Elsevier

Aquatic Toxicology

Volume 59, Issues 3–4, 24 September 2002, Pages 209-223
Aquatic Toxicology

Algal esterase activity as a biomeasure of environmental degradation in a freshwater creek

https://doi.org/10.1016/S0166-445X(01)00254-5Get rights and content

Abstract

This study investigated the potential for using algal esterase activity of Microcystis aeruginosa and Selenastrum capricornutum as a rapid measure of the biological effects of acid mine drainage (AMD) in a South Australian stream (Australia) also affected by sewage pollution and dry-land salinity. Algal bioassays were based on the non-fluorescent substrate, fluorescein diacetate (FDA) which is metabolised by esterases to the fluorescent product, fluorescein. Esterase activity was interpreted as the mean rate of conversion of FDA to fluorescein and expressed as a percentage of the rate achieved by control algae (%FDAC). Flow cytometry was used to measure the fluorescence of individual algal cells, enabling differentiation of three esterase activity states (low=S1, normal and stimulated) and calculation of the percentage of algal cells in each activity state relative to that found for control algae (e.g. %S1). Algal esterase activity responded rapidly to AMD-affected water but also to increased conductivity (associated with dry-land salinity) and nutrient concentrations (associated with sewage). Exposure to AMD-affected water for 1 h reduced %FDAC by 30–70%, and increased %S1 by 60–90%, a depression of esterase activity that was maintained over 24 h. A similar depression of esterase activity occurred in both algae exposed to comparatively high-conductivity water (ca. 20 mS cm−1) for 1 h but the algae recovered from this ‘shock’ within 24 h. The %FDAC of S. capricornutum increased from 66 to 158% of control values after a 24 h exposure to nutrient-enriched water sampled downstream from a sewage treatment plant, despite the fact that the alga was grown in nutrient-sufficient culture. The combination of cyanobacterial (M. aeruginosa) and green (S. capricornutum) algal cultures with exposure times of 1 and 24 h was successful in distinguishing between the three types of pollution. Correlation of esterase activity measures with water quality parameters indicated that the clearest and least equivocal biological measure of AMD for the study area was the %S1 for M. aeruginosa after a 24 h exposure. The use of the flow cytometer to define a low esterase activity state was therefore successful in clarifying the response to AMD-affected water. The study demonstrates the successful application of algal esterase activity bioassays, in combination with flow cytometry, to rapidly assess the toxicity of AMD-affected waters and to differentiate this response from the effects of other pollutants (increased nutrients and conductivity).

Introduction

Laboratory-based toxicity tests using algae generally rely on endpoints, e.g. growth rate or death, which take days to determine. Potentially more rapid bioassays measure the metabolic activity of an organism. Such measurements are focussed on a small part of the more complex sequence of metabolic processes that determine an organism's growth or death. The metabolic activity of micro-algae has been assessed via measurements of photosynthesis, respiration and ATP synthesis (Gilbert et al., 1992, Greene et al., 1994). A relatively new technique measures the activity of enzymes (e.g. peroxidases, β-galactosidase, esterases) using fluorogenic probes (Arsenault et al., 1993, Blaise and Menard, 1998). Fluorescein diacetate (FDA) is a suitable metabolic probe, because, it is readily absorbed by cells and metabolised by esterases. The resultant conversion to fluoroscein has been used as a measure of micro-algal esterase activity in, for example, Microcystis (Geary et al., 1997) and Selenastrum (Arsenault et al., 1993, Gala and Giesy, 1994, Blaise and Menard, 1998). The rate of FDA conversion to fluorescein is correlated with photosynthesis (Brookes et al., 2000a) and nutrient-limited growth (Brookes et al., 2000b), which validates the use of this assay to assess the metabolic activity of phytoplankton cells.

Flow cytometry is a powerful tool for rapidly measuring various optical properties of individual microscopic particles (Platt, 1989). With its extension to uses beyond the field of human medicine (Yentsch and Horan, 1989), has come the potential for viewing micro-algal populations according to, amongst other things, the fluorescent attributes of individual cells. The combined use of metabolic probes, such as FDA, with the analytical speed and flexibility of flow cytometry holds real potential for quicker tests of algal responses to toxic substances in water.

Acidification of natural freshwater ecosystems impacts at all trophic levels (Baker et al., 1990) and simplifies the food web (Locke, 1992). Acid leachate from sulfidic mine wastes is a special case insofar as it can have lower pH than is generally associated with natural acidic drainage (e.g. from peat) or acid rain. Also, this comparatively low pH is coupled with high concentrations of dissolved metals (Kelly, 1988). The ecological effects of acid mine drainage (AMD) are frequently, locally severe. Further, AMD represents a very important ‘export’ of environmental detriment from the mine site into drainage systems that are a crucial public amenity, from the viewpoint of water and food supply and in aesthetic terms.

This study evaluates algal FDA bioassays, coupled with flow cytometry, as a technique for measuring the short-term responses of algae to AMD sampled from a polluted Australian stream, and for distinguishing these responses from those associated with nutrient enrichment and increased conductivity/salinity.

Section snippets

Study area

Dawesley Creek is situated approximately 40 km east of Adelaide in South Australia (Australia) and drains via Mt Barker Creek into the Bremer river (Fig. 1). Dawesley Creek is polluted with secondarily treated sewage that can represent a major proportion of flow in the upper creek during the dry summer period. AMD, from a disused pyrite mine, enters the creek at Brukunga and, further downstream, the Bremer river is affected by dry-land salinity.

Several studies have shown that the biota (e.g.

Chemical characteristics of the Dawesley–Bremer catchment

Selected water quality parameters for the Dawesley–Bremer catchment are shown in Fig. 2(a–c). The impact of AMD from the Brukunga mine site on stream pH is clearly evident (Fig. 2a). The pH decreased to three immediately downstream of the mine site and remained less than 3.5 for at least a further 6 km to site 7. Subsequent field observations show that the pH probably remained low throughout the approximately 13 km stream section from the mine to the confluence of Nairne and Dawesley Creeks (

Esterase activity as a measure of the effects of AMD

Esterase activity assays are not yet widely used to examine the impact of water pollution on freshwater algae. In this study, algae representing the two essential cell-types, prokaryote and eukaryote, were exposed to a complex pollutant mix and their esterase activity determined after 1 and 24 h with the aid of flow cytometry. Two related measures of esterase activity were determined relative to control algae: conversion rate of FDA to fluorescein (%FDAC) and the number of cells in a ‘low

Conclusion

An in vivo bioassay, based on the metabolism of FDA to fluorescein, reliably measured the esterase activity of M. aeruginosa and S. capricornutum and reflected the short-term metabolic response of these algae when exposed to polluted water. The use of flow cytometry provided a rapid method of quantifying both the rate of FDA conversion to flourescein and the proportion of algal cells in each of three esterase activity states (reduced, normal and stimulated). These states were effective in

Acknowledgements

We thank the Australian Nuclear Science and Technology Organisation's Managing Mine Wastes Project for supporting this study. We thank Adam Sincock (Department of Geographical & Environmental Studies, The University of Adelaide) for valuable assistance in the field. Water quality analyses were undertaken by the South Australian Water Quality Centre (AWQC). Thanks also to Gordon Radcliffe and Peter Schultz of the AWQC for their assistance with the water quality methods and flow data,

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