Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods.

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.     

Capillary electrochemical enzyme immunoassay (CEEI) for phenobarbital in serum

A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 microl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product (p-aminophenol) from the catalytic conversion of the substrate (p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 microg l(-1) (2.8 pmoles or 0.65 ng) and a range of 30-3000 microg l(-1). The assay could be used to determine the phenobarbital serum concentration in a 4 microl clinical serum sample without pretreatment.     

Effects of cyanobacterial biomass and purified microcystins on malformations in Xenopus laevis: teratogenesis assay (FETAX)

Xenopus laevis (African clawed frog) embryos in a 96-h teratogenesis assay (FETAX) were exposed to 0-250 microg/L and 500 microg/L of purified microcystin-LR (MCYST-LR) for the estimation of lethality, as well as to equivalent concentrations of biomass containing MCYST-LR (natural water bloom dominated by Microcystis aeruginosa) and biomass without MCYST-LR (bloom dominated by Microcystis wesenbergii). The highest tested concentrations of purified MCYST-LR caused up to 30% lethality after a 96-h exposure, corresponding to a LC(25) of 380 microg/L. Cyanobacterial biomass containing MCYST-LR caused significant lethality up to 50% at the highest tested concentrations (300 mg/L, i.e., 250 microg/L of MCYST-LR). The estimated 96-h LC(25) values varied from 125 mg/L (biomass containing MCYST-LR) up to 232 mg/L (biomass without MCYST-LR). A statistically significant increase in the number of malformed embryos was observed after exposure to cyanobacterial samples. Purified MCYST-LR at and above 25 microg/L significantly increased the number of malformations, with 53% of surviving embryos malformed in the highest tested concentration, 250 microg/L (EC(25) = 27 microg/L). Exposure to the highest concentration of MCYST-LR containing biomass resulted in more than 60% of the embryos being malformed and an EC(25) of 52 mg/L (i.e., 43 microg of MCYST-LR/L). Cyanobacterial biomass with no natural microcystin also induced substantial malformations-about 50% aberrant embryos at the highest concentration, 300 mg/L (EC(25) = 75 mg/L). External additions of purified MCYST-LR to the biomass that was originally without microcystins resulted in a slight additional increase in the rate of malformations (80% at the highest concentration, 300 mg of biomass plus 250 microg of MCYST-LR per liter). A comparison of lethality and effects on malformations (teratogenic index, TI = LC(25)/EC(25)) showed that all samples had significant teratogenic potential in the FETAX assay (TI(MCYST-LR) = 14; TI for biomass with and without microcystin ranged between 2.4 and 3.1, respectively). We conclude that cyanobacterial water blooms can significantly alter the normal development of amphibian embryos.     

Effects of microcystin-LR on mouse lungs.

Toxic cyanobacteria blooms in drinking water supplies have been an increasing public health concern all over the world. Human populations can be exposed to microcystins, an important family of cyanotoxins, mainly by oral ingestion. However, inhalation from recreational water and hemodialysis can represent other routes. This study investigated changes in respiratory mechanics, histology, protein phosphatase (PP) 1 and 2A activity and microcystin in lung of adult mice injected intraperitoneally (i.p.) with microcystin-LR. Thirty-six mice were divided into control (CTRL) and test (CYANO) groups. CTRL group received an i.p. injection of saline and the CYANO group received 40 microg MCYST-LR/kg i.p. After 2 and 8 h, and 1, 2 and 4 days after toxin injection, six mice from each group were sampled for analyses. Resistive and viscoelastic pressures, static and dynamic elastances augmented at 2 h in CYANO and so remained until day 4. Alveolar collapse and inflammatory cell infiltration were found 2h after the injection, reaching peak values at 8 h. However, no microcystin or inhibition of PPases could be detected in mice lungs. In conclusion, MCYST-LR led to a rapid increase in lung impedance and an inflammatory response with interstitial edema and inflammatory cell recruitment in mice.     

Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels.

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate (p-NPP) and the bioluminescence assay using luciferin phosphate (L-P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC50 values of 0.9 and 6 nM using L-P and p-NPP respectively. CDP-star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC50 for the colorimetric method (IC50=2 nM [p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations < 1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels < or = 1 microg/100 g of mussel tissue which is well below the limit of 20 microg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to78% (p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC-MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC-MS all scored false negative results compared to those for the mouse bioassay in the range 20-40 microg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay > ELISA > PP-2A > LC-MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 microg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.     

A new morphospecies of Microcystis sp. forming bloom in the Cheffia dam (Algeria): seasonal variation of microcystin concentrations in raw water and their removal in a full-scale treatment plant

Toxic cyanobacterial blooms are an increasing problem in Algeria. The production of cyanotoxins (microcystins) and their presence in drinking water represent growing hazards to human health. In this study, seasonal variations in the concentrations of total microcystins and physicochemical parameters (pH, temperature, dissolved oxygen, nitrate, orthophosphate, and chlorophyll-a) were analyzed in the Cheffia dam (Algeria), mainly used to supply drinking water. The removal of cyanobacterial cells and microcystins was also evaluated in full-scale plant associated with the Cheffia reservoir. The levels of microcystins (MCYSTs) in both raw and drinking water were evaluated using the protein phosphatase type 2A (PP2A) inhibition test as MCYST-LR equivalents. Identification of microcystin variants was achieved by LC/MS/MS. During the period of study (March-December 2004), microscopic observation showed the dominance in the autumn months (September-November) of a new morphospecies of Microcystis sp. The MCYST-LR equivalent concentrations in raw water varied between 50.8 and 28,886 ng L(-1). The highest level of toxins was observed in October 2004 and was significantly correlated with the chlorophyll-a. Three variants of microcystins assigned as microcystin-YR (MCYST-YR), microcystin-LR (MCYST-LR), and 6Z-Adda stereoisomer of MCYST-LR were observed in the crude extract of the Microcystis sp. bloom sample. During the bloom period, total elimination of Microcystis sp. and toxins were achieved through a classical treatment plant comprised of coagulation and flocculation, powdered activated carbon at 15 mg L(-1), slow sand filtration and chlorination before storage.     

A colorimetric protein phosphatase inhibition assay for the determination of cyanobacterial peptide hepatotoxins based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1

A colorimetric protein phosphatase inhibition assay based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1 was developed for analysis of waters for cyanobacterial hepatotoxins. The phosphate released in the assay was determined using a malachite green reagent. Good agreement with toxin concentrations determined by HPLC was obtained. The assay was capable of determining these toxins at concentrations around 1 μg/L with high precision and without sample concentration. This is of considerable benefit as the World Health Organisation specifies a provisional guideline of 1 μg/L for microcystin‐LR. There was evidence, however, that the sample matrix might affect quantification, leading to false positive results. Thus the assay should be viewed as a screening procedure, and confirmatory analyses by an alternative procedure should be carried out for positive results. Further work is required to resolve the question of matrix interferences if phosphatase inhibition assays are used directly for measuring toxin levels in water, especially if this information is used to check compliance with water quality guidelines. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 242–252, 2001     

Exposure of beef cattle to sub-clinical doses of Microcystis aeruginosa: toxin bioaccumulation, physiological effects and human health risk assessment.

Yearling beef cattle were fed 1 x 10(5) cells ml(-1) of toxic Microcystis aeruginosa in their drinking water for a period of 28 d. Feed and water intakes of four control and four treated animals remained unchanged following introduction of M. aeruginosa into the drinking water of the treatment animals, and there were no significant differences in feed and water intakes between control and treated animals. We tested the blood plasma of both control and treated animals twice each week for elevated concentrations of the liver enzymes gamma-glutamyl transferase (GGT), glyceraldehyde dehydrogenase (GADH), amino aspartate transferase (AST) and bilirubin. All tests were negative indicating no measurable liver dysfunction resulting from microcystin intoxication. We also tested for the presence of free microcystin in the liver and blood plasma by HPLC and ELISA and for total microcystin (free+bound) in the liver by GC-MS. If all the ingested microcystin was bioaccumulated within the liver, the concentration would have exceeded 3 microg MCYST-LR g(-1) fresh weight. HPLC and GC-MS analysis of the liver tissue and blood plasma from treated animals failed to detect any microcystins. ELISA analysis of liver tissue extracts from the treated animals indicated microcystin concentrations as high as 0.92 microg MCYST-LR equivalents g(-1) fresh weight although none was indicated in the blood plasma. The microcystin concentrations measured by ELISA in livers of treated animals were more than 1000 times higher than the limit of quantification by HPLC and GC-MS indicating the ELISA results were almost certainly due to cross reactivity with something other than intact MCYST-LR. Based on the detection limits of the HPLC and the per capita daily consumption of beef in Australia, it appears that consumption by beef cattle of water containing M. aeruginosa cell concentrations up to 1 x 10(5)cells ml(-1) for 4 weeks would not produce concentrations of microcystin within the liver or blood plasma that would present an unacceptable risk to human health based on World Health Organization protocols for determining such risks.     

Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels.

The fluorescence protein phosphatase (PP-2A) inhibition assay detects okadaic acid (OA) and DTX-1 in mussels down to 1 microg/100 g of mussel tissue. It is more sensitive than the mouse bioassay (detection limit, 20 microg/100 g) or ELISA using the SCETI DSP check kit (detection limit, 10 microg/100 g). A drawback of the PP-2A assay method has been its lack of sensitivity towards the ester derivatives of OA and DTX-1. This has been addressed by including a hydrolysis step in the pretreatment of extracts which allows these derivatives to be converted to either okadaic acid or DTX-1 prior to the DSP assay. The method has been applied to the analysis of DSP in 19 samples of naturally contaminated mussels and the results from the PP-2A inhibition assay compared to those for HPLC. A good correlation was obtained for OA determined by the two methods in both unhydrolysed and hydrolysed samples. The new procedure will substantially reduce the incidence of false negatives in the DSP assay.     

Influence of drinking water composition on quantitation and biological activity of dissolved microcystin (cyanotoxin)

Toxic cyanobacteria in aquatic environments have been implicated in many poisoning incidents of livestock, wildlife, and domestic animals. Microcystins (MCYSTs) in water supplies represent a risk to public health. This work investigated the effect of water composition on the quantitation and biological activity of MCYSTs analyzed by different methods (HPLC, ELISA, and protein phosphatase 1 inhibition assay). Different MCYST concentrations were added to deionized water and quantified, confirming the efficiency of these analytical methods. MCYST concentrations diluted in drinking water had reduced detection by all methods tested. The drinking water used contained a free chlorine concentration of 2.5 mg/L and an Fe concentration of 0.45 mg/L, and the conductivity was 69.8 microS cm(-1), whereas in deionized water, free chlorine and Fe were not detectable, and the conductivity was 1.6 microS cm(-1). Drinking water also interfered with the biological activity of MYCSTs, as these toxins showed reduced protein phosphatase-1 inhibition. A free chlorine concentration of 2.5 mg/L in deionized water was completely effective in preventing any detection of 10 microg/L of added MCYSTs. Fe and Al ions also were very effective in reducing MCYST detection. The chemical composition of drinking water thus affected MCYST detection, indicating a significant reduction in quantitation of this molecule either because of its decomposition or through complexation with metal ions.     

Recovery of MC-LR in fish liver tissue

Cyanotoxins, particularly microcystins (MCs), have been shown to be a hazard to human health. MCs accumulate in aquatic organisms probably as a result of irreversible binding to liver protein phosphatases. The aim of this study was to describe the recovery of MC from fish liver using various detection methods, with MC-LR as the representative congener. These findings are discussed in conjunction with the current procedures and limit values used for human risk assessment. Following incubation of liver homogenates with various MC-LR concentrations, the homogenates were extracted by a water/methanol/butanol mixture via different treatments and subsequently analyzed via the colorimetric protein phosphatase inhibition assay (cPPA), HPLC, and anti-Adda ELISA. Detection via cPPA appeared to yield the highest recovery of MC-LR, although the presence of unspecific background may have resulted in overestimation of the true recovery. The recoveries determined via HPLC and anti-Adda ELISA were comparable to each other. The limits of detection were 0.01-2.4 microg MC-LR/g liver tissue, depending on the method used. Maximum MC-LR recovery from samples incubated with 10 and 100 microg MC-LR/g ranged between 44% and 101%. Recovery from samples incubated with 1 microg MC-LR/g liver tissue was below 3%. Lower recovery is assumed to result from irreversible, covalent MC protein binding, as confirmed by Western blotting of liver homogenates with anti-Adda immunoprobing. The results demonstrate that further investigation of and improvement in routinely applied MC methods for fish tissue and/or food analyses are needed for a reliable risk assessment.     

The repeatability of two HPLC methods and the PP2A assay in the quantification of diarrhetic toxins in blue mussels (Mytilus edulis).

Repeatability in the quantification of diarrhetic shellfish toxins was investigated for two fluorometric HPLC methods and a colorimetric PP2A assay, using agreement analysis. Blue mussels (Mytilus edulis) from the Sognefjord on the southwest coast of Norway were sampled during the periods of July to October, 1996, March to November, 1997, and October to December, 1998. The results from the first and the duplicate measurement were found to be very similar for both HPLC methods using two fluorogenic reagents, 4-bromomethyl,7-methoxycoumarin and 9-anthryl diazomethane, and the colorimetric PP2A inhibition assay in detection of diarrhetic shellfish toxins. The levels of agreement between the measurements were satisfactory for all three methods. Significant correlations were found between the level of observation and the absolute difference between the two measurements and were mainly due to concentrations of OA/DTX-1 higher than 100 microg/100 g mussel meat. However, the precision of quantifying DSP toxins in the upper level was not found to be less than in the lower level. Both the HPLC methods and the PP2A assay were found to give repeatable results and thereby found to be reliable.     

Development of an integrated laboratory system for the monitoring of cyanotoxins in surface and drinking waters

A system of analytical processes has been developed in order to serve as a cost-effective scheme for the monitoring of cyanobacterial toxins on a quantitative basis, in surface and drinking waters. Five cyclic peptide hepatotoxins, microcystin-LR, -RR, -YR, -LA and nodularin were chosen as the target compounds. Two different enzyme-linked immunosorbent assays (ELISA) were validated in order to serve as primary quantitative screening tools. Validation results showed that the ELISA methods are sufficiently specific and sensitive with limits of detection (LODs) around 0.1 μg/L, however, matrix effects should be considered, especially with surface water samples or bacterial mass methanolic extracts. A colorimetric protein phosphatase inhibition assay (PPIA) utilizing protein phosphatase 2A and p-nitrophenyl phosphate as substrate, was applied in microplate format in order to serve as a quantitative screening method for the detection of the toxic activity associated with cyclic peptide hepatotoxins, at concentration levels >0.2 μg/L of MC-LR equivalents. A fast HPLC/PDA method has been developed for the determination of microcystins, by using a short, 50 mm C18 column, with 1.8 μm particle size. Using this method a 10-fold reduction of sample run time was achieved and sufficient separation of microcystins was accomplished in less than 3 min. Finally, the analytical system includes an LC/MS/MS method that was developed for the determination of the 5 target compounds after SPE extraction. The method achieves extremely low limits of detection (<0.02 μg/L), in both surface and drinking waters and it is used for identification and verification purposes as well as for determinations at the ppt level. An analytical protocol that includes the above methods has been designed and validated through the analysis of a number of real samples.     

Monthly variations in diarrhetic toxins and yessotoxin in shellfish from coast to the inner part of the Sognefjord, Norway.

Monthly concentrations of diarrhetic shellfish poisoning (DSP) toxins and yessotoxin (YTX) in mussels from the coast to the inner part of the Sognefjord were determined. Mussels from nine locations were sampled from March to November 1997. The DSP toxins and YTX were analysed by a colorimetric protein phosphatase 2A (PP2A) inhibition assay or fluorometric HPLC, respectively. The mouse bioassay for DSP toxins was performed including either chloroform or diethyl ether in the final step of extraction. Using ether in the final step normally facilitated extraction of the DSP toxins, okadaic acid (OA) and dinophysis toxin-1 (DTX-1), while chloroform extraction included a wider spectrum of toxins, including YTX and a fast acting toxin(s) with neurotoxic effects. The concentrations of DSP toxins and YTX in mussels increased with distance from the coast. The highest concentrations of YTX (574 microg YTX/100 g mussel meat) and diarrhetic toxins (349 microg OA equivalents/100 g mussel meat) were measured in May and August, respectively, at locations in the inner part of the fjord. Since concentrations of DSP toxins and YTX in mussels increased with distance from the coast, the locations for mussel farming in the Sognefjord close to the coast, seem to be preferable.     

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases.

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.     

Residential exposure to drinking water arsenic in Inner Mongolia, China

In the Ba Men region of Inner Mongolia, China, a high prevalence of chronic arsenism has been reported in earlier studies. A survey of the arsenic contamination among wells from groundwater was conducted to better understand the occurrence of arsenic (As) in drinking water. A total of 14,866 wells (30% of all wells in the region) were analyzed for their arsenic-content. Methods used to detect arsenic were Spectrophotometric methods with DCC-Ag (detection limit, 0.5 microg of As/L); Spot method (detection limit, 10 microg of As/L); and air assisted Colorimetry method (detection limit, 20 microg of As/L). Arsenic-concentrations ranged from below limit of detection to 1200 microg of As/L. Elevated concentrations were related to well depth (10 to 29 m), the date the well was built (peaks from 1980-1990), and geographic location (near mountain range). Over 25,900 individuals utilized wells with drinking water arsenic concentrations above 20 microg of As/L (14,500 above 50 microg of As/L-the current China national standard in drinking water and 2198 above 300 microg of As/L). The presented database of arsenic in wells of the Ba Men region provides a useful tool for planning future water explorations when combined with geological information as well as support for designing upcoming epidemiological studies on the effects of arsenic in drinking water for this region.     

Characterization of 9H-(1,3-dichlor-9,9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP)

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157–9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.     

Elimination of microcystin peptide toxins from water by reverse osmosis

The elimination of microcystin-LR (MCYST-LR) and microcystin-RR (MCYST-RR) by reverse osmosis from tap and salt (3000 ppm NaCl) water was examined. The average retention rates for the microcystins of three different membrane types were determined by measuring the MCYST-LR and MCYST-RR concentrations in both the permeate and the retentate. The effectiveness of the reverse osmosis apparatus was assured by conductivity measurements. Using high performance liquid chromatography analysis the detection limit for both MCYST-LR and MCYST-RR was 0.2 μg/L and the quantification limits were 0.9 and 0.8 μg/L for MCYST-LR and MCYST-RR, respectively. Under the experimental conditions applied, the average retention rates for MCYST-LR and MCYST-RR in tap water were in the range of 96.7% to 99.9% and in salt water between 98.5 and 99.9%. There was no significant difference between salt and tap water for either compound. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13: 143–148, 1998     

HPLC determination of naproxen in plasma

An assay method using isocratic HPLC with fluorometric detection for the determination of naproxen sodium in plasma is presented. A reverse phase Microbondapack column was used with a mobile phase consisting of 42% acetonitrile and 58% water adjusted to pH 3 using phosphoric acid. The fluorometric detector with an excitation wavelength of 270 nm and emission wavelength of 340 nm provided high sensitivity and no interferences from plasma constituents. Plasma samples were injected to HPLC without any extraction. The method was precise and reproducible as was demonstrated by replicate analysis of pooled plasma sample containing 0.5-80 microg/ml naproxen sodium.     

Seasonal production and molecular characterization of microcystins in Oneida Lake, New York, USA

Oneida Lake, northeast of Syracuse, New York, in the United States, is a shallow eutrophic lake with a well-established toxic cyanobacterial population. Samples for DNA, toxin, and phycological analyses were collected from six stations throughout the summers of 2002 (78 samples) and 2003 (95 samples). DNA was amplified by PCR using primer sets specific to the nonribosomal microcystin synthetase complex (mcyB and mcyD). PCR analysis in 2002 indicated that the microcystin genes were present in the water column from mid-June through October, as 88% of the samples tested positive for mcyB and 79% of the samples tested positive for mcyD. In both years the onset of microcystin production was detected as early as mid-July by the protein phosphatase inhibition assay, reaching a maximum in 2002 of 2.9 microg L(-1) and in 2003 of 3.4 microg L(-1). Beginning in mid- to late August of both years the microcystin level at all six stations was in excess of the World Health Organization (WHO) advisory level of 1.0 microg L(-1). In the present study we compared microcystin occurrence and potential production at the six stations using protein phosphatase inhibition assay, high-performance liquid chromatography, and polymerase chain reaction analyses.