Analysis of microcystins in alum water treatment sludges: holding times, temperatures, linearity of response, and sensitivity to pre-coagulation cell titers

Abstract

ELISA assays are a potential tool to screen for dissolved or cell bound microcystins in drinking water treatment sludges. In order to evaluate this potential more thoroughly, experiments were performed in alum sludges to: (1) evaluate the impacts of sample storage times, temperatures, and sludge composition on spiked microcystin-LR recovery by ELISA; (2) examine the linearity of ELISA responses to spiked microcystin-LR as a function of sludge composition; and (3) examine the sensitivity ELISA and LC/MS/MS to five different concentrations of microcystin-producing cyanobacteria entrained in sludges of two different compositions. During storage experiments, microcystin recovery efficiencies ranged from 85% to 125% across the range of 12 storage time and temperature combinations with recovery efficiencies in 7 of the 12 combinations falling into the 90% to 110% range. During the linearity experiments, linear models fit ELISA responses in all sludge compositions with R² values ≥ 0.95. During the sensitivity studies, simple freeze/thaw/centrifugation processing combined with ELISA or LC/MS/MS analyses resulted in detection of microcystins in thickened sludges derived from pre-coagulation cell suspensions of 10²-10⁶ cells/mL. In addition, the relationships between toxin concentrations in sludges and the original cell titers were linear regardless of analytical method.

Keywords: ELISA; LC/MS/MS; cyanobacteria; microcystin; sludge.

Figure 1.

Holding time and temperature results. For each sludge composition, holding time, and temperature combination, seven replicate samples were spiked with 1ug/L of MC-LR. Bars and error bars represent mean recovery efficiencies (n = 7) and 95% confidence intervals around the means, respectively.

Figure 2.

Linearity of response results. MC-LR was spiked into thickened sludge at 0.5, 1.0, 2.0, 3.0, and 4.0 μg/L. Data points and error bars represent the means of duplicate spiked samples and average deviations about the means, respectively. Each panel represents a different water quality prior to coagulant addition.

Figure 3.

Impacts of ELISA calibration curve non-linearity and between-plate variability on calculated MC concentrations during the sludge linearity of response experiments. Each curve represents the calibration curve for one ELISA plate run during one of the four linearity experiments. Variations in calculated MC concentrations are summarized by their coefficients of variation (CV) where CV = std. dev / mean. The CVs were calculated at blank-normalized absorbances of 0.6 and 0.3.

Figure 4.

Sensitivity experiment results – artificial water. TOC = 2.7 mg/L, Turb. = 2.6 NTU, Alum dose = 40 mg/L. Toxin producing cyanobacterial cells were spiked into artificial water at five different concentrations in five separate jar tests. Phycocyanin fluorescence was measured in uncoagulated water. The coagulation sludge was collected, thickened, freeze/thaw processed, and analyzed for extra + intracellular microcystins by ELISA and extra + intracellular microcystin-LR by LC/MS/MS.

Figure 5.

Sensitivity experiment results – Ohio River water. TOC = 3.5 mg/L, Turbidity = 51 NTU, Alum dose = 40 mg/L. Toxin producing cyanobacterial cells were spiked into Ohio River water at five different concentrations in five separate jar tests. Phycocyanin fluorescence was measured in spiked but uncoagulated water. The coagulation sludge was collected, thickened, freeze/thaw processed, and analyzed for extra + intracellular microcystins by ELISA and extra + intracellular microcystin-LR by LC/MS/MS.