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Article history:
Received 12 June 2015
Received in revised form
18 December 2015
Accepted 21 December 2015
Available online 29 December 2015
Cyanobacterial harmful algal blooms occur in freshwater lakes, ponds, rivers, and reservoirs, and in
brackish waters throughout the world. The wide variety of cyanotoxins and their congeners can lead to
frequent exposure of humans through consumption of meat, sh, seafood, blue-green algal products and
water, accidental ingestion of contaminated water and cyanobacterial scum during recreational activities,
and inhalation of cyanobacterial aerosols. Cyanotoxins can also occur in the drinking water supply. In
order to monitor human exposure, sensitive analytical methods such as enzyme linked immunosorbent
assay and liquid chromatography-mass spectrometry are often used. Regardless of the analytical method
of choice, some problems regularly occur during sample collection, treatment, storage, and preparation
which cause toxin loss and therefore underestimation of the true concentration. To evaluate the potential
inuence of sample treatment, storage and preparation materials on surface and drinking water samples,
the effects of different types of materials on toxin recovery were compared. Collection and storage
materials included glass and various types of plastics. It was found that microcystin congeners LA and LF
adsorbed to polystyrene, polypropylene, high density polyethylene and polycarbonate storage containers, leading to low recoveries (<70%), cylindrospermopsin and saxitoxin did not adsorb to the containers tested. Therefore, this study shows that glass or polyethylene terephthalate glycol containers are
the materials of choice for collection and storage of samples containing the cyanotoxins cylindrospermopsin, microcystins, and saxitoxin. This study also demonstrated that after 15 min chlorine
decreased the concentration of microcystin LR to <40%, microcystin LA and saxitoxin to <15%, therefore
quenching of drinking water samples immediately upon sample collection is critical for accurate analysis.
In addition, the effect of various drinking water treatment chemicals on toxin recovery and the behavior
of those chemicals in the enzyme linked immunosorbent assays were also studied and are summarized.
2016 Elsevier Ireland Ltd. All rights reserved.
Keywords:
ELISA
Microcystins
Cylindrospermopsin
Saxitoxin
Adsorption to storage vessels
ADDA
1. Introduction
Cyanobacterial harmful algal blooms (CyanoHABs) occur in
freshwater lakes, ponds, rivers, and reservoirs, and in brackish
waters throughout the world. Organisms responsible include an
Abbreviations: ADDA, (2S, 3S, 8S, 9S, 4E, 6E)-3-amino-9-methoxy-2, 6, 8trimethyl-10-phenyl-4, 6-decadienoic acid); CyanoHABs, cyanobacteria harmful
algal blooms; ELISA, enzyme linked immunosorbent assay; gpg, grains per gallon;
HDPE, high density polyethylene; LC-MS, liquid chromatography-mass spectrometry; LOD, limit of detection; MC, microcystin; PETG, polyethylene terephthalate
glycol; PC, polycarbonate; PP, polypropylene; PS, polystyrene; RT, room temperature; SOP, standard operating procedure.
* Corresponding author.
E-mail address: frubio@abraxiskits.com (F. Rubio).
http://dx.doi.org/10.1016/j.cbi.2015.12.016
0009-2797/ 2016 Elsevier Ireland Ltd. All rights reserved.
46
Fig. 1. Structures of various common microcystin congeners. The variable amino acids
(labeled circle) largely determine the relative hydrophobicity of the whole molecule
and are also reected in the name and abbreviation of the respective microcystincongener.
the Fall of 2014. Drinking water samples were treated with 100 mg/
L of sodium thiosulfate as described in the Ohio EPA- ADDA by
ELISA Analytical Methodology SOP [23]. Samples were aliquoted
into glass bottles and fortied individually with 1 mg/L of MC-LR,
MC-LA or MC-LF; 0.5 mg/L cylindrospermopsin; or 0.1 mg/L saxitoxin. In samples fortied with saxitoxin, the Abraxis saxitoxin
sample preservation solution was added at a ratio of 1:10 prior to
toxin addition. Solutions were further aliquoted into pre-rinsed (3
times with fortied sample) glass, HDPE, PC, PETG, PP, or PS storage
containers, then tested immediately (within 2 h of spiking and
aliquoting into containers) as well as after storage at 4 C for 24, 48,
and 96/120 h.
2.3. Sodium thiosulfate quenching study
Samples were collected in glass containers. Immediately after
collection, drinking water samples were fortied with MC-LR or
MC-LA at 1.0 mg/L, cylindrospermopsin at 0.5 mg/L, or saxitoxin
(after sample preservation) at 0.1 mg/L. Samples were then split, one
set treated with 100 mg/L sodium thiosulfate, and both sets stored
and tested for the time periods specied above.
2.4. Drinking water chemicals assay tolerance
Chemicals commonly used in the water treatment process
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Fig. 2. Effect of type of storage container on microcystin congener recovery. Waters fortied with 1 mg/L Microcystin-LR, LA, or LF (from left to right, respectively) and stored in
polyethylene terephthalate glycol (PETG), polystyrene (PS), polypropylene (PP), high density polyethylene (HDPE), and polycarbonate (PC) containers for 0, 24, 48, and 120 h Fig. 2a
deionized water, 2b. drinking water, and 2c. surface water. All container results reported as % recovery compared to recovery in glass.
Fig. 3. Effect of chlorine on cyanotoxin degradation over time. Effect of dissolved chorine at 0.7 ppm (without quenching) on the degradation of Microcystin-LR, -LA (1 mg/L) and
saxitoxin (0.1 mg/L) (Fig. 3a); and cylindrospermopsin (0.5 mg/L) (Fig. 3b) over time. Error bars represent SD from the mean of two individual experiments (n 2).
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Fig. 5. pH sample tolerance on cyanotoxin ELISAs. Recovery (%) of cyanotoxin ELISAs on surface water samples at various pH levels. Microcystin-LR was fortied with 1 mg/L,
cylindrospermopsin with 0.5 mg/L, and saxitoxin with 0.1 mg/L. Error bars represent SD from the mean of two individual experiments (n 2).
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Table 1
Microcystins-ADDA, microcystins (ADDA)-DM, saxitoxin, and cylindrospermopsin ELISAs water treatment chemicals assay tolerances.
Water treatment
chemical
Microcystins-ADDA
Microcystins (ADDA)-DM
Saxitoxin
Cylindrospermopsin
Aluminum Sulfate
9 gpg
10 gpg
10 gpg
25 gpg
25 gpg
25 gpg
10 ppm
10 ppm
250 ppm
250 ppm
Sodium Carbonate
(Soda Ash)
Potassium
Permanganate
Calcium Oxide (Lime)
Sodium Silicouoride
Sodium Chlorite
10 ppm with quenching using 1 mg 10 ppm with quenching using
sodium thiosulfate per 1 mL sampleb 1 mg sodium thiosulfate per 1 mL
sampleb
2000 gpg with pH adjustment
4 gpg
using ascorbic acid to within assay
pH tolerancea
10 ppm
10 ppm
10 ppm with quenching using
5 ppm with quenching using
0.1 mg sodium thiosulfate per 1 mL 0.1 mg sodium thiosulfate per
sampleb
1 mL sampleb
250 ppm
250 ppm
Sodium
Hexametaphosphate
2 ppm with ltering at time of
Carbonc
samplingc
a
b
c
Natural pH of solution outside of the tolerance level of the assay, therefore chemical tolerance levels determined after adjustment to within the pH tolerance of the assay.
Strong oxidizer which may affect toxin, therefore chemical tolerance level of assay determined after quenching solutions with sodium thiosulfate.
Carbon is commonly used to remove toxins from water samples, therefore tolerance level due to effect of carbon on toxin, not on assay performance.
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