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marine drugs
Article
Application of Six Detection Methods for Analysis of
Paralytic Shellfish Toxins in Shellfish from Four
Regions within Latin America
Andrew D. Turner 1 ,* , Sophie Tarnovius 1,2 , Robert G. Hatfield 1, Mickael Teixeira Alves 1,
Maggie Broadwater 3, Frances Van Dolah 3, Ernesto Garcia-Mendoza 4, Dinorah Medina 5,
Maria Salhi 5, Alejandra B. Goya 6, Fernanda Barrera 7, Daniel Carrasco 7, Ignacio Rubilar 7
and Benjamin A. Suarez-Isla 7
1Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, The Nothe, Weymouth,
Dorset DT4 8UB, UK; sophie.tarnovius@gmx.de (S.T.); Robert.Hatfield@Cefas.co.uk (R.G.H.);
Mickael.teixeiraalves@cefas.co.uk (M.T.A.)
2Technische Universität München, Walther-Meißner-Straße 3, 85748 Garching, Germany
3National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science Stressor
Detection and Impacts Division, Charleston, SC 29412, USA; maggie.broadwater@noaa.gov (M.B.);
franvandolah@gmail.com (F.V.D.)
4Departamento de Oceanografía Biológica, Centro de Investigación Científica y de Educación Superior de
Ensenada. Carr. Ens-Tij 3608, Ensenada, Baja California 22860, Mexico; ergarcia@cicese.mx
5Dirección Nacional de Recursos Acuáticos, PO Box 1612, Constituyente 1497, Montevideo 11200, Uruguay;
dmedina@mgap.gub.uy (D.M.); msalhi@mgap.gub.uy (M.S.)
6Marine Biotoxin Department, Mar del Plata Regional Laboratory, Agri-food Health and Quality National
Service (Senasa), Mar del Plata 7600, Argentina; agoya@senasa.gov.ar
7Laboratory of Marine Toxins, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile,
Santiago 8320000, Chile; mferbarr@uchile.cl (F.B.); dcarrasco@uchile.cl (D.C.); irubilar@med.uchile.cl (I.R.);
bsuarez@u.uchile.cl (B.A.S.-I.)
*Correspondence: Andrew.turner@cefas.co.uk; Tel.: +44-(0)-1305206636
Received: 13 November 2020; Accepted: 30 November 2020; Published: 3 December 2020
Abstract:
With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish
for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt
non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have
been developed which have been subjected to single-laboratory and multi-laboratory validation
studies, gaining acceptance as official methods of analysis and approval for use in some countries
as official control testing methods. The majority of validation studies conducted to date do not,
however, incorporate shellfish species sourced from Latin America. Consequently, this study sought
to investigate the performance of five alternative PSP testing methods together with the MBA,
comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included
a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD)
methods including both pre-column and post-column oxidation, liquid chromatography with tandem
mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total
of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay
were assessed. For the majority of samples, qualitative results compared well between methods.
Good statistical correlations were demonstrated between the majority of quantitative results, with a
notably excellent correlation between the current EU reference method using pre-column oxidation
LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins,
although the findings of high numbers of false-positive results and two false negatives highlighted
that some caution is still needed when interpreting results. This study demonstrated that effective
replacement methods are available for countries that no longer wish to use the MBA, but highlighted
Mar. Drugs 2020,18, 616; doi:10.3390/md18120616 www.mdpi.com/journal/marinedrugs
Mar. Drugs 2020,18, 616 2 of 30
the importance of comparing toxin data from the replacement method using local shellfish species of
concern before implementing new methods in official control testing programs.
Keywords: paralytic shellfish poisoning (PSP); LC-FLD; LC-MS/MS; MBA; RBA; toxin profiles
1. Introduction
Paralytic shellfish toxins (PSTs) are harmful neurotoxins originating from phytoplankton of the
genera Gymnodinium,Pyrodinium and Alexandrium that periodically accumulate in shellfish through
filter feeding. These may result in sickness and even fatalities following human consumption of
contaminated shellfish products [
1
,
2
]. The toxins are members of the saxitoxin family, which contain
over 55 structurally related compounds [
3
] (Figure 1). Toxicity relates to the action of the toxins on
voltage-gated sodium channels, leading to numbness, tingling sensations and nausea, with high doses
causing paralysis and death by asphyxiation [
4
,
5
]. The hydrophilic saxitoxins consist of three different
groups of compounds, as classified by their chemical structure: N-sulfocarbamoyl, decarbamoyl and
the carbamoyl toxins [
6
]. Other hydrophobic congeners are also known to exist, most notably in
bivalves exposed to Gymnodinium catenatum [7], including those from South America [8].
Mar. Drugs 2020, 18, x 2 of 32
use the MBA, but highlighted the importance of comparing toxin data from the replacement method
using local shellfish species of concern before implementing new methods in official control testing
programs.
Keywords: paralytic shellfish poisoning (PSP); LC-FLD; LC-MS/MS; MBA; RBA; toxin profiles
1. Introduction
Paralytic shellfish toxins (PSTs) are harmful neurotoxins originating from phytoplankton of the
genera Gymnodinium, Pyrodinium and Alexandrium that periodically accumulate in shellfish through
filter feeding. These may result in sickness and even fatalities following human consumption of
contaminated shellfish products [1,2]. The toxins are members of the saxitoxin family, which contain
over 55 structurally related compounds [3] (Figure 1). Toxicity relates to the action of the toxins on
voltage-gated sodium channels, leading to numbness, tingling sensations and nausea, with high
doses causing paralysis and death by asphyxiation [4,5]. The hydrophilic saxitoxins consist of three
different groups of compounds, as classified by their chemical structure: N-sulfocarbamoyl,
decarbamoyl and the carbamoyl toxins [6]. Other hydrophobic congeners are also known to exist,
most notably in bivalves exposed to Gymnodinium catenatum [7], including those from South America
[8].
Figure 1. Structures of saxitoxin analogues incorporated into testing methods from this study.
Occurrences of PSP-producing harmful algal blooms have been recognised along the Pacific and
Atlantic coasts of Latin America (LA) for many years [9–14]. In Argentina, Gymnodinium catenatum
was initially recorded in 1961-62 around Mar del Plata [15] and currently occurs in the northern
regions of the country. Alexandrium catenella (formerly A. tamarense) was identified as the cause of
Figure 1. Structures of saxitoxin analogues incorporated into testing methods from this study.
Occurrences of PSP-producing harmful algal blooms have been recognised along the Pacific and
Atlantic coasts of Latin America (LA) for many years [
9
–
14
]. In Argentina, Gymnodinium catenatum
was initially recorded in 1961-1962 around Mar del Plata [
15
] and currently occurs in the northern
regions of the country. Alexandrium catenella (formerly A. tamarense) was identified as the cause of
PSP outbreaks in 1980 and has been recorded annually from southern Argentina up to the coast
Mar. Drugs 2020,18, 616 3 of 30
of Uruguay [
16
–
20
]. Intense blooms of A. catenella have also been measured within the Beagle
Channel in southern Argentina [
21
–
23
]. Further up the Atlantic coast in Uruguay, repeated blooms of
A. catenella and G. catenatum have been identified regularly since 1991 and 1992, respectively [
24
–
26
],
with the subsequent identification of A. fraterculus [
9
]. Harmful algal blooms also severely impact the
Pacific coastal regions of North and Central America [
12
,
27
,
28
], where G. catenatum and Pyrodinium
bahamense var. compressum are typically associated with outbreaks [
29
–
32
]. Alexandrium species
including A. catenella have also been detected in Mexico [
32
–
34
], although none associated with
shellfish contamination [
35
,
36
]. In Chile, A. catenella has been reported since the 1970s [
9
,
11
]. HABs of
A. catenella have increased in their frequency, extension, duration and intensity [37,38].
High levels of PSTs in Latin American shellfish have resulted in a significant number of PSP
outbreaks. PSTs have been measured in a wide range of bivalve molluscs as well as other marine species
including gastropods [
39
–
44
]. During 1980 in Argentina, the PSP toxicity of mussels was reported
at levels equivalent to 312,048
µ
g STX eq./kg [
44
,
45
], with repeated events reported throughout the
next 15 years [
18
,
19
,
46
–
48
]. The toxicity of mussels harvested in Patagonia during 1992 was found
to reach a maximum of 1,272,000
µ
g STX eq./kg [
21
,
49
]. Consequently, the waters along the Atlantic
and Pacific coasts of LA are periodically affected by PST accumulation resulting in human health
risks [
19
,
45
,
50
–
58
]. PSTs have also been observed in Uruguayan shellfish, with maximum levels
recorded at 82,850
µ
g STX eq./kg during a 1991 bloom of A. tamarense [
59
–
61
] and at 14,780
µ
g STX
eq./kg following a 1992 bloom of G. catenatum [
26
], Medina, unpublished data]. Human illness due
to PSP-contaminated shellfish is also well known along the Pacific coast, with incidents reported as
far back as the 1970s in Mexico [
62
,
63
] with over 40 deaths and 1200 intoxications recorded between
1976 and 2002 [
12
,
34
]. Bivalve species implicated in outbreaks include oysters, clams, mussels and the
Geoduck clam, with impacts not only on human shellfish consumers, but also animal and ecosystem
health [
64
]. In Chile, high PSP toxicity has been measured in shellfish since the first outbreak in 1972 in
the south, causing intoxications and fatalities [
21
,
48
,
65
,
66
]. Since then, outbreaks have been found
on an annual basis between southern Patagonia and the more northerly regions around Chile [67,68].
Between 1972 and 2004, PSP-contaminated shellfish was responsible for the intoxication of 527 people in
Chile, with 32 fatalities [
65
] with additional impacts including mass mortalities of invertebrates [
68
] and
substantial socio-economic costs [
69
]. In recent years, blooms of A. catenella have been reported to have
expanded spatially as well as resulting in catastrophic incidents of human poisonings, including the
2016 “Godzilla-Red tide event” triggered by exceptional El Niño conditions with consequent drastic
socio-economic impacts [70,71].
To ensure consumer protection, monitoring of toxic phytoplankton and PSTs in shellfish is
a statutory requirement, including those countries exporting shellfishery products to the EU [
72
].
The statutory limit for PSTs in flesh is 800
µ
g saxitoxin equivalents (STX eq.) per kg of shellfish flesh [
73
]
as described by EC Regulation 853/2004. For many years, the official reference method in the EU and
LA for detecting PSTs has been the PSP mouse bioassay (MBA) [
74
,
75
]. The method has provided a
useful quantitative monitoring tool, although the method is known to be affected by low sensitivity,
poor reproducibility and is subject to matrix interferences [
5
,
76
]. In recent years, alternative chemical
or biomolecular methods have been tested and validated for PST detection. In 2006, a pre-column
oxidation (PreCOX) liquid chromatography with fluorescence detection (LC-FLD) [
73
,
77
–
79
] method
was validated and accepted as an alternative method of analysis for official control testing within the
EU (Regulation EC 2074/2005 as amended) [
80
] and since 1 January 2019 has become the EU reference
method [81,82].
Similarly, a post-column oxidation (PCOX) LC-FLD method was validated [
83
,
84
] and both LC-FLD
methods have been adopted by the AOAC as Official First Action methods (AOAC 2005.06 [
74
] and
AOAC 2011.02 [
85
]). The PreCOX method is implemented into the official control testing programmes
of European member states, the UK and New Zealand [
82
,
86
–
89
] with the PCOX method approved
for use in the US and Canada by the Interstate Shellfish Sanitation Conference [
90
]. In 2011, a PSP
receptor binding assay (RBA) was validated [
91
,
92
] and adopted by the AOAC as a first action method
Mar. Drugs 2020,18, 616 4 of 30
(AOAC 2011.27 [
93
]) for the analysis of mussels and clams. More recently, a method using hydrophilic
interaction liquid chromatography with tandem quadrupole mass spectrometry (HILIC-MS/MS) has
been developed and validated for PST testing in 12 different shellfish species [
94
,
95
] and has undergone
successful interlaboratory validation [
96
], with implementation into regulatory monitoring programmes
in New Zealand and Australia. Finally, various commercial antibody-based assays exist which are
capable of either qualitative or semi-quantitative determination of PSP in shellfish extracts. One of
these, produced by Scotia Rapid Testing Ltd., based upon an immunochromatographic format, is a
lateral flow assay (LFA) that has been single-laboratory validated [
97
] and tested on a range of shellfish
samples [98], and is utilised in the US and Mexico under certain scenarios for shellfish screening.
Some of these methods established as alternatives for PSP testing may be used by laboratories in
LA for routine official control testing. However, most regions in LA still rely on the MBA. Reasons for
this include the considerable efforts and expense required to set-up, validate and implement these
alternatives. The choice of method can be confusing as it is also highly dependent on both method
performance and the intended export market destination. LA regions intending to export mussels to
the US, for example, will be required to use either the MBA, RBA or PCOX LC-FLD method. In addition,
within the US legislation, there is the option for use of the Scotia PSP rapid immunochromatographic
assay for PSP screening, which offers countries exporting to the US another opportunity for cost-effective
toxin testing as part of their regulatory control system. Whilst the EU reference method for PSP is
now AOAC2005.06, LA regions have not changed national laws to enforce the implementation of
non-animal-based alternative methods.
A wide range of validation studies have been published for PSP testing methods in recent years,
but to date very few of these have been focussed on shellfish species typically harvested in LA.
Additionally, various authors have assessed the advantages and disadvantages of each of these PSP
testing methods, incorporating aspects of sample throughput and turnaround, method performance,
financial costs, practicalities and reagent/instrumentation/training requirements [
5
,
82
,
97
–
105
], but again
with a primary focus on regions where analytical costs are achievable.
Consequently, there is a need to establish comparative performance between potential regulatory
testing methods for PSP utilising shellfish samples harvested from LA in order to evaluate alternative
methods to the MBA which are appropriate for the species of relevance in the region. This study
therefore evaluated the alternative methods available for monitoring PSTs in shellfish from four
different countries within LA. A large range of shellfish species were assessed including mussels,
oysters, clams, cockles, scallops and marine gastropods. The total sample toxicities were assessed
following quantitation of PSTs using the MBA in comparison with the PreCOX LC-FLD
(AOAC 2005.06)
,
the RBA (AOAC 2011.27), the PCOX LC-FLD (AOAC 2011.02), the HILIC-MS/MS [
94
–
96
] and the LFA
manufactured by Scotia Rapid Testing Ltd. (chester, Canada) [97,98].
2. Results
2.1. Toxicities
Total PST concentrations were determined by PreCOX, PCOX and LC-MS/MS together with
PSP toxicities assessed directly by both MBA and RBA. Table S1 in the Supplementary Materials
tabulates all the results obtained for each individual sample. Out of the 349 shellfish samples analysed
by PreCOX LC-FLD in this study, total toxicities were found to vary enormously, with 62 samples
showing PSP <16
µ
g STX eq./kg and toxicities reaching a maximum of >400,000
µ
g STX eq./kg in a
mussel sample originating from Argentina. Table S1 illustrates the high toxicities found in mussels,
scallops and snails from Argentina, as well as mussels and clams from Chile, and mussels from Uruguay.
In total, 137 samples were found to contain total PSP above the regulatory maximum permitted limit
of 800 µg STX eq./kg (39%) as determined by PreCOX LC-FLD.
Analysis of a large number of shellfish exhibiting low or no detectable PST presence enabled
the assessment of toxin/toxicity results between multiple methods across a wide geographical extent.
Mar. Drugs 2020,18, 616 5 of 30
In total, 80 samples returned an MBA result of not detected, evidencing either an absence of toxicity or
total PST below the MBA LOD of ~320
µ
g STX eq./kg. All of these samples were analysed by PreCOX,
with a mean total PST of 259
µ
g STX eq./kg, with the results skewed by five samples with PreCOX total
PST >1000
µ
g STX eq./kg, all from Argentinean mussels and scallops. High toxicities were confirmed
in these samples by other methods, showing issues with the original MBA or storage/transportation
issues relating to toxin stability issues of the samples, rather than the performance of the non-MBA
alternatives (Table S1). Out of these 80 samples, 57 were also analysed by LC-MS/MS, with a mean total
PST concentration of 306
µ
g STX eq./kg, still below the LOD of the MBA. In total, 43 samples returned
not detected results following PreCOX, with all of these also showing not detected by MBA and RBA.
A total of 39 of these samples were also analysed by LC-MS/MS, with 27 of these (69%) returning total
PST <16
µ
g STX eq./kg and the remaining positive results reaching a maximum of 200
µ
g STX eq./kg.
2.2. Quantitative Comparison and Toxin Profiles
An initial visual assessment focused on shellfish samples containing total PST concentrations
above a 160
µ
g STX eq./kg threshold, equating to 224 samples run by the EU reference method (PreCOX
LC-FLD). Of these samples, 173 returned positive MBA results, 160 PCOX, 199 LC-MS/MS and 100 RBA.
A wide variety of toxin profiles were also evidenced across all samples assessed. Table S2 summarises
the toxin profile data in terms of saxitoxin equivalents for each sample as determined by LC-MS/MS.
The most commonly occurring PST analogue was STX, quantified in 99% of PST-positive shellfish
samples. GTX2&3, C1&2, dcSTX, GTX1&4, NEO and GTX5 were also detected in large numbers of
samples (85%, 83%, 71%, 70%, 66%, 63%, respectively). K-means clustering analysis highlighted the
presence of three main toxin profile types from the dataset, based on the analysis of quantitative data
following LC-MS/MS. This method was chosen given its ability to quantify the largest number of
PST analogues, with each epimeric pair quantified separately. Figure 2a illustrates the mean profiles,
showing profile 1 to be dominated by STX (65
±
35%) followed by GTX2&3
(22 ±27%)
, with low/trace
relative concentrations of GTX1&4, NEO, C1&2 and GTX5 (all <2% each). On the other hand, profile 2
showed a near total presence of gonyautoxins, with 72
±
23% GTX1&4,
15 ±14%
GTX2&3 and just
5±11% STX
, with low/trace proportions of NEO, dcGTX2&3, C1&2, GTX5 and M toxins (Table 1)
(all <2% each). Finally, cluster profile 3 represented a larger mix of toxin analogues, with no clear
dominance of any one toxin and mean proportions showing significant presence of other less-commonly
encountered toxins. Mean profiles included most notably 32
±
24% dcSTX,
15 ±14%
C1&2, 14
±
17%
dcGTX2&3 and 11
±
12% GTX5. Profile 3 also incorporated higher relative proportions of M toxins, with
a total mean proportion of 18
±
24% for M1-4 summed. Low/trace proportions of GTX6, dcGTX1&4,
GTX1&4 and GTX2&3 were also detected (<2% each). Supplemental Figure S1 illustrates the three
profile types in relation to both country of origin and shellfish species. Samples exhibiting cluster
1 were found only in Argentina and Chile, with cluster 2 samples found to dominate Argentinean
samples as well as Uruguay and Chile. All Mexican geoduck samples were associated with cluster
3 profile. Overall, mussels were most commonly associated with cluster 2, with the majority of
gastropods exhibiting profile 1. No other clear patterns were evident from the distribution of data
shown in Figure S1.
Mar. Drugs 2020,18, 616 6 of 30
Mar. Drugs 2020, 18, x 6 of 32
Figure 2. Summary of toxin profiles in terms of saxitoxin equivalents for each of the three profile
clusters determined in LA shellfish samples following (a) LC-MS/MS analysis, (b) PreCOX LC-FLD
and (c) PCOX LC-FLD.
Figure 2.
Summary of toxin profiles in terms of saxitoxin equivalents for each of the three profile
clusters determined in LA shellfish samples following (
a
) LC-MS/MS analysis, (
b
) PreCOX LC-FLD
and (c) PCOX LC-FLD.
Table 1.
Summary of mean PSP toxicities (
µ
g STX eq./kg) calculated for each shellfish type in the four
LA regions based upon 57 samples for which all five quantitative methods were used.
Region Shellfish n MBA PreCOX PCOX LC-MS/MS RBA
Argentina
Clams 9 1020 1174 959 2001 1544
Mussels 11
107,912
103,352 10,899 111,426 86,292
Scallops 4 10,427 1480 3277 5064 4147
Snails 11 5622 6775 4377 3719 9283
Chile Clams 3 40,717 2018 1933 1675 1829
Mussels 8 3902 1756 1039 1547 2332
Oysters 1 1070 870 523 859 1016
Scallops 2 339 349 339 145 501
Mexico
Geoduck (w)
1 4120 1582 1277 3680 792
Uruguay Clams 2 2315 356 259 785 380
Mussels 5 5536 2395 1904 4743 2641
Mar. Drugs 2020,18, 616 7 of 30
2.2.1. Comparison of Five Quantitative Methods
Across all quantitative data points, mean ratios between the total PST results obtained from each
method in comparison with the PreCOX LC-FLD were 0.88 (PCOX), 1.34 (LC-MS/MS), 1.65 (RBA) and
3.91 (MBA). However, different numbers of analyses were performed for each method, so method
comparison continued with the analysis of samples where either five or four quantitative method
results were generated. Tables summarises the mean PSP toxicities for each shellfish type (mussels,
clams, oysters, scallops, geoduck and miscellaneous) in each of the four LA regions, using five testing
methods. In total, 57 shellfish samples contained total PST results for all of the five quantitative
methods—PreCOX, PCOX, LC-MS/MS, RBA and MBA. The data were found to be skewed, but the
log transformation successfully normalised the data and results, with similar median values and
homogeneous variances being observed across the five test methods (Figure 3, Table S3).
Mar. Drugs 2020, 18, x 8 of 32
Figure 3. Distribution of test result values across five different test methods (MBA, PreCOX, PCOX,
LCMSMS and RBA) with (a) raw data and (b) log-transformed data.
The scatter plots showed that the five methods were strongly correlated (Figure 4). The
correlation coefficients were high and all of them were significant at the level of 5%. PCOX had,
however, the lowest correlation coefficients (0.68 < r < 0.78), while all the other methods exhibited
correlation coefficients higher than 0.80.
Figure 3.
Distribution of test result values across five different test methods (MBA, PreCOX, PCOX,
LCMSMS and RBA) with (a) raw data and (b) log-transformed data.
The scatter plots showed that the five methods were strongly correlated (Figure 4). The correlation
coefficients were high and all of them were significant at the level of 5%. PCOX had, however, the lowest
correlation coefficients (0.68 <r<0.78), while all the other methods exhibited correlation coefficients
higher than 0.80.
Mar. Drugs 2020,18, 616 8 of 30
Mar. Drugs 2020, 18, x 9 of 32
Figure 4. Correlation coefficients (upper half), scatter plots (lower half) and distributions (diagonal)
for five different test methods (MBA, PreCOX, PCOX, LC-MS/MS and RBA) with samples taken
within the same shellfish.
The repeated-measures ANOVA demonstrated that the different test methods explained a
significant amount of the variability observed in the dataset (p = 0.004). Though the difference
between the mean values obtained via each test method appeared small (Table S3), pairwise
comparisons suggested that PCOX results were significantly different to the results of all the other
methods, while PreCOX differed from the RBA (Table 2). The pairwise comparisons indicated no
significant difference between the other methods at a level of 5%.
Table 2. p-values from pairwise comparisons using paired t-tests, with underlined results showing
significant differences between method data.
MBA PreCOX PCOX LCMSMS
PreCOX 0.225
PCOX 0.00035 0.006
LCMSMS 0.766 0.884 0.004
RBA 0.884 0.019
0.00003 0.884
Overall, mean values determined for each species/country combination compared well between
each method, with some exceptions. MBA data were found to be high in comparison to other method
results for Argentinean scallops (n = 4), Chilean clams and mussels (n = 11), and Uruguayan samples
(n = 7). Whilst the PCOX results compared generally well with those from other methods for the
majority of samples, for Argentinean mussels, PCOX data were significantly lower than others.
Whilst mussels from Argentina and Chile were associated most commonly with toxin profile 2,
dominated by GTX1&4, other samples showing high relative MBA results were associated with toxin
profile 1. High mean RBA values were obtained for Argentinean snails (n = 11), with the geoduck low
RBA associated with just one sample.
Figure 4.
Correlation coefficients (upper half), scatter plots (lower half) and distributions (diagonal) for
five different test methods (MBA, PreCOX, PCOX, LC-MS/MS and RBA) with samples taken within the
same shellfish.
The repeated-measures ANOVA demonstrated that the different test methods explained a
significant amount of the variability observed in the dataset (p=0.004). Though the difference between
the mean values obtained via each test method appeared small (Table S3), pairwise comparisons
suggested that PCOX results were significantly different to the results of all the other methods,
while PreCOX differed from the RBA (Table 2). The pairwise comparisons indicated no significant
difference between the other methods at a level of 5%.
Table 2.
p-values from pairwise comparisons using paired t-tests, with underlined results showing
significant differences between method data.
MBA PreCOX PCOX LCMSMS
PreCOX 0.225
PCOX 0.00035 0.006
LCMSMS 0.766 0.884 0.004
RBA 0.884 0.019 0.00003 0.884
Overall, mean values determined for each species/country combination compared well between
each method, with some exceptions. MBA data were found to be high in comparison to other
method results for Argentinean scallops (n =4), Chilean clams and mussels (n =11), and Uruguayan
samples
(n =7)
. Whilst the PCOX results compared generally well with those from other methods
for the majority of samples, for Argentinean mussels, PCOX data were significantly lower than
others. Whilst mussels from Argentina and Chile were associated most commonly with toxin profile 2,
dominated by GTX1&4, other samples showing high relative MBA results were associated with toxin
profile 1. High mean RBA values were obtained for Argentinean snails (n =11), with the geoduck low
RBA associated with just one sample.
2.2.2. Comparison of Four Quantitative Methods
The previous analysis was repeated with the dataset excluding RBA, resulting in the comparative
assessment of a larger total number of shellfish samples (n =115). With approximately double the
number of shellfish samples incorporated, differences were induced in the statistics of the method
Mar. Drugs 2020,18, 616 9 of 30
results compared to the previous dataset (Figure 5, Table S4). Table 3summarises the mean PSP toxicity
data obtained from each of the four methods. The scatter plots showed that the four methods were
strongly correlated (Figure 6), which was confirmed with the high positive correlation coefficients.
All of them were significant at the level of 5%.
Mar. Drugs 2020, 18, x 10 of 32
2.2.2. Comparison of Four Quantitative Methods
The previous analysis was repeated with the dataset excluding RBA, resulting in the
comparative assessment of a larger total number of shellfish samples (n = 115). With approximately
double the number of shellfish samples incorporated, differences were induced in the statistics of the
method results compared to the previous dataset (Figure 5, Table S4). Table 3 summarises the mean
PSP toxicity data obtained from each of the four methods. The scatter plots showed that the four
methods were strongly correlated (Figure 6), which was confirmed with the high positive correlation
coefficients. All of them were significant at the level of 5%.
Table 3. Summary of mean PSP toxicities (µg STX eq/kg) calculated for each shellfish type in the four
LA regions based upon 115 samples for which four quantitative methods were used.
Region Shellfish N MBA PreCOX PCOX LC-MS
/
MS
Argentina Clams 10 1020 1174 959 2001
Mussels 34
62,878
53,633 11,240 59,801
Scallops 9 13,508 4516 3912 7816
Snails 23 5993 5591 4506 3586
Chile Clams 5 24,692 1343 1204 1271
Mussels 19 4981 2275 1886 2071
Oysters 1 1070 870 523 859
Scallops 3 336 414 439 143
Mexico Geoduck (w) 1 4120 1582 1277 3680
Uruguay Clams 3 1810 307 200 679
Mussels 7 6667 5663 7284 9223
Figure 5. Distribution of test result values across four different test methods (MBA, PreCOX, PCOX
and LC-MS/MS) with log-transformed data.
Figure 5.
Distribution of test result values across four different test methods (MBA, PreCOX, PCOX
and LC-MS/MS) with log-transformed data.
Table 3.
Summary of mean PSP toxicities (
µ
g STX eq/kg) calculated for each shellfish type in the four
LA regions based upon 115 samples for which four quantitative methods were used.
Region Shellfish n MBA PreCOX PCOX LC-MS/MS
Argentina Clams 10 1020 1174 959 2001
Mussels 34 62,878 53,633 11,240 59,801
Scallops 9 13,508 4516 3912 7816
Snails 23 5993 5591 4506 3586
Chile Clams 5 24,692 1343 1204 1271
Mussels 19 4981 2275 1886 2071
Oysters 1 1070 870 523 859
Scallops 3 336 414 439 143
Mexico
Geoduck (w)
1 4120 1582 1277 3680
Uruguay Clams 3 1810 307 200 679
Mussels 7 6667 5663 7284 9223
Mar. Drugs 2020, 18, x 11 of 32
Figure 6. Correlation coefficient (upper half), scatter plot (lower half) and distribution (diagonal) for
four different test methods (MBA, PreCOX, PCOX and LC-MS/MS) with samples taken within the
same shellfish.
The analysis of variance assessment demonstrated that the different test methods explained a
significant amount of the variability observed in the dataset (p = 0.0005). Pairwise comparisons
suggested that PreCOX results were not significantly different to the results of the LC-MS/MS
method, while all the other pairwise comparison were significantly different at the level of 5% (Table
4). The conclusions from this analysis differed from the ones obtained with the dataset for the five
methods. The larger number of samples is known to increase the power of tests used here. Therefore,
a greater confidence was obtained with this second analysis. Table S4 summarises the mean total PST
data generated using the four methods on the larger number of samples. High MBA data seen from
the assessment of all five quantitative methods in Table 1 were confirmed using a larger number of
samples with four testing methods in Argentinean scallops (n = 9), Chilean clams and mussels (n =
24) and Uruguayan clams (n = 3), although mussels from Uruguay showed a better comparison with
a larger dataset (n = 7) (Table 3). Mean values again compared well with the other chemical detection
methods with the exception of Argentinean mussels (n = 34).
Table 4. Summary of p-values from pairwise comparisons using paired t tests, with underlined results
showing significant differences between method data.
MBA PreCOX PCOX LC-MS
/
MS
PreCOX 6.50 × 10−5
PCOX 7.20 × 10−10 1.20 × 10−5
LC-MS/MS 0.0007 0.331 1.50 × 10−5
Figure 6.
Correlation coefficient (upper half), scatter plot (lower half) and distribution (diagonal) for
four different test methods (MBA, PreCOX, PCOX and LC-MS/MS) with samples taken within the
same shellfish.
Mar. Drugs 2020,18, 616 10 of 30
The analysis of variance assessment demonstrated that the different test methods explained
a significant amount of the variability observed in the dataset (p=0.0005). Pairwise comparisons
suggested that PreCOX results were not significantly different to the results of the LC-MS/MS method,
while all the other pairwise comparison were significantly different at the level of 5% (Table 4).
The conclusions from this analysis differed from the ones obtained with the dataset for the five methods.
The larger number of samples is known to increase the power of tests used here. Therefore, a greater
confidence was obtained with this second analysis. Table S4 summarises the mean total PST data
generated using the four methods on the larger number of samples. High MBA data seen from the
assessment of all five quantitative methods in Table 1were confirmed using a larger number of samples
with four testing methods in Argentinean scallops (n =9), Chilean clams and mussels (n =24) and
Uruguayan clams (n =3), although mussels from Uruguay showed a better comparison with a larger
dataset (n =7) (Table 3). Mean values again compared well with the other chemical detection methods
with the exception of Argentinean mussels (n =34).
Table 4.
Summary of p-values from pairwise comparisons using paired t tests, with underlined results
showing significant differences between method data.
MBA PreCOX PCOX LC-MS/MS
PreCOX 6.50 ×10−5
PCOX 7.20 ×10−10 1.20 ×10−5
LC-MS/MS 0.0007 0.331 1.50 ×10−5
2.2.3. Comparison of Toxin Profiles between Methods
Figure 2a–c illustrate the mean toxin profiles determined for each of the three main profile clusters
(1 to 3 inclusive) using all three chemical detection methods. Overall the profiles determined by
LC-MS/MS are similar to those generated using both LC-FLD methods, with a clear dominance of
STX in profile 1, GTX1&4 in profile 2 and with a similar spread of dcGTX2&3, C1&2 and dcSTX in
samples associated with profile 3. The main difference observed relates to the additional incorporation
of M-toxins into the LC-MS/MS, which are not detected by either LC-FLD method.
2.3. LFA vs. PreCOX LC-FLD
A total of 250 shellfish extracts were analysed by Scotia LFA, with test cassettes interpreted visually
by two analysts, resulting in 100% agreement between analysts. Two samples returned an invalid test
strip result, with 199 positive results and 49 negative. Consequently, a dataset of 248 qualitative results
were assessed in comparison with the reference PreCOX LC-FLD method. PreCOX values associated
with both LFA positive and negative results were highly skewed. A log transformation normalised
the data (Figure 7a). Welch’s two-sample t-test showed that PreCOX results were significantly higher
in the group of positive LFA samples compared to the LC-FLD values determined in negative LFA
samples at the level of 5%.
Figure 8illustrates the qualitative LFA results obtained in comparison with the total PST
concentrations determined by PreCOX LC-FLD. A total of 21 samples were found to contain no
detectable levels of toxins by PreCOX, with 100% of these returning a negative LFA result. Further,
26 out of a total of 28 more negative LFA results exhibited total PST concentrations of <120
µ
g STX
eq./kg by PreCOX LC-FLD, although the remaining two were highly toxic, with total PST of 3483 and
3914
µ
g STX eq./kg for a clam and gastropod sample, respectively. Out of the 199 positive LFA results
following visual confirmation by two analysts, total PST concentrations determined by LC-FLD ranged
from not detected to just below 400,000
µ
g STX eq./kg. A total of 106 LFA positive samples contained
<800
µ
g STX eq./kg toxicity by PreCOX, with a further 77 samples <400
µ
g STX eq./kg which equates
to the approximate, but profile-dependent limit of detection of the Scotia assay (Laycock et al., 2010).
Consequently, out of 254 LFA tests conducted, 77 (30%) of these resulted in false-positive LFA results,
Mar. Drugs 2020,18, 616 11 of 30
with two false negatives (0.8%) (Table 5). The highest proportion of false-positive results were found in
samples of Geoduck (66% of geoduck samples), followed by mussels and clams (both 20%), scallops
(19%) and snails (9%).
Mar. Drugs 2020, 18, x 12 of 32
2.2.3. Comparison of Toxin Profiles between Methods
Figure 2a–c illustrate the mean toxin profiles determined for each of the three main profile
clusters (1 to 3 inclusive) using all three chemical detection methods. Overall the profiles determined
by LC-MS/MS are similar to those generated using both LC-FLD methods, with a clear dominance of
STX in profile 1, GTX1&4 in profile 2 and with a similar spread of dcGTX2&3, C1&2 and dcSTX in
samples associated with profile 3. The main difference observed relates to the additional
incorporation of M-toxins into the LC-MS/MS, which are not detected by either LC-FLD method.
2.3. LFA vs. PreCOX LC-FLD
A total of 250 shellfish extracts were analysed by Scotia LFA, with test cassettes interpreted
visually by two analysts, resulting in 100% agreement between analysts. Two samples returned an
invalid test strip result, with 199 positive results and 49 negative. Consequently, a dataset of 248
qualitative results were assessed in comparison with the reference PreCOX LC-FLD method. PreCOX
values associated with both LFA positive and negative results were highly skewed. A log
transformation normalised the data (Figure 7a). Welch’s two-sample t-test showed that PreCOX
results were significantly higher in the group of positive LFA samples compared to the LC-FLD
values determined in negative LFA samples at the level of 5%.
Figure 7. Comparison of log-transformed PreCOX test results associated with Scotia LFA negative (−)
and positive (+) results with (a) visual interpretation “Int1” and (b) automated scan interpretation
method “Scan.Int”.
Figure 8 illustrates the qualitative LFA results obtained in comparison with the total PST
concentrations determined by PreCOX LC-FLD. A total of 21 samples were found to contain no
detectable levels of toxins by PreCOX, with 100% of these returning a negative LFA result. Further,
26 out of a total of 28 more negative LFA results exhibited total PST concentrations of <120 µg STX
eq./kg by PreCOX LC-FLD, although the remaining two were highly toxic, with total PST of 3483 and
3914 µg STX eq./kg for a clam and gastropod sample, respectively. Out of the 199 positive LFA results
Figure 7.
Comparison of log-transformed PreCOX test results associated with Scotia LFA negative (
−
)
and positive (+) results with (
a
) visual interpretation “Int1” and (
b
) automated scan interpretation
method “Scan.Int”.
Mar. Drugs 2020, 18, x 13 of 32
following visual confirmation by two analysts, total PST concentrations determined by LC-FLD
ranged from not detected to just below 400,000 µg STX eq./kg. A total of 106 LFA positive samples
contained < 800 µg STX eq./kg toxicity by PreCOX, with a further 77 samples < 400 µg STX eq./kg
which equates to the approximate, but profile-dependent limit of detection of the Scotia assay
(Laycock et al., 2010). Consequently, out of 254 LFA tests conducted, 77 (30%) of these resulted in
false-positive LFA results, with two false negatives (0.8%) (Table 5). The highest proportion of false-
positive results were found in samples of Geoduck (66% of geoduck samples), followed by mussels
and clams (both 20%), scallops (19%) and snails (9%).
Table 5. Summary of Scotia LFA performance in comparison with PreCOX LC-FLD reference method
highlighting for each shellfish species the total number of tests, invalid tests, disagreements, positive
results, negative results, false-negative and false-positive results.
Mussel Clam Oyste
r
Geoduck Snail Scallop Misc Total
Total tests 84 41 6 73 32 16 2 254
Invalid tests 1 0 0 0 1 0 0 2
Disagreements
a
0 1 0 0 2 1 0 4
Total Pos 81 24 1 51 27 13 2 199
Total Neg 2 16 5 22 2 2 0 49
False Neg
b
0 1 0 0 1 0 0 2
False Pos
c
17 8 0 46 3 3 0 77
a
Disagreement between visual and automated test strip interpretation.
b
PreCOX LC-FLD > MPL, with
negative LFA result.
c
PreCOX LC-FLD < 0.5 MPL with positive LFA result.
Mar. Drugs 2020, 18, x 14 of 32
Figure 8. Total PST concentrations determined by PreC OX LC- FLD i n comp ariso n with (a) Scotia LFA
qualitative results, highlighting MPL and (b) LFA scan number.
A total of 56 LFA results were also assessed using the automated scanner, with LFA scan results
confirming positive LFA results in 29 samples, with negative results in 27. Overall, there was good
agreement between the visual and automated LFA results, with just four samples deemed positive
by visual assessment, confirmed as negative by the automated approach (Table 5). PreCOX values
associated with both LFA positive and negative results were again log transformed for normalising
data (Figure 7b). However, the variance between the two groups remained heterogeneous, with much
greater variability in PreCOX values observed in the negative LFA samples as compared to the
positive group. Welch’s two-sample t-test, which does not require equal variances between groups,
showed that PreCOX results were significantly higher in the LFA positive samples compared to the
negative group at the level of 5%.
Scan numbers were produced for each of the automated scanner interpretation results from each
of the LFA determinations. A regression was plotted between the scan numbers and PreCOX LC-
FLD toxin concentrations, using 16 µg STX eq./kg for LC-FLD results showing no detectable toxins.
Results showed the LFA scan numbers to decrease significantly with higher toxicity samples, with
the regression showing a logarithmic correlation between the two parameters (y = −0.15 ln +
1.1719) with a correlation coefficient r2 = 0.7712 (Figure 8b).
3. Discussion
3.1. PST Outbreaks in Latin America and Social Impacts
PST-producing harmful algal blooms are widely reported on an annual basis throughout Latin
America, with regular occurrences of regiospecific toxigenic outbreaks of A. catenella, G. catenatum
and P. bahamense. PST concentrations can periodically reach extraordinarily high levels in some
regions resulting not only in extreme levels of risks to human health, but also significant impacts on
animal health as exemplified by mass mortalities of marine mammals, and consequent socio-
economic impacts [57,64,69]. The social impacts of this type of phenomenon are especially relevant
in the coastal communities of southern Chile, whose traditions, gastronomy and subsistence are
based on the ancestral relationship between the coastal communities and the sea. In this sense,
precautionary closures make it impossible for coastal communities to use seafood as a source of food
Figure 8.
Total PST concentrations determined by PreCOX LC-FLD in comparison with (
a
) Scotia LFA
qualitative results, highlighting MPL and (b) LFA scan number.
Mar. Drugs 2020,18, 616 12 of 30
Table 5.
Summary of Scotia LFA performance in comparison with PreCOX LC-FLD reference
method highlighting for each shellfish species the total number of tests, invalid tests, disagreements,
positive results, negative results, false-negative and false-positive results.
Mussel Clam Oyster Geoduck Snail Scallop Misc Total
Total tests 84 41 6 73 32 16 2 254
Invalid tests 1 0 0 0 1 0 0 2
Disagreements a0 1 0 0 2 1 0 4
Total Pos 81 24 1 51 27 13 2 199
Total Neg 2 16 5 22 2 2 0 49
False Neg b0 1 0 0 1 0 0 2
False Pos c17 8 0 46 3 3 0 77
a
Disagreement between visual and automated test strip interpretation.
b
PreCOX LC-FLD >MPL, with negative
LFA result. cPreCOX LC-FLD <0.5 MPL with positive LFA result.
A total of 56 LFA results were also assessed using the automated scanner, with LFA scan results
confirming positive LFA results in 29 samples, with negative results in 27. Overall, there was good
agreement between the visual and automated LFA results, with just four samples deemed positive
by visual assessment, confirmed as negative by the automated approach (Table 5). PreCOX values
associated with both LFA positive and negative results were again log transformed for normalising
data (Figure 7b). However, the variance between the two groups remained heterogeneous, with much
greater variability in PreCOX values observed in the negative LFA samples as compared to the positive
group. Welch’s two-sample t-test, which does not require equal variances between groups, showed that
PreCOX results were significantly higher in the LFA positive samples compared to the negative group
at the level of 5%.
Scan numbers were produced for each of the automated scanner interpretation results from
each of the LFA determinations. A regression was plotted between the scan numbers and PreCOX
LC-FLD toxin concentrations, using 16
µ
g STX eq./kg for LC-FLD results showing no detectable toxins.
Results showed the LFA scan numbers to decrease significantly with higher toxicity samples, with the
regression showing a logarithmic correlation between the two parameters (
y=−
0.15
ln x+
1.1719)
with a correlation coefficient r2=0.7712 (Figure 8b).
3. Discussion
3.1. PST Outbreaks in Latin America and Social Impacts
PST-producing harmful algal blooms are widely reported on an annual basis throughout Latin
America, with regular occurrences of regiospecific toxigenic outbreaks of A. catenella,G. catenatum
and P. bahamense. PST concentrations can periodically reach extraordinarily high levels in some
regions resulting not only in extreme levels of risks to human health, but also significant impacts on
animal health as exemplified by mass mortalities of marine mammals, and consequent socio-economic
impacts [
57
,
64
,
69
]. The social impacts of this type of phenomenon are especially relevant in the
coastal communities of southern Chile, whose traditions, gastronomy and subsistence are based on
the ancestral relationship between the coastal communities and the sea. In this sense, precautionary
closures make it impossible for coastal communities to use seafood as a source of food and small-scale
marketing. Chile is the sixth-largest exporter of fish products in the world [
106
], an industry which
is also a source of work and income for thousands of families, who are directly or indirectly related
to the industry. For this reason, since 1995 the Shellfish National Sanitation Programme (PSMB),
which is dependent on the National Fisheries and Aquaculture Services (Sernapesca), has maintained a
surveillance system in the extraction areas and cultivation centres, whose products are mainly destined
for export. In the year 2016, the worst PSP toxic event of all those recorded in terms of geographical
extension and species affected occurred in the south of Chile. A total of 1700 people were unemployed
due to HABs because of the inactivity of the processing plants and the ban on shellfish harvesting.
Additionally, during this toxic episode, the highest mass mortality of invertebrates and vertebrates
Mar. Drugs 2020,18, 616 13 of 30
recorded was observed [
70
]. A particularly harmful case was observed on Cucao beach located on
the Pacific Ocean coast of Chilo
é
island, where one of the most important commercial benthic fishing
species was affected (macha: Mesodesma donacium) during the toxic outbreak, the main resource of the
local economy of fishermen from the Huilliche ethnic group [70].
Consequently, in Chile and other LA countries, there has been an ongoing urgent need to ensure
shellfish harvesting areas are monitored both routinely and effectively for the presence of potentially
toxic microalgae and for biotoxins in shellfish flesh. Whilst the MBA has provided an effective
monitoring tool for rapid assessment of PSP risks in shellfish for many decades, international laws
are changing, resulting in the need for LA regions wishing to export shellfish to Europe and other
regions to utilise non-animal bioassays for official control testing. For the internationally-validated
methods to be used by LA countries, they must also be verified in house for applicability to the shellfish
species of relevance, to generate performance characteristics and determine the size of measurement
uncertainty. As part of these assessments, the comparison of multiple PSP detection methods would
provide insights into the relative performance of each method, thereby benefitting the end users.
To date, various authors have reported the extent anddistribution of total shellfish toxicity in each of the
regions included in this study in a wide range of bivalve mollusc species [
12
,
19
,
21
,
45
,
50
–
59
,
64
,
65
,
67
–
69
],
with more recent work describing PST analogues present in each of the four LA regions included in
this study; Mexico, Uruguay, Chile and Argentina [
44
,
57
,
61
,
64
]. Analogues of saxitoxin reported are
wide ranging including all the main gonyautoxins, carbamoyl and decarbamoyl congeners available as
commercial reference materials, in addition to M toxins more recently reported [
44
,
57
,
61
,
64
]. Whilst the
M toxins currently have no assigned toxicity equivalence factors (TEFs), the remaining analogues all
contribute to total sample toxicity to varying extents through the continent, so any monitoring methods
should be capable of detecting and quantifying the analogues of importance in each region.
3.2. Method Comparison
3.2.1. MBA and RBA
Both the MBA and RBA should theoretically provide a direct and accurate determination
of the total toxicity of any given shellfish sample. Whilst exhibiting low sensitivity, the MBA
is generally thought to return similar total toxicity results when compared to chemical detection
methods (e.g., [
77
,
78
,
86
,
99
]. There are well reported instances, however, where matrix effects cause
significant underestimation of total toxicity by MBA, most notably in oysters containing naturally
high concentrations of zinc [
76
,
88
,
107
,
108
], or where differences occur between methods related to
different extraction steps and/or certain specific-source phytoplankton species such as Gymnodinium
catenatum [103].
From the four LA regions studied here, statistical analysis demonstrated the MBA to correlate
equally well with the RBA, PreCOX and LC-MS/MS methods, but with a lower correlation with PCOX
(Figure 4). The correlation coefficient between MBA and PreCOX of 0.84 was higher than that reported
previously in a multi-method assessment of PST concentrations in samples from Alaska [
99
] but similar
to that from UK mussels [
86
]. Here, apparent differences in total PST following MBA in comparison
with others were identified in certain species from some geographical regions, notably Chilean clams
and mussels, Argentinean scallops and Uruguayan clams (Tables 1and 3). It is noted, however, that the
MBA analyses were conducted several years prior to the other analyses, so there may be issues relating
to either sample storage and/or transportation to other testing facilities. This does not explain, however,
several instances of extreme outliers (defined here as results showing differences greater than an
order of magnitude) for the MBA, specifically in three Argentinean mussels, one Argentinean scallop,
two Chilean and one Uruguayan clam, all of which returned MBA results more than 10-fold higher
than those determined using all other assays (Table S1).
The RBA, measuring toxicity through the direct measurement of the affinity of toxins to the
sodium channel receptor, has been shown previously to compare generally well with the MBA for
Mar. Drugs 2020,18, 616 14 of 30
Chilean mussel and clam samples (r
2
=0.94, n =41; [
109
]). Interestingly, an alternative functional
electrophysiological assay for saxitoxins applied to a different set of 30 Chilean mussel and gastropod
samples showed good correlation with the MBA (r
2
=0.90) but with the MBA consistently returning
higher results [
109
]. During the RBA AOAC single-laboratory validation, which incorporated some
shellfish samples from Chile, RBA toxicities were found to show a degree of overestimation in
comparison to the MBA and PreCOX LC-FLD [
91
], as confirmed by more recent work based on
mussels [
110
] and oysters [
105
], with the latter study confirming the RBA to be unaffected by high
concentrations of zinc and other metals. In this study, RBA data further confirm its applicability
to a wide range of shellfish species from wide-ranging geographical regions, with generally good
correlation with the chemical detection methods, in particular with excellent correlations against
both LC-MS/MS and PreCOX methods (Figure 4). Overall, these findings compare well with those
from samples taken from Alaska, where the correlation between RBA and PreCOX was excellent
(r2=0.95 [99])
, although the RBA returned, on average, total toxicity results lower than those estimated
from PreCOX LC-FLD analysis. Conversely, the mean RBA to PreCOX ratio in this study was 1.65,
confirming the findings of [92,105,110].
3.2.2. Instrumental Methods
As opposed to the MBA and RBA detection methods, all three instrumental chemical assays utilise
external calibration standards for each individual toxin analogue to quantify toxin concentrations,
subsequently estimating total saxitoxin equivalents through application of published TEFs. It is
therefore important for any chemical assay utilised to be developed and validated to facilitate analytes
which are likely to be present in any given region. In Latin American countries, it is therefore
important for assays to be capable of incorporating analogues derived from all three genera of
PST-producing microalgae, resulting in the requirement for including almost all known congeners,
currently commercially available as certified reference materials. Table 6summarises the PST analogues
which are incorporated into each of the three chemical testing methods utilised in this study. All PST
analogues incorporated into the LC-MS/MS method were detected in samples from this study, but to
varying extents, resulting in three major profile clusters.
Table 6. Summary of PST analytes incorporated into each chemical testing method.
Analogue PreCOX PCOX LC-MS/MS
STX y y y
GTX2 y y y
GTX3 y y y
GTX1 y y y
GTX4 y y y
GTX5 y y y
GTX6 y - y
NEO y y y
C1 y y y
C2 y y y
C3 y y y
C4 y y y
dcSTX y y y
dcGTX2 y y y
dcGTX3 y y y
dcGTX1 - - y
dcGTX4 - - y
dcNEO y - y
doSTX - - y
M1 - - y
M2 - - y
M3 - - y
M4 - - y
y=analyte is incorporated into method; - =analyte is not incorporated into method.
Mar. Drugs 2020,18, 616 15 of 30
The LC-MS/MS assay is capable of analysing all analytes listed, although the importance of
including M toxins and doSTX remains unclear given the absence of experimentally determined TEFs
for these analogues. In this study, total PST concentrations correlated excellently with the current EU
reference method, PreCOX LC-FLD with a Pearson correlation coefficient of 0.95 (Figure 6) and no
significant differences between the method data (Tables 2and 4), with an overall mean LC-MS/MS to
PreCOX ratio of 1.34. This confirms previous work which has also described an excellent agreement
between the LC-MS/MS and PreCOX method [94].
The PreCOX LC-FLD is validated for all other remaining PSTs with the exception of dcGTX1&4,
which to the authors knowledge has to date only been reported in certain specific clam species capable
of enzymatic conversion to these congeners from GTX1&4 [
111
–
113
]. In this study, dcGTX1&4 was
detected in only eleven samples, representing only 5% of the total samples exhibiting total PST toxin
concentrations >16
µ
g STX eq./kg, with the maximum contribution to toxicity found in one clam
sample containing 10% dcGTX1&4. Overall, therefore, the absence of dcGTX1&4 monitoring for
these samples would not greatly affect the accuracy of the final results. doSTX was quantified in
47% of samples, but at concentrations so low, the overall contribution to the total sample toxicity
was negligible, with a mean and maximum total toxin proportion of 0.01% and 0.21%, respectively
(Table S2). M toxins, detected in 80% of the samples currently have no TEF data formally assigned,
but using current assumptions, total M toxin contributions to sample toxicity averaged 3.5%, with
a maximum of 33%. Whilst there is consequently the potential for total PST concentrations to be
underestimated using non-mass spectrometric instrumental methods such as PreCOX, further work
is required to determine the correct TEFs for these analogues. Overall, however, whilst the PreCOX
method showed statistically significant differences from results returned by other methods other than
LC-MS/MS, the correlation coefficients associated with the pairwise comparisons were high, ranging
from 0.76 to 0.96 (Figure 4).
Total toxin concentrations determined by PCOX LC-FLD also correlated well with results obtained
by the other quantitative methods, although the correlation coefficients were lower than between the
other methods, ranging from 0.68 to 0.78 (Figure 4) and with data deemed significantly different to those
generated by all other methods (Tables 2and 4). Most notably, large differences were found for samples
of Argentinean mussels, where PCOX total PST concentrations were lower than those calculated
for all other methods (Tables 1and 3). The correlation coefficients determined here are similar to
those reported from previous studies incorporating multiple shellfish matrices [
83
], although Alaskan
researchers have shown a correlation coefficient (r
2
) of 0.96 between PCOX and MBA, with an average
200% toxicity result for PCOX in comparison to the bioassay [
114
]. Higher toxicity results from the
PCOX in comparison to PreCOX LC-FLD methods have also been described [
115
], although the opposite
has been reported by other authors when assessing PST concentrations in oysters [
104
]. The mean
PCOX to PreCOX ratio in this study was 0.88 showing a close similarity in total PST concentrations
overall, as determined by the two FLD methods. Whilst the method is fully validated for the majority
of analogues, in addition to dcGTX1&4, notable exceptions include dcNEO and GTX6, both of which,
along with other analogues can contribute significantly to total STX equivalents in shellfish associated
with G. catenatum [
27
,
116
–
119
]. Consequently, it is important to assess local toxin profiles to help
determine whether intended methods of analysis will successfully incorporate all present and relevant
analogues. Samples from this study typically contained low relative proportions of dcNEO and GTX6,
found in a total of 13% and 27% of samples, respectively (Table S2). Mean total toxin contributions from
these two analogues were low (0.1% and 0.6%), reaching a maximum of 5.3% and 15% for dcNEO and
GTX6, respectively. Consequently, there is the potential for underestimation of toxicity using PCOX
for any samples containing high relative proportions of dcNEO and GTX6, given that the method
used in this study was not extended to these two analogues. Overall, however, other than the findings
with Argentinean mussels, the low PCOX to PreCOX ratio observed in samples in this study were not
associated with any specific shellfish species, geographical region, or toxin profile cluster, so no clear
patterns were found which may explain the observed differences between these methods.
Mar. Drugs 2020,18, 616 16 of 30
3.2.3. LFA
For LA regions wishing to export shellfish products to the US, the Scotia LFA can be used in
certain situations as a screen for PST presence, hence there was a need to assess the comparative
performance of this assay against validated regulatory methods. In this study, the Scotia LFA was
assessed in direct comparison with the current EU reference method, PreCOX LC-FLD (n =248).
From a total of 49 LFA negative test results, all except two contained total PST concentrations <15%
of the 800
µ
g STX eq./kg MPL, although the two remaining negative LFA results related to samples
containing PSP at more than 4-fold higher than the MPL. As such, whilst 96% of negative LFA results
compared well with the PreCOX LC-FLD, the two false-negative LFA results were of concern given the
high levels of toxins present. The two samples concerned were A48 and A54, a clam and snail sample
from Argentina, respectively. Both samples were confirmed to contain high toxin concentrations by all
other detection methods, evidencing the problem was not due to a PreCOX false positive. In addition,
the LFA analysis was repeated on each sample, with the same findings. Toxin profiles for both the
false-negative samples were assessed, with A48 containing 74% GTX1&4 together with a mix of C1&2
and GTX2&3, whereas toxicity in sample A54 resulted from 92% STX. Sample A54, containing almost
exclusively STX would therefore not be expected to result in a false-negative LFA, given that the
antibody was for STX. On the other hand, the high proportion of GTX1&4 in sample A48 may have
been a factor. However, the refined LFA protocol was used, whereby GTX1&4 is transformed into
an analogue with higher cross reactivity, resulting in a much lower chance of false-negative assay
results [
98
]. Moreover, many other additional shellfish samples from this study contained very high
proportions of GTX1&4 and did not result in false-negative LFA results.
All other samples containing PSTs as evidenced by PreCOX were found to result in a positive
LFA result. A total of 30% of the LFAs conducted were, however, found to be false-positive results,
with PreCOX showing toxins were either not detected or quantified at concentrations well below the
MPL. Consequently, there is a risk that positive LFA results may be associated with sample batches that
are safe for human consumption. These findings are similar to those reported previously by authors
assessing the Scotia LFA in comparison to LC-FLD methods in samples from Alaska [
99
], the UK [
98
],
the US [120,121] and Hong Kong [122].
Previous work also illustrated a logarithmic correlation between LFA scan number and total
PST concentrations determined by PreCOX LC-FLD, with scan numbers <0.3 equating to toxicities
above the MPL [
98
]. Such a relationship was also evident overall in this study (Figure 8b). However,
three samples, two scallops and one mussel, with total PST higher than twice the MPL, returned scan
values >0.3, making use of the semi-quantitative aspect of the scanner more risky. Overall, therefore,
use of the Scotia LFA would be useful for reducing the number of quantitative confirmatory analyses
required, particularly as a product batch test, but a fully validated quantitative assessment using an
appropriate method would still be required for regulatory testing, noting the risks resulting from a
high proportion of false-positive and the potential for occasional false-negative LFA results in samples
with significant toxin loads.
3.3. Method Implementation
The EU is the main destination market for bivalve molluscs from South America [
58
], so the
recent changes to EU shellfish safety legislation and the global move away from reliance on the MBA
have placed pressures on LA regions to conduct official control testing using alternative methods.
These pressures arise from the ethical aspects of utilising animal experimentation for food safety
testing, and the legal aspects associated with exporting seafood to the EU and other regions where
the MBA is no longer accepted as a regulatory monitoring tool [
123
,
124
]. In Europe, parts of North
America and Australasia, alternative PSP detection methods have been used for both regulatory official
control and commercial testing for many years. The PreCOX method is perhaps the most widely
implemented currently, given its status as the EU reference method for PSP determination and its
application nowadays in the UK and the majority of European member states [
82
] with developments
Mar. Drugs 2020,18, 616 17 of 30
to increase throughput and performance being adopted [
125
]. The PCOX method has been used for
many years in Canada, and more recently selected by US States, given its acceptance by the Interstate
Shellfish Sanitation Conference (ISSC) for shellfish growing area classification [
126
]. The RBA can also
be used for regulatory control in mussels and for screening in clams and scallops [
126
]. More recently,
the LC-MS/MS method has become the main official control method in New Zealand and Australia and
the Scotia LFA is approved for use as a shellfish screening tool in the US [
126
]. Consequently, there is
currently no single testing method available which is suitable for official control monitoring of bivalves
destined for global export. Within LA, some countries have been working towards and achieving
validation and accreditation of alternative testing methods for some time. In Mexico, where US exports
are of high importance, PCOX (or alternative post-column protocols) has been utilised by some research
laboratories since 2014 [
64
] but still this method still is not recognised in Mexican National Shellfish
Sanitation Program. In contrast, the Scotia LFA was officially incorporated into this program in 2015
and used routinely for monitoring PSTs in shellfish. To date, however, its effectiveness has not been
evaluated. Whilst the PreCOX method has been set up in monitoring laboratories in Chile, it has not
been implemented for official controls. Similarly, both Uruguay and Argentina still utilise the MBA for
official control testing, although work is underway to prepare for potential future implementation of
chemical detection methods in Argentina.
Through the comparison of testing methods, this comparative study has helped to further
demonstrate that across a wide range of shellfish species and geographical regions, the various
alternative methods for PSP testing in bivalve molluscs are suitable for official control use. For those
countries yet to move away from reliance on MBA, however, there is much work required in order
to implement any such alternative methods. Replacement methods must be formally validated in
each official control laboratory, with a particular focus on the species of commercial importance.
In order to obtain accreditation to the relevant national or international quality standards, a robust
series of controls and management processes also need to be developed and implemented [
82
].
Numerous challenges exist to LA regions, such as the four studied here, including the expense of
instrumentation, often required in multiple establishments given the wide geographical expanse of
many regions [
60
], the cost of consumables and toxin standards, instrument maintenance charges and
the costs associated with training and maintaining the skills and experience of laboratory analysts.
4. Materials and Methods
4.1. Samples
Shellfish samples were obtained from four countries within LA-Argentina, Chile, Mexico and
Uruguay. The samples chosen for analysis included the major species of commercial importance to
each country and were harvested between 1986 and 2012. These consisted of four species of mussels,
oysters, five clam species, three scallop species, two marine snails and a selection of miscellaneous
marine organisms (Table 7). A total of 263 samples were collected and analysed by the MBA before
being transported to the UK for additional testing. An additional 86 samples were sent to the UK which
were not subjected to MBA, making a total of 349 samples associated with this study. All samples were
analysed by quantitative PreCOX LC-FLD, with other methods of analysis applied to selected samples
as described below.
Mar. Drugs 2020,18, 616 18 of 30
Table 7.
Summary of shellfish species analysed during the current study including shellfish type,
species, common name and geographical source.
Samples Species Common Name Source
Mussels Mytilus edulis Blue mussels Uruguay, Argentina
Mytilus chilensis Chilean blue mussels Chile
Aulacomya ater Ribbed mussels Argentina
Brachidontes rodriguezii Scorched mussels Argentina
Oysters Crassostrea gigas Pacific oysters Chile
Clams Gari solida Gari clams Chile
Venus antiqua Venus clams Chile, Argentina
Donax hanleyanus Wedge clams Uruguay, Argentina
Mesodesma mactroides Yellow clams Argentina
Panopea globosa Gulf of California
geoduck clam Mexico
Scallops Argopecten purpuratus Scallops Chile
Zygochlamys patagonica Patagonian scallops Argentina
Aequipecten tehuelchus Tehuelche scallops Argentina
Marine snails Adelomelon beckii Beck’s volute sea snail Argentina
Zidona dufresnei Angular volute sea snail Argentina
Squid Illex argentinus Mantle squid Argentina
Miscellaneous Exact species unknown Limpets, shrimp heads Argentina
4.2. Reagents and Chemicals
Chemicals were LC-MS-reagent grade where possible, with sample preparation and solid-phase
extraction reagents of HPLC grade (Rathburns, Walkerburn, UK). Mobile phases were prepared from
LC-MS-grade solvents (Fisher Optima, ThermoFisher, Loughborough, UK). Certified reference materials
for purified toxin standards of saxitoxin (STX), gonyautoxins 1–5 (GTX1-5), neosaxitoxin (NEO),
decarbamoylsaxitoxin (dcSTX), N-sulfocarbamoyl gonyautoxin-2&3 (C1&2), decarbamoylneosaxitoxin
(dcNEO) and decarbamoylgonyautoxin-2&3 (dcGTX2&3) were obtained from the Institute of
Biotoxin Metrology, National Research Council Canada (NRCC, Halifax, Nova Scotia, Canada).
Additional non-certified reference material standards of N-sulfocarbamoyl gonyautoxin-1&4 (C3&4),
decarbamoylgonyautoxin-1&4 (dcGTX1&4) and gonyautoxin-6 (GTX6) were obtained from the NRCC.
A reference standard for deoxydecarbamoylsaxitoxin (doSTX) was obtained from Cawthron Natural
Compounds (CNC; Nelson, New Zealand).
For quantitation of PSTs by PreCOX LC-FLD, CRMs were diluted in ~4.5 mL water to form
concentrated stock standard solutions and subsequently diluted in 0.1 mM acetic acid to form instrument
calibration standards. Toxin standard mixes were prepared as recommended [
73
]. For PCOX LC-FLD
analysis, concentrated stock solutions were prepared following AOAC 20011.02 [
85
], with primary
GTX and STX toxin standards diluted in ~4.5 mL 0.3 mM HCl. C1&2 primary standards were
diluted in ~4.5 mL pH5 water. Instrumental calibrants were prepared following further dilution in
the same reagents. For HILIC-MS/MS analysis, 100
µ
L of each CRM was pipetted to form a mixed
stock, containing C1&2, dcGTX2&3, GTX1-5, dcSTX, dcNEO, STX, NEO and doSTX. This solution
was subsequently used to prepare calibration standards for HILIC-MS/MS by diluting the mixed
stock solution into a diluent of carbon SPE-cleaned mussel extract, diluted as per samples to give a
concentration of 80% acetonitrile (MeCN) with 0.25% acetic acid [95].
[
3
H] STX for the RBA was obtained from American Radiolabeled Chemicals (St. Louis,
MO 63146, USA). Saxitoxin standard curves were prepared from 3 mM HCl dilutions of STX diHCl
standard (NIST RM8642, US National Institute of Standards and Technology). The RBA buffer was
100 mM MOPS/100 mM choline chloride, pH 7.4. Rat brain membranes were prepared in bulk according
to [
92
] and stored at
−
80
◦
C until use. Optiphase liquid scintillation cocktail (Perkin-Elmer Life Sciences,
Downers Grove, IL USA) was used for scintillation counting.
No additional reagents were required for running the Scotia PSP testing method, other than those
supplied in the testing kit.
Mar. Drugs 2020,18, 616 19 of 30
4.3. Methods of Analysis
4.3.1. Shellfish Extraction and MBA
Shellfish were shipped after sampling to the regional laboratories, where the molluscs were
shucked and fleshy tissues extracted following AOAC Official Method 959.08 [
74
]. An amount of 100 g
shellfish homogenates was mixed with 100 mL 0.1 M hydrochloric acid, with the pH adjusted to <4.0.
The mixture was boiled gently for 5 min before cooling, re-adjusting the pH to 3.0–4.0 if required and
centrifugation prior to analysis. The supernatant fluids were used for the assays. The MBAs were
performed at each laboratory following individual laboratory standard operating procedures (SOPs),
following the guidance of [
74
]. Sample toxicities were calculated from the median death times of
the mice and expressed in terms of
µ
g STX eq./kg shellfish flesh. The limit of detection (LOD) of the
bioassays was between 300 and 350
µ
g STX eq./kg across all laboratories. The established guideline of
800 µg STX eq./kg was applied to determine whether harvesting areas were to be open or closed.
After completion of the MBA, remaining tissues and/or HCl extracts were stored frozen
(<−15 ◦C)
until required for further analysis. Subsamples of tissues and extracts were transported frozen under
temperature-controlled conditions to Cefas. Samples were received from each laboratory after a
maximum of five days transportation. Upon arrival in the UK, samples were checked and stored at
−
20
◦
C until required for analysis. Samples received as shellfish tissue homogenates were thawed and
extracted in 1% acetic acid, following the double extraction procedure detailed by AOAC 2005.06 [
73
]
and as standardised [86].
MBA data were generated in the relevant LA-based organisation soon after the shellfish samples
had been obtained. After shipment of samples to Cefas, both PreCOX and PCOX LC-FLD analyses
were conducted over a period of four months, in multiple batches, followed by LFA. Sample extracts
were subsequently stored for a further four years before the LC-MS/MS was conducted at Cefas. At the
same time, samples were sent to NOAA for RBA testing.
4.3.2. PreCOX LC-FLD
Acetic acid and HCl extracts from the 349 shellfish samples were cleaned up using reverse-phase
solid-phase extraction (SPE) with C18-bonded cartridges (C18-T SPE, Phenomenex, Manchester, UK).
Eluants were adjusted to pH 6.5
±
0.5 and diluted to 4.0 mL. C18-cleaned extracts were fractionated
using a refined ion-exchange (COOH) SPE clean up [
86
]. C18-cleaned extracts of each sample were
subjected to periodate oxidation [
79
] before qualitative LC-FLD analysis. All samples were subsequently
quantified against 5 level calibration standards following peroxide oxidation of C18-cleaned extracts.
Periodate oxidation of fractions F1-F3 was conducted prior to additional LC analysis for samples
showing the potential presence of N-hydroxylated PSTs. Un-oxidised C18-cleaned extracts were also
analysed with peak areas of any naturally-fluorescent chromatographic peaks subtracted from the
toxin peak areas at the same retention time within the oxidised sample.
Liquid chromatography was conducted according to [
86
]. Mobile phases were those described
by AOAC 2005.06 [
73
]. Agilent (Manchester, UK) 1200 series LC systems were used to deliver the
mobile phase at a flow rate of 2 mL/min. Gemini C18 reversed-phase columns (150 mm
×
4.6 mm,
5
µ
m; Phenomenex, Manchester, UK) were with a Gemini C18 guard pre-column (both held at 35
◦
C).
The chromatographic gradient was as described by [
86
]. Agilent fluorescence detectors (1200 model
FLD) were used for the detection of the PSP toxin oxidation products, with excitation and emission
set to 340 and 395 nm, respectively. PST analogues incorporated into the PreCOX LC-FLD detection
method are summarised in Table 6. Toxicity equivalence factors (TEFs) were taken from those published
by EFSA [127].
4.3.3. PCOX LC-FLD
For PCOX LC-FLD analysis, acidic extracts of 165 shellfish samples were deproteinated and
analysed following AOAC 2011.02 [
85
]. The samples chosen were those found to contain total PST
Mar. Drugs 2020,18, 616 20 of 30
above 200
µ
g STX eq./kg. The post-column system consisted of an Agilent 1200 LC-FLD instrument
with a quaternary LC pump, with the addition of two Agilent 1260 isocratic pumps and an external
column oven. PST concentrations in sample extracts were quantified against five-point calibration
standards with individual toxin concentrations and total saxitoxin equivalents determined. As per
PreCOX LC-FLD, toxicity equivalence factors (TEFs) were taken from those published by EFSA [
127
].
Concentrations of individual toxins were calculated in units of STX di-HCl eq./kg and concentrations
summed to estimate sample toxicities in terms of
µ
g STX di-HCl eq./kg. PST analogues incorporated
into the PCOX LC-FLD detection method are summarised in Table 6.
4.3.4. UHPLC-HILIC-MS/MS
UHPLC-HILIC-MS/MS was conducted following the method described by [
94
] and validated
by [
95
,
96
]. Crude sample extracts from 277 samples were cleaned up to remove salt-based interferences,
using Supelclean ENVI-Carb 250 mg/3 mL SPE cartridges. De-salted extracts were collected in 20%
MeCN +0.25% acetic acid and further diluted in MeCN in polypropylene autosampler vials.
A Waters (Manchester, UK) Xevo TQ-S tandem quadrupole mass spectrometer (MS/MS) coupled
to a Waters Acquity UPLC I-Class was used for analysis. Chromatography was conducted using a
1.7
µ
m, 2.1
×
150 mm Waters Acquity BEH Amide UPLC column with a Waters VanGuard BEH Amide
guard cartridge. The columns were held at +60
◦
C, with samples held in the autosampler at +4
◦
C.
The mobile phases, column treatment and analysis gradient were all as described by [
94
]. All Waters
Xevo TQ-S parameters were as detailed by [95].
Quantitation was conducted against the response factors calculated for 14 PSTs present in the
five-point calibration standards available as certified reference standards. The additional toxins (C3,
C4, dcGTX1, dcGTX4 and GTX6) were quantified using experimentally determined relative response
factors (RRF) [
94
]. Toxin concentrations were adjusted for recovery based on the recoveries determined
in matrix spikes [
95
]. Toxicity equivalence factors (TEFs) for STX, NEO, dcSTX, dcNEO, dcGTX2&3,
GTX1-6, C2 and C4 were taken from EFSA recommendations [
127
]. TEFs for other congeners C1,
C3, dcGTX1&4, doSTX were taken from other published evidence [
6
,
89
,
95
]. Semi-quantitation of
M-toxins was conducted using a RRF of 1.0 in comparison to the calibration response generated by the
nearest structurally similar analogue with TEFs taken as 0.1 (M1, M3 and M5) and 0.3 (M2 and M4) as
derived from EFSA TEF data for GTX5&6 and 11-hydroxy STX, respectively [
95
]. Individual toxin
concentrations and total sample toxicity were calculated as above. A summary of the PST analogues
incorporated into the HILIC-MS/MS detection method are summarised in Table 6.
4.3.5. Lateral Flow Assay (LFA)
A total of 250 shellfish extracts were subjected to the PSP LFA [
97
] following the test kit instructions
provided. Higher toxin concentrations added to the cassette result in the test line (T line) becoming
fainter, with the intensity of the T line providing a visual qualitative indication of sample toxin content.
The test sensitivity is approximately 250
µ
g STX eq./kg tissue in terms of the boundary between a
positive and negative test result [
97
,
100
]. A refined version of the assay was utilised for the testing of
samples in this study, as described and assessed previously by [
95
]. The modified protocol incorporated
an additional hydrolysis step to convert GTX1&4 into NEO, thereby improving the detection of GTX1&4
toxins which normally have a very low cross reactivity [
128
]. A volume of 200
µ
L of acidic shellfish
extracts were mixed with the hydrolysis reaction powder provided in the test kit and left standing
at room temperature for 60 min. A volume of 100
µ
L of the solution was subsequently transferred
into 400
µ
L of test kit buffer solution and mixed. A volume of 100
µ
L of the resulting solution was
pipetted into the sample well on the test kit cartridge and left to develop for 30 min before interpreting
the results. All 250 cassettes were interpreted visually, following the guidance provided on the test kit
instructions, with assessments conducted by two independent analysts [
95
]. For a smaller number of
samples, a scanner provided by Scotia Rapid Testing Ltd. was used for automatically interpreting
Mar. Drugs 2020,18, 616 21 of 30
qualitative results and providing a numerical result for each test (n =56). Positive samples were those
recording a scanner result <0.5.
4.3.6. Receptor Binding Assay (RBA)
RBAs were performed on a subset of 117 samples. Analyses were performed as per AOAC
OMA-2011-27 [
93
], with all assays conducted in a 96-well microtiter filter plate format with type GF/B
glass fibre filters and 0.65
µ
m pore size Durapore support membranes (Millipore, Bedford, MA, USA)
as described by [
105
]. Samples were run in triplicate, with each plate containing a seven-point STX
calibration curve and a QC check sample in addition to samples. Assay components were added
to each well in the following order: 35
µ
L assay buffer; 35
µ
L STX standard, QC check, or sample
extract; 35
µ
L [
3
H] STX; and 105
µ
L membrane preparation. Assay plates were subsequently covered
and incubated at 4
◦
C (1 h), filtered and rinsed twice with ice cold assay buffer while under vacuum.
After removal of residual buffer, 50
µ
L Optiphase scintillation cocktail was added per well and the
top of the plate sealed prior to incubation (30 min at room temperature) and counted using a Wallac
Microbeta II microplate scintillation counter for 1 min/well.
Quality control criteria were applied as per Turner et al., 2018. Curve fitting was performed in
Prism (Graph Pad Software, Inc., San Diego, CA, USA) using a sigmoidal dose–response curve with a
variable slope. Sample quantification was carried out only on sample dilutions that fell on the linear
part of the curve (B/B
o
of 0.2–0.7), where B represents the bound [
3
H]STX (in counts per minute, CPM)
in the sample and B
o
represents the maximum bound [
3
H]STX (in CPM) in the absence of sample or
cold STX standard. Where more than one dilution fell within 0.2–0.7 on the standard curve, all sample
wells corresponding to these dilutions are used to calculate sample concentrations.
4.4. Data Assessment
4.4.1. Toxin Results
Total PST concentrations generated by the two LC-FLD methods and LC-MS/MS were used to
estimate sample toxicities, which could be compared with total PSP toxicities determined by MBA
and RBA. Furthermore, the concentrations quantified by the three LC-based methods were used to
assess toxin profiles throughout the dataset, with K-means clustering used to assess groups of similarly
clustered profile patterns [129].
4.4.2. Statistical Assessment
Statistical analysis was performed using R statistical software (R Core Team, 2016). The aim
was first to compare the total toxin levels in shellfish measured with the five different quantitative
methods—MBA, PreCOX, PCOX, LC-MS/MS and RBA. Measurements were assessed on samples for
which toxins have been detected by all five methods, thereby precluding results where samples had
not been analysed by one or more methodologies. Initially, the dataset was filtered to incorporate all
values higher than 32
µ
g STX eq./kg only. Any sample for which a method did not detect toxins or
for which a method detected a toxin at a level lower than this limit was removed from the dataset.
Fewer samples were analysed with RBA than with the other methods, which lead to a small dataset of
55 samples. Therefore, a second assessment was conducted incorporating the four remaining methods
with a larger filtered dataset of 112 samples. A third analysis aimed to evaluate the performance of
Scotia LFA for discriminating positive and negative results. For this purpose, the results obtained by
the LFA were compared with the quantitative results obtained by the PreCOX LC-FLD method.
For each dataset, relationships between the methods were compared graphically using
box-and-whisker and scatter plots. The strength and significance of the relationships were assessed by
Pearson’s correlation. Results obtained by each test method applied to each shellfish sample in the
dataset were compared using a repeated-measures analysis of variance (ANOVA). Test results were
assumed to be nested within shellfish samples in the model error structure. This analysis therefore
Mar. Drugs 2020,18, 616 22 of 30
focused on assessing the variability observed between test methods as opposed to between subjects.
Where the test method was found to explain a significant amount of variability observed within
the dataset at the level of 5%, pairwise t-tests using the ‘holm’ method of adjusting p-values for the
effect of multiple comparisons were used to determine which tests produced results that significant
differed from one another. The comparison of the results obtained by the LFA and PreCOX LC-FLD
methods was based on a box-and-whisker plot and a Welsh two-sample t-test. The same approach was
performed for qualitative LFA results obtained using both visual and automated interpretation.
5. Conclusions
Three hundred and forty-nine shellfish samples sourced from four regions within Latin
America—Argentina, Mexico, Chile and Uruguay—were subjected to analysis for PSP toxins using
six different validated detection methods, the MBA, RBA, PreCOX and PCOX LC-FLD, LC-MS/MS
and the Scotia LFA. Total sample toxicities determined in a large number of different shellfish species
harvested between 1986 and 2012 were found to vary enormously, ranging from no detectable toxin
concentrations to total sample toxicities more than 500-fold higher than the regulatory MPL of 800
µ
g
STX eq./kg, with wide-ranging toxin profiles also determined. Qualitatively, the methods generally
compared well. Whilst datasets for some method comparisons were deemed significantly different,
strong correlations were determined in the total PST data calculated from each of the quantitative
methods. Notably, an excellent comparison was demonstrated between the current EU reference
method, PreCOX LC-FLD, and the LC-MS/MS method, with conversely an apparent overestimation
in PSP when using the MBA in comparison to other methods for some shellfish species. The rapid,
portable LFA from Scotia was shown to be effective for detecting PSTs in the majority of PST-positive
samples, although two false-negative test strip results in samples more than 4-foldhigher than the
MPL and the high proportion of false positives determined showed that there are limitations in the
applicability of the assay for official control testing. Overall, the data determined have shown the
potential for numerous alternative methods for PSP testing in shellfish to be applied to samples from
selected regions within LA. Any such replacement method needs to be formally validated and a range
of quality management processes developed before it can be implemented into any routine monitoring
programme within the region.
Supplementary Materials:
The following are available online at http://www.mdpi.com/1660-3397/18/12/616/s1;
Table S1. Summary of PST toxicities determined by MBA, PreCOX, PCOX, LC-MS/MS and RBA in Latin American
shellfish samples, with Scotia LFA results summarised according to both visual and automated assessment
including LFA scan number. (v) =viscera only; (s) =siphon only; (w) =whole tissues; nd =not detected;
Pos =positive LFA results; Neg =negative LFA result. Scan value =numerical output from scanner (no units).
Sample id prefix denote country of origin: A =Argentina, C =Chile, M =Mexico, and U =Uruguay. Blank cells
infer no test conducted. Yellow shaded cells highlight extreme outliers (more than order of magnitude difference
compared with mean of other results). Table S2. Summary of total PST (
µ
g STX eq/kg) determined by LC-MS/MS,
associated toxin profiles (in terms of proportion saxitoxin equivalents), cluster types and total number of toxin
analogue detections for all Latin American shellfish samples exhibiting total PST >16
µ
g STX eq/kg. Table S3.
Summary of statistics (minimum, maximum, mean, median and variance) obtained from log-transformed PST
toxicity data generated by the five quantitative testing methods (MBA, PreCOX, PCOX, LC-MS/MS and RBA).
Table S4. Summary of statistics (minimum, maximum, mean, median and variance) obtained from log-transformed
PST toxicity data generated by the four quantitative testing methods (MBA, PreCOX, PCOX and LC-MS/MS).
Figure S1. Bar charts illustrating sample types associated with each toxin profile cluster, in relation to a) country
of origin and b) shellfish species. Arg =Argentina, Mex =Mexico, and Uru =Uruguay. M =mussel, Cl =clam,
Sc =scallop, G =gastropod, Ge =geoduck, O =oyster, and Misc =miscellaneous.
Author Contributions:
Conceptualisation, A.D.T., E.G.-M., A.B.G., B.A.S.-I.; methodology, A.D.T.; software,
A.D.T. and M.T.A.; formal analysis, A.D.T., S.T., R.G.H., M.B., F.V.D., D.M., M.S., and I.R.; data curation, A.D.T.;
writing—original draft preparation, A.D.T.; writing—review and editing, A.D.T., R.G.H., M.T.A., F.V.D., M.B.,
E.G.-M., A.B.G., D.C., F.B., I.R. and B.A.S.-I. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments:
We wish to thank Michael Rychlik from the Technische Universität München for funding
the time of Sophie Tarnovius during her Masters studies at Cefas. The work described in this article has been
Mar. Drugs 2020,18, 616 23 of 30
conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for
animal experiments. We thank Tod Leighfield, Christina Mikulski, and Jennifer Maucher-Fuquay (NOAA/NCCOS)
and Ben Maskrey (Cefas) for helping to review this manuscript. These data and related items of information have
not been formally disseminated by the US National Oceanic and Atmospheric Administration (NOAA), and do
not represent any agency determination, view, or policy. We also thank Dorothy Easy and May Anne Donovan
(Scotia Rapid Testing) for their kind and generous provision of the LFA kits for use in this study.
Conflicts of Interest:
The authors have no conflict of interest. LFA results were discussed with Scotia Rapid
Testing at the time of this study. No employees or ex-employees of Scotia Rapid Testing have had any input into
or seen any copy of this manuscript prior to submission.
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