Environ. Sci. Technol. 1994, 28, 753-755
COMMUNICATIONS Quantitation of the Microcystin Hepatotoxins in Water at Environmentally Relevant Concentrations with the Protein Phosphatase Bioassay Timothy W. Lambert,'vt Marion P. Boiand,* Charles F. B. Holmes,* and Steve E. Hrudeyt
Environmental Health Program, Department of Public Health Sciences, and MRC Protein Structure and Function Group, Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Introduction Toxic cyanobacteria (blue-green algae) blooms have been documented in lakes and drinking water sources in Europe, China, Australia, western Canada, and the American Midwest (1). A primary cause of cyanobacterial toxicity has been attributed to the microcystin class of cyclic heptapeptides. The microcystin toxins are very potent hepatotoxins causing death to a variety of animals, and they have shown evidenceof being potent tumor promoters (1-3). The overall toxicity of a bloom can be uncertain because of variations in toxin concentration over a short time and spatially within a water body experiencing a bloom (4). Several incidents of human illness have been attributed to the presence of cyanobacterial toxins in drinking water in Australia, Africa, and the United States (5-8). Water treatment studies conducted at laboratory and pilot plant scale have concluded that activated carbon and ozone are capable of removing microcystins from drinking water below the detection limit of the mouse bioassay and high performance liquid chromatography using UV absorbance detection (HPLC/UV) (9-14). Conventional water treatment practices (coagulation/sedimentation,filtration, and chlorination) have been found to be ineffective at removing the toxins (9-11, 14). Because of the lack of a sensitive analytical technique, the water treatment studies have been conducted at concentrations higher than would likely be encountered at treatment facilities. Microcystin-LR is one of the most common cyanobacterial hepatotoxins, but there are at least 40 structural variations ( I ) . Microcystin-LR has been shown to be a potent inhibitor of the catalytic subunits of the serine/ threonine protein phosphatases PP-1and PP-2A (termed PP-lc and PP-2Ac) (15-18), and this probably underlies its toxicity to animals (I 7,18). Microcystin-LR inhibited either PP-lc or PP-2Ac at concentrations of -0.1 nM when assays were performed at phosphatase concentrations of 0.2 munit/mL (16). There are currently two methods for the quantitation of microcystin toxins in water at low levels; the protein phosphatase (PP) bioassay (19) and an enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of microcystins (20). This paper presents an application of the protein phosphatase (PP) bioassay (19) for the
* To whom correspondence should be addressed. + Department of t
Public Health Sciences. Department of Biochemistry.
0013-936X/94/0928-0753$04.50/0
0 1994 American Chemical Soclety
quantitation of microcystin toxins in drinking water at concentrations likely to be found in natural waters. Standard curves for microcystin-LR at various concentrations of PP-lc were prepared. The dependence of the microcystin-LR inhibition of PP-lc activity in the assay upon the amount of PP-lc used in the assay was evaluated. The PP bioassay was then used to quantify microcystin in raw water and finished drinking water at concentrations as low as 0.1 pg/L microcystin-LR.
Experimental Section Enzymes and reagents were prepared as described by Holmes (19). PP-lc was purified from rabbit skeletal muscle and stored in a 60% (v/v) glycerol solution at -4 OC. The PP bioassay was performed as described by Holmes (19) using 32P-radiolabeled phosphorylase a (a known physiological substrate) as the substrate. PP-lc (- 1munit/mL) (10 pL) and a standard (10 pL) of varying concentration of microcystin-LR in Tris-HC1 buffer (50 mM, pH 7.0) were incubated at 30 "C for 10 min. A 10-pL sample of 32P-radiolabeledphosphorylase a (3.5 mg/mL in Tris-HC1, 50 mM, pH 7.0) was added to start the reaction, and the mixture was incubated at 30 OC for 10 min. The reaction was stopped by precipitation of phosphorylase a by adding 200 pL of 20% (w/v) trichloroacetic acid (TCA), and the samples were placed in ice for 2 min. The samples were centrifuged for 2 min, and 200 pL of the supernatant (containing TCA soluble 32Pradiolabeled phosphate) was added to 1mL of scintillation fluid (ACS) and counted (counts per minute, cpm) on a Pharmacia 1209 Rackbeta liquid scintillation counter. Standard microcystin-LR was obtained from Calbiochem (San Diego, CA). The purity of the standard was assessed by HPLC, with a Vydac C-18 analytical column, 10 mM ammonium acetate/acetonitrile mobile phase. The concentration of the standard was verified by amino acid analysis. A stock solution of 5 pg/pL standard microcystinLR was prepared. Standard solutions of 4 3 ,and 2 pg/pL were prepared with fresh Tris-HCl(50 mM, pH 7.0) buffer for each assay. Raw drinking water samples were collected after the microscreen from the intake tap within the water treatment facilities. Treated drinking water was collected from a drinking water tap within the treatment facilities. Both raw and treated water samples were collected in the fall of 1992. A 1-mL aliquot of each sample was dried down in a Savant Speed Vac SC 100 and resuspended in an Environ. Sci. Technol., Vol. 28, No. 4, 1994
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appropriate amount of distilled water such that the sample inhibited PP-lc activity in the assay by -50%. The identity of the microcystin analogue was determined by high-performance liquid chromatography (HPLC) analysis.
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Calculation of Microcystin-LR Concentration The inhibition of PP-lc and PP-2Ac by microcystinLR has been shown to have a sigmoidal response curve when plotting percent activity of PP-lc vs log microcystinLR concentration (15-17) (Figure 1). The curve is linear in the 50% PP-lc activity range, and therefore microcystinLR sample concentrations can be quantified in this region of PP-lc activity. Standard solutions of microcystin-LR were used to calculate the equation of the linear portion of the PP-lc activity curve. Water samples were diluted or concentrated such that they inhibit PP-lc activity by approximately 50 % . The PP-lc activity of all samples, microcystin-LR standard, and water sample were calculated by the equation: (sample - blank) X 100% % PP-lc activity = 100 (control - blank) where (a) control cpm is the maximum PP-lc activity; control is the maximum activity of the PP-lc enzyme, 100% activity, which contained the following: 10 pL of Tris-HC1 buffer, 10 pL of phosphatase, PP-lc, and 10 pL of phosphorylase a; (b) blank or background cpm; blank is the free [32P]phosphatein the phosphorylase a solution, which contained the following: 20 pL of Tris-HC1 buffer, 0 pL of phosphatase, PP-lc, and 10 pL of phosphorylase a; (c) microcystin-LR standard sample cpm, which contained the following: 10 pL of standard microcystin-LR in Tris-HC1 buffer, 10 pL of phosphatase, PP-lc, and 10 FL of phosphorylase a; and (d) water sample cpm, which contained the following: 10 pL of sample (1-3 pL) TrisHC1 buffer (7-9 pL), 10 pL of phosphatase, PP-lc, 10 p L of phosphorylase a. The amount of inhibition of PP-lc and PP-2Ac by a standard concentration of microcystin-LR depends on the concentration of phosphatase in the assay (16). The amount of enzyme in the assay can be related to the amount of 32P released from phosphorylase a during the assay relative to the total amount of 32Ppresent. The percent
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(control cpm - blank cpm) X 1.15 X 100 (total cpm - blank cpm) where total cpmis the concentration 0f32P in the substrate; total is [32Plphosphorylasea and free [32Plphosphateand 10 pL substrate + 200 pL scintillation fluid; and 1.15 is a dilution factor:
---(200) total reaction contents in assay (230) The change in PP-lc activity with the percent of 32P released was evaluated by plotting percent of PP-lc activity vs percent of S2Prelease a t 2, 3, and 4 pg/L microcystinLR (Figure 2) (3 pg/pL is not shown to provide clarity). =--volume of reaction contents counted
Results and Discussion For each assay, fresh solutions of PP-lc and [32P]phosphorylase a were prepared from stock solutions. Therefore, the concentration of PP-lc in each assay varied (observed by changes in percent of 32Preleased from phosphorylase a ) and the amount of PP-lc activity observed with a standard concentration of microcystin-LR varied (Figure 2). An elevated PP-lc activity is representative of lower levels of inhibition by microcystins. In practice, a detectable change in inhibition of PP-lc was observed at 2, 3, and 4 pg/pL microcystin-LR. The slope [-1.5 ( % PPICactivity)/(% 32Prelease)] was approximately the same for these three microcystin concentrations (Figure 2). To account for the variation in PP-lc activity, it was therefore essential that a standard microcystin-LR curve be established for each assay (Figure 3). The PP bioassay was applied to the evaluation of raw and treated water samples for the presence of microcystins. To carry out routine analysis, the water sample was diluted or concentrated so that the activity of PP-lc was within the linear bounds of the microcystin-LR standard curve. It was found that the water samples required no preparation before analysis and that a collected sample volume of 5 mL was adequate for quantitation. Reproducible results were obtained with both raw and treated drinking water samples collected in the fall of 1992,from Ferintosh and Camrose, Alberta, suggesting that natural organic
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Table 1. Raw and Treated Total Microcystin Concentrations, Late Summer/Fall 1992
date Aug 26 Aug 27 Aug 31 Sep 2 Sep 4 Sep 8 Sep 10 Sep 14 Sep 16 Sep 18 Sep 22 Sep 24 Sep 28 Sep 30 Oct 2
total microcystin (pg/L of microcystin-LR equivs) raw treated standard standard av deviation av deviation 0.15 0.87 0.86 0.67 0.12 0.62 0.73 0.35 0.29 0.26 0.27 0.15 0.17 0.22 0.15
r0.041 r0.271 C0.081 r0.161 [0.011 10.081 rO.lO1 [0.071 10.031 L0.071 C0.031 10.031 10.031 [0.071 10.021
by Sim and Mudge (23)concerning the PP bioassay. This method provides a means of early detection of the presence of microcystin in drinking water before concentrations increase to hazardous levels. If high levels are detected, the problem of false positives can be overcome by linking the PP bioassay to an instrumental analytical technique (e.g., HPLC or capillary electrophoresis) as was performed for confirmation in this study. With careful monitoring, the health risk that may be associated with drinking water containing microcystins can be minimized and the likelihood of a serious exposure incident reduced.
0.11 0.09 0.13 0.10 0.14 0.18 0.17 0.15 0.14 0.14 0.18 0.10 0.14 0.15 0.14
[0.031 10.031 C0.051 [0.041 10.031 [0.03] [0.011 [0.041 10.051 [0.031 C0.081 E0.041 10.051 [O.Q3l 10.031
matter present in the samples did not interfere with the assay (Table 1). Although a large number of microcystin analogues were observed in a concomitant cyanobacterial bloom present in the lake supplying the water used for analysis (211, microcystin-LR was identified by HPLC as the principal microcystin in the water samples. The PP bioassay does not distinguish between microcystin analogues and measures the total PP-lc inhibition of the water. Therefore, the quantity of toxin in the water is reported in units of microcystin-LR equivalents per volume. The inhibition of PP-lc by microcystin-LR, microcystin-RR, and nodularin differs slightly for each toxin (17). The effect of varying concentrations of microcystin analogues present in a water sample has not been evaluated. The toxicity of the microcystins is probably mediated through inhibition of the protein phosphatases (17,18); therefore, the measured PP-lc inhibition of the water sample with the PP bioassay is representative of the toxicity to biological systems. With the PP bioassay, it is now feasible for the concentration of microcystin toxins to be evaluated at water concentrations relevant to observed environmental conditions in western Canada. Microcystin-LR was quantified in treated drinking water at levels down to 0.1 pg/L. An inherent limitation of any enzyme-linked bioassay procedure concerns the possibility for false positives. This potential problem has been fully addressed
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