Elsevier

Toxicon

Volume 150, August 2018, Pages 13-16
Toxicon

Short communication
Benthic periphyton from Pennsylvania, USA is a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a)

https://doi.org/10.1016/j.toxicon.2018.05.002Get rights and content

Highlights

  • Periphyton samples were collected from Pennsylvania, USA in 2016.

  • ELISA, Individual Variant Analysis (LC-MS/MS) & MMPB were used to measure Microcystins/Nodularins.

  • LC-MS/MS was used to characterize anatoxin analogs.

  • Anatoxin-a, Homoanatoxin-a were confirmed present in some samples.

  • Dihydrohomoanatoxin-a and Epoxyanatoxin-a were also tentatively identified.

Abstract

In 2016, the Pennsylvania Department of Environmental Protection conducted a limited survey of streams in the Susquehanna River basin in Pennsylvania, USA, to screen for microcystins/nodularins, anatoxin-a (ATX) and homoanatoxin-a (HTX). Testing revealed the presence of HTX in samples collected from the Pine Creek basin, with ATX present at lower levels. Microcystins/nodularins (MCs/NODs) were also tested and found to be concomitant, with NOD-R confirmed present by LC-MS/MS.

Introduction

Anatoxin-a (ATX) and homoanatoxin-a (HTX) have been responsible for animal poisonings around the world (Osswald et al., 2007). Due to its acute toxicological action and association with other waterborne toxins and pathogens, it is suspected that ATX related poisonings go underreported (Backer et al., 2013). Similarly, many monitoring programs are focused on the analysis of ATX, with HTX and associated analogs (e.g. epoxy and dihydro derivatives) largely ignored. ATX and HTX are both considered highly acute toxins, with an i. p. LD50 of 0.25 mg kg−1 (Rogers et al., 2005; Skulberg et al., 1992). Although the toxicity of the ATX related derivatives is considered much lower (Wonnacott et al., 1991), the concentrations of these derivatives can sometimes be much higher than their ATX/HTX counterparts (Heath et al., 2010; Mann et al., 2012).

Reported ATX and HTX producers include cyanobacterial genera Anabaena/Dolichospermum (Beltran and Neilan, 2000; Brown et al., 2016), Arthrospira (Ballot et al., 2004), Blennothrix (Méjean et al., 2010b), Cuspidothrix (Wood et al., 2007), Cylindrospermopsis (Vehovszky et al., 2009), Cylindrospermum (Sivonen et al., 1989), Microcoleus/Phormidium/Kamptonema (Gugger et al., 2005; Heath et al., 2010; Hemscheidt et al., 1995), Oscillatoria (Cadel-Six et al., 2009; Méjean et al., 2010a), Planktothrix (Viaggiu et al., 2004), Raphidiopsis (Hodoki et al., 2013), and Tychonema (Shams et al., 2015). Of which, benthic cyanobacteria (e.g. Microcoleus/Phormidium/Kamptonema, Oscillatoria, Blennothrix) are represented in literature as significant HTX producers. Benthically derived ATX and HTX have been reported geographically in areas such as California, USA (Puschner et al., 2008), New Zealand (Wood et al., 2010) and Europe (Cadel-Six et al., 2007; Edwards et al., 1992; Faassen et al., 2012), but have not been reported in the mid-Atlantic region of the United States. Since benthic mats can detach from their substrate and become available to animals along shorelines, including humans, the presence of ATX/HTX in the benthos is of toxicological and ecological importance.

In 2017, the Pennsylvania Department of Protection (PADEP) reported on periphyton collections made in 2013 and 2015 from Susquehanna, Ohio, and Delaware watersheds that were analyzed for microcystins/nodularins (MCs/NODs) (Foss et al., 2017). Periphyton was found to contain up to 5000 ng g−1 d. w. MCs/NODs (ELISA) with NOD-R confirmed present in many of the collections. Additionally, NOD-R was confirmed present in smallmouth bass juveniles and adult livers, indicating possible trophic transfer. The original investigation was briefly expanded to test samples collected from the Susquehanna River watershed in 2016 for MCs/NODs in the same manner, but to also include ATX and HTX analysis. The goal was to determine the potential for multiple cyanotoxin presence in periphyton mats.

Section snippets

Sample collection, preparation and extraction

Periphyton collections, preparations and extractions were all conducted as previously described, with slight modifications (Foss et al., 2017). A total of 8 periphyton collections were made from 8 sites July 26–28, 2016 (Table 1). Samples were lyophilized to dryness and homogenized to a powder. Extractions for free MCs/NODs and ATX/HTX were conducted on 0.10 gram subsets using 75% methanol in 0.1 M acetic acid (5 mL, 2×). MeOH was removed from extracts (60 °C N2) followed by dilution with

Results and discussion

This work revealed the presence of multiple cyanotoxins in benthic periphyton collected from the Susquehanna River (PA, USA). In addition to NOD, MCs were confirmed present in this system (Table 2). ATX/HTX were detected in Pine Creek samples, with one sample (Hamilton Bottom) containing ppm levels of both HTX and NOD. All sites positive for HTX also tested positive for dhHTX and epoxyHTX, while epoxyATX or dhATX were not detected. Chromatograms and spectra illustrating native ATX, HTX, and

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

The authors thank Kamil Cieslik and Sarah Fuller for their assistance in sample preparation and analysis. Also, the authors thank PADEP interns Kala Topping, Devin Radel, and Clair Swink for their field assistance. Funding for this work was provided by the Pennsylvania Department of Environmental Protection.

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