Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs

Highlights

• Benthic hepatotoxins were detected in temperate Australian reservoirs.

• Phormidium was shown to produce cylindrospermopsins.

• Nostoc linckia was highlighted as a microcystin-producer in benthic mats.

Abstract

Cyanobacteria represent a health hazard worldwide due to their production of a range of highly potent toxins in diverse aquatic environments. While planktonic species have been the subject of many investigations in terms of risk assessment, little is known about benthic forms and their impact on water quality or human and animal health. This study aimed to purify isolates from environmental benthic biofilms sampled from three different drinking water reservoirs and to assess their toxin production by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR). Microscopic observation of the isolates allowed the identification of various filamentous cyanobacterial genera: Anabaena (benthic form), Calothrix and Nostoc from the Nostocales and Geitlerinema, Leptolyngbya, Limnothrix, Lyngbya, Oxynema, Phormidium and Pseudanabaena representing non-heterocystous filamentous cyanobacteria. The Phormidium ambiguum strain AWQC-PHO021 was found to produce 739 ng/mg of dry weight (d/w) of cylindrospermopsin and 107 ng/mg (d/w) of deoxy-cylindrospermopsin. The Nostoc linckia strain AWQC-NOS001 produced 400 ng/mg (d/w) of a microcystin analogue. This is the first report of hepatotoxin production by benthic cyanobacteria in temperate Australian drinking water reservoirs. These findings indicate that water quality monitoring programs need to consider benthic cyanobacteria as a potential source of toxins.

Introduction

Planktonic cyanobacteria can challenge drinking water management and treatment processes by growing exponentially and forming a large biomass also called a bloom. In addition, these phototrophic bacteria have the ability to produce toxic secondary metabolites that are released into the water after cell death or during water treatment (Falconer and Humpage, 2005, Falconer and Humpage, 2006). Over the past twenty-five years, knowledge of toxigenic cyanobacteria has developed to the stage that they are now recognised as a major risk factor for drinking water quality in Australia and worldwide (Falconer and Humpage, 2005, Falconer and Humpage, 2006, Hudnell, 2008).

While planktonic cyanobacteria have been the subject of many investigations in terms of risk assessment, little is known about benthic species and their impact on water quality or human and animal health. Benthic cyanobacteria are a part of biofilms growing on the surface of the sediment in water bodies. They are organised in mats or clusters, forming, with the help of other autotrophic microbes, the photosynthetic layer of epilithic biofilms (Quiblier et al., 2013). In these mats, thin filamentous micro-algae are woven into several juxtaposed layers creating a complex matrix. During periods of increased photosynthetic activity, air bubbles form within the biofilm, causing some segments to detach and float. The accumulation of floating mats close to banks has led to the consumption of high levels of toxins by animals, causing death in many cases (Gugger et al., 2005, Mez et al., 1997). The presence of these toxins at high concentrations in floating mats also represents a threat to human health (i.e. through cutaneous exposure and accidental ingestion) as the water sources investigated were mainly used for recreational purposes.

Studies worldwide established the production of known cyanotoxins by benthic species. In New Zealand and Europe, the highly potent neurotoxin anatoxin-a was detected in cyanobacterial mats (Cadel-Six et al., 2007, Edwards et al., 1992, Gugger et al., 2005, Wood et al., 2011).

Similarly, hepatotoxins have been detected in benthic cyanobacteria worldwide (Mez et al., 1997, Mohamed et al., 2006; Izaguirre et al., 2007, Seifert et al., 2007, Bormans et al., 2014, Fetscher et al., 2015). Microcystin-LR and -YR were identified in benthic mats in the Nile River and connected irrigation canals, demonstrating potential toxic issues for the use of this water as a source for irrigation and drinking water (Mohamed et al., 2006). In one instance, the production of microcystin (MC) by benthic Oscillatoriales was associated with cattle death in eleven separate alpine pasture sites in Switzerland (Mez et al., 1997). Occasionally MCs were detected in benthic mats in drinking water reservoirs (Izaguirre et al., 2007). Benthic cyanobacterial taxa also produce cylindrospermopsin (CYN) (Seifert et al., 2007, Bormans et al., 2014). Lyngbya wollei, a benthic cyanobacterial species, was established as the producer of CYN and deoxy-cylindrospermopsin (deoCYN) in Queensland (Australia) freshwater ecosystems (Seifert et al., 2007). While MCs are considered to be mostly retained inside the cell (Rapala et al., 1997), CYNs are excreted by cyanobacterial cells more extensively and extracellular concentrations often exceed intracellular content (Bormans et al., 2014). This makes the potential production of CYN by benthic species of particular interest. Indeed, the routine monitoring of the risk associated with cyanotoxins is primarily achieved by enumeration of problematic planktonic species in water samples. In the commonly applied alert level framework monitoring program, toxin analyses are conducted only if cell densities are above the Alert Level (i.e. 6500 cells/mL for Microcystis spp. and 15,000 cells/mL for Cylindrospermopsis raciborskii (Chorus, 2012)). In the absence of potential toxigenic culprits in the water column the presence of toxins is not routinely investigated. Therefore, in the absence of extensive toxicological data and a regular monitoring program tailored for toxigenic benthic cyanobacteria, the risk they represent to human and animal health is unknown and needs to be evaluated.

For the purpose of evaluating the risk associated with the production of highly potent hepatotoxins by benthic cyanobacteria in three Australian drinking water reservoirs, this study aimed to purify isolates from environmental benthic biofilms sampled from three different water bodies and to assess their production of known cyanobacterial hepatotoxins by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR).