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| This article will appear in a forthcoming issue of Trends in Biotechnology. | |
Abstract
Toxicity tests based on growth inhibition of algae and bacteria have long been used in conjunction with invertebrate and fish tests to assess the environmental impact of contaminants in aquatic systems [1]. The data from such measurements are used in ecological risk assessments, in the derivation of sediment and water quality guidelines, to investigate the bioavailability of contaminants, and to establish cause-effect relationships for particular toxicants [1].
Standard algal-growth inhibition tests have several limitations. These include:
Flow cytometry is a rapid method for the quantitative measurement of individual cells in a moving fluid. Thousands of individual cells are passed through a light source (lamp or laser) and measurements of light scatter and fluorescence properties are collected simultaneously. Although it is widely used in medical and oceanographic applications, flow cytometry has only recently been applied to ecotoxicological studies [2,3]. Microalgae are ideal for flow cytometric analysis because they are single-celled and contain photosynthetic pigments such as chlorophyll a, which autofluoresce when excited by blue light. Most previous studies, however, have used unrealistically high concentrations of toxicants (e.g. metals) or algal cells to obtain a measurable algal response. Here, we describe recent research on the development of more environmentally relevant toxicity tests with marine and freshwater unicellular algae.
Development of Multi-endpoint Toxicity Tests
Flow cytometry can perform multi-parameter analysis on a wide range of cell properties by measuring algal cell densities, light scatter signals and chlorophyll a autofluorescence, after excitation at 488 nm. Shifts in forward-angle light scatter (<15°) and side-angle light scatter (15-85°) indicate cell size and shape or granularity. Similarly, chlorophyll α fluorescence is detected at 660-700 nm, after setting thresholds to remove non-algal particles.
Through the use of biochemically specific fluorescent dyes, flow cytometry can also provide information about the physiological status of cells and the mechanisms of action of toxicants. For example, algal esterase activity can be determined by detecting cell fluorescence from 530-560 nm, after staining cells with fluorescein diacetate (FDA). Healthy cells take up FDA, which is then hydrolysed inside the cell by esterases, to produce fluorescent fluorescein, which is hydrophilic and retained by viable cells. Decreased fluorescein fluorescence indicates impaired enzyme activity or loss of cell membrane integrity and toxicity is expressed as a decrease in cell numbers in the healthy cell region (S2) as a percentage of controls [4]. Figure 1 shows the shifts in FDA fluorescence in the freshwater green alga Selenastrum capricornutum (now called Pseudokirchneriella subcapitata) in response to increasing concentrations of copper (added as copper sulfate) after a 3 and 24-h exposure.
Concentrations of copper that affect 50% of the cells or cause a 50% effect (EC50) for example a 50% shift of the cell population outside a defined healthy control region, for a range of endpoints determined by flow cytometry, are compared in three algal species (Table 1). Of the endpoints examined, cell division rate was the most sensitive to copper with 48 and 72-h EC50 values of <20 µg Cu l-1. This chronic test endpoint was, in general, more sensitive than the acute test endpoints at shorter exposure times including cell size changes, and inhibition of chlorophyll a and FDA fluorescence [5-7].
Development of Sediment Toxicity Tests
Before the use of flow cytometry, whole sediment bioassays with benthic algae had not been possible because the algal response could not be separated from the background sediment particles. Pore waters from sediments could only be tested with aquatic species but the results of these tests were often confounded by metal speciation changes from pore water oxidation during collection and storage.
Recently, toxicity tests based on esterase activity (FDA fluorescence) in the benthic alga Entomoneis cf punctulata were developed in our laboratory using flow cytometry to enable assessment of contaminant bioavailability in sediments. Although cell division rate is a more sensitive endpoint to copper than FDA fluorescence, it is often stimulated by ammonia released from sediments and this can mask the toxic effects of contaminants. By contrast, FDA fluorescence is rarely stimulated and is therefore a more suitable endpoint for whole sediment and pore water tests.
The test uses a benthic diatom Entomoneis cf punctulata, which is tolerant to a wide range of sediment physicochemical characteristics such as pH, salinity, particle size, ammonia and sulfide, but is sensitive to metals such as copper. Cells are exposed to either clean or contaminated sediment for 3 or 24 h, after which FDA is added and fluorescein fluorescence in cells is compared using flow cytometry (M.S. Adams, Bachelor Applied Sciences Honours Thesis, University of Technology, Sydney, Australia, 2000). Healthy cells plus FDA (no sediment) and dead algal cells plus FDA are used as additional controls. Sediments are ranked as toxic if fluorescence is <80% of the response in clean control sediments. This bioassay has been applied to testing a range of whole sediments and sediment pore waters collected from metal-contaminated areas in Australia. An in situ test, in which algae are placed in acrylic dialysis chambers, deployed with the membrane face down at the sediment-water interface, are currently being developed to avoid chemical and bioavailability changes to sediment during collection and storage. Such flow cytometry-based sediment tests with algae, in conjunction with higher organism tests, will enable better assessment of hazard and risk from contaminated sediments.
Development of Environmentally Relevant Aquatic Toxicity Tests
Flow cytometry has sufficient sensitivity to analyse cells at the low cell densities (100 cells ml-1) more typical of algal population densities in fresh and coastal marine waters. Standard algal bioassays, which use high initial cell densities of >104 cells ml-1, not only lack environmental realism but also might underestimate contaminant toxicity owing to the effects of the algae themselves on the speciation of contaminants in the test solutions. Flow cytometry-based toxicity tests at low cell densities, however, are more likely to indicate contaminant bioavailability and toxicity, without the confounding effects of changes in contaminant speciation.
We have shown that as the initial algal cell density increases, the toxicity of copper (added as copper sulfate) to the freshwater algae Chlorella sp. and Selenastrum capricornutum decreases [8]. Measured concentrations of intracellular and extracellular copper at 103, 104 and 105 cells ml-1 were determined after a 48-h and 72-h exposure by washing cells in dilute ethylene diamine tetraacetic acid (EDTA) to remove extracellular copper [8,9]. As cell density increased, less copper was bound at the cell surface, leading to less copper uptake into the cell and consequently less disruption of cell division. At higher cell densities, copper binding to algal cells and exudates led to a depletion of copper in solution and consequently lower toxicity. Standard algal tests should therefore use low cell densities to avoid these confounding effects on contaminant bioavailablity.
Development of Multi-species Tests
Flow cytometry could also be used to distinguish living algae from particulate matter, dead cells and other algal populations. In aquatic systems, algae rarely occur as single species populations (except in algal blooms) but as mixtures of different species. Apart from the effects of grazers, contaminant toxicity might be more influenced by algal-algal interactions than by contaminant speciation alone [1]. We are currently undertaking studies with cultured algae [10] to assess the effect of copper on different algal mixtures, versus the effects on single species. Flow cytometry enables separation of each algal population on the basis of their characteristic fluorescence signal, so that effects of contaminants can be assessed in multi-species bioassays.
Conclusions
The application of flow cytometry to ecotoxicology is enabling the development of more environmentally relevant aquatic and sediment toxicity tests with unicellular algae. Multiparameter, multispecies tests at low cell densities are now available to better assess the bioavailability of contaminants in aquatic systems. In future, these techniques could be extended to the development of other microbial bioassays using bacteria to assess sediment and water quality.
Current and Future Applications of Flow Cytometry in Aquatic Microbiology - reviews recent advances. From FEMS Microbiology Reviews, 2000, 24:4:429-448.
Go with the Flow: Use of Flow Cytometry in Environmental Microbiology - reviews current applications of flow cytometry. From FEMS Microbiology Ecology, 1997, 24:2:93-101.
Bioremedial Potential of Microbial Mechanisms of Metal Mobilization and Immobilization - details recent advances in understanding mechanisms of microbial metal transformations. From Current Opinion in Biotechnology, 2000, 11:271-279.
The Australasia Society for Ecotoxicology - offers conference and seminar news, articles, and links.
Purdue University Cytometry Laboratories - an extensive resource including meeting information, journals, protocols, and links on Flow Cytometry and Microbiology.
Archives of Environmental Contamination and Toxicology - provides online abstracts of articles.