Environ. Sci. Technol. 2007, 41, 2617-2621
Single Cell Level Microalgal Ecotoxicity Assessment by Confocal Microscopy and Digital Image Analysis YARLAGADDA V. NANCHARAIAH, MOHANRAJ RAJADURAI, AND VAYALAM P. VENUGOPALAN* Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Bhabha Atomic Research Center, BARC Facilities, Kalpakkam, Tamil Nadu 603102, India
In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dosedependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDA technique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multiparameter bioassay using confocal microscopy.
Introduction Environmental contaminants arising out of industrial activities are directly or indirectly discharged to natural aquatic systems. Environmental stress effects are most easily and rapidly manifested in unicellular organisms (1). Accordingly, the effects of environmental contaminants are generally assessed using microalgae as model organisms, as they form the basis of the aquatic food chain. Marine phytoplankton * Corresponding author phone: +91 44 274 80 203; fax: +91 44 274 80 097; e-mail:
[email protected]. 10.1021/es0627390 CCC: $37.00 Published on Web 03/06/2007
2007 American Chemical Society
are especially useful as markers for environmental monitoring (1). Tests relying on growth inhibition of microalgae have long been used for assessing the environmental impact of toxic contaminants in aquatic systems (2-4). Data obtained from such tests are used in ecological risk assessments for formulating discharge guidelines for various industrial effluents. But standard algal growth inhibition tests have several limitations such as (i) the use of environmentally unrealistic high cell density and toxicant concentration, (ii) the inability to distinguish between live and dead cells, and (iii) the inability to provide information on sublethal effects and mechanisms of toxicity (3). Moreover, such growth-based methods are time-consuming, often requiring 72 h or more and severely underestimate the effect, particularly when dealing with oxidizing biocides (5). Therefore, it is imperative to use rapid and more effective methods for the assessment of toxicity. Measurement of fluorescence properties of microalgae allows development of rapid and sensitive ecotoxicity methods (6, 7). Phototrophic organisms are autofluorescent due to their pigments and permit noninvasive in situ analysis with no external fluorescence labeling or staining (8). Recently, techniques to collect cellular fluorescence from single-celled phototrophic organisms for phylogenetic identification have been reported (9, 10). Earlier, chlorophyll-fluorescence-based assay has been used as a simple, rapid, nondestructive method for observing effects of pollutants on plants (11). Confocal laser scanning microscopy (CLSM) allows in situ quantitative assessment of fluorescence properties of algal cells, with the added advantage that visualization and quantification are possible at single-cell level. As environmental changes directly impact cells before populations are affected, measurements at singlecell level are desirable (1). As an added advantage, CLSM technique also allows in situ monitoring of attached microbial or microalgal (periphyton) communities in four dimensions (x, y, z, and t) (8). Earlier, CLSM has been employed to study the distribution of microalgal community in complex photoautotrophic biofilms (12, 13). Chlorine is one of the most common oxidizing biocides used for controlling micro- and macro-fouling in industrial cooling water systems (14, 15). Chlorine affects metabolic and physiological processes of microorganisms by causing damage to cell membrane, proteins, and nucleic acids (5, 16, 17). Phototrophic microalgae, a major constituent of the coastal primary producer community, may suffer damages arising out of chlorine stress from power plant discharges (18, 19). However, standard growth assays have limitations when used to study the effect of oxidizing chemicals such as chlorine on microalgae. Mean fluorescence intensity (MFI) determined by means of flow cytometry has been used as an endpoint for determining the effect of copper on microalgae (3). The objective of the present study was to test the hypothesis that MFI of individual microalgal cells can be used as an endpoint for toxicity assessment. For the first time, CLSM in conjunction with digital image analysis was employed in the present study to quantitatively determine MFI per cell in order to assess the effect of chlorine on marine diatom cells. In addition, the effect on metabolic activity of the diatom cells was studied using fluorescein diacetate (FDA) staining. The study presents a technique combining autofluorescence inhibition and esterase activity to understand the effect of chlorine on unicellular marine microalgae using short-term exposure studies. VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Materials and Methods Algal Culture and Growth Conditions. The diatom Cocconeis scutellum Ehrenberg 1833 was chosen as the test organism for the study. It is one of the commonest diatoms present in the coastal waters of Kalpakkam (east coast of India), which is used as source water for an operating electric power plant. This power plant employs chlorination (at residuals in the range 0.2-0.5 mg/L) for biofouling control. The diatom was isolated from sediments near the intake point of the power station (19). The diatom was cultured in f/2 medium (20) prepared in sterile seawater at 28 °C under light (ca. 40 µE m-2 s-1; 12 h light: 12 h dark photocycle). Double filtered (0.22 µm) UV sterilized aged seawater (henceforth referred to as sterile seawater) was used for the preparation of f/2 algal growth medium. Aged seawater was prepared from nearsurface water samples collected from the coastal Bay of Bengal. The seawater samples were filtered and stored in dark containers for 4 to 6 weeks before use. Cells from the exponential growth phase were harvested and used in algal toxicity assays. Toxicity Tests. Exponentially growing cells were harvested by centrifugation at 1157g for 5 min and washed three times with sterile seawater. Fifty milliliters of sterile seawater was dosed with different concentrations of chlorine (1.0, 1.2, 3.6, and 4.6 mg L-1; corresponding total residual oxidant levels were 0.2, 0.4, 2.8, and 3.8 mg L-1) and inoculated with prewashed algal cell suspension at a final density of 2.0 × 106 cells mL-1. Test and control experiments were performed at 30 °C on a rotary shaker set at 100 rev min-1. Samples were drawn after 5, 15, and 60 min posttreatment and imaged immediately for chlorophyll autofluorescence. Separate samples were stained with FDA (see below) for metabolic activity assessment. At the end of 60 min of exposure, the cells were harvested by centrifugation, washed with sterile seawater before suspending in sterile f/2 growth medium. These cell suspensions were re-incubated under normal culture conditions for about 18 h and subsequently imaged for chlorophyll fluorescence. The treated cells were reincubated to determine whether the reduction in MFI per cell was a transient or permanent phenomenon. Experiments were carried out in duplicates. Biocide. Test chlorine solution was prepared by diluting sodium hypochlorite (Ranbaxy, Mumbai, India) in sterile seawater. Aliquots of the treatment solution were sampled periodically to monitor the residual chlorine concentration. The concentration of chlorine was determined by using the N, N-diethyl-p-phenylenediamine (DPD) method (21). Staining and CLSM. Control and biocide-treated diatom cells were stained with FDA (Molecular Probes, U.S.) as per Franklin et al. (22). Prior to imaging, the sample containing diatom cells was placed on glass slide and covered with a glass slip. A confocal laser scanning microscopy system TCS SP2 AOBS equipped with an inverted microscope DMIRE2 (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) was used to image the diatom cells. A water immersion lens (63×/1.2 NA) was used for obtaining all images. A 488 nm Ar laser was used for excitation. Emission was collected by setting the detection bandwidth between 500 and 530 nm for FDA fluorescence and 630-750 nm for chlorophyll autofluorescence. Images were acquired at 2 µm z-intervals, with a 2.2× zoom factor and two-frame averaging. Image acquisition parameters including PMT settings were optimized initially and not changed during the acquisition of subsequent images. Digital Image Analysis. Digital image analysis was performed using the freeware ImageJ 1.29×, downloadable from the site http://rsb.info.nih.gov/ij. For mean fluorescence intensity (MFI) analysis, all images were acquired using identical CLSM settings. The projections were obtained from 2618
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xy-confocal slices of an image stack to display them in a two-dimensional image and to determine MFI. Maximum intensity projections of confocal slices of an image stack were obtained using Leica confocal software. These projections were used directly for MFI determination per diatom cell using ImageJ, without further image processing. The given MFI values indicate average of MFI values of a minimum of 50 diatom cells. Statistical Analysis. Statistical comparison analysis of MFI means between control and treated, different exposure times, and chlorine doses was performed using paired-t-tests (Microsoft Excel 2002, Microsoft, Redmond, WA). Differences between MFI means for control and treated suspensions were considered significant at a p-level below 0.05.
Results and Discussion Development of rapid and multi-parameter-based bioassays in place of conventional growth-based inhibition assays is desirable for ecotoxicity assessment and for understanding the mechanism of toxicity. The toxicity methods should be rapid when dealing with oxidizing chemicals. Oxidizing biocides such as chlorine are extremely nonspecific and can attack several oxidizable organic and inorganic substances present in water. Actively growing algal cultures exhibit high chlorine demand due to the presence of algal exudates, which may interfere with the toxicity assay. Assays with mixed microalgal populations (e.g., natural phytoplankton), which consist of several species with varying sensitivity to the stressor, may generate data that is difficult to interpret (23). In the present study, CLSM along with digital image analysis was employed for understanding the effect of chlorine on diatom cells at single-cell level. CLSM has been widely applied in biomedical and bioscience research but its potential in ecotoxicological studies have not been fully explored (24, 25). CLSM allows rapid and multi-parametric analysis in microalgal toxicity assays and in the present instance, chlorophyll autofluorescence and FDA fluorescence were used as end points. Autofluorescence Inhibition. In a maximum intensity projection obtained from a stack of xy-confocal slices, the maximum intensity value is assigned to each pixel location as the representative of all values within the stack. The image is displayed as a two-dimensional image, which is used to determine MFI. Confocal images (projections) depicting chlorine-induced decrease in chlorophyll fluorescence in individual diatom cells can be seen in Figure 1. The hollow region at the center of individual diatom cells (Figure 1) can be clearly seen when maximum intensity projections of chlorophyll fluorescence of treated cells were made. Mean MFI calculated from the individual cells (n g 50) decreased in a dose-dependent manner (Figure 2). Quantitative analysis of MFI per diatom cell before and after exposure to chlorine is shown in Figure 3. The data clearly show that reduction in chlorophyll fluorescence is chlorine-dose- and exposuretime-dependent. The percentage reduction in MFI per diatom cell was 4 and 16% immediately (5 min) and increased to about 35 and 50% after 60 min of treatment with 1.0 and 1.2 mg L-1 chlorine dose, respectively (Figure 2). Diatom cells subjected to 1 h term exposure were shifted to normal culture conditions to check whether the chlorophyll fluorescence recovered. MFI analysis after 18 h of incubation in f/2 growth medium or sterile seawater under normal culture conditions revealed no recovery but a further decrease in MFI to about 68% (Figure 3). Difference in MFI between control population and diatom cells subjected to 1.0 mg L-1 chlorine exposure for 5 min was statistically significant (n g 50; two-tail P ) 0.04). The difference in MFI between control population and diatom cells exposed to 1.0 mg L-1 chlorine dose for more than 5 min and 1.2 mg L-1 for 5 min and more was extremely significant (two-tail P < 0.005). The difference in MFI between
FIGURE 1. CLSM images showing reduction in autofluorescence in marine diatom Cocconeis scutellum cells exposed to 1.0 mg L-1 chlorine dose. One hour after treatment, the cells were transferred to fresh medium and imaged after 18 h incubation. A 63× objective was used for imaging. Excitation: Ar 488 nm. Emission: 630-750 nm. Images are maximum intensity projections of xy-slices obtained at 2 µm intervals. Scale bar ) 10 µm.
FIGURE 2. Reduction in mean fluorescence intensity per diatom cell after 15 min of exposure to different chlorine doses (error bars represent ( SD; n g 50). diatom cells treated with 1.0 and 1.2 mg L-1 chlorine dose for different exposure periods was also significant (two-tail P ) 0.02). However, when the diatom cells exposed to 1.0 and 1.2 mg L-1 were further incubated for 18 h (Figure 3), the difference in MFI between the two doses was not statistically significant (two-tail P ) 0.5). This shows that transfer to fresh medium, subsequent to chlorine insult, does not result in recovery of the cells independently from the biocide dose (1.0 or 1.2 mg L-1). Results based on in situ analysis at single-cell level, therefore, clearly demonstrate that chlorine causes significant autofluorescence inhibition in diatom cells. Transmission and fluorescence confocal microscopic images of individual diatom cells before and after chlorine exposure (1 mg L-1, 1 h) are shown in Figure 4. Damage to intracellular organization was clearly seen in cells subjected to chlorine exposure. Apart from the reduction in MFI, apparent crumbling and condensation of thylakoid structure in cells exposed to chlorine was evident (Figure 4). Surface plots of chlorophyll fluorescence collected from individual diatom cells prior to and after chlorine exposure are shown in Figure 5. The figure clearly shows that decrease in pixel
FIGURE 3. Mean fluorescence intensity after 0, 5, 15, and 60 min of contact time and subsequent transfer to fresh medium (error bars represent ( SD; n g 50).
FIGURE 4. Transmission and fluorescence confocal images of individual diatom cells before and after exposure to chlorine. A and B are control while C and D are cells exposed to 1.0 mg L-1 chlorine dose for 1 h. A and C are transmission images while B and D are maximum intensity projections of fluorescence confocal xy-slices. Arrows indicate sites of cellular damage. A 63× objective was used for imaging. Excitation: Ar 488 nm. Emission: 630-750 nm. intensities resulting from chlorine exposure gave a splintered appearance to the cellular structure of individual diatom cells. Chlorine is a nonselective strong oxidant and reacts rapidly with cell-components and affects metabolic processes (26). Intact chlorophyll molecules in the living cells exhibit red fluorescence, and the irreversible damage to cells is connected with a gradual disappearance of fluorescence (27). From the microscopic images, the reduction in MFI can be directly linked to the effect of chlorine on biomolecules, especially chlorophyll. Though no attempt was made in the present study to determine the targeted regions or degradation products of biomolecules, it is apparent that chlorination causes marked inhibition in chlorophyll fluorescence, inevitably leading to significant cellular damage. Esterase Activity. Cellular damage was further confirmed by metabolic imaging. Esterase activity in control and treated cells was studied by fluorescence imaging after staining with FDA, a technique increasingly being used to probe cell membrane integrity. Esterases are enzymes essential for normal cell functioning (22, 27, 28). FDA enters freely into the cells and is then hydrolyzed to brightly fluorescing fluorescein by intracellular esterases in viable cells. Fluorescein is retained only by the cells that have a fully functional VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Surface plots of chlorophyll fluorescence in single diatom cells. (A) Control cell and (B) treated cell (1.0 mg L-1 chlorine dose for 1 h). The 3-D plots were generated for single diatom cells from a stack of confocal xy-slices. X and Y axes are in µm. The Z-axis is pixel intensity value. A 63× objective was used for imaging. Excitation: Ar 488 nm. Emission: 630-750 nm.
FIGURE 6. Esterase activity in Cocconeis scutellum cells before and after treatment with chlorine. Images are an overlay of maximum intensity projections of autofluorescence (red) and FDA fluorescence (green) signals. Excitation: Ar 488 nm. FDA emission: 500-530 nm. Autofluorescence emission: 630-750 nm. A 63× objective was used for imaging. (A) Control cells before chlorine treatment, (B) cells treated with 1.2 mg L-1 chlorine dose for 1 h (Scale bar ) 10 µm). cell membrane. Any reduction in fluorescein fluorescence indicates either impaired esterase activity or loss of cell membrane integrity. In the present study, almost all cells in the control samples were FDA positive when stained and imaged using the CLSM (Figure 6A). This suggests that all cells of the control population were apparently healthy and metabolically active. Nevertheless, the fluorescence intensity of FDA varied among individual cells of the control population, possibly indicating heterogeneity in their metabolic status. In contrast, only a fraction of the treated (1.2 mg L-1 chlorine dose) cells was FDA positive (Figure 6B). The number of FDA positive cells decreased with an increase in chlorine dose and an increase in exposure time (Table 1). The reduction in mean percent FDA positive cells during biocide treatment was significantly different from that of the control
population (Table 1). This is a clear indication that metabolic activity of cells was severely affected by chlorine exposure. In contrast, the reduction in mean percent FDA positive cells treated with 1.0 mg‚L-1 chlorine dose was not statistically different from that of cells treated at 1.2 mg‚L-1 chlorine dose (two tail p-value)0.62). Moreover, a small subset of treated cells displayed higher fluorescence intensity of FDA per cell after 5 min exposure to 1.0 mg L-1 as compared to the control cells (data not shown). Other researchers have also observed a modest stimulation in FDA fluorescence when microalgae were exposed to copper (22, 23). It was proposed that the increased FDA fluorescence in diatom cells could be either due to a mere increase in the uptake of FDA due to cell membrane hyperpolarization or due to increased esterase activity. Since chlorine is known to change the cell membrane permeability (26), it is likely that stimulation in FDA fluorescence observed in the present study could be due to increased uptake of FDA in some cells. However, further experimentation is required to understand the cellular level changes brought about by chlorine in microalgae. The CLSM-based technique, reported here for the first time for ecotoxicity assessment, has the following advantages as compared to the standard growth inhibition tests: (i) quantifying the effect at single-cell level, (ii) localization of damage sites, (iii) ability to distinguish metabolically active and inactive cells, (v) use of environmentally relevant concentrations of toxicant, and (iv) use of dilute algal suspensions (or natural water samples). With the help of metabolic imaging using FDA, it is possible to monitor delayed mortality or posttreatment recovery of cells, as the case may be. In this study, we examined the effect of in-use concentrations of chlorine, an oxidizing biocide, on microalgae using a diatom as a model to understand the effect at single-cell level. Additional experiments employing other
TABLE 1. Percentage of Diatom Cells Stained by Fluorescein Diacetate (FDA) after Exposure to Chlorine chlorine dose (mg L-1)
incubation time (minutes)
% FDA positive cellsa
total number of cells counted (n)
two tail p-valueb
0 (control) 1.0
0-15 5 15 5 15 5 15
100 (0.0) 46 (5.3) 25 (0.6) 46 (3.2) 37 (11.2) 8 (0.1) 4 (0.8)
101 70 53 63 52 90 86
0.09 0.003 0.02 0.08 0.0005 0.003
1.2 3.6 a
The values indicate mean (SD in parentheses).
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The decrease in FDA positive cells was compared with control population using paired t-tests.
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stressors and microalgal species are necessary before the method can be fully developed into an acceptable ecotoxicological protocol. An added advantage is that this technique may allow assessment of toxicity to intact attached microalgae (biofilms or periphyton) in a nondestructive noninvasive manner. In such cases, CLSM can also allow assessment of the effect of toxicants in x, y, z, and t dimensions (spatial distribution of effect and temporal changes). Moreover, with the combination of specific fluorophores, the scope of assay can be further broadened to include quantitative changes in metabolic activity, membrane damage, exopolymeric substances production, and damage to chloroplasts. Our current research is aimed at the use of microalgal biofilm as model for ecotoxicity assessment using confocal microscopy.
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Received for review November 16, 2006. Revised manuscript received January 25, 2007. Accepted February 1, 2007. ES0627390
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