- Email: [email protected]
homoanatoxin-a)
Accepted Manuscript Benthic periphyton from Pennsylvania, USA are a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxin-a) Amanda J. Foss, Jeffery Butt, Mark T. Aubel PII:
S0041-0101(18)30177-6
DOI:
10.1016/j.toxicon.2018.05.002
Reference:
TOXCON 5883
To appear in:
Toxicon
Received Date: 23 February 2018 Revised Date:
1 May 2018
Accepted Date: 2 May 2018
Please cite this article as: Foss, A.J., Butt, J., Aubel, M.T., Benthic periphyton from Pennsylvania, USA are a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxina), Toxicon (2018), doi: 10.1016/j.toxicon.2018.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Benthic Periphyton from Pennsylvania, USA are a source for both hepatotoxins (Microcystins/Nodularin) and neurotoxins (Anatoxin-a/Homoanatoxin-a)
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Amanda J. Foss1*, Jeffery Butt2, Mark T. Aubel1
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GreenWater Laboratories/CyanoLab, 205 Zeagler Drive, Palatka, Florida 32177; [email protected]
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Pennsylvania Department of Environmental Protection, Bureau of Clean Water, Rachel Carson State Office Building, 400 Market Street Harrisburg, PA 17101; [email protected]
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* Corresponding Author: [email protected]; Tel.: +1-386-328-0882
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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 and 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.
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Keywords: anatoxin-a, homoanatoxin-a, microcystins, nodularin, benthic cyanobacteria, periphyton
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1. Introduction
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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
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association with other waterborne toxins and pathogens, it is suspected that ATX related
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poisonings go underreported (Backer et al., 2013). Similarly, many monitoring programs are
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focused on the analysis of ATX, with HTX and associated analogs (e.g. epoxy and dihydro
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derivatives) largely ignored. ATX and HTX are both considered highly acute toxins, with an
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i.p. LD50 of 0.25 mg kg-1 (Rogers et al., 2005; Skulberg et al., 1992). Although the toxicity of
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the ATX related derivatives is considered much lower (Wonnacott et al., 1991), the
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concentrations of these derivatives can sometimes be much higher than their ATX/HTX
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counterparts (Heath et al., 2010; Mann et al., 2012).
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Reported ATX and HTX producers include cyanobacterial genera
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Anabaena/Dolichospermum (Beltran and Neilan, 2000; Brown et al., 2016), Arthrospira
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(Ballot et al., 2004), Blennothrix (Méjean et al., 2010b), Cuspidothrix (Wood et al., 2007),
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Cylindrospermopsis (Vehovszky et al., 2009), Cylindrospermum (Sivonen et al., 1989),
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Microcoleus/Phormidium/Kamptonema (Gugger et al., 2005; Heath et al., 2010; Hemscheidt et
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al., 1995), Oscillatoria (Cadel-Six et al., 2009; Méjean et al., 2010a), Planktothrix (Viaggiu et
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al., 2004), Raphidiopsis (Hodoki et al., 2013), and Tychonema (Shams et al., 2015). Of which,
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benthic cyanobacteria (e.g. Microcoleus/Phormidium/Kamptonema, Oscillatoria, Blennothrix)
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are represented in literature as significant HTX producers. Benthically derived ATX and HTX
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have been reported geographically in areas such as California, USA (Puschner et al., 2008),
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New Zealand (Wood et al., 2010) and Europe (Cadel-Six et al., 2007; Edwards et al., 1992;
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States. Since benthic mats can detach from their substrate and become available to animals
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along shorelines, including humans, the presence of ATX/HTX in the benthos is of
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toxicological and ecological importance.
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In 2017, the Pennsylvania Department of Protection (PADEP) reported on periphyton collections made in 2013 and 2015 from Susquehanna, Ohio, and Delaware watersheds that
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were analyzed for microcystins/nodularins (MCs/NODs) (Foss et al., 2017). Periphyton was
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found to contain up to 5000 ng g-1 d.w. MCs/NODs (ELISA) with NOD-R confirmed present
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in many of the collections. Additionally, NOD-R was confirmed present in smallmouth bass
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juveniles and adult livers, indicating possible trophic transfer. The original investigation was
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briefly expanded to test samples collected from the Susquehanna River watershed in 2016 for
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MCs/NODs in the same manner, but to also include ATX and HTX analysis. The goal was to
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determine the potential for multiple cyanotoxin presence in periphyton mats.
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2.
Materials and Methods
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2.1.Sample collection, preparation and extraction
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Periphyton collections, preparations and extractions were all conducted as previously
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described, with slight modifications (Foss et al., 2017). A total of 8 periphyton collections were
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made from 8 sites July 26-28, 2016 (Table 1). Samples were lyophilized to dryness and
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homogenized to a powder. Extractions for free MCs/NODs and ATX/HTX were conducted on
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0.10 gram subsets using 75% methanol in 0.1 M acetic acid (5 mL, 2x). MeOH was removed
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from extracts (60°C N2) followed by dilution with deionized water (DI; 5 mL). ATX/HTX
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extracts were adjusted to pH>10 with 2 M NaOH directly prior to loading on pre-conditioned Page 3 of 13
ACCEPTED MANUSCRIPT Strata X SPE (PN 8B-S100-FCH, Phenomenex, Torrance, CA, USA). MCs/NODs were also
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clarified using Strata X, but without pH adjustment. Columns were rinsed with 2 mL 5%
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methanol and eluted using 90% acetonitrile. Elutions were blown to dryness, reconstituted in
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one mL deionized water and filtered (0.20 µm PVDF).
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Oxidation for MMPB formation was conducted on 0.10 gram subsets at final
concentrations of 0.2 M K2CO3, 0.1 M KMnO4 & 0.1 NaIO4 (5 mL) for 2 hours, stopped with
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sodium bisulfite (40% w/v) and centrifuged 10 min 1500 xg. Pellets were rinsed with 2 mL
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deionized water and pooled supernatant was sent through Strata X as previously described.
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Dried elutions were reconstituted in DI (1.5 mL), pH adjusted (<3) with 1 M HCl and loaded
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onto simplified liquid extraction columns. Elutions (10 mL ethyl acetate) were blown to
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dryness, reconstituted (1 mL; 5% MeOH) and filtered (0.20 µm PVDF).
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Quality control included pre-extraction matrix spikes for free MCs/NODs via ELISA &
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individual variant analysis (n=2, 100 ng g-1 MC-LR; National Research Council Canada,
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Halifax, Nova Scotia, CA), individual variant analysis internal standard fortification (n= 5;
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d5-MC-LF; Abraxis Kits, Warminster, PA, USA), MMPB (n=5; 5-1000 ng g-1 MC-LR), ATX
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(n=3; 10 – 50 ng g-1 ATX; National Research Council Canada, Halifax, Nova Scotia, CA), and
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HTX (n=3, 20 – 100 ng g-1 HTX; Novakits, Nantes, FR). Duplicate extractions of samples
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positive for HTX were conducted. Standard addition (matrix) curves were utilized in
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quantitation of ATX, HTX and total MCs/NODs via MMPB (MC-LR, pre-oxidation). The
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internal standard method was utilized to quantitate individual variants in extracts. Dilutions on
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samples were conducted, as needed, in DI in order to remain within standard curve ranges for
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each method.
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2.2. Analysis Techniques
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Analysis for MCs/NODs were conducted as in Foss et al. (2017). An Adda ELISA (Abraxis Kits P/N 520011, Warminster, PA) was used to screen samples. Samples with
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detections (≥15 ng g-1) were analyzed for total MCs/NODs (via MMPB) and individual
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variants using LC-MS/MS. Separations for both analyses were conducted using a Thermo
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Scientific Surveyor HPLC system and Phenomenex Kinetex™ 2.6 µm C18 100 Å, LC Column
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(150 x 2.1 mm). Individual variant analysis was conducted using the same gradient described
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in Foss et al. (2017). MMPB was separated using mobile phase A (5% methanol in 0.05%
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acetic acid) and B (95% methanol in 0.05% acetic acid) at 0.2 mL min-1 as follows; 25% A 0-2
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min, 25-0% A over 3 minutes, back to 25% A over 3 minutes and held at 25% A for two
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minutes. A TSQ Quantum Access MAX Triple quadrupole mass spectrometer system was used
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in MMPB analysis (m/z 207131) and a LTQ XL™ Linear Ion Trap Mass Spectrometer was
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used to analyze 14 variants of microcystin ([DAsp3]MC-RR, MC-RR, MC-YR, MC-HtYR,
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MC-LR, [DAsp3]MC-LR, [Dha7]MC-LR, MC-HilR, MC-WR, [Leu1]MC-LR, MC-LY,
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MC-LA, MC-LW, MC-LF) and nodularin-R (NOD-R) as previously described.
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A LTQ XL™ Linear Ion Trap Mass Spectrometer was used in ATX and HTX analyses.
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Transitions monitored were m/z 166149, 131, 107, 91 (24% CE) for ATX and m/z 180163,
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145, 95, 80, 57 (26% CE) for HTX. Quantification ions for ATX (m/z 91) and HTX (m/z 57)
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were utilized. Separation was achieved using a XSelect HSS T3 column (2.1 x 150 mm,
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3.5-µm, PN 186006466, Waters Corp., Millford, MA, USA) with mobile phase C (0.2% acetic
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acid in DI) and D (0.2% acetic acid in MeOH) at 0.2 mL min-1 as follows: C-95% over 2
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minutes, C-95% - 60% over 7 minutes, back to C-95% over 2 minutes and held for 2 minutes. Page 5 of 13
ACCEPTED MANUSCRIPT Additional experiments were conducted to detect derivatives of ATX and HTX. LC-MS/MS
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scans of EpoxyATX (18250-200), dhATX (16850-170), epoxyHTX (19650-200) and
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dhHTX (18250-200) were conducted (CE 30-32%) and fragmentation patterns of major
118
peaks were compared to James et al. (2005).
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3. Results and Discussion
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This work revealed the presence of multiple cyanotoxins in benthic periphyton
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collected from the Susquehanna River (PA, USA). In addition to NOD, MCs were confirmed
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present in this system (Table 2). ATX/HTX were detected in Pine Creek samples, with one
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sample (Hamilton Bottom) containing ppm levels of both HTX and NOD. All sites positive for
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HTX also tested positive for dhHTX and epoxyHTX, while epoxyATX or dhATX were not
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detected. Chromatograms and spectra illustrating native ATX, HTX, and peaks corresponding
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to suspected dhHTX and epoxyHTX (MS/MS scans) can be viewed in Figure 1. When
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ATX/HTX spikes were compared to external curves (Figure 2), it was determined that all
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spikes were returned <40%,
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ATX has been shown to trophically transfer to fish and to elicit concentration dependent toxic effects (Carneiro et al., 2015; Osswald et al., 2013; Pawlik-Skowrońska et al.,
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2012). Evidence of periphyton derived NOD-R transfer to fish in this system has been shown
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(Foss et al., 2017), implicating the potential for ATX/HTX transfer as well. Since a
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combination of toxins are present in periphyton, the impact on animals exposed to this source
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may be considerable. Synergistic toxicological responses in animals exposed to cellular
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extracts or MC/ATX mixtures have been reported in other studies (Fitzgeorge et al., 1994;
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Osswald et al., 2009). Therefore, additional investigations in the extent of this concomitant
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presence are substantiated.
138 Acknowledgments: The authors thank Kamil Cieslik and Sarah Fuller for their assistance in
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sample preparation and analysis. Also, the authors thank PADEP interns Kala Topping, Devin
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Radel, and Clair Swink for their field assistance. Funding for this work was provided by the
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Pennsylvania Department of Environmental Protection.
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Conflicts of Interest: The authors declare no conflict of interest.
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Table 1: Samples Sites that were targeted in this work are listed below, including the dates
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collected and latitude/longitude information. All the samples were collected from the
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Susquehanna River watershed over a 3-day period. Latitude
Longitude
Lycoming Potter Snyder Clinton Lycoming Clinton Lycoming Clinton
7/27/2016 7/28/2016 7/26/2016 7/26/2016 7/26/2016 7/27/2016 7/27/2016 7/26/2016
41.310166 41.500484 40.810796 40.982519 41.283624 41.358845 41.343413 41.074561
-77.362892 -77.770576 -77.178595 -77.461336 -77.320901 -77.625531 -77.397010 -77.478045
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Little Pine Creek - Waterville Kettle Creek - Cross Fork Moyers Mill Run - near Walker Lake Fishing Creek - Summer Mt. Rd bridge Pine Creek - at Ramsey Run Hyner Run - Hyner Run State Park Pine Creek - at Browns Run Fishing Creek - Rt 2004 bridge
County
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Σ MCs/NODs
Total MCs/NODs
Σ MCs/NODs
Variants
ATX
HTX
dhHTX
epoxyHTX
ELISA
MMPB
MS/MS
Confirmed
MS/MS
MS/MS
MS/MS
MS/MS
87
+
+
Little Pine Creek - Waterville
ND
nt
nt
Kettle Creek - Cross Fork
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23.5
6.0
Moyers Mill Run - near Walker Lake
ND
nt
Fishing Creek - Summer Mt. Rd bridge
ND
nt
2,718
2,035
113
43.3
3,530
1,615
Hyner Run - Hyner Run State Park Pine Creek - at Browns Run Fishing Creek - Rt 2004 bridge MDL:
21.0
3.5
15
2.0
ND = Not detected above the method detection limit (MDL)
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a = above MDL but below method quantification limit (MQL) *congener dependent
a
a
nt
5
NOD-R
ND
ND
nt
nt
nt
nt
ND
ND
nt
nt
nt
nt
ND
ND
nt
nt
912
NOD-R
ND
97
+
+
41.2
-RR, -YR, -LR
ND
ND
nt
nt
945
NOD-R
15
7,685
+
+
none
ND
ND
nt
nt
5
10
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Pine Creek - at Ramsey Run
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Table 2: The below table illustrates the toxins detected in the periphyton samples collected in 2016. This includes the sum of microcystins/nodularins (MCs/NODs) as measured using an Adda ELISA, measured via MMPB and the sum of targeted MCs/NODs detected as intact variants. The variants detected (including NOD) are also listed. Anatoxins are also shown, including anatoxin-a (ATX), homoanatoxin-a (HTX) levels quantitated using standard addition. The tentatively positive (+) analogs dihydrohomoanatoxin-a (dhHTX) and epoxyhomoanatoxin-a (epoxyHTX) are also listed.
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ND
0.1-2.0*
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Figure 1: Extracted ion chromatograms (EIC) of native analytes detected in the Pine Creek Hamilton Bottom sample. ATX and HTX are shown with MS/MS spectra and were verified and quantitated using standard addition. MS/MS scans of other potential derivatives were conducted, with EpoxyHTX and dhHTX tentatively identified in the sample. Since standards are not currently available to confirm this observation, a comparison of spectra to previous literature (James et al., 2005) was utilized in the assessment.
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158 159 160 161 162 163
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Figure 2: Standard curves generated throughout the course of the study for ATX and HTX. Due to losses during extraction, lower responses were observed with matrix curves (solid lines; R2 = 0.9786 for ATX and R2 = 0.8256 for HTX) when compared to external curves (dashed). The matrix spikes (n=3) were analyzed twice and utilized in quantification of native ATX and HTX in samples. Low spike returns highlight the need for standard addition quantification techniques in complex matrices.
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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
S0041-0101(18)30177-6
DOI:
10.1016/j.toxicon.2018.05.002
Reference:
TOXCON 5883
To appear in:
Toxicon
Received Date: 23 February 2018 Revised Date:
1 May 2018
Accepted Date: 2 May 2018
Please cite this article as: Foss, A.J., Butt, J., Aubel, M.T., Benthic periphyton from Pennsylvania, USA are a source for both hepatotoxins (microcystins/nodularin) and neurotoxins (anatoxin-a/homoanatoxina), Toxicon (2018), doi: 10.1016/j.toxicon.2018.05.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Benthic Periphyton from Pennsylvania, USA are a source for both hepatotoxins (Microcystins/Nodularin) and neurotoxins (Anatoxin-a/Homoanatoxin-a)
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Amanda J. Foss1*, Jeffery Butt2, Mark T. Aubel1
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GreenWater Laboratories/CyanoLab, 205 Zeagler Drive, Palatka, Florida 32177; [email protected]
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Pennsylvania Department of Environmental Protection, Bureau of Clean Water, Rachel Carson State Office Building, 400 Market Street Harrisburg, PA 17101; [email protected]
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* Corresponding Author: [email protected]; Tel.: +1-386-328-0882
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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 and 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.
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Keywords: anatoxin-a, homoanatoxin-a, microcystins, nodularin, benthic cyanobacteria, periphyton
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1. Introduction
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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
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association with other waterborne toxins and pathogens, it is suspected that ATX related
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poisonings go underreported (Backer et al., 2013). Similarly, many monitoring programs are
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focused on the analysis of ATX, with HTX and associated analogs (e.g. epoxy and dihydro
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derivatives) largely ignored. ATX and HTX are both considered highly acute toxins, with an
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i.p. LD50 of 0.25 mg kg-1 (Rogers et al., 2005; Skulberg et al., 1992). Although the toxicity of
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the ATX related derivatives is considered much lower (Wonnacott et al., 1991), the
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concentrations of these derivatives can sometimes be much higher than their ATX/HTX
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counterparts (Heath et al., 2010; Mann et al., 2012).
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Reported ATX and HTX producers include cyanobacterial genera
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Anabaena/Dolichospermum (Beltran and Neilan, 2000; Brown et al., 2016), Arthrospira
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(Ballot et al., 2004), Blennothrix (Méjean et al., 2010b), Cuspidothrix (Wood et al., 2007),
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Cylindrospermopsis (Vehovszky et al., 2009), Cylindrospermum (Sivonen et al., 1989),
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Microcoleus/Phormidium/Kamptonema (Gugger et al., 2005; Heath et al., 2010; Hemscheidt et
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al., 1995), Oscillatoria (Cadel-Six et al., 2009; Méjean et al., 2010a), Planktothrix (Viaggiu et
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al., 2004), Raphidiopsis (Hodoki et al., 2013), and Tychonema (Shams et al., 2015). Of which,
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benthic cyanobacteria (e.g. Microcoleus/Phormidium/Kamptonema, Oscillatoria, Blennothrix)
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are represented in literature as significant HTX producers. Benthically derived ATX and HTX
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have been reported geographically in areas such as California, USA (Puschner et al., 2008),
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New Zealand (Wood et al., 2010) and Europe (Cadel-Six et al., 2007; Edwards et al., 1992;
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States. Since benthic mats can detach from their substrate and become available to animals
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along shorelines, including humans, the presence of ATX/HTX in the benthos is of
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toxicological and ecological importance.
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In 2017, the Pennsylvania Department of Protection (PADEP) reported on periphyton collections made in 2013 and 2015 from Susquehanna, Ohio, and Delaware watersheds that
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were analyzed for microcystins/nodularins (MCs/NODs) (Foss et al., 2017). Periphyton was
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found to contain up to 5000 ng g-1 d.w. MCs/NODs (ELISA) with NOD-R confirmed present
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in many of the collections. Additionally, NOD-R was confirmed present in smallmouth bass
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juveniles and adult livers, indicating possible trophic transfer. The original investigation was
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briefly expanded to test samples collected from the Susquehanna River watershed in 2016 for
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MCs/NODs in the same manner, but to also include ATX and HTX analysis. The goal was to
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determine the potential for multiple cyanotoxin presence in periphyton mats.
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2.
Materials and Methods
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2.1.Sample collection, preparation and extraction
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Periphyton collections, preparations and extractions were all conducted as previously
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described, with slight modifications (Foss et al., 2017). A total of 8 periphyton collections were
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made from 8 sites July 26-28, 2016 (Table 1). Samples were lyophilized to dryness and
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homogenized to a powder. Extractions for free MCs/NODs and ATX/HTX were conducted on
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0.10 gram subsets using 75% methanol in 0.1 M acetic acid (5 mL, 2x). MeOH was removed
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from extracts (60°C N2) followed by dilution with deionized water (DI; 5 mL). ATX/HTX
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extracts were adjusted to pH>10 with 2 M NaOH directly prior to loading on pre-conditioned Page 3 of 13
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clarified using Strata X, but without pH adjustment. Columns were rinsed with 2 mL 5%
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methanol and eluted using 90% acetonitrile. Elutions were blown to dryness, reconstituted in
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one mL deionized water and filtered (0.20 µm PVDF).
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Oxidation for MMPB formation was conducted on 0.10 gram subsets at final
concentrations of 0.2 M K2CO3, 0.1 M KMnO4 & 0.1 NaIO4 (5 mL) for 2 hours, stopped with
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sodium bisulfite (40% w/v) and centrifuged 10 min 1500 xg. Pellets were rinsed with 2 mL
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deionized water and pooled supernatant was sent through Strata X as previously described.
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Dried elutions were reconstituted in DI (1.5 mL), pH adjusted (<3) with 1 M HCl and loaded
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onto simplified liquid extraction columns. Elutions (10 mL ethyl acetate) were blown to
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dryness, reconstituted (1 mL; 5% MeOH) and filtered (0.20 µm PVDF).
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Quality control included pre-extraction matrix spikes for free MCs/NODs via ELISA &
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individual variant analysis (n=2, 100 ng g-1 MC-LR; National Research Council Canada,
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Halifax, Nova Scotia, CA), individual variant analysis internal standard fortification (n= 5;
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d5-MC-LF; Abraxis Kits, Warminster, PA, USA), MMPB (n=5; 5-1000 ng g-1 MC-LR), ATX
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(n=3; 10 – 50 ng g-1 ATX; National Research Council Canada, Halifax, Nova Scotia, CA), and
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HTX (n=3, 20 – 100 ng g-1 HTX; Novakits, Nantes, FR). Duplicate extractions of samples
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positive for HTX were conducted. Standard addition (matrix) curves were utilized in
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quantitation of ATX, HTX and total MCs/NODs via MMPB (MC-LR, pre-oxidation). The
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internal standard method was utilized to quantitate individual variants in extracts. Dilutions on
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samples were conducted, as needed, in DI in order to remain within standard curve ranges for
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each method.
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2.2. Analysis Techniques
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Analysis for MCs/NODs were conducted as in Foss et al. (2017). An Adda ELISA (Abraxis Kits P/N 520011, Warminster, PA) was used to screen samples. Samples with
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detections (≥15 ng g-1) were analyzed for total MCs/NODs (via MMPB) and individual
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variants using LC-MS/MS. Separations for both analyses were conducted using a Thermo
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Scientific Surveyor HPLC system and Phenomenex Kinetex™ 2.6 µm C18 100 Å, LC Column
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(150 x 2.1 mm). Individual variant analysis was conducted using the same gradient described
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in Foss et al. (2017). MMPB was separated using mobile phase A (5% methanol in 0.05%
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acetic acid) and B (95% methanol in 0.05% acetic acid) at 0.2 mL min-1 as follows; 25% A 0-2
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min, 25-0% A over 3 minutes, back to 25% A over 3 minutes and held at 25% A for two
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minutes. A TSQ Quantum Access MAX Triple quadrupole mass spectrometer system was used
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in MMPB analysis (m/z 207131) and a LTQ XL™ Linear Ion Trap Mass Spectrometer was
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used to analyze 14 variants of microcystin ([DAsp3]MC-RR, MC-RR, MC-YR, MC-HtYR,
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MC-LR, [DAsp3]MC-LR, [Dha7]MC-LR, MC-HilR, MC-WR, [Leu1]MC-LR, MC-LY,
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MC-LA, MC-LW, MC-LF) and nodularin-R (NOD-R) as previously described.
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Transitions monitored were m/z 166149, 131, 107, 91 (24% CE) for ATX and m/z 180163,
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145, 95, 80, 57 (26% CE) for HTX. Quantification ions for ATX (m/z 91) and HTX (m/z 57)
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were utilized. Separation was achieved using a XSelect HSS T3 column (2.1 x 150 mm,
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3.5-µm, PN 186006466, Waters Corp., Millford, MA, USA) with mobile phase C (0.2% acetic
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acid in DI) and D (0.2% acetic acid in MeOH) at 0.2 mL min-1 as follows: C-95% over 2
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minutes, C-95% - 60% over 7 minutes, back to C-95% over 2 minutes and held for 2 minutes. Page 5 of 13
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scans of EpoxyATX (18250-200), dhATX (16850-170), epoxyHTX (19650-200) and
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dhHTX (18250-200) were conducted (CE 30-32%) and fragmentation patterns of major
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peaks were compared to James et al. (2005).
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3. Results and Discussion
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This work revealed the presence of multiple cyanotoxins in benthic periphyton
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collected from the Susquehanna River (PA, USA). In addition to NOD, MCs were confirmed
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present in this system (Table 2). ATX/HTX were detected in Pine Creek samples, with one
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sample (Hamilton Bottom) containing ppm levels of both HTX and NOD. All sites positive for
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HTX also tested positive for dhHTX and epoxyHTX, while epoxyATX or dhATX were not
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detected. Chromatograms and spectra illustrating native ATX, HTX, and peaks corresponding
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to suspected dhHTX and epoxyHTX (MS/MS scans) can be viewed in Figure 1. When
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ATX/HTX spikes were compared to external curves (Figure 2), it was determined that all
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spikes were returned <40%,
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ATX has been shown to trophically transfer to fish and to elicit concentration dependent toxic effects (Carneiro et al., 2015; Osswald et al., 2013; Pawlik-Skowrońska et al.,
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2012). Evidence of periphyton derived NOD-R transfer to fish in this system has been shown
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(Foss et al., 2017), implicating the potential for ATX/HTX transfer as well. Since a
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combination of toxins are present in periphyton, the impact on animals exposed to this source
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may be considerable. Synergistic toxicological responses in animals exposed to cellular
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extracts or MC/ATX mixtures have been reported in other studies (Fitzgeorge et al., 1994;
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Osswald et al., 2009). Therefore, additional investigations in the extent of this concomitant
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presence are substantiated.
138 Acknowledgments: The authors thank Kamil Cieslik and Sarah Fuller for their assistance in
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sample preparation and analysis. Also, the authors thank PADEP interns Kala Topping, Devin
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Radel, and Clair Swink for their field assistance. Funding for this work was provided by the
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Pennsylvania Department of Environmental Protection.
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Conflicts of Interest: The authors declare no conflict of interest.
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Table 1: Samples Sites that were targeted in this work are listed below, including the dates
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collected and latitude/longitude information. All the samples were collected from the
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Susquehanna River watershed over a 3-day period. Latitude
Longitude
Lycoming Potter Snyder Clinton Lycoming Clinton Lycoming Clinton
7/27/2016 7/28/2016 7/26/2016 7/26/2016 7/26/2016 7/27/2016 7/27/2016 7/26/2016
41.310166 41.500484 40.810796 40.982519 41.283624 41.358845 41.343413 41.074561
-77.362892 -77.770576 -77.178595 -77.461336 -77.320901 -77.625531 -77.397010 -77.478045
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Little Pine Creek - Waterville Kettle Creek - Cross Fork Moyers Mill Run - near Walker Lake Fishing Creek - Summer Mt. Rd bridge Pine Creek - at Ramsey Run Hyner Run - Hyner Run State Park Pine Creek - at Browns Run Fishing Creek - Rt 2004 bridge
County
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Σ MCs/NODs
Total MCs/NODs
Σ MCs/NODs
Variants
ATX
HTX
dhHTX
epoxyHTX
ELISA
MMPB
MS/MS
Confirmed
MS/MS
MS/MS
MS/MS
MS/MS
87
+
+
Little Pine Creek - Waterville
ND
nt
nt
Kettle Creek - Cross Fork
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23.5
6.0
Moyers Mill Run - near Walker Lake
ND
nt
Fishing Creek - Summer Mt. Rd bridge
ND
nt
2,718
2,035
113
43.3
3,530
1,615
Hyner Run - Hyner Run State Park Pine Creek - at Browns Run Fishing Creek - Rt 2004 bridge MDL:
21.0
3.5
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2.0
ND = Not detected above the method detection limit (MDL)
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a
nt
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NOD-R
ND
ND
nt
nt
nt
nt
ND
ND
nt
nt
nt
nt
ND
ND
nt
nt
912
NOD-R
ND
97
+
+
41.2
-RR, -YR, -LR
ND
ND
nt
nt
945
NOD-R
15
7,685
+
+
none
ND
ND
nt
nt
5
10
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Pine Creek - at Ramsey Run
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Table 2: The below table illustrates the toxins detected in the periphyton samples collected in 2016. This includes the sum of microcystins/nodularins (MCs/NODs) as measured using an Adda ELISA, measured via MMPB and the sum of targeted MCs/NODs detected as intact variants. The variants detected (including NOD) are also listed. Anatoxins are also shown, including anatoxin-a (ATX), homoanatoxin-a (HTX) levels quantitated using standard addition. The tentatively positive (+) analogs dihydrohomoanatoxin-a (dhHTX) and epoxyhomoanatoxin-a (epoxyHTX) are also listed.
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ND
0.1-2.0*
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Figure 1: Extracted ion chromatograms (EIC) of native analytes detected in the Pine Creek Hamilton Bottom sample. ATX and HTX are shown with MS/MS spectra and were verified and quantitated using standard addition. MS/MS scans of other potential derivatives were conducted, with EpoxyHTX and dhHTX tentatively identified in the sample. Since standards are not currently available to confirm this observation, a comparison of spectra to previous literature (James et al., 2005) was utilized in the assessment.
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Figure 2: Standard curves generated throughout the course of the study for ATX and HTX. Due to losses during extraction, lower responses were observed with matrix curves (solid lines; R2 = 0.9786 for ATX and R2 = 0.8256 for HTX) when compared to external curves (dashed). The matrix spikes (n=3) were analyzed twice and utilized in quantification of native ATX and HTX in samples. Low spike returns highlight the need for standard addition quantification techniques in complex matrices.
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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