings when compared to the controls. These results were consistent with seedlings exposed to pure MCLR at 10 μg/l. Seedlings exposed to 1 μg/l MCLR showed a significant decrease in root development from day 2 to day 6. Glutathione S-transferase and glutathione peroxidase activities were also significantly raised in plants from days 5 and 4, respectively, at both toxin levels investigated.

Determination of microcystins in lake water using reusable immunoaffinity column

A reusable immunoaffinity column for purification of microcystins in lake water was prepared by coupling anti-microcystin-LR monoclonal antibodies to immunoaffinity support. Thanks to spherical shape of the immunoaffinity support Formyl-Cellulofine® used in this study, applied solutions passed the column smoothly even when used repeatedly. Reusability of the column was examined by determining the recoveries of spiked microcystins-RR, -YR and -LR (100ng each) from lake water. After extraction with a Sep-Pak PS2® cartridge containing styrene–divinylbenzene copolymer, the extract was purified with the immunoaffinity column. The immunoaffinity column was regenerated by washing with Tris–HCl buffer containing bovine serum albumin for repeated uses. Recoveries of spiked microcystins from the first use of the column were 87–88%, and 83–88% from the second and third uses, and the recoveries gradually dropped to 63–77% from the 4–5th uses, the results of which indicated that the column could be used repeatedly for three times. The present method was applied to determine microcystins in water collected from three different lakes in Japan in 1999. In a sample from Lake Suwa, microcystins-RR and -LR were determined by high performance liquid chromatography with photodiode array detection and electrospray ionization-liquid chromatography/mass spectrometry.

Trace Analysis of Microcystins in Environmental Samples

Microcystins, the cyclic heptapeptide toxins produced by cyanobacteria such as Microcystis sp., show potent hepatotoxicity and tumor-promoting activity through inhibition of protein phosphatases 1 and 2A. A toxic incident resulting in the death of 50 people in Brazil in 1996 was due to microcystins in water used for hemodialysis. Microcystins are now threatening human health and life, but many problems associated with microcystins remain unsolved. This report describes how to analyze trace amounts of microcystins in complicated matrixes such as liver tissue, lake water, and sediments.

Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia, Germany)

The presence of microcystins in cyanobacterial samples collected from the Bleiloch reservoir, formerly an important drinking-water supply in Thuringia, Germany, was proven by application of a combination of recently developed analytical methods. The raw extracts were cleaned by size-exclusion chromatography (SEC) or solid-phase extraction (SPE). The determination of microcystins was achieved by different HPLC separation followed by the application of alternative detection methods (UV, diode array detection (DAD), and mass spectrometry (MS), respectively). Furthermore, the different results of clean-up by SPE and SEC are demonstrated. The identity of microcystins was verified by MS/MS measurements. In the cyanobacterial sample from 1998, microcystin-RR, -LR and -YR were found, whereas in 1999 only microcystin-LR and -YR were detectable. In addition to detection of cell-bound microcystins, in 1999 traces of dissolved microcystins in water from the Bleiloch reservoir were detected. It can be assumed that not only the Bleiloch reservoir is contaminated with hepatotoxins but also many similar lakes still used for drinking water supply.

Direct colorimetric assay of microcystin using protein phosphatase

A new direct colorimetric assay of microcystin in water and algal samples is proposed consisting of two procedures as follows: 1) the elimination of phosphorus in the sample and concentration of microcystin using a C18 cartridge, 2) the detection of the released phosphorus by the ascorbic acid method and determination of protein phosphatase (PP) inhibition by microcystin. The optimum amounts of phosphorylase α and PP-1 in 50 μL concentrated sample were 50 μg/50 μL buffer and 1.0 unit/50 μL buffer, respectively, for the best assay. The pH for the maximum activity of PP-1 was 8. The minimum detectable concentration for this method was about 0.02 μg/L, which is sufficient to meet the proposed guideline level of 1 μg microcystin/L in drinking water. Consequently, it would seem that the proposed direct colorimetric assay using PP is a rapid, easy, and convenient method for the detection of microcystin in water and algal samples.

Immunoaffinity purification method for detection and quantification of microcystins in lake water

We have developed a new clean-up method, which consisted of solid-phase extraction on a Sep-Pak PS-2 (styrene–divinylbenzene copolymer) or Excelpak SPE-GLF (polymethacrylate) cartridge instead of conventional ODS silica gel and silica gel together with following immunoaffinity purification using anti-microcystin-LR monoclonal antibodies. This newly developed method was demonstrated to eliminate co-existing substances and to concentrate microcystins in the lake water. The recoveries from lake water (1 liter) spiked with 100 ng each of microcystins-RR, -YR and -LR were 85.5, 89.2 and 92.2%, respectively, with coefficients of variation of 3.3–7.6%. Only 3 h were required to complete the total procedures starting from the microcystin extraction, the immunoaffinity purification, and the quantification using HPLC. The detection limits for all of the 3 microcystins in lake water were 0.005 μg/l. Applicability of this method has been demonstrated by measuring the concentrations of microcystins in water samples collected from lakes where water blooms occurred, which turned out to be 0.012–0.177 μg/l of total microcystins.

Total analysis system for tumor promoter microcystins produced by cyanobacteria

Microcystins, produced by freshwater cyanobacteria, are cyclic peptide hepatotoxins and tumor promoters. An outbreak of human poisoning attributed to microcystins has been reported in Caruaru, Brazil in 1996, where exposure through renal dialysis led to the death of 50 patients. Although such severe acute effects on human health seem to be rare, microcystins poses problems to human health which could result from low-level, chronic exposure to microcystins in drinking water. It is therefore important to monitor the levels of microcystins in water reservoirs where cyanobacterial blooms occur. We have developed a total analysis system for microcystins using GC-MS and LC-MS. This comprises initial screening of samples to check for the presence of microcystins by detecting 2-methyl-3-methoxy-4-phenylbutyric acid as an ozonolysis product using thermospray interface LC-MS and electron ionization/GC-MS. If a sample is positive in a screening test, it will be necessary to follow through with identification and quantification. Frit-FAB interface LC-MS allowed the rapid identification of microcystins in cyanobacteria and lake water, and also enabled us to identify microcystins and their metabolites formed in vivo in mouse liver. Finally, Frit-FAB/LC-MS using selected ion monitoring could be used for quantitative analysis of microcystins in lake water in the low nanogram range. The total analysis system proposed in the present study should be applicable to studies of the metabolism of microcystins, of their detoxification, and those of the mechanism(s) of the accumulation in the food chain.

Survey of microcystins in water between 1995 and 1996 in Paraná, Brazil using ELISA.

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml(-1)) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming-fishing sites at Itaipu dam, 6380 and 10,000 pg ml(-1)) and human supplies (6627 pg ml(-1)) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl- reduced MCs levels, although 267 pg ml(-1) remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region.

Survey of Microcystins in water between 1995 and 1996 in Paraná, Brazil using ELISA

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml−1) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming–fishing sites at Itaipu dam, 6380 and 10 000 pg ml−1) and human supplies (6627 pg ml−1) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl− reduced MCs levels, although 267 pg ml−1 remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region. Copyright © 1999 John Wiley & Sons, Ltd.

Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay.

An epidemiological survey for the causes of a high incidence of primary liver cancer (PLC) in Haimen city, Jian-Su province and Fusui county, Guangxi province in China, found a close correlation between the incidence of PLC and the drinking of pond and ditch water. With an aim to clarify whether microcystins (MC), a hepatotoxic peptide produced by water bloom algae, contaminate the drinking water in the endemic areas of PLC in China, a highly sensitive enzyme-linked immunosorbent assay with a detection limit of 50 pg/ml, was introduced to monitor the MC. Three trials to survey the drinking water were carried out in 1993-1994. Samples, 1135 in total, were collected from different sources such as: ponds, ditches, rivers, shallow wells and deep wells in Haimen city. The first survey in September 1993 found that three out of 14 ditch water specimens were positive for MC, with a range of 90-460 pg/ml. Several toxic algae such as Oscillatoria agardhii were present in some of the ditches. In the second trial, samples were collected from five ponds/ditches, two rivers, two shallow wells and two deep wells monthly for the whole year of 1994. These data showed that MC was highest in June to September, with a range of 62-296 pg/ml. A third trial on the 989 different water samples collected from the different types of water sources in July 1994 revealed that 17% of the pond/ditch water, 32% of the river water, and 4% of the shallow-well water were positive for MC, with averages of 101, 160 and 68 pg/ml respectively. No MC was detected in deep well water. A similar survey on 26 drinking water samples in Fusui, Guangxi province, demonstrated a high contamination frequency of MC in the water of ponds/ditches and rivers but no MC in shallow and deep wells. These data support a hypothesis that the blue-green algal toxin MC in the drinking water of ponds/ditches and rivers, or both, is one of the risk factors for the high incidence of PLC in China. Based on previous findings on the epidemiology of PLC and the present results from the mass screening of MC in the drinking water, an advisory level of MC in drinking water was proposed to below 0.01 microg/l. The combined effect of a potent hepatocarcinogen AFB1 and an intermittent intake of MC in drinking water in the summer season was discussed as an etiology of PLC.

Detection of cyanobacterial toxins (microcystins) in waters of northeastern wisconsin by a new immunoassay technique

The development of reliable, sensitive immunoassay techniques for detection of microcystins in water is becoming increasingly important. We have developed an enzyme-linked immunosorbent assay (ELISA) potentially able to detect microcystins at concentrations as low as 95 pg microcystin/ml water. The procedure uses antibodies extracted from the eggs of immunized chickens, eliminating the need to collect blood from laboratory rabbits. The antibody is able to recognize microcystin-LR, and -RR, and may recognize other forms of microcystin. The newly developed ELISA technique was utilized to measure the amount of microcystin in waters of northeastern Wisconsin. Of the water samples analyzed, 87% contained measurable amounts of microcystin (0.2–200 ng/ml). Organisms of the genus Microcystis were identified most frequently from microcystin-containing waters. The distribution of microcystin-producing cyanobacterial strains was apparently random throughout the sampling area.

A clean-up method for analysis of trace amounts of microcystins in lake water

A clean-up method using high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) was developed to pursue trace amounts of microcystins in lake water. The method consisted of the combined usage of octadecyl silanized (ODS) silica gel and silica gel cartridges. In the first clean-up process, the retention behavior of microcystin RR on ODS silica gel cartridge was carefully observed together with microcystin LR, and 10% water-methanol was chosen as the best solvent system to elute microcystins from the ODS silica gel cartridge. Because many impurities still remained in the desired fraction from the raw water even after the clean-up with ODS silica gel, an additional clean-up process was developed using various cartridges. As a result of extensive experiments, the second clean-up process using silica gel cartridge was established, and the impurities were effectively eliminated. The present method including a tandem cartridge system allowed a precise analysis of microcystins in water samples from three different lakes at a 0.02 ppb level.