Environmental Bioindicators, 2:264272, 2007

Copyright Taylor & Francis Group, LLC

ISSN: 1555-5275 print/ 1555-5267 online
DOI: 10.1080/15555270701693612

Use of Perna viridis as a Bioindicator of Paralytic

1555-5267
1555-5275
UEBI
Environmental Bioindicators,
Bioindicators Vol. 2, No. 4, October 2007: pp. 127

Shellfish Toxins at Low Pyrodinium bahamense var

compressum Density using a Radioreceptor Assay

E.Z. SOMBRITO,1 M.C.V. HONRADO,1 A. DE VERA,1

Use of Perna
Sombrito et al.
viridis as a bioindicator

R.S. TABBADA,1 MA.L. RAADA,1 J. RELOX JR.,2 AND

M.DC. TANGONAN1
1
Philippine Nuclear Research Institute, Diliman, QC, Philippines
2
Bureau of Fisheries and Aquatic Resources, Diliman, QC, Philippines

A radioassay method, i.e. the Receptor Binding Assay, was used for studying the uptake
of Paralytic Shellfish Poisoning (PSP) toxins in the green bay mussel Perna viridis highly
consumed in the Philippines. This method allowed working at low cell density of Pyrod-
inium bahamense var compressum (~102-103 cells/L) representative of early stages of
toxic algal blooms. The results indicated that within 16 hours toxic levels of PSP are
reached in the tissues of P. viridis, confirming the suitability of the green mussel as an
indicator organism for paralytic shellfish toxicity in bivalves during the early stages of
the bloom. Results also demonstrated that the weight-specific toxicity significantly
increased with mussel size reduction. This method, based on the competition between the
labeled and unlabeled toxin for the sodium channel receptor, offers better sensitivity than
the mouse bioassay method. With an increasing amount of toxins in the sample, the
amount of radiolabeled toxin binding with the receptor decreases. The amount of radio-
labeled toxin (tritiated saxitoxin) is then measured using liquid scintillation counting.
Quantification is based on a competition curve established by measuring competitive
binding on a rat brain membrane with known concentrations of saxitoxin.

Keywords: Pyrodinium bahamense var. compressum, Perna viridis, paralytic

shellfish poisoning toxins, receptor binding assay

INTRODUCTION
Green bay mussel (Perna viridis) is grown commercially in many embayments in the
Philippines. During large blooms of toxic Pyrodinium bahamense var. compressum (PbC),
the mussel becomes toxic through continuous intake of PbC cells. An association of PSP
with the presence of PbC cells has long been observed in the Philippine bays. The country
has experienced a total of 42 toxic PbC bloom outbreaks between 1983 and 2001, which
resulted in a total of 2107 PSP cases with 117 fatalities (Furio and Gonzales 2002).
The toxic substance is a suite of compounds collectively called saxitoxins. More than
20 structurally related saxitoxin congeners have so far been identified in saxitoxin-producing
dinoflagellates and in filter-feeding bivalves that consume them. These saxitoxin conge-
ners vary widely in their potency or biological activity (Hall et al 1990). They can be
present in different proportions in the same dinoflagellate species and are subject to

Address correspondence to E.Z. Sombrito, Philippine Nuclear Research Institute,

Commonwealth Ave., Diliman, QC, Philippines 1101. E-mail: ezsombrito@pnri.dost.gov.ph

264
 Use of Perna viridis as a Bioindicator 265

interconversions and changes in relative proportions as they are transferred through the
food chain (Bricelj and Shumway 1998).
Saxitoxin (STX) elicits its effects by binding with high affinity (Kd ~ 2 nM) to site 1 on
the voltage-dependent sodium channel, inhibiting channel conductance and thereby causing
blockade of neuronal activity The primary site of STX action in humans is the peripheral
nervous system, where its binding results in paralytic shellfish poisoning characterized by
tingling and numbness of the perioral area and extremities, loss of motor control, drowsi-
ness, incoherence, and in the case of high doses, respiratory paralysis (Van Dolah 2000).
The saxitoxins readily accumulate in tissues of filter-feeding shellfish to concentra-
tions that can be lethal to humans following ingestion of contaminated shellfish. To
prevent ill health effects among shellfish consumers in the country, the PbC cell concen-
tration in seawater and shellfish toxicity are regularly monitored. Green bay mussel, if cul-
tured in the area, serves as the indicator organism for shellfish safety. Commercial mussel
harvest and consumption are not allowed if the toxicity assay exceeds 40 g 100 g-1 meat.
The mussels are grown on bamboo poles immersed in seawater in a commercial mussel
farm. The mussels are growing at different heights of the bamboo and are of different sizes
and maturity. The resulting level of toxicity in the mussel is correlated with PbC cell density
but many other factors may also affect the level of toxicity in the mussel, such as its size and
the depth at which the mussel attaches itself on the vertical bamboo pole. Toxicity differences
may also exist among mussel cultures growing upstream or downstream of water flow. The
rate of uptake and depuration of the toxins also affects the net toxicity in the mussel.
In this study, the suitability of commercial Perna viridis as an indicator of toxicity in
shellfish during the early stages of the toxic algal bloom is assessed by determining mussel
toxicity exposed to low densities of the toxic algae. The most commonly used method for
direct toxicity assay is the mouse bioassay (MBA). However, the MBA is not the appro-
priate method because of the low level of toxicity that can be present in the mussel during
the early stages of the bloom. Thus, it will also not be useful for studying the uptake of the
toxins in the mussel before it reaches the no-harvest, no-consumption level.
The receptor binding assay method (RBA) has a detection limit about a hundred times better
than the mouse bioassay method. This assay method showed remarkable sensitivity, measuring
saxitoxin at less than 1 ng/ml. (Davio and Fontelo 1984; Vieytes 1993). RBA is based on the
competition between the labeled and unlabeled toxin (3HSTX) for the sodium channel receptor.
With increasing amount of toxins in the sample, the amount of radiolabeled toxin binding with
the receptor decreases. The amount of radiolabeled toxin (tritiated saxitoxin) is then measured
using liquid scintillation counting. Quantification is based on a competition curve established by
measuring competitive binding on a rat brain membrane with known concentrations of saxitoxin.
The microplate format receptor-binding assay increased the sample throughput
(Doucette et al. 1997). The sensitivity and high sample throughput of the microplate RBA
method afforded the study of saxitoxin uptake in mussels at levels of toxicity below the
regulatory level through feeding at consistently low levels of Pyrodinium bahamense var.
compressum cells under natural conditions.

MATERIALS AND METHODS

Exposure Protocol
A site in Juag Lagoon, Matnog, Sorsogon (Figure 1) provided a natural laboratory for
studying the uptake of PSP toxin in mussels as a function of time. The 20-ha lagoon opens
to the sea through a narrow channel. The deepest part of the lagoon is about seven meters.
266 Sombrito et al.

LUZON

VISAYAS

MINDANAO

Figure 1. The study site is in Juag Lagoon, Matnog, Sorsogon, located at the southernmost tip of Luzon
Island about 20 ha in size and experiencing recurrent blooms of Pyrodinium bahamanse var. compressum.

This area is experiencing recurring bloom of Pyrodinium bahamense var. compressum.

Though there is no commercial rearing of green mussels in the area, wild shellfish is abundant.
Mussels that are free from STX contamination were placed in small clusters in several
fishnet bags and immersed at a depth of one meter in the experimental stations. At defined
time intervals, mussels were removed from the test sites and the weights and lengths of
mussels were taken individually. The meat was shucked in the field, stored in ice and ana-
lyzed in the laboratory. Experiments were done in triplicates.

Receptor Binding Assay

The toxicity of mussels was quantified using the RBA method. Meat was drained,
weighed and homogenized and processed according to the Association of Official Analyt-
ical Chemists (AOAC) mouse bioassay method (1). Briefly, the toxin was extracted with
0.1 N HCl (of volume equal to sample weight) in boiling water for five minutes at pH
between 24 and preferably around 3. The extract was centrifuged and an aliquot of the
supernatant was used for the receptor-binding assay.
Binding competition was performed in 96-well incubation plates. The standard curve
was prepared from mixtures of 35 l [3H] STX, 35 l unlabeled standard STX (10-10 to 10-6 M)
and 140 l rat brain membrane preparation (1 mg/ml protein concentration). For analysis of
 Use of Perna viridis as a Bioindicator 267

unknowns, samples were diluted with binding buffer as needed so that analysis results fall on
the linear part of the competition curve. The mixtures were incubated at 4C for 1 hr and then
harvested using a Packard Filtermate Harvester on a 96-well microplate with bonded GF/C
filter (Packard Unifilter-96). The plate was washed twice with cold 200 l buffer. The plate
bottom was sealed and 40 l Microscint 20 scintillant was added to each well. A topseal was
placed on the plate and allowed to sit for 30 min before counting on a microplate scintillation
counter. Data was analyzed using the four parameter logistic method of MultiCalc (Wallac)
and nonlinear regression of GraphPad Prism 3 (GraphPad Software, Inc.).

STX Concentration in Pyrodinium Bahamense var. Compressum

The concentration of saxitoxin (the main contributor to PSP toxicity) in PbC cells was
measured by high-pressure liquid chromatography method (HPLC). A plankton net was
used for collecting the cells. Several hauls of seawater were taken and placed in 20-L
carboys. The seawater was then filtered using filter membranes. Cells on the filters were
then resuspended with dilute acetic acid and sonicated. Crude extracts were refiltered to
separate debris from filtrate. Portions were passed in syringe filters to further clean the
filtrates before injecting into the HPLC. Aliquots were then analyzed by HPLC.
The volume of seawater in cm3 was computed from the area of the plankton net (cm2)
multiplied by the hauling depth (cm).

RESULTS

Uptake of PSP Toxins in Mussel

The uncontaminated mussels were exposed in different concentrations of PbC during the
stationary phase of the bloom.
During the first field experiment, the PbC cell concentration was in the order of
2 103 PbC cells/liter. Exposure of uncontaminated mussels to these PbC cells exhibited
uptake of saxitoxin within sixteen (16) hours as shown in Figure 2. The slope of the linear
regression obtained was 2.4 0.9 (R2 =0.87).

100
Toxicity (ug STX eq / 100 g)

80

60

40

20

0
0 5 10 15 20 25
Exposure Time (hours)

Figure 2. Toxicity levels (mean SD) in mussels exposed to Pyrodinium bahamense var. compres-
sum at concentrations ~103 cells/liter.
268 Sombrito et al.

18

16

Toxicity (ug STX eq / 100 g)

14

12

10

0
2 4 6 8 10 12 14 16 18
Exposure Time (hours)

Figure 3. Toxicity levels (mean SD) in mussels exposed to Pyrodinium bahamense var. compres-
sum at concentrations ~102 cells/liter.

In the second field experiment, the PbC cell concentration was generally lower than
that of the first field experiment by one tenth and was in the order of 3 x 102. Figure 3
represents the average toxicity value for at least three individual mussels, taken at 1 m
depth as a function of time exposure to PbC cells. The slope of the linear regression
obtained was 0.55 0.12 (R2 =0.88).
The trend lines obtained assume a linear increase of toxicity with time during the first
16 hours of exposure. Considerable inter-individual variability of STX toxicity was
observed. Though there is a large scatter in the values brought about by the natural vari-
ability in the mussel population accentuated by the small sample size taken from the lot,
an increasing level of toxicity even at these low levels of concentrations of toxic algal
cells was observed.

Effect of Mussel Weight on Specific Toxicity Attained by the Mussel

The specific toxicity is representative of the toxin content of the organism. Figures 4 and 5
show how toxicity per unit weight varies with increasing mussel weight. Three sets of data
are presented, where each set represents different exposure time. The decreasing specific
toxicity with mussel size as indicated by the mussel weight was clearly observed for the
data set representing a 3-hour exposure time (Figure 4). The correlation coefficient for the
other sets is not significant but a decreasing trend can still be observed.
The relationship was expressed here as a linear function but the relationship may be
more complicated and can be shown by the analysis of more samples.

Estimate of STX Content in the Algal Cells

During the second field experiment, PbC cells were collected to get an estimate of the
average PbC cell count and average PbC cell toxicity in the study site.
The cell density at the place where the PbC cells were collected was 300 cells/liter.
The volume of the seawater from which the cells were collected was estimated from the
 Use of Perna viridis as a Bioindicator 269

14

12

Toxicity (ug STX eq / 100 g)

10

0
4 5 6 7
Mussel Weight (grams)

Figure 4. Relationship between specific toxicity levels (mean SD) reached in mussels and weight
of the mussels exposed for 3 hours to Pyrodinium bahamense var. compressum cells at a density of
102 cells/liter.

10

9
Toxicity (ug STX eq / 100 g)

4
4 h exposure
5 h exposure
3 regression (4 h)
regression (5 h)
2
4 6 8 10
Mussel Weight (grams)

Figure 5. Relationship between toxicity levels (mean SD) and weight of mussels exposed for 4
and 5 hours to Pyrodinium bahamense var. compressum cells.

area of the plankton net and the depth of hauling. The cell extracts containing the saxitoxin
were analyzed by HPLC giving an average saxitoxin content of 3.7 2.0 pg STX/cell.

DISCUSSION

Radiometric Receptor Binding Assay for Low PSP Toxicity Levels

The most commonly used method to monitor the saxitoxin toxicity in shellfish is based on
a time-of-death mouse bioassay. This method is easy to perform and requires no special
270 Sombrito et al.

equipment. However, its detection limit of 40 g 100 g-1shellfish is not suitable for
studying the uptake of STX toxicity at low Pyrodinium cell density. There is also a large
variability of results (20% variability) due to differences in the reaction of individual
mice, and it requires a large number of animals to test for a few samples.
The radiometric receptor binding assay method for the saxitoxins in shellfish has sen-
sitivity much better than that of the mouse bioassay (detection limit is 0.4 g STX 100 g-1
meat). This method is based on the competition between saxitoxin labeled with tritium
(3H) and saxitoxin present in the sample on the sodium channel receptor site. It can be
used in microplate format for high throughput capability.
This method demonstrated its performance when measuring low toxin contents cor-
responding to early stages of toxic PbC blooms. Therefore, it can be a useful method for
providing an early warning signal of toxicity, thus early harvest prior to shellfish har-
vest ban can be made or a timely warning can be issued to protect the public health. It
can also alert the monitoring agency for the conduct of a more intensive monitoring
scheme that will contribute to an effective control of PSP cases in the shellfish-consuming
public.
The receptor binding assay is also a useful tool to study the increase of toxicity levels
during the onset of the bloom, where toxic PbC cell concentrations are still very low, and
even more during the first few hours of exposure in the marine environment.

PSP Toxin Uptake in Mussel

Major factors believed to be controlling the level of toxin in bivalves include physio-
logical processes in the bivalves and the kinetics of PSP toxins in bivalves (Choi et al
2003). The physiological processes will include the clearance rate defined as the
volume of water cleared per unit time, the filtration rate (the food biomass filtered by
the bivalves), and the ingestion rate (actual amount of algal cells ingested by the
bivalves). The kinetics of PSP toxin deals with the net uptake of the toxins as deter-
mined by the absorption, assimilation, biotransformation and depuration of the toxin
in the bivalves.
Mussels do not have Na-channels targeted by saxitoxin, which means that their
physiological processes are not much affected by the ingestion of PSP toxic foods (Bricelj
and Shumway 1998). Mussels also have elevated filtration rates compared to other shell-
fish. These two factors probably determine their faster rate of phycotoxin accumulation
compared with other bivalves (Morono et al 2001).
In this study we observed that the rate of increase in saxitoxin toxicity levels at higher
PbC cell concentration (slope=2.4 g 100 g-1 hr-1) is higher than that of the lower cell
concentration (slope=0.55 g 100 g-1 hr-1). If the filtration rate remains the same irrespec-
tive of the amount of toxic algae in the diet, it is expected that the amount of toxin ingested
will increase with increasing concentration of the toxic cells. However, the rate of increase
should remain the same if the physiological processes in the organism remain the same. It
may also be inferred from the results obtained that increasing the proportion of toxic food
in the diet of the mussel increases the rate of the assimilation of the toxin in the mussel.
However, there is no basis for assuming an increase in assimilation rate but it can be due
to higher amount of unassimilated toxic cells in the viscera as the density of the toxic cell
increases. This may partially explain the increasing STX uptake rate with cell density
observed in this study.
The data also showed that PbC cell density as low as 103 cells/L are enough to raise
the toxicity concentration in the mussels to 40 g 100 g-1 within 16 hours.
 Use of Perna viridis as a Bioindicator 271

Variation of Specific Toxicity With Mussel Weight

Reports (Morono et al. 2001; Bricelj and Shumway 1998) have cited the negative effect of size
on weight-specific filtration. It is thus expected that the weight-specific toxicity of bivalves (in
g STXeq 100 g1) will vary inversely with body size. For the uptake of the toxin in mussels,
the assimilation rate may also affect the weight-specific toxicity; however, further studies are
needed to determine if a difference in assimilation rate has an effect on weight-specific toxicity.
Our results indicated that there is a linear negative correlation of weight-specific tox-
icity with mussel size. However, increasing the number of samples analyzed would help in
confirming the results obtained.

Estimate of STX Content in the Algal Cells

The results showed that the toxin production of the PbC cells in the study area was high
and that toxicity observed in the mussels that were analyzed in the experiments indeed
came from the ingestion of the PbC cells through the filtration process.

Conclusions and Recommendations

This study confirms that the commercial green bay mussel, Perna viridis, is a good bioin-
dicator of PbC toxicity especially because it immediately responds to the presence of the
toxic PbC cells by taking up PSP toxins within the first sixteen (16) hours of exposure.
The use of uniformly small-sized mussels in national monitoring programs will give a
conservative estimate of PSP toxicity for public health purposes.
The nuclear-based receptor-binding assay provides a tool for screening toxicity at the
onset of the bloom just before the toxicity level reaches the regulatory limit for mussel
harvest and consumption. It can be used for early warning of increasing levels of PSP
toxicity in mussels serving as bioindicators.
There are refinements that must be done to validate the results of the two field studies
conducted. As stated above, we observed an increasing rate of uptake with increasing cell
density. The assumption of PbC cell density being a measure of cell toxicity must be
validated with further studies and at a higher cell density level. Other variables that may
determine the uptake rate like seston volume, vertical and horizontal movement of the
toxic cells in time and space will be defined in future field experiments.

ACKNOWLDEGEMENTS

The IAEA Interregional Project INT/7/016 provided the labeled toxin. The funding support of the
Department of Science and Technology and the Internation Atomical Energy Agency (IAEA)
through the Research Contract CRP 12563 is gratefully acknowledged. Authors thank Mr. Val
Macasaed (BFAR), Mr. Efren J. Sta Maria and Mr. Ryan U. Olivares (PNRI) and Ms. Aileen de
Leon University of the Philippines Marine Science Institute for their assistance to the project.

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