VARIED ASSAYS FOR PSP TOXINS IN HEAT SHOCKED

PHILIPPINE GREEN MUSSELS (PERNA VZRDZS)

MA. PATRICIA V. AZANZA'g3, RHODORA V. AZANZA' and

SHARON R. VENTURA'

'Department of Food Science and Nutrition

College of Home Economics
University of the Philippines
Diliman 1101, Quezon City, Philippines

'Marine Science Institute

College of Science
University of the Philippines
Diliman 1101, Quezon City, Philippines

Accepted for Publication October 20, 2003

ABSTRACT

Toxin assays namely: the mouse bioassay, the receptor binding assay (RBA)
and, the immuno-chromatography assay using MIST AlertTMrapid test kit were
used to determine the concentrations of Paralytic Shellfish Poisoning (PSP)
toxinsfrom untreated and heat shocked Philippine green mussels, Perna viridis
contaminated with Pyrodinium bahamense var. compressum. Toxin levels
rangingfrom 4-15 pg STXeq/lOO g sample were quantified in the mussel samples
analyzed using RBA. Higher levels of PSP toxins at about 30 pg STX eq/lOO g
sample were recorded using mouse bioassay, which was attributed to integering
factors that could induce mouse death resulting in false positive reactions. The
MISTAlertTMtest kit showedpositive reaction in the samples evaluated based on
the reported average profile of PSP toxin analogues at about 40 pg STX eq/100
g sample. The test heat treatments did not elicit definitive change in the PSP
toxin profiles of heat shocked mussels relative to the untreated samples.

Corresponding Author: Department of Food Science and Nutrition, College of Home Economics,
University of the Philippines, Diliman 1101, Quezon City, Philippines. TEL: (+632) 920-5301
loc. 6552 or 9273828; FAX: ( + 6 3 2 ) 9262813 or 9215967; EMAIL:
ma-Patricia .azanzaaup.edu .ph or mpv-azanza@ y ahoo .com

Journal of Food Safety 23 (2003) 249-261. All Rights Reserved.

'Copyright 2003 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 249
250 AZANZA, R.V. AZANZA and S.R.VENTURA
M.P.V.

INTRODUCTION

Surveillance of Paralytic Shellfish Poisoning (PSP) toxins in edible

molluscan bivalves is part of the National Level Seafood Safety Program of the
Philippines (NRTT'F 1999). Local edible marine molluscan bivalves are
considered at risk of being contaminated with the PSP toxins from the toxic
dinoflagellate Pyrodinium bahamense var. compressum during toxic algal blooms
(Azanza 1997). Historical accounts of PSP outbreaks due to consumption of
contaminated bivalves in the Philippines have been recorded since 1983
(Corrales and Gomez 1990: Corrales and Maclean 1995; Azanza 1997).
Pyrodinium bahamense var. compressum has been the primary etiological agent
of toxic red tide occurrences in the Philippines and in other countries in the
lndo-Pacific (Shumway 1990; Azanza and Taylor 2001). The crippling economic
repercussions of Pyrodinium red tide blooms to coastal communities associated
with mariculture, harvest and marketing of local edible oysters and mussels have
been experienced (Maclean 1989).
The traditional technique used for the detection of PSP toxins in edible
Philippine bivalves is the mouse bioassay. However, this method is prone to
yield unreliable results due to interfering factors (McCulloch ef al. 1989). Also,
ethical considerations have also been raised for the use of mice in the assay
(Mackintosh and Smith 2002). Newer analytical assays for PSP toxins that are
considered as alternatives to mouse bioassays are based on imuno-chromatog-
raphy and competition binding techniques (Laycock et al. 2001; Doucette et al.
1997). The MIST AlertT" rapid test kit is an example of an immuno-chromatog-
raphy assay that can detect the presence or absence of toxins at lower limits
from 20-60 pg STXeq/100 g sample in various shellfish extracts (Laycock et al.
2001). The receptor binding assay is a competition binding assay that can
quantify the levels of PSP toxins in contaminated samples based on the amount
of reduced radiolabeled toxin-receptor complex as a function of the amount of
toxin in an unknown sample analyzed (Doucette and Powell 2002).
Heat shock treatment is a commercial postharvest processing procedure for
bivalves, which facilitate shellstock shucking (Banks and House 1958; Saralaya
and Nagaraj 1978; Cook and Ruple 1994). Heat shock treatment of molluscan
shellfish is defined as a process using any form of heat such as steam, hot water
or dry heat for a short time to facilitate molluscan meat detachment from the
shell (FAO/WHO 2000). In Thailand, comparatively only small amount of green
mussels reach the final consumer as shellstocks because the heat shock
treatment, in tandem with shucking and low temperature storage, are currently
used by the commercial mollusc industry (Vakily 1986).
In the Philippines, the heat shock treatment is being proposed as a primary
processing protocol for the production of value-added molluscan products.
Although the heat shock treatment is technically accepted as a minimal
 ASSAYS FOR PSP TOXINS IN PHILIPPINE GREEN MUSSELS 25 1

processing method that is not supposed to bring about any changes in the quality
of the molluscan meats relative to the fresh untreated samples (Cook and Ruple
1994), scientific data to verify the effect of treatment to PSP toxins in molluscan
bivalves should still be provided if its commercial application is to be pursued.
National food safety surveillance agencies of the government, as well as
consumer advocacy groups, may require such information.
The objective of this study was to evaluate analytical techniques for the
detection of PSP toxins in fresh and heat shocked Philippine green mussels. The
standard mouse bioassay (AOAC 1990), RBA (Doucett et al. 1997; PNRI 2000)
and immuno-chromatography assay using Jellet Bioteks MIST AlertTMTest Kit
(Laycock et al. 2001) were used in monitoring the PSP toxin levels of naturally
contaminated fresh and heat treated green mussels.

METHODOLOGY

Collection of Samples
Three batches of naturally contaminated green mussels were collected
during recorded occurrences of toxic algal bloom of Pyrodinium bahamense var.
compressurn last January 24, January 28 and February 5, 2002 in Limay,
Bataan; Manila Bay, Metro Manila and; Bolinao, Pangasinan, Philippines,
respectively. The harvested mussel shellstocks from these sites were stored in
Styrofoam containers and transported from the environmental sites to the
laboratory for testing within 6 h of harvest. The freshly collected mussel
shellstocks were declustered and washed with tap water under high-pressure to
remove the dirt and byssal threads of the mussels. The gaped shellstocks and
those exhibiting foul odors were sorted out and discarded.

Heat Shock Treatment of Mussels

The naturally contaminated mussel shellstocks were heat shocked at lOOC
for 0.33 min using boiling water and 3 min using steam and dry heat. These
protocols were previously established based on the experiments on heat shock
treatment of mussels that produced high sensory acceptability ratings and 100%
gaping in heat shocked green mussel. After the heat shock treatment, the green
mussel shellstocks were manually shucked and subjected to PSP toxicity
analysis. Paralytic shellfish poisoning toxicity was evaluated using mouse
bioassay (AOAC 1990), RBA (Doucette et al. 1997; PNRI 2000) and immuno-
chromatography assay using MIST AlertTMrapid test kit (Laycock et al. 2001)
for PSP toxicity analysis.
252 M.P.V. AZANZA, R.V. AZANZA and S.R. VENTURA

Heat Penetration Studies

The temperature in the mussel meats was monitored using the Hanna-Check
Temperature thermometer (Singapore) to determine the mussel internal
temperature attained during the various heat shock treatments. To facilitate the
insertion of the thermocouple probe into the mussel meats during the tempera-
ture monitoring, the mussel shellstocks were suspended in solutions containing
2% (w/v) NaCl and 1% (w/v) sucrose to induce spontaneous but transitory
gaping of the mussels using procedures described by Gillies (1975). The
stainless thermometer probe was inserted at the thickest portion of a gaped
mussel beside the posterior adductor muscle into the centermost portion. The
mussel shellstock with the thermometer probe was placed in the centermost
position of the batch of raw mussel shellstocks that were subjected to heat
treatment.
The monitoring of the internal temperature of the monitored mussel
shellstocks and the temperature of the heating medium during the various heat
treatments were done every 5 s for 0.33 min for boiling and, 15 s for 3 min at
100C for steam and dry heat, respectively. Heat penetration profiles for each
heat shock treatment were done in 3 trials.

Acid Extraction of Toxins

Toxins from the heat treated meats of naturally contaminated mussels were
extracted using the AOAC (1990) method. From the heat treated meats, 100-150
g were weighed and homogenized in a blender for 2 min at high speed. One
hundred grams of the homogenized samples was mixed with 100 mL of 0.1 N
HCl. The pH of the mixture was adjusted to pH 3-4 by dropwise addition of 5
N HCl or 0.1 N NaOH with constant stimng. Then the mixture was boiled
gently in a water bath at lOOC for 5 min and cooled to room temperature. The
pH was adjusted back to pH 3-4 when the mixture has cooled down to room
temperature. The mixture was diluted to 200 mL by adding distilled water and
mixed thoroughly. The extract was centrifuged for 5 min at 3000 rpm and the
supernatant (acid extract) was collected. The acid extracts were kept refrigerated
at 4C until use in the mouse bioassay, RBA and, immuno-chromatography assay
using MIST AlertTMrapid test kit.

Mouse Bioassay

Mouse bioassay was done using AOAC (1990). Laboratory test mice
weighing 11-21 g that were acquired from the Bureau of Animal Industries
(BAI) in Quezon City, Philippines were intraperitoneally injected with 1 mL of
the HC1 extract. This procedure was applied for each of the test samples. The
time of death was estimated from the exact moment of injection to the last
 ASSAYS FOR PSP TOXINS IN PHILIPPINE GREEN MUSSELS 253

gasping breath of the mice. The median death times of mice were determined
and the corresponding numbers of mouse unit were drawn from the Sommers
Table. For the test mice weighing less than 19 g or more than 21 g, a weight
correction factor (CF) from the Sommers Table was multiplied to the
corresponding mouse unit of the test mice. Mouse units were converted to pg
saxitoxin equivalent/mL by multiplying with CF value. The PSP toxin level was
converted to pg STX equivalent/100 g meat.

Receptor Binding Assay

The RBA was conducted at the Chemistry Department of the Philippine
Nuclear Research Institute (PNRI) in Diliman. Quezon City, Philippines.
Shellfish acid extracts were prepared according to standard AOAC (1990)
procedures. The receptor binding assay used was prepared and used as described
by Doucette er al. (1997). Briefly, the assay was conducted using a 96-well
microtiter filter plate (Millipore, Bedford, MA; cat. No. MHFC NOB 50). The
filter plate was filled with 35 pL radioligand [3H] STX (0.85 nM in assay
concentration), 35 pL STX standard and 140 pL rat brain synaptosome
preparation in the order indicated. The synaptosome membrane preparation was
added to provide 0.5 mg/mL of protein in the assay. The plate was incubated
at 4C for 1 h. After incubation, the 96-well plate was dried for 2-5 s in a
Multiscreen vacuum manifold. Then each well was rinsed with 200 pL ice-cold
HEPES/NaCl using Multichannel pipette to remove any unbound labeled or
unlabeled toxin. The equivalent STX count was determined using a scintillation
counter. The analysis was done in triplicate and the mean counts were
determined.
The concentration of PSP toxins (nM STX Equivalent) in samples was
determined by fitting the data from a competitive binding of standard STX toxin
solution (B/Bo) using a logit transformation and graphing the resulting values.
The competition curve was generated using a computer program called Ligand
(Biosoft, Milltown, NJ). A line of best fit was generated using a simple linear
regression and the STX concentrations (nM STX Equivalent) for the sample
unknowns were obtained by solving the regression equation for x using the logit
transformation of B/Bo for the sample (y). From the curve fitting data, the
sample concentration was determined by multiplying the calculated in-assay STX
concentration (x) by the assay dilution factor and sample dilution factors used.
The calculated values of PSP toxins were reported as pg STX equivalent/l00 g
meat.

Immuno-Chromatography Assay
The third assay used to screen PSP toxins in the study was the immuno-
chromatography assay using the MIST AlertTMrapid test kit (Jellett Biotek,
254 M.P.V. AZANZA. R.V. AZANZA and S.R. VENTURA

Dartmouth, Canada). The acid extract was prepared using the AOAC (1990)
method. Test strips consisting of an absorption pad, a membrane containing a
mixture of toxin analogues as indicated by the T line and an antibody detection
reagent as indicated by the C line, a sample pad and a conjugate pad containing
the antibodies, were used for this assay.
In the analysis, 100 pL of the shellfish acid extracts were added to 400 pL
MIST AlertTMbuffer solution. The buffer and the sample extract were mixed by
pipetting the mixture up and down for at least 3x. Then 100 pL of the buffer-
acid extract mixture was placed into the sample hole of the test strips. After 20
min at ambient condition, the test strips were analyzed for the toxin. A partial
or complete fading of the T line in the test strip indicated positive reaction of the
PSP toxins. The visible T line manifests the absence of toxin in a sample
(Laycock et al. 2001). Results obtained are reported as +/- reaction to the
average profile of PSP toxin analogue of 40 pg STX eq/100 g sample.

RESULTS AND DISCUSSION

Although technically, the heat shock process should not impart any
significant quality change to the green mussel meat, scientific data to verify this
information should be made available to the seafood industries, consumer
protection groups and concerned government agencies. The heat penetration data
obtained from mussels exposed to various test heat shock treatments are shown
in Fig. 1. The boiling water treatment effected the highest internal temperature
of 75C in the heat shocked mussels while steam and dry heat effected maximum
values of 65 and 59C, respectively. The attained internal temperatures in the
mussels during the heat shock treatments were adequate to cause gaping of the
mussels due to the relaxation of the adductor muscles of bivalves. Studies on
heat shock treatment of oysters using steam showed that an internal temperature
of only about 40C already facilitated the opening of bivalves (Cook and Ruple
1994).
The effects of the various heat shock treatments employed to green mussels
using boiling water, steam and dry heat on the PSP toxin levels were determined
using the 3 techniques namely: mouse bioassay (AOAC 1990), RBA (Doucette
ef al. 1997; PNRI 2000) and immuno-chromatography assay using the MIST
AlertTMrapid test kit (Laycock et al. 2001). The PSP toxin concentrations in
naturally contaminated P. vin'dis shellstocks were determined during natural
blooms of P. bahamense var. compressum during the first quarter 2002 in
Central and Northern Luzon. Table 1 shows the results of the assays on PSP
toxin levels in the test green mussel samples.
 ASSAYS FOR PSP TOXINS IN PHILIPPINE GREEN MUSSELS 255

Boiling water
&--Time of 100% gaping of shenstock
Temperatwe of boiling water

80 eat temperature dwing air cooling (25-27 C)

60

40 Meat temperature during heat shocking

20

0
0 50 100 150 200 250 300 350 400 450

h Steam
0
O
- 120 Temperatwe of steam
Time of 100% gaping of shelktock
E=!I 100
+, 80
60

E
a
40
2o
+ o 7

450

Dry heat
T e m e r a l u e of drv heat
+.--Erne of 100% gaping of shenstock
Meat temperatme duina air coolina (25-27 C )
80
60
40

20
0 L---- I
_-

0 50 100 150 200 250 300 350 400 450

Time (sec)

FIG. 1. HEAT PENETRATION CHARACTERISTICS OF HEAT SHOCKED MUSSELS

USING BOILING WATER, STEAM AND DRY HEAT AT ABOUT lOOC
( A Trial 1. 0 Trial 2, 0 Trial 3)
 N
TABLE 1 VI
Q\
RESULTS OF PSP TOXIN ASSAYS IN FRESH A N D HEAT SHOCKED PHILIPPINE GREEN MUSSELS NATURALLY CONTAMINATED
WITH P bahamense var compressum
.---- - - _I
- - ___ -- -
Sampling Site Heat Treatments' Test Assay **
--------- ----_ ~ _ _
MA RBA MIST
(pg STXeq/lOOg) (pg STXeq/lOOg) (+/- reacfion, 640 pg STXeq
- - - - - .___ . __ __ - -.___ detectlo? IimiUlOOg) P
Llmay, Bataan ?J
(January 24,2002) c
Control PSP Symptoms"' 8.57 f 1 50 ++ 9
N
Bailing Water PSP Symptoms 8.95 f 2.00 ++ 9
Steam PSP Symptoms 9 78 f 1 40 ++ 3
Dry Heat Nd ND ND -9
F
Manlla Bay, Metro Manila c
(January 28,2002)
Control PSP Symptoms 5.44 f 0 50 ++
Boiling Water PSP Symptoms 529f010 ++
Steam PSP Symptoms 4 22 i 0 40 ++
Dry Heat PSP Symptoms 9 79 f 0 20 ++

Bollnao, Pangaslnan
(February 5,2002) c
Control 20.90 f 1.50 15.98 f 1 00 ++ m
Boiling Water 20.05 f 2.70 13.14 f 1.50 ++ 3
Steam 29.60 f 0.90 6.29 i 0.30 ++
Dry Heat 29.19 f 4.60 15.52 i 0 80 ++ B
- , X~ . .,._ - ._-____.- ",
,,(,, ~
,,,, .- ,

* Heat treatments at IOOC for 0.33 min (boiling) and 3 min (steam and dry heat)
** Test assay for MA (mouse bioassay) and RBA (receptor binding assay) are means of 3 trials and, MIST (Maritime In Vitro Shellfish Test) AlertT"
rapid test kit was done in '2 trials
*** PSP Symptoms: general weakness, muscular paralysis, loss of balance, red and chinky eyes, nonfatal
ND, Not Determined
 ASSAYS FOR PSP TOXINS IN PHILIPPINE GREEN MUSSELS 257

Only samples collected from Bolinao, Pangasinan on February 5, 2002

produced quantifiable levels of toxin using mouse bioassay. The PSP toxin levels
of the naturally contaminated samples ranged from 28.85-29.60 pg STXeq/l00
g meat. These values were below the reported reliable detection limit for PSP
toxins using mouse bioassay as a PSP detection tool, which is about 32-58 pg
STXeq/100 g meat (Bricelj and Shumway 1998). The 2 samples from Bataan
and Manila Bay elicited only PSP symptoms in injected mice but with no
recorded fatalities in the test animals. The PSP symptoms in mice include:
general weakness, dizziness, immobility, rapid breathing, loss of balance and,
development of chinky eyes. A related study made by Azanza et al. (2000)
showed that the required cumulative uptake of vegetative cells P. bahamense
var. compressumcell by green mussels was at least lo7dinoflagellate cells/g live
mussel meat to elicit PSP symptoms in laboratory test mice in mouse assay.
The other method used in the toxicity analysis was the immuno-chromatog-
raphy assay using the MIST AlertTMrapid test kit (Laycock et al. 2001). This
method is only a qualitative technique that can provide positivehegative results
within its lowest detection limit in less than 20 min. Jellett ef al. (2003) reported
that the test kit can detect the presence or absence of toxins at low levels that
may vary from 20-60 pg STX eq/100 g in various shellfish extracts. It is able
to detect the 36 different chemical analogs of PSP group of toxins in a variety
of shellfish matrices in shellfish tissue (Hoyle 2000). The results of toxicity
analysis showed that the toxicity levels of naturally contaminated mussels
evaluated were all within the detectable limit of the MIST AlertTMtest kit and
that the heat shock treatments used did not effect a reduction in PSP toxin levels
below its minimum limit. The MIST AlertTMtest for PSP toxins yielded positive
results in all the mussel samples evaluated, even for the samples that provided
negative results in the mouse bioassay. The differing sensitivities to different
toxin profiles of this antibody based assay may be responsible for the inconsis-
tency between the results of the MIST AlertTMrelative to the mouse bioassay.
Mackintosh and Smith (2002) reported that extracts containing high levels of
low-toxicity toxins such as C toxins may yield positive results in the MISTTM
Alert test for PSP but may yield negative results in the mouse bioassay.
The third technique used to analyze PSP toxin in heat shocked and control
samples was RBA. This method was reported to measure up to C 1 ng STX/mL
based on a competition binding assay in which the toxin samples compete with
radiolabeled toxin for a given number of binding sites in a rat brain synaptosome
preparation to form a complex (Van Dolah 2000; Doucette ef al. 1997). The
amount of reduced radiolabeled toxin-receptor complex is a function of the
amount of toxin in an unknown sample analyzed (Doucette and Powell 2002).
The results of RBA in this study demonstrated no conclusive change in the PSP
toxin levels of the heat treated mussels relative to the untreated control, except
for the samples from Bolinao, Pangasinan and Manila Bay that were heat
258 M.P.V. AZANZA, R.V. AZANZA and S.R.VENTURA

shocked using steam and dry heat, respectively. The results of this study showed
that the RBA detected toxicity levels in the mussel samples ranging from 4.22-
15.98 pg STXeq/100 g sample. The RBA has been cited to have a detection
limit which is several orders of magnitude more sensitive than mammalian
bioassays (Doucette and Powell 2002).
The data gathered from the 3 assays generally showed that the test heat
shock treatments effected no definitive change in the PSP toxin levels in
naturally contaminated green mussels. The results obtained were not surprising
since the maximum temperatures recorded in each of the 3 heat shock techniques
employed was not high enough to induce PSP toxin biotransformations. Oshima
(1 990) and Azanza et al. (2000) separately reported that the PSP toxin profiles
of vegetative cells of P . baharnense var. compressum in the Philippines and also
in Pyrodinium-contaminated shellstocks consisted primarily of STX, dcSTX and
B1. It has been reported that STX thermal degradation occurs only when heated
up to 100-120C with 30-60 min exposure times (Nagashima ef al. 1991;
Indrasena and Gill 2000). On the other hand, B1 was reported to be more heat
resistant than STX and dcSTX (Nagashima et al. 1991; Motohiro 1992). The
range of mussel internal temperature during heat shocking was 59-75C. The
results of the assays also validate the premise that heat shock processes should
not change the quality characteristics of the heat shocked molluscs but should
rather just facilitate meat extraction from gaped shellstocks (Cook and Ruple
1994; FAO/WHO 2000).
The results of the mouse bioassay of the samples from Bolinao, Pangasinan
demonstrated noticeably higher levels of PSP toxin concentrations relative to the
values established using RBA. This disparity could perhaps be explained by
some reliability defects in the mouse bioassay (McCulloch ef al. 1989). The
presence of interfering factors like heavy metal pollutants was reported to induce
mouse death with apparent neurotoxic symptoms similar to PSP in mouse
bioassay. This affects the measurement of total toxicity in mammalian bioassay
(McCulloch et al. 1989). Barile (1990) confirmed the presence of heavy metal
contamination in Philippine green mussel growing waters.
In summary, the study established that the various heat shock treatments
employed were not able to effect any definitive change in the PSP toxin profiles
of heat treated mussels. The RBA technique was shown to be the more sensitive
assay for monitoring the PSP toxin levels relative to mouse bioassay and MIST
AlertTMrapid test kit for fresh and heat treated samples. This technique can
detect quantifiable levels of PSP toxins at concentrations much lower than the
reported reliable detection limit of the mouse bioassay. On the other hand, the
MIST AlertTMrapid test kit could qualitatively and variably detect the presence
or absence of PSP toxins within its reported detection level.
 ASSAYS FOR PSP TOXINS IN PHILIPPINE GREEN MUSSELS 259

ACKNOWLEDGMENT

This study has been made possible through the funding of the Department
of Agriculture-Bureau of Agricultural Research, Diliman, Quezon City,
Philippines. We would also like to acknowledge the assistance of Philippine
Nuclear Research Institute, Diliman, Quezon City, Philippines and Jellett Biotek,
Dartmouth, Canada.

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