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Please cite this article as: Rundberget, T., Gustad, E., Samdal, I.A., Sandvik, M., Miles, C.O. AConvenient and Cost-Effective Method for Monitoring Marine Algal Toxins with PassiveSamplers, Toxicon (2009), doi: 10.1016/j.toxicon.2009.01.010
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Accepted Manuscript
Title: A Convenient and Cost-Effective Method for Monitoring MarineAlgal Toxins with Passive Samplers
Authors: Thomas Rundberget, Eli Gustad, Ingunn A. Samdal, Morten
Sandvik, Christopher O. Miles
PII: S0041-0101(09)00046-4
DOI: 10.1016/j.toxicon.2009.01.010
Reference: TOXCON 3404
To appear in: Toxicon
Received Date: 3 July 2008
Revised Date: 6 January 2009Accepted Date: 16 January 2009
http://dx.doi.org/10.1016/j.toxicon.2009.01.010 -
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A Convenient and Cost-Effective Method for Monitoring Marine Algal
Toxins with Passive Samplers
Thomas Rundberget1, Eli Gustad
2, Ingunn A. Samdal
1, Morten Sandvik
1,
Christopher O. Miles1,3
1National Veterinary Institute, PB 8156 Dep., NO-0033 Oslo, Norway
2
Institute of Marine Research, Fldevigen Research Station, Fldevigen, N-4817 His, Norway
3AgResearch Ltd., Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand
*Corresponding author: National Veterinary Institute
Tel: +47 2321-6231; Fax: +47 2321-6201
E-mail address: thomas.rundberget@vetinst.no
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Abstract
Passive sampling disks were developed based on the method of MacKenzie et al. (2004) and
protocols were formulated for recovering toxins from the adsorbent resin via elution from
small columns. The disks were used in field studies to monitor in situ toxin dynamics during
mixed algal blooms at Fldevigen in Norway. Examples are given from time-integrated
sampling using the disks followed by extraction and high performance liquid
chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid
analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the
disks and toxin profiles in blue mussels ( Mytilus edulis) were compared with the relative
abundance of toxin-producing algal species. Results obtained showed that passive sampling
disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to
produce and convenient to use and, when combined with HPLC-MS or enzyme-linked
immunosorbent assay (ELISA) analysis, provides detailed time-averaged information on the
profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool
for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.
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Keywords:Dinophysis, okadaic acid, dinophysistoxin, azaspiracid, passive sampling,
shellfish toxin, algal toxin
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1. Introduction
Over the last decade there has been an increase in the commercial cultivation and exploitation
of shellfish along the Norwegian coast. Contamination of shellfish with biotoxins from micro-
algae can be a problem for public health not only in Norway, but world wide (Hallegraeff,
1993; Toyofuku, 2006; Camacho et al., 2007), and many countries regulate the biotoxins in
shellfish (FAO/WHO/IOC, 2005). The Norwegian marine biotoxin monitoring programme
involves phytoplankton identification and enumeration, together with analysis of shellfish
flesh. The Norwegian Food Safety Authorities have a public surveillance program for algal
toxins in mussels. During the 2007/2008 season the algal monitoring was performed weekly
while chemical analysis of shellfish was performed monthly, from February to December and
only at selected places (3540 locations), and the programme is not able to cover all of the
vast Norwegian coastline.
Analysis of biotoxins in the shellfish flesh is required to determine the safety of the product
for consumption. However, analysis of shellfish is time consuming, technically demanding
and expensive, so it is not ideal as a tool for monitoring the progress of toxigenic blooms. In
addition, many of the toxins are metabolised in shellfish during digestion and assimilation,
and the increased variety and complexity of the metabolite profile makes toxin quantification
even more challenging. Phytoplankton monitoring involves collecting a concentrated sample
of the algae, shipping the sample to a suitable laboratory, and then enumerating the
identifiable toxigenic species (Lund et al., 1958). Phytoplankton monitoring has the ability to
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monitoring and shellfish analysis has historically provided a reasonable degree of protection
to shellfish consumers in Norway and elsewhere (Hallegraeff, 1993; van Egmond et al., 1993;
Batoreu et al., 2005).
Recently, alternatives have been sought to improve marine biotoxin monitoring. Of these,
passive sampling methods have shown much promise as tools for measuring aqueous
concentrations of a wide range of priority pollutants. The first passive sampling methods were
aimed at monitoring the concentrations of dissolved inorganic compounds in surface water
(Benes and Steinnes, 1974). Since then, there has been a rapid development in the use of
passive sampling devices (Huckins et al., 1990; Sodergren, 1990; Alvarez et al., 2004). Some
of the general features of different passive sampling devices have previously been reviewed
(Vrana et al., 2005; Stuer-Lauridsen, 2005). In comparison to traditional water sampling,
passive samplers offer the ability to integratively sample a range of environmental
contaminants over an exposure period, mimicking biological uptake while potentially
avoiding the heterogeneity and clean-up problems implicit with biological matrices (Verhaar
et al., 1995; Kot-Wasik et al., 2007). Recently, MacKenzie et al. (2004) introduced the idea of
monitoring algal toxins by passively adsorbing them directly from seawater using solid-phase
adsorbents. These so-called solid-phase adsorption toxin tracking (SPATT) devices,
consisting of bags sewn from polyester mesh containing activated polystyrydivinylbenzene
resin, adsorb lipophilic algal toxins dissolved in seawater. The SPATT bags provide a more
convenient means to perform time-averaged sampling prior to, or during, algal blooms than
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present in the sample extracts. Also, since the devices adsorb toxins released directly from the
algae into the water, the toxin profile is much simpler than the metabolite profile usually
found in shellfish. This results in easier assays, fewer toxins to quantify, and lower detection
limits for the targeted toxins. The resin used in the SPATT bags was tested and validated by
MacKenzie et al. (2004) for a range of algal toxins found in New Zealand (pectenotoxin-2
(PTX-2), PTX-2 seco acid (PTX-2 SA), yessotoxin (YTX), ocadaic acid (OA) and
dinophysistoxin-1 (DTX-1)).
The suitability of this approach for monitoring algal toxins in Norwegian waters was
investigated. As part of the study, the practicality of the device was improved by introducing a
frame in which the HP-20 resin is restrained to form a passive sampling disk. This design is
simple, cheap, more easily assembled and disassembled than the sewn SPATT bags, and is
well suited to high throughput processing of samples. In the trials, results obtained from
analysis of the passive sampling disks were compared to those from shellfish analyses and
phytoplankton monitoring at Fldevigen in Norway. Because passive sampling devices
containing the HP-20 resin have been validated for analysis of PTXs, YTX, OA/DTXs and
azaspiracids (AZAs) (MacKenzie et al., 2004; Fux et al., 2008), no attempt was made to
perform validation in the present investigation. Parts of the work have been reported in a
preliminary form in an earlier communication (Rundberget et al., 2006).
2. Materials and Methods
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2004a), PTX-2 SA (Miles et al., 2004b), PTX-2 (Miles et al., 2004c), OA, DTX-1 and DTX-2
(Larsen et al., 2007) and a semi purified mixture of AZA-1, -2 and -3 (unpublished) were
available in our laboratory from previous work.
2.2 Passive sampling disks.
Passive samplers were constructed from 100-m nylon mesh (Sefar AG, Heiden, Switzerland)
folded in half, a 75 mm diameter plastic embroidery frame (Permin, Copenhagen, Denmark)
and HP-20 resin (DIAION HP-20, Mitsubishi Chemical Corporation, Tokyo, Japan). The
resin (3.0 g) was placed between the two layers of nylon mash, and clamped tightly in the
embroidery frame so as to form a thin layer of resin between the layers of mesh. A No. 2
fishing swivel (Mustad, Gjvik, Norway) was attached to the outer ring of the embroidery
frame to provide a point of attachment during deployment (Figure 1). The resin was activated
by soaking the packed disk in methanol for 15 min and washing in deionised water, as
described in the resin-manufacturers instructions. The activated passive sampling disks were
placed in an air-tight plastic bags and stored cold (but not below 0 C) prior to and after
deployment in the sea.
2.3 Extraction of toxins from disks.
The embroidery ring was opened, and the used resin was quantitatively transferred to a 25 mL
Varian Bond-elute reservoir fitted with a 20 m nylon frit (Varian, Palo Alto, CA) and
washed free of salts with 3050 mL deionized water. Excess water was drawn from the
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dryness in vacuo. The residue was dissolved in 1.0 mL 80% MeOH, centrifuged, and the
supernatant analyzed by HPLC-MS. Alkaline hydrolysis was performed by mixing 200 L of
5 M NaOH with 0.8 mL methanolic HP-20 extract. The mixture was left to react at 37 C for
45 min, followed by addition of 210 L of 5 M HCl. Samples were filtered through 0.2 m
Spin-X filters prior to chromatographic analysis.
2.4 Field trials.
Trials were performed at the Marine Research Institute, Fldevigen, on the south-west coast
of Norway. Passive sampling disks were taken from their packaging and deployed by
attaching them to a fixed point at 1 m depth, and leaving them for the required time. The disks
were then rinsed briefly with fresh tap water, sealed in an air-tight plastic bag, and shipped to
the laboratory for analysis. Simultaneously, shellfish were harvested weekly and kept at 20
C prior to analysis, while algal cell counting was performed 3 times weekly.
2.5 Shellfish samples.
Frozen samples of blue mussels (Mytilus edulis) were thawed, and the flesh was removed
from the shells and homogenized using an Ultra Turrax
homogenizer (IKA
, Werke GmbH
& Co. KG, Staufen, Germany). The homogenates were stored at 20 C until extracted.
Homogenized shellfish (2 g) was extracted three times with 6 mL methanol by vortex mixing
for 2 min, and centrifuged at 2500 g for 5 min between extractions. The three extracts were
combined in a 20 mL volumetric flask, and the volume was adjusted to 20 mL with methanol.
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2.6 Algal cell counts.
Phytoplankton is routinely monitored in Fldevigen Bay three times per week. Every
Monday, Wednesday, and Friday, samples for enumeration and identification of
phytoplankton were taken as an integrated sample using a flexible hose, from 03 m depth,
from the same location as mussels and passive samplers. The water sample was preserved
using neutral Lugols solution. Smaller flagellates and algae in high concentration were
counted under the light microscope using a PalmerMaloney chamber (200 magnification),
with a detection limit of 104
cells/L (Palmer and Maloney, 1954). Larger dinoflagellates were
counted on semitransparent filters according to the description of Fournier (1978).
Examination in light microscope (100 magnification) was performed on 50 mL of the
sample that was gently filtered onto the filter for cell-counting, giving a detection limit of 20
cells/L.
2.7 HPLC-MS analysis.
Liquid chromatography was performed on a Symmetry C18 column (3 m, 50 2.1 mm)
(Waters, Milford, MA) using a Waters 2670 HPLC module. Separation was achieved by
linear gradient elution, starting from acetonitrilewater (35:65 v/v, both containing 5 mM
ammonium formate and 0.01% formic acid) rising to 100% acetonitrile over 10 min, held for
5 min, then switched back to the start-eluent. The HPLC system was coupled to a Quattro
Ultima Pt triple quadrupole mass spectrometer operating with an electrospray ionization (ESI)
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collision energy settings were optimized while continuously infusing (syringe pump) 20
ng/mL of the toxin standards at 3 L/min. Detection of the analytes was performed by
multiple reaction monitoring (MRM) in either positive (AZA-1 842.5>672.5, AZA-2
856.5>672.5, AZA-3 828.5>658.5, PTX-2 876.5>823.5, PTX-2 SA 894.5>823.5, PTX-12
874.5>821.5, 20-methylSPX-G/SPX-C 706.5>164.2) or negative (OA/DTX-2 803.5>255.1,
DTX-1 817.5>255.1, YTX 1141.5>1061.5) ionization mode. Except for PTX-12 and 20-
methylSPX-G/SPX-C, and DTX-2, which were quantified from calibration curves of PTX-2
and OA, respectively, all toxins were quantified using external calibration curves of standard
specimens dissolved in 80% MeOH.
3. Results and discussion
3.1 Practical aspects of the improved disks
The HP-20 resin used in the disks has been tested and validated for a range of lipophilic
biotoxins by others (MacKenzie et al., 2004; Fux et al., 2008), and no attempts were made to
perform validation in this study. The main improvement over the SPATT bags of MacKenzie
et al. (2004) lies in the design of the frame in which the HP-20 resin is retained. This design
simplifies the preparation of the activated disks, their deployments and the subsequent toxin
extraction compared to the sewn SPATT bags. The new design was quick and easy to use, the
frames and algal mesh could be washed and reused, and the frames hold the resin in a
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water in much the same way as mussels might. However, the disks have the advantage that
there is no toxin metabolism, they are more easily stored and cheaper to transport, and provide
a much cheaper and cleaner extract for the analytical laboratory.
The adsorption rate of lipophilic toxins from sea water by HP-20 resin is fast. MacKenzie et
al. (2004) found that after only 3.5 h exposure, significant amounts of toxins were adsorbed
on the resin even though theDinophysis cell numbers were low (100 cells/L). In the
Norwegian trial there was a mixed bloom containing high amounts of different flagellates and
microalgae, typically ca 13 106
cells/L and theD. acuta andD. acuminata numbers ranged
from 100360 cells/L. In this period the HP-20 material also became dark green, indicating a
high concentration of algal pigments in the water. It can not be ruled out that the HP-20
material can become saturated or that the 100 m nylon mesh can clog during the exposure
time and consequently the toxin levels can be underestimated. This needs further
investigation.
3.2 Sample preparation.
Recovery of the lipophilic algal toxins from the HP-20 resin was straight forward. A fresh
water rinse was necessary, prior to elution with MeOH, to remove salts which may disturb
ionization in the HPLC-MS. The ESI interface on the mass spectrometer is susceptible to salt
effects (Gustavsson et al., 2001), and a high salt content can influence the relative intensities
of the H+, NH4
+, and Na
+adducts ions used for MRM quantitation of the toxins. The resulting
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It was found necessary to elute the 3 g of HP-20 resin with at least 2 10 mL of methanol to
fully recover the adsorbed toxins and this is in accordance with the findings of Fux et al.
(2008). It was also important to use a low flow rate through the column, typically 12 bed-
volumes per hour (Manufacturers recommendation). Elution with 23 mL of solvent gave a
diluted sample, but a concentration step can be included depending on the required detection
limits and the sensitivity of the HPLC-MS system. The detection limits obtained with the
instrument used in this work were typically 0.10.3 ng/disk, depending on the toxin. By
omitting the concentration step and adjusting the extract volume to 25 mL, detection limits of
about 25 ng/disk were obtained.
3.3 Toxin profile of disks versus cell counts and blue mussels.
The OA/DTX concentrations in the disks and blue mussels, andDinophysis spp. (D. acuta
andD. acuminata) cell concentrations in the water, are shown in Figure 2. The amount of
OA/DTXs in the disks fluctuated from 120 to 660 ng/g disk, with maxima in weeks 30, 34
and 38. The cell numbers ofD. acuta andD. acuminata also fluctuated during this period,
with numbers ranging from 0 to 360 cells/L and one major peak around week 29. In shellfish,
the sum of both free OA and DTX and their esters was about 65 ng/g at the beginning of the
trial and about 220 ng/g when the trial ended, with peaks at weeks 34 and 40. During the
monitoring period (weeks 2841), three events can be described. The first was the increase of
Dinophysis cell densities and OA/DTX levels in the disks around week 30, but the toxin levels
in the shellfish did not show a corresponding increase (Figure 2). The reason for this might be
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In the second incident, in weeks 3334, levels of OA/DTXs in the disks increased to ca 650
ng/g, and theDinohpysis counts also rose to a moderate level (ca 100 cells/L) in weeks 33 and
34 (Figure 2). The level of OA/DTXs in the shellfish reached a peak in week 34, as did the
levels of OA/DTXs in the disks. OA/DTXs decreased in the shellfish in the following weeks
(3536), when theDinophysis cell numbers and toxin level in the disks also declined.
Depuration of algal toxins from shellfish is poorly understood (Duinker et al., 2007). Passive
samplers could be a useful tool in studies of the depuration of toxins in shellfish through
improved monitoring of the toxin-exposure of the investigated shellfish.
The third event occurred when OA/DTXs in the disks and levels ofDinophysis in the water
reached a maximum in week 38 and 39, respectively (Figure 2). Levels of OA/DTXs in the
shellfish increased in week 39 and reached a maximum in week 40. During this period the
Dinophysis numbers were moderate (ca 140 cells/L) but the amount of other algae was lower
(typically ca 1 106
cells/L) than earlier in the period when the algae population was typically
23 106 cells/L.
Based on these three events, it is difficult to recommend the passive samplers as an early
warning tool. The first incident had increased toxin levels in the disks, with no corresponding
increase in the shellfish. However, in the second and third events there was a marked increase
in OA/DTXs in the disks (weeks 33 and 38) some time before the levels in the shellfish were
observed to increase (weeks 34 and 39).
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week 34 and 38 had 120 (120D. acuminata and 0D. acuta) and 140 (20D. acuminata and
120D. acuta) cells/L, respectively. Algal cell-counting precision is usually good at high cell
densities if over 200 cells of the target species are counted, but poor at low cell densities when
fewer cells are counted (Lund et al., 1958). Except for in weeks 29 and 30, levels of
Dinophysis were below 200 cells/L (corresponding to only 10 cells counted), and this may
account for the lack of a precise correlation between theDinophysis cell counts and the toxin
levels in the disks and shellfish. Lindal, et al 2008 reported substantial variations in toxin
content of bothD. acuta and D. acuminata due to population density and environmental
variations and this may also affect the toxin levels found in the disks and shellfish compared
to the numbers of algae counted.
One difficulty with algal counting as a monitoring tool is that algal blooms can be short-lived
and mobile, and thus occur between algal samplings. This is especially so at locations prone
to tidal flows and/or exposed to wind and wave motion such as in Fldevigen where this trial
was performed. Passive sampling disks should be a valuable tool at such locations, where the
algal counts can change quickly from noDinophysis, up to 360 cells/L and back to a few
cells/L again during one week (Figure 2B). With passive samplers, the water column is
continuously being sampled and hence provides an integrated measurement of toxin levels
throughout the exposure period.
Another problem with algal counting is that it can only provide effective monitoring for toxins
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HP-20 resin has been shown to adsorb OA/DTXs, PTXs, and YTX in New Zealand waters
(MacKenzie et al., 2004) and recently Fux et al. (2008) detected AZAs in Irish waters using
HP-20. In northern European waters, the AZA group is commonly detected in shellfish and is
often present at levels that make shellfish unsuitable for consumption (Hess et al., 2005;
Aasen et al., 2006). During the summer of 2005, blue mussels at Fldevigen contained low
levels of AZA-1, AZA-2, AZA-3 and AZA-6, in a ratio of approximately 3:1:1:0.3
respectively, with concentrations of 2050 g/kg (Figure 4). In the disks, however, only
AZA-1 and AZA-2 (in a ratio of ca 5:1), and no AZA-3 or AZA-6, were detected (Figure 4).
Similarly, Fux et al. (2008) found AZA-1 and AZA-2 in a ratio of ca 4:1 together with traces
of AZA-3, and recently Krock et al. (2008) isolated and cultured an alga producing AZA-1,
AZA-2 and an isomer of AZA-2 but not AZA-3 or AZA-6. This suggests that AZA-3 and
AZA-6 may be produced by metabolism of ingested AZA-1 and AZA-2. Little is known
about the formation and metabolic transformation of the AZAs, but in shellfish a whole range
of AZA analogues has been identified (Satake et al., 1998; Ofuji et al., 1999; Ofuji et al.,
2001; James et al., 2003; Rehmann et al., 2008).
3.5 Detection of SPXs in the disks
A spirolide, most likely 20-methylSPX-G (Aasen et al., 2005), was also detected in the disks
throughout the trial, but only at low levels (ca 540 ng/disk, relative to PTX-2). The MRM
transition of 706.5>164.2, which corresponds to 20-methylSPX-G and SPX-C, was chosen
based on the findings of (Aasen et al., 2005), where 20-methylSPX-G was found to be the
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3.6 PTX-2 and PTX-2 SA in disks.
The detection of both PTX-2 and PTX-2 SA in the disks shows that formation of seco acids
can take place outside of the shellfish, before the algal cells and their PTX-2 are ingested.
PTX-2 SA has previously been observed as a constituent ofDinophysis (Daiguji et al., 1998;
James et al., 1999; Suzuki et al., 2001; MacKenzie et al., 2002)and it appears that conversion
of PTX-2 into PTX-2 SA can be mediated by enzymes present in the algae (MacKenzie et al.,
2002). Esterases responsible for the seco acid formation may leak from damaged algal cells
together with PTX-2, resulting in hydrolysis before adsorption to the HP-20 resin in the disk.
The ratio of PTX-2 to PTX-2 SA in the disks was typically 1:1, compared to 10:1 PTX-2 in
the trial of MacKenzie et al. (2004) in New Zealand, showing that the degree of PTX-2
conversion can vary greatly.
3.7 OA/DTXs and their esters in the disks.
Dinophysis spp. can contain OA diol esters (Suzuki et al., 2004; Miles et al., 2004b; Miles et
al., 2006). However, basic hydrolysis of extracts from passive samplers indicated that little or
no OA or DTX esters were present in the disks. MacKenzie et al. (2004) performed basic
hydrolysis on some of their samples to determine the levels of esterified DTXs, and found
only low amounts of esterified forms (030%) in their extracts. Miles et al. (2004b) isolated a
substantial amount of OA C8-diol ester from harvestedD. acuta cells, and this diol ester was
converted rapidly to OA by a homogenate from the hepatopancreas of the green-lipped mussel
(Perna canaliculus). Also it is known that the complex OA-ester DTX-4 is very short lived
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Concluding remarks.
The passive sampling disk system is cheap to produce and convenient to use and, when
combined with HPLC-MS or ELISA analysis, provides detailed time-averaged information on
the profile of lipophilic toxin analogues in the water. The passive sampling disks have now
been shown to accumulate azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins
and spirolides. The HP-20 resin in the samplers should also be able to accumulate other
lipophilic algal toxins such as brevetoxin and ciguatoxins. Passive sampling disks have the
potential to be a convenient tool for monitoring the exposure of shellfish and other bivalves to
toxigenic algae containing lipophilic toxins, and may also be useful for monitoring exposure
of aquatic ecosystems to these compounds as well as to a range of lipophilic pollutants.
Acknowledgement
This study was supported by the Norwegian Research Council grant 139593/140, by the
BIOTOX project (partly funded by the European Commission, through the 6th Framework
Programme contract no. 514074, priority Food Quality and Safety, and by the New Zealand
Foundation for Research, Science and Technology (FRST) International Investment
Opportunities Fund (IIOF contract number C10X0406).
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Captions for Figures
Figure 1. Fully assembled passive sampling disk (E), and its component parts: (A) 100 m
nylon mesh; (B) HP-20 resin; (C) inner and (D) outer rings of a 75 mm diameter embroidery
ring with (F) a No. 2 fishing swivel attached.
Figure 2. A) Concentrations of OA/DTXs in passive sampling disks and shellfish (ng/g), and
B)D. acuta +D. acuminata concentration (cells/L) in water for weeks 2841 of 2005.
Figure 3. Typical MRM HPLC-MS chromatogram of toxins in an extract from a passive
sampling disk (week 30) containing 20-methyl-SPX-G, AZA-1, AZA-2, OA, DTX-1, DTX-2,
PTX-2, PTX-12 and YTX.
Figure 4. Chromatogram of AZA profile in extracts of: (A) a passive sampling disk and: (B)
blue mussels (M. edulis).
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A
B
C
D
E
F
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0
100
200
300
400
500
600
700
Concentration
(ng/g) Sum DTX ng/g
disks
Sum DTX ng/g inshellfish
A
0
100
200
300
400
Cellco
unts(cell/L)
28 29 30 31 32 33 34 35 36 37 38 39 40 41
week
BD. acuta + D. acuminata
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0 5 10 15 20
20-methyl-SPX-G
PTX-2SA
OA, DTX-2
PTX-2
AZA-1
AZA-2
DTX-1
YTX
PTX-12a,b
Time (min)
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