Feeding behavior of individuals and groups of king scallops (Pecten maximus)
contaminated experimentally with PSP and detoxified
S. Bougrier (1), P. Lassus (2), B. Beliaeff (2), M. Bardouil (2), P. Masselin (2), F. Mornet (1), P. Truquet (2), F. Matignon (2)
(1) CNRS-IFREMER, CREMA, BP 5, 17137 L'Houmeau, France
(2) IFREMER, Centre de Nantes, BP 21105, 44311 Nantes Cedex 3, France
Objectives
Introduction
After initial events in 1988 [1], [2] several areas along the Northern Brittany coast have
been subjected almost annually to intense blooms of Alexandrium minutum Halim, a
PSP toxin producer. St. Brieuc Bay, close to these blooming areas, is the most important
king scallop harvesting center. Although only oysters and mussels have been
contaminated to date by PSP toxins, the risk of a transfer of A. minutum cysts or
vegetative forms along the Northern Brittany coasts cannot be excluded. Therefore,
further studies are required to determine the physiology of
contamination/detoxification patterns in scallops.
As only the adductor muscle and gonad of the scallop are generally eaten by
consumers, some national monitoring networks for phycotoxins assess toxicity in the
digestive gland (considered to concentrate the most toxin) and not in whole meat or
each separate tissue. A toxin threshold in digestive gland, different from the
international safety threshold used for whole tissue (80 µg. STX eq.100 g-1), can then
be used, provided that the relative toxin ratio between digestive gland and muscle is
known. However, gonad toxicity may be difficult to predict on the basis of the toxic
level in digestive gland [3] since no correlation was found between the toxicity of
gonad and that of viscera in wild populations of sea scallops (Placopecten
magellanicus). It would also appear that PSP toxins accumulated by scallops are
eliminated very slowly [4] Thus, the uptake, tissue sequestration and biotransformation
of toxic compounds, as well as the detoxification pathways, are of particular interest in
scallops, especially for improved assessment and monitoring.
Toxic
algae
tank
TURNER designs
fluorimeter
Flow-meter
VISUAL
BASIC
software
ADCLONE
data-loger
Material and Methods
1) what proportion of toxin from ingested algal food is durably accumulated in
scallop tissues (Tbe = toxin bioaccumulation efficiency, or the ratio of real toxin
to estimated absorbed toxin)?
2) is toxin body burden correlated with the filtration rate?
3) are GTX2/GTX3 (the major toxin analogs in the French strain of A. minutum)
hydrolyzed or transformed through enzymatic pathways. As to the feeding
behavior the effect of different non-toxic diets upon PSP-contaminated
scallops detoxification rates was investigated at two levels: the immediate
“food effect” following diet switch and the trend observed in ecophysiological
parameters during detoxification phase.
MP1
Draining
TURNER designs
fluorimeter
Biodeposits
Membrane
Flow-meter
switch
Thermo
regulation
800 l h -1
Pump Pump
1 m3/h 2 m3/h
Data recorder
photo C. Daniel, 1999
Fig 1 : Simplified diagramatic view of a complete recirculated water system showing the 100-L rearing tank
(raceway), the 30-L thermoregulated "buffer" tank, the micro-pump-delivered (MP) algal culture set and the
"individual- size" derived system.
Results
140
FTA %
Mean FTA
Absorbed PSP toxins (µg STXeq.)
100
90
80
70
60
50
C1
40
C2
30
C3
20
C4
10
C1
C2
C3
C4
C5
120
100
4
5
6
7
1
Biodeposition Rate (mg.h-1.g d.w.-1)
Absorbed PSP toxins (µg STXeq.)
20
15
10
2
3
4
5
6
7
8
Days
9
Fig 3: Cumulated amounts of PSP toxins in the whole meat of each scallop during the
contamination phase.
0.4
0.2
Isochrysis
0.4
0.2
5
0
5
6
7
8
Days
Fig 4 : Daily variations in absorbed PSP toxins for each of the 5 experimented scallops during
the contamination phase.
9
0
5
10
15
20
25
Time (day)
Fig. 5: Biodeposition rates for scallop populations
first contaminated by A.minutum and
then detoxified with either Tetraselmis- or Isochrysis-based diets.
A classical distribution was found for mean toxin body burden per tissue (or bulk of tissues) compared to total wet weight of tissue (100%), i.e. digestive gland > >
kidneys > other tissues. However, it was noteworthy that the contribution of kidneys to total toxin body burden reached 20%. Mean toxin absorption efficiency (Tae or
the ratio of absorbed toxin to ingested toxin), was 42%, and mean Tbe (Toxin Bioaccumulation Efficiency) was 19%, which means that only 42% of the filtered toxins
was absorbed, including 19% PSP toxins accumulated in tissues. Toxin bioaccumulation yield (Tby), calculated as Tby = Tox/TFR, represented the ratio of toxin actually
accumulated in tissues to the uptake of toxins contained in algal food. This percentage was quite low (8%).
Population-size experiment
Characteristic curves for biodeposition rates (BR) recorded during detoxification periods based on Tetraselmis or Isochrysis diets (Fig. 5) were analyzed, as well as
equivalent curves obtained for clearance (CR) and filtration rates (FR). The typical example given for BR shows two different kinds of feeding behavior at the population
level: (i) when the non-toxic Tetraselmis diet was supplied to a scallop population previously fed A. minutum, there was no detectable “food effect” at the time of the shift
in diets (BR gradually increased up to a roughly constant level); and (ii) the non-toxic Isochrysis diet, when supplied to scallop population actively feeding on A.minutum,
produced a marked decrease of BR just at the time when the diets were switched.
These two different behaviors were analyzed with a general linear model (Table 1). A “food effect” was observed only for the switch from an A. minutum to an Isochrysis
diet, which produced a marked decrease in each of the three ecophysiological parameters. No particular trend was detected during detoxification with this alga,
except for a slight positive trend in BR. Conversely, the results were variable for Tetraselmis depending on the parameter observed, i.e. negative and positive trends
during detoxification for CR and BR respectively and a significant “food effect” only for CR and FR, with an increase in each of these parameters.
Clearance rate
Filtration rate
Biodepositionrate
Food
Slope
Food
Slope
Food
Slope
Tetraselmis
- 0.31 (0.0013)
- 0.014 (0.0028)
- 0.18 (0.0067)
0.010 (0.0123)
0.010 (0.0123)
Isochrysis
0.20 (0.0023)
0.09 (0.0011)
0.38 (0.0017)
0.018 (0.0027)
Detoxification pathways
The detoxification kinetics for each of the two non-toxic diets (Fig. 6) showed wide individual variations within each of the 5 scallop samples, especially at day 9 of
contamination. This corroborates observations for the individual-size experiment. The safety threshold was not reached, even after 14 days of detoxification in each
case.
ANCOVA showed no significant difference between each kinetic result (P-value: 0.12) and a significant slope was found only for the “Isochrysis”
detoxification curve, although the most appropriate mathematical model describing the detoxification pathway was a linear relationship and not an exponential
function [11], [12].
The relative ratio of STX and GTX2/GTX3 gonyautoxins was evaluated in kidneys during the detoxification time-course to estimate the bioconversion of toxins in the
excretory system. This ratio increased continuously between day 0 and 8 of detoxification before reaching and maintaining a steady level of 25%.
References
4
6
8
10
12
14
16
Days
C1
C2
C3
C4
C5
4
2
Fig. 6: Detoxification kinetics of PSP toxin concentrations in whole shellfish meat and
for the Isochrysis-based diet (blue) and the Tetraselmis-based diet (red).
Tetraselmis
3
200
0
30
2
250
0
0
1
300
Days
Fig. 2: Feeding time activities recorded for each experimental scallop in the “individual-size”
system during the contamination phase.
25
Raceway 2
350
50
40
20
3
Raceway 1
400
100
60
C5
2
450
150
80
0
1
500
µg STXeq 100 g-1
Individual-size experiment
Feeding time activities (FTA) showed wide variations among all 5 scallops (Fig. 2) during the 9-day contamination phase. FTAs were between 90 and 98% for two, 66%
for one, and 37 and 43% for the other two. In terms of PSP toxins absorbed, cumulated curves (Fig. 3) showed the greatest amounts in the scallop with the highest FTA
value(C4), whereas scallops with low or mean FTA (C1, C2, C3) showed no more than 63 µg STX.eq at the end of contamination. Curiously, the C5 scallop reached the
same score despite high FTA. Individual variations in daily amounts of absorbed PSP toxins during the contamination time-course (Fig. 4) were another characteristic
feature. Two peaks were detected at days 2 and 6 for C1, C2 and C3, but only one peak at day 6 for C5 and at day 5 for C4 (the animal with the highest FTA and
cumulated PSP toxin values).
The recirculated flume used (Fig 1) was identical to that previously described [6] for a
long-term experiment on oysters.
Cell concentrations during contamination and detoxification phases were
-1
equivalent to 0.5Êmg.l total particulate matter (TPM), i.e. the quantity of A.
minutum required to induce a toxic concentration in bivalves higher than the
-1
salubrity threshold (80 µg.eq.STX, 100 g of meat) at the end of the exposure period.
Biodeposits (feces and pseudofeces) of each 40 scallops populations were
collected twice a day and total renewal of the water circuit was performed daily to
avoid high concentrations of ammonia. Total Particulate Matter in water (TPMwater)was
measured in 8-L samples obtained every morning [7].
Apart from the “population size system,” another experimental device was used to
investigate individual responses of scallops to A. minutum feeding. For that purpose,
6 1-L boxes (5 live scallops and a control with an empty shell) were continuously
supplied (input) with seawater directly pumped into the 100-L raceway. Each
experimental box was connected to a micropump and an electroswitch such as
every 1 mn an aliquot of sea water + algae was pumped through a Turner
spectrofluorometer and analyzed for fluorescence level. The 6-electroswitch
opening and closing process was controlled by a computer + a data-logger set.
Thus, the seawater of each experimental box was analyzed for 1 min every 6 min.
Active feeding was the result of a significant difference in fluorescence levels
between assay and control boxes. The feeding time activity (FTA) could then be
defined as the percentage of time that the animal spent filtering during the
experiment.
The impact of a change of food in a raceway in given experimental conditions was
assessed using a general linear model [8] which allowed testing for two effects: (i) a
“foodÊeffect,” when the diet was switched; and (ii) a linear time effect obtained by
fitting a simple regression line to the data.
For PSP toxin analysis, five scallops were randomly collected during the
detoxification period. Digestive gland, kidneys and the remaining part of each
individual were dissected and ground with 0.1N CH3COOH at 4°C on a v/w basis.
After centrifugation (3,000 x g, 15 min, 4°C), the pH of extracts was adjusted to 3.03.5 using glacial acetic acid to prevent excessive dilution. After half-dilution,
supernatants were ultrafiltrated (20,000 Da, Sartorius Centrisart) and then stored at
4°C until analysis.
Analysis were performed by reverse-phase ion-pairing high-performance liquid
chromatography (IP-HPLC) according to the method of Oshima et al. [9] and molar
-1
concentrations were converted into µg STXeq.100 g scallop meat using the
conversion factors of Oshima [10].
Acknowledgements
Discussion and Conclusions
Wide individual variations were observed in FTA, daily toxin uptake and total toxin
body burdens at the end of the contamination phase, which is in agreement with
the findings of Shumway and Cembella [12] who noted that
"problems in monitoring scallops for PSP toxicity are exacerbated by high variability
in toxicity among individual specimens from the same location."
One of the most surprising results was the variation in the daily uptake of PSP toxins,
which seemed unrelated to the daily FTA pattern. However, elevated FTA values
were consistently associated with high PSP toxin content in the meat of at least one
specimen.
The distribution of PSP toxins in tissues followed a classical pattern, with the highest
amounts in digestive gland and the lowest in other tissues, except kidneys (which
represented 20% of total toxin body burden). The amount in kidneys was much
higher than in a previous study (11%) which involved a static system, larger adult
king scallops and a more toxic Alexandrium species [13]
The toxin bioaccumulation yield was relatively low (only 8% of that contained in
ingested algal food), but sufficient (at least for actively feeding animals) to exceed
the safety threshold after 6 days of continuous feeding on an A. minutum diet at a
mean cell concentration level of 150 cells.ml-1.
The results for population-size experiments were more difficult to analyze. A food
effect or a marked change in the level of at least two physiological parameters
(clearance and filtration rates) was apparent, regardless of the non-toxic diet
used. Both clearance and filtration seemed to be enhanced with Tetraselmis, but
decreased with Isochrysis. In previous experiments on the Pacific oyster using the
same experimental system [6] the Isochrysis-based diet showed no detectable
food effect or detoxification slope for CR, FR and BR, whereas the Tetraselmisbased diet led to increases in all three parameters (particularly marked for BR).
Although the Tetraselmis-based diet appears to be more suitable for restoring
healthy feeding activity, further experiments are needed, especially with diatombased diets. With respect to detoxification pathways, it is noteworthy that STX toxin,
which was absent from the algal food used as a toxin vector, was detected in
shellfish tissue, particularly in kidneys, and showed an increased ratio during the
detoxification time-course. This finding is in agreement with previous results [13]
showing a very slow decreasing trend of PSP toxin concentration in king scallop
kidneys as well as an increase in neoformed STX during detoxification.
Similar results to ours have been reported by Shumway and Cembella [12] for
another scallop species, Patinopecten yessoensis. Once contaminated with PSP
toxins, a 6-month detoxification period for these scallops resulted in a parallel
decrease in GTX1/4 and an increase in NeoSTX and STX in kidneys. It has been
suggested that the conversion of GTX2/3 into STX could be due to sulfhydril
reductants that cause a loss of hydroxysulfate groups in C11.
The fact that Isochrysis-based detoxification kinetics was better described by a
linear relationship than an exponential function, and that the safety threshold was
not reached after 14 days of detoxification, suggests that king scallops might be
assimilated to “ slow detoxifiers ” rather than “ fast detoxifiers,”
as reported by Bricelj and Shumway[14] for Placopecten magellanicus and
Patinopecten yessoensis. If confirmed, these observations could indicate a
possible physiological homogeneity for the group.
The authors wish to thank E.Erard-Le Denn for supplying the AM 89 BM strain of Alexandrium minutum and C.Mingant for collecting scallops in the Bay of Brest.
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Institut Français de Recherche pour l'Exploitation de la Mer - Direction de l'Environnement et de l'Aménagement Littoral
Département Microbiologie et Phycotoxines, BP 21105, 44311 Nantes Cedex 3, France