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Aquatic Toxicology 146 (2014) 205– 211

Contents lists available at ScienceDirect

Aquatic Toxicology

jou rn al hom ep age: www.elsev ier .com/ locate /aquatox

ptake, transfer and elimination kinetics of paralytic shellfish toxinsn common octopus (Octopus vulgaris)

anessa M. Lopesa,b, Miguel Baptistaa, Tiago Repolhoa, Rui Rosaa, Pedro Reis Costab,∗

Laboratório Marítimo da Guia, Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, Av. Nossa Senhora do Cabo, 939,750-374 Cascais, PortugalIPMA – Instituto Português do Mar e da Atmosfera, Avenida de Brasília, 1449-006 Lisboa, Portugal

r t i c l e i n f o

rticle history:eceived 29 August 2013eceived in revised form1 November 2013ccepted 17 November 2013

eywords:axitoxinctopusarmful algaeccumulationepurationarine toxinseurotoxinSP

a b s t r a c t

Marine phycotoxins derived from harmful algal blooms are known to be associated with mass mortal-ities in the higher trophic levels of marine food webs. Bivalve mollusks and planktivorous fish are themost studied vectors of marine phycotoxins. However, field surveys recently showed that cephalopodmollusks also constitute potential vectors of toxins. Thus, here we determine, for the first time, the timecourse of accumulation and depuration of paralytic shellfish toxins (PSTs) in the common octopus (Octo-pus vulgaris). Concomitantly, the underlying kinetics of toxin transfer between tissue compartments wasalso calculated. Naturally contaminated clams were used to orally expose the octopus to PSTs during 6days. Afterwards, octopus specimens were fed with non-contaminated shellfish during 10 days of depu-ration period. Toxins reached the highest concentrations in the digestive gland surpassing the levels inthe kidney by three orders of magnitude. PSTs were not detected in any other tissue analyzed. Net accu-mulation efficiencies of 42% for GTX5, 36% for dcSTX and 23% for C1+2 were calculated for the digestivegland. These compounds were the most abundant toxins in both digestive gland and the contaminatedshellfish diet. The small differences in relative abundance of each toxin observed between the prey and

the cephalopod predator indicates low conversion rates of these toxins. The depuration period was betterdescribed using an exponential decay model comprising a single compartment – the entire viscera. It isworth noting that since octopuses’ excretion and depuration rates are low, the digestive gland is able toaccumulate very high toxin concentrations for long periods of time. Therefore, the present study clearlyshows that O. vulgaris is a high-potential vector of PSTs during and even after the occurrence of these toxic algal blooms.

. Introduction

Marine toxins produced by harmful algal blooms (HAB), gen-rally as secondary metabolites, alter cellular processes of otherrganisms from plankton to humans. Bioaccumulated toxins cane transferred up through the marine food web and ultimatelyause events of mass mortality of top predators, such as marineammals and sea birds [see reviews in Landsberg, 2002; Lopes

t al., 2013]. Bivalve molluscs and planktivorous fish are tradition-lly the most studied vectors of toxins in marine food webs. Recenteld observations have reported cephalopods as highly potentialoxin vectors as well (Costa et al., 2004, 2005a, b, 2009; Costa andereira, 2010; Monteiro and Costa, 2011). These mollusks are vora-

ious and opportunistic predators that occupy a key position in theoastal marine food webs. Cephalopods prey mostly on crustaceans,mall fish and other mollusks, including cannibalism (Hanlon and

∗ Corresponding author. Tel.: +351 21 3027072; fax: +351 21 3015948.E-mail address: prcosta@ipma.pt (P.R. Costa).

166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aquatox.2013.11.011

© 2013 Elsevier B.V. All rights reserved.

Messenger, 1996; Nixon, 1985; Rodhouse and Nigmatullin, 1996).At the same time they constitute important items in the diets ofmarine mammals (Clarke and Goodall, 1994; Daneri et al., 2000;Pauly et al., 1998) and top predatory fish (Brock, 1985; Stillwelland Kohler, 1982).

Among the panoply of known marine toxins, the highly potentand neurotoxic paralytic shellfish toxins (PSTs), produced by threedinoflagellate genera, Gymnodinium, Pyrodinium and Alexandrium,have been reported to accumulate in three different cephalopodspecies, namely the common octopus (Octopus vulgaris), the Hum-boldt squid (Dosidicus gigas) and the Australian octopus [Octopus(Abdopus) sp. 5] (Braid et al., 2012; Costa et al., 2009; Monteiroand Costa, 2011; Robertson et al., 2004). Mass mortality eventsattributed to PSTs were also shown to affect top oceanic predatorcephalopods, namely the Humboldt squid. Evidence points towardmarine toxins (PSTs and/or domoic acid) as the cause of strandings

of these voracious predators (Braid et al., 2012; Lopes et al., 2013).

PSTs block conduction of electrical impulses in axons byreversely binding to voltage-gated sodium channels, inhibitingneuronal transmission (Henderson et al., 1973). In other marine

2 oxicology 146 (2014) 205– 211

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sttSsb2stbith

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2

2

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Table 1Toxin profile of Donax clams given to octopus (mean ± SD).

Toxin Concentration, �g g−1 (SD) Molar fraction, % (SD)

dcGTX2,3 0.23 (0.06) 3.2 (1.1)C1+2 0.92 (0.13) 9.1 (2.2)dcSTX 1.35 (0.20) 24.3 (1.2)

06 V.M. Lopes et al. / Aquatic T

rganisms, such as fish, PSTs are known to cause impairmentsf sensorimotor function and decreased larval and adult survivalsLefebvre et al., 2005; Robineau et al., 1991; Samson et al., 2008;

hite, 1981). Numerous compounds belong to the PST family,hich are most commonly divided into three groups, based on

heir chemical structure: carbamoyl (saxitoxin – STX, neosaxitoxin NEO, gonyautoxins – GTX 1–4), (ii) decarbamoyl (derivatives ofTX, NEO and GTX), and (iii) sulfamate (C-toxins 1–4, B1 – GTX5, B2

GTX6) toxins. A hierarchy of PST toxicity was established based onheir neurotoxicity potential with the carbamoyl group containinghe most toxic compounds [STX, neosaxitoxin (NEO), gonyautoxinsGTX 1–4)], followed by the decarbamoyl group that includes theecarbamoyl derivatives of STX, GTX1-4 and NEO. The sulfamateroup is the least toxic and comprises the four C-toxins and also B1GTX5) and B2 (GTX6) (Oshima, 1995).

From the few PST-related studies conducted in cephalopodso far, it is known that the PST levels are highest in the diges-ive gland (DG). For instance, in O. vulgaris, PSTs accumulateo the greatest extent in DG (ranging from 390 to 2680 �gTX equiv. kg−1) � kidneys > stomach > branchial hearts > posterioralivary glands > gills. The respective toxin profile was constitutedy C1+2, dcSTX, GTX2+3, B1, STX and dcNEO (Monteiro and Costa,011). As for the Humboldt squid, PSTs were detected only in thetomach and DG, with the values of latter tissue ranging from 2910o 4830 �g STX equiv. kg−1. The most predominant toxin was STX,ut low levels of dcSTX were also found (Braid et al., 2012). Last,

t is worth noting that significant levels of STX were detected inhe arms of the Australian octopus, which may represent healthazards for human consumption (Robertson et al., 2004).

Toxin kinetics and depuration dynamics in cephalopods aretill unknown. On the other hand, they are well documented inivalve mollusks (Blanco et al., 2003; Botelho et al., 2010; Briceljnd Shumway, 1998; Galimany et al., 2008; Li et al., 2005; Silvertnd Cembella, 1995; Yu et al., 2007) and some fish (Costa et al.,011; Kwong et al., 2006). The uptake, transformation and elim-

nation are simultaneous processes, thus, it is difficult to directlyeasure them. One- and two-compartment kinetic models have

een developed to describe changes in phycotoxins concentration,ncluding the PSTs, in shellfish based upon the balance betweennput and output rates (Blanco et al., 2003; Silvert and Cembella,995; Yu et al., 2007). The one-compartment model assumes thatoxin elimination occurs at a constant rate following an exponentialecrease throughout the depuration period. The second categoryses a multi-compartment distribution kinetic with a rapid initialhase, usually associated with an organ/tissue with high elimina-ion rates, followed by a period of slower toxin loss as a result ofesidual toxin concentrations that may be retained or bound to par-icular organs/tissues, thus a two-compartment model. Within thisontext, here we assessed, for the first time, the time course of accu-ulation and depuration of PSTs under laboratory conditions in O.

ulgaris and determined the kinetics behind the transfer of toxinsetween compartments.

. Materials and methods

.1. Collection and laboratorial maintenance of octopus

Thirty three specimens of juvenile octopus (O. vulgaris; ran-ing from 115 to 331 g weight and from 5.1 to 10.5 cm length)ere obtained from traps employed by local fishermen between

ebruary and March 2013 in Cascais, Portugal. After collection,

rganisms were transferred to the aquaculture facilities of Labo-atório Marítimo da Guia (Cascais). They were placed in individual

L seawater aquaria connected in parallel to a 270 L sump equippedith a wet-dry filter with bioballs (assuring biological filtration), a

B1 5.19 (0.71) 63.1 (2.2)STX 0.02 (0.00) 0.3 (0.1)

protein skimmer and one 36 W ultraviolet sterilizer. Natural sea-water was 1 �m filtered, with salinity being maintained at 34 ± 1through the regular addition of freshwater purified by reverseosmosis and temperature was kept stable through the use of Haileaheating/cooling systems. The tanks were illuminated from abovewith a photoperiod of 14 L:10 D. Ammonia and nitrite were mon-itored every other day and maintained below detectable levels.Nitrate and pH showed average values (±standard deviation, SD)of 7 (±2.5) mg L−1 and 8.1 (±0.1), respectively.

2.2. Preparation of contaminated octopus diet

Naturally contaminated clams (Donax sp.) were used to exposeoctopus to PSTs through a dietary route. Clams (0.64 ± 0.16 g) werecollected in Olhão (South coast of Portugal) in September 2012 dur-ing a bloom of Gymnodinium catenatum. Clams were checked for thepresence of PSTs by means of liquid chromatography. Thirty clamswere divided in 3 samples containing 10 clams each. The PSP tox-icity measured was 2665 ± 330 �g STX equiv. kg−1. Toxin profile ispresented in Table 1.

2.3. PST exposure experiments

Previous to the exposure experiments, three specimens wereselected for PSTs analysis. Having checked that octopuses werenot naturally contaminated with PSTs, octopuses were fed with10 contaminated clams every day for 6 days. Daily ingestion rateswere 3.2 ± 0.9% octopus body weight. In the following 10 days non-contaminated shellfish replaced the toxic diet. Every 24 h afterfeeding three specimens were randomly sampled and the diges-tive gland (DG), kidney, branchial hearts, salivary glands, gills andmantle were carefully dissected, weighed and prepared for toxinanalysis. Although PST has been only associated with organs/tissuesfrom the viscera of this octopus species, a portion of the mantle fromeach specimen was analyzed to confirm whether toxins reachedthis tissue.

2.4. Toxin extraction and quantification

Toxins from the organ homogenate were heat-extracted in1% acetic acid, vortexed, and centrifuged (15,000 × g) for 10 min.Extracts followed a solid-phase extraction (SPE) with octadecylbonded phase silica (Supelclean LC-18 SPE cartridge, 3 mL, Supelco,USA). Periodate and peroxide oxidations of PSTs were carriedout and toxins were immediately quantified by high performanceliquid chromatography with fluorescence detection (HPLC-FLD)based on the precolumn oxidation method developed by Lawrenceand Niedzwiadek (2001). The HPLC-FLD equipment consisted ofa Hewlett-Packard/Agilent Model 1050 quaternary pump, Model1100 in-line degasser, autosampler, column oven, and Model 1200fluorescence detector. The PSTs oxidation products were separatedusing a reversed-phase Supelcosil LC-18, 15 × 4.6, 5 �m column

(Supelco, USA). The mobile phase gradient consisted of 0–5% B(0.1 M ammonium formate in 5% acetonitrile, pH 6) in the first5 min, 5–70% B for the next 4 min and back to 0% B in the next2 min. Then 100% mobile phase A (0.1 M ammonium formate, pH 6)

oxicology 146 (2014) 205– 211 207

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U

D

D

FlTptd

FrmT

Table 2Parameters used in kinetics equations.

Parameter Description Unit

CDG Toxin concentration in digestive gland (DG) �g g−1

Ci Toxin concentration in the i-th compartment(KD, BH, SG, GL, MT)

�g g−1

Net accumulation effiency %F Feeding rate g d−1

Cfeed Toxin concentration in food �g g−1

kel DG Elimination rate from digestive gland d−1

kel i Elimination rate from i-th compartment d−1

KT i Transfer coefficient from digestive gland to thei-th compartment

d−1

qDG Toxin concentration in digestive gland at initialconditions of depuration

�g g−1

qi Toxin concentration in the i-th compartment �g g−1

V.M. Lopes et al. / Aquatic T

sed for 3 min before the next injection. Flow rate was 1 mL min−1

nd the detection wavelength set to 340 nm for excitation and95 nm for emission. Instrumental limits of detection (S/N = 3) were

ng g−1 dcSTX, 9 ng g−1 STX, 12 ng g−1 B1, 19 ng g−1 for dcGTX2+3nd GTX2+3, 34 ng g−1 C1+2. Certified calibration solutions for PSTsere purchased from the Certified Reference Materials Program

f the Institute for Marine Biosciences, National Research Coun-il, Canada (STX-e, NEO-b, GTX2+3-b, GTX1+4-b, dcSTX, dcGTX2+3,TX5-b (B1), C1+2 and dcNEO-b). For the digestive gland andantle three replicates per day were used, resulting in individual

nalyses for each specimen, however, for the other tissues pooledamples were used due to their reduced mass.

.5. Modeling

The model developed for the present study was based on thenowledge that the DG is the prime site of digestive absorptionnd storage of numerous substances, including PSTs (Monteirond Costa, 2011). Toxins are then transferred to the other tissueompartments (i.e. kidney, branchial hearts, salivary glands, gillsnd mantle). The model included six compartments, the DG beinghe first and the other five the remaining tissue compartmentsi = kidney – KD, branchial hearts – BH, salivary glands – SG, gills

GL and mantle – MT) shown in Fig. 1. The biokinetics of uptakend depuration of the six compartments were modeled by first-rder kinetic models. The equations used to fit experimental dataf PSTs in octopus tissues are:

ptake in digestive gland : CDG = ˛FCfeed

kel DGek

el DGt (1)

ptake in the i-th compartment : Ci = KTi˛FCfeed

kel DGek

el it (2)

epuration in digestive gland : CDG = qDGe−kel DGt (3)

epuration in the i-th compartment : Ci =qie−k

el it +KTiqDGe−kel DGt

(4)

The description of the parameters involved in Eqs. (1)–(4) andig. 1 is provided in Table 2. Octopus growth was assumed to be neg-igible due to the short span of the experimental period (16 days).

he experimental data were fitted using the non-linear curve fitrogram of SigmaPlot 11.0 (Systat Software Inc.). The exponen-ial growth model for best fit was selected after calculating theetermination coefficients, R2, and examining the residuals.

ig. 1. Schematic view for the five compartments of Octopus vulgaris. The solid linesepresent the transfer coefficients from the digestive gland to the other compart-ents. The dashed lines represent the elimination rates of the five compartments.

he other parameters used are described in Table 2.

at initial conditions of depurationt Time days

3. Results

3.1. Feeding behavior, survival and toxin distribution

The naturally contaminated clams provided to octopus as feedwere always consumed within the first hour and there was no mor-tality or behavioral changes during the experimental period. Notsurprisingly, the highest concentrations of PSTs were detected indigestive gland (DG) (Fig. 2). A clear exponential increase of toxinconcentrations in the DG was observed during the uptake period,i.e., the first 6 days (Fig. 2). The sum of all toxin analogs at the endof uptake period was 52.2 �g g−1, corresponding in terms of toxic-ity to 1249.2 �g STX equiv. kg−1. The toxin profile of the DG wasdominated, in terms of molar fraction, by GTX5 (53%), followedby dcSTX (35%), C1+2 had intermediate abundance (8.5%), and theleast abundant toxins were dcGTX2+3 (2.2%), GTX2+3 (1.1%) andSTX (0.3%). This toxin profile resembles that of the clams ingested(Table 1). The occurrence of GTX2+3 in octopus DG, a toxin whichwas not detected in the donax clams, is noteworthy. Afterwards,the shift from contaminated diet to non-contaminated one resultedin an exponential decay of toxin concentrations, although toxinswere not completely eliminated during the depuration period. Therelative abundance of PSTs was similar to that in the uptake period.

Paralytic shellfish toxins were also found in the kidney, althoughwith levels three orders of magnitude lower than in the digestivegland (Fig. 3). The progressive increase of these toxins throughoutthe uptake period did not show a clear exponential growth as inthe DG. The kidney profile of toxins was restricted to three toxins,namely GTX5, dcSTX and C1+2, which were the dominant com-pounds in DG. The kidney toxin profile was not dominated by GTX5,as in the DG, since it only accounted for 11% in terms of molar frac-tion. In contrast, dcSTX was the most abundant toxin (45%) followedby C1+2 (39%). Toxin concentrations showed an exponential decaytrend during the depuration period, and at the end of this periodthey were still detectable. PSTs were not detected in the remainingtissues analyzed, namely branchial hearts, salivary glands, gills andmantle.

3.2. Model fitting

The experimental data fitted to the dynamic model provided agood description of toxin kinetics in octopus DG (Table 3 and Fig. 2).Using the calculated toxin concentrations at the initial conditions ofthe depuration period (qDG, Tables 2 and 3) and the elimination rate

(kel DG, Tables 2 and 3) of the uptake period, the net accumulationefficiency (˛) for each toxin was estimated (Tables 2 and 3). Netaccumulation efficiency ranged between 18 and 42% for STX andGTX5, respectively.

208 V.M. Lopes et al. / Aquatic Toxicology 146 (2014) 205– 211

Fig. 2. Concentration (�g g−1) of (a) GTX5, (b) dcSTX, (c) C1+2, (d) dcGTX2+3, (e) GTX2+3 and (f) STX in octopus digestive gland throughout the experimental period. Dotsand error bars represent experimental data (mean based on three replicate samples). The dashed line represents the outputs of the best fit model.

Table 3Net accumulation efficiency (˛, %), initial concentration in digestive gland at beginning of depuration (qDG). elimination rate (kel DG, d−1) (standard deviation) and coefficientof determination R2 for each PST determined in octopus digestive gland during uptake and depuration.

Toxin Uptake Depuration

� kel DG (d−1) R2 qDG (�g g−1) kel DG (d−1) R2

dcGTX2+3 23% 0.311 (0.018)* 0.991 1.1 (0.02)* 0.131 (0.008)* 0.995C1+2 23% 0.243 (0.040)* 0.924 5.6 (0.62)* 0.229 (0.052)* 0.958dcSTX 36% 0.269 (0.054)* 0.895 11.4 (0.83)* 0.193 (0.029)* 0.9788GTX2+3 – 0.292 (0.042)* 0.941 0.2 (0.03)* 0.113 (0.045) 0.846GTX5 42% 0.336 (0.048)* 0.948 40.6 (3.29)* 0.220 (0.035)* 0.979

13 0.1 (0.01) 0.099 (0.072) 0.945

c(oePtph

Table 4Toxin transfer rate from digestive gland to kidney (KT, d−1), elimination rate (kel KD,d−1) (standard deviation) and coefficient of determination R2 for each PST deter-mined in octopus kidney during uptake.

Toxin Uptake

−1 2

STX 18% 0.244 (0.043)* 0.9

* Values within the confidence limit (P < 0.05).

The toxin transfer coefficient from DG to the kidney (KT) wasalculated using the obtained kel DG and the above values in Eq.2). Considerable low values of transference for each toxin werebtained (Table 4). This finding together with the poor fit of thexperimental data obtained for kidney, and due to the fact that

STs were not detected in the remaining tissues, another approacho describe the accumulation/depuration dynamics of PST in octo-us was adopted. Instead of a two compartment model initiallyypothesized, we assumed a single-compartment model in which

KT kel KD (d ) R

C1+2 0.0927 0.148 (0.199) 0.349dcSTX 0.0207 0.147 (0.099) 0.424GTX5 0.0018 0.165 (0.124) 0.388

V.M. Lopes et al. / Aquatic Toxicology 146 (2014) 205– 211 209

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Table 5Toxin concentration at initial depuration conditions (q0) elimination rate (kel V, d−1)(standard deviation) and coefficient of determination R2 for each PST determinedfollowing one compartment model.

Toxin Depuration

q0 kel V (d−1) R2

C1+2 4.9 (0.6)* 0.245 (0.056)* 0.958dcSTX 10.4 (0.6)* 0.205 (0.026)* 0.986

* *

ig. 3. Concentration (�g g−1) of (a) GTX5, (b) dcSTX and (c) C1+2 in octopus kidneyhroughout the experimental period. The dashed line represents the outputs of theest fit model.

ll tissues analyzed correspond to the viscera of octopus (wherehe toxins are allocated). If the viscera are considered to be one sin-le compartment, toxin concentrations during depuration can bealculated by:

tissues = mDGqDG + mKDqKD

mDG + mKD(5)

ith mDG and mKD being the wet mass of the digestive gland andidney, respectively, and qDG and qKD being toxin concentration inhe digestive gland and kidney, respectively. Eq. (4) can be simpli-ed to describe the depuration of the single compartment:

V = q0e−kel Vt (6)

here CV is the toxin concentration in octopus viscera, q0 is theoxin concentration at the initial conditions of the depurationeriod. The kel V denotes the toxin elimination rates from visceraTable 5).

. Discussion

Bioaccumulation of marine toxins in cephalopods is still poorlynderstood. Analyses of PSTs performed in specimens of O. vul-aris collected during blooms of G. catenatum suggested a greatapacity for accumulation of these compounds (Costa et al., 2009;

GTX5 36.6 (2.5) 0.233 (0.031) 0.986

* Values within the confidence limit (P < 0.05).

Monteiro and Costa, 2011). Here we provided the first conclusiveevidence of such capacity under laboratory (controlled) conditions.More specifically, we found very high PST levels in octopus DG aftersix days of ingestion of contaminated clams. The toxin concentra-tions, in terms of saxitoxin equivalents, reached similar or highervalues than those reported in the field observations (Costa et al.,2009; Monteiro and Costa, 2011). The DG is the largest octopusorgan (with the exception of female gonads at the peak of matu-rity) (Grisley and Boyle, 1988), and the prime site for substancestorage (Rosa et al., 2004, 2005). For this reason, PSTs as well asother marine toxins, such as domoic acid, have been found at highlevels in this tissue (Costa et al., 2004, 2009; Costa and Pereira,2010; Lage et al., 2012; Monteiro and Costa, 2011). The profile ofPSTs in the DG was dominated by the same toxins as present in thenaturally contaminated clams provided here as prey to the octo-puses. This indicates low toxin conversion between the bivalve preyand the cephalopod predator. On the contrary, biotransformation ofthe toxins was perceptible after feeding experiments involving fish(white seabream Diplodus sargus) and contaminated cockles (Ceras-toderma edule) (Costa et al., 2011). In this case, conversion of C1+2into their analogs, namely GTX5 was suggested (Costa et al., 2011).C1+2 are the less stable toxins and were the foremost dominanttoxin analogs in fish feed with a molar fraction of about 84%, whilein the present study C1+2 molar fraction did not reach 9%. Inter-estingly, the octopus DG revealed, although low, levels of GTX2+3,a toxin not detected in the clams. GTX2+3 was probably presentin clams at levels below the HPLC-FLD detection limit (19 ng g−1)and was then concentrated in the octopus digestive gland afteringestion of the 10 clams provided daily.

The fact that the toxin concentrations in the kidney were threeorders of magnitude lower than in the DG, confirms low toxin trans-ference between these tissues. The presence of PSTs in kidney, evenif in low levels, may indicate that the renal processes are a routefor toxin excretion, as was suggested in field studies (Monteiro andCosta, 2011). Three toxins were detected in the kidney, namely:GTX5, dcSTX and C1+2. They were the ones in highest abundancein the DG. PSTs were not detected in any other tissue, suggest-ing that longer periods of exposure and depuration are requiredto detect toxins in the remaining tissues (branchial hearts and sali-vary glands). As in previous studies, PSTs were not detected in themantle indicating that toxins are allocated exclusively in the vis-ceral tissues and are gradually released without being assimilated(Monteiro and Costa, 2011).

First-order linear differential equations governing the kinet-ics of uptake and depuration are commonly used to predict theconcentration of a given compound in marine organisms, bothin invertebrates (bivalves, cephalopods) and vertebrates (fish)(Barber, 2008; Bustamante et al., 2002; Kwong et al., 2006; Liet al., 2005; Yu et al., 2007). Here we show that the accumulationand depuration of PSTs in the common octopus can effectively be

described by such models. Net accumulation efficiencies for theDG ranged between 18 and 42% for STX and GTX5, respectively.Comparing to other studies, O. vulgaris has shown to have higheraccumulation efficiencies than fish [D. sargus – accumulating 1.7

2 oxico

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10 V.M. Lopes et al. / Aquatic T

nd 5% of GTX5 and dcSTX (Costa et al., 2011)], and lower thanivalves [mussels; 67% of the GTX4 ingested (Blanco et al., 2003)].limination rates were calculated for both uptake and depurationeriod, with the uptake period presenting slightly higher valueshan depuration. With these results, sequestration or binding ofhe toxins can be suggested (Yu et al., 2007), which is in accor-ance with the elevated capacity for the common octopus to retainSTs. On the other hand, when higher elimination rates are obtaineduring depuration, one may suggest the occurrence of conversionf toxins into their analogs (Costa et al., 2011; Yu et al., 2007). Thisas not the case in this study, in addition, identical profile of toxinsas found in clams and octopuses.

After describing the toxin distribution profile, it was not sur-rising to observe low toxin transfer coefficients from the DG tohe kidney (Table 5; Eq. (2)). It is noteworthy that toxin transferoefficients were considerably lower than toxin elimination ratescalculated for the DG), which suggests that other routes or mech-nisms are associated with their elimination. Although the toxinsere found in the octopus DG and in the kidney, the anatomic dis-

ribution of PSTs may not be restricted to these two tissues. It isupposed that toxin transfer may occur from digestive gland tother compartments, such as branchial hearts and salivary glands,ut at even lower rates than that calculated for kidney. There-ore, discrepancies between elimination rates and toxins transferoefficients to the kidney can be explained, in part, by eliminationhrough transfer to other compartments at rates lower than those

easurable by the analytical detection methods used. Moreover,STs may be metabolized through biotransformation enzymes thatre usually found in higher concentrations in digestive gland ofctopus (Tang et al., 1994). The activity of detoxification enzymesas been investigated in amphipods and fish liver exposed toST, showing increasing activities of glutathione-s-transferase GSTCosta et al., 2012; Gélinas et al., 2013; Gubbins et al., 2000).

One- or two-compartment exponential decay models are usu-lly used in toxin depuration (Bricelj and Shumway, 1998; Costat al., 2011; Kwong et al., 2006; Li et al., 2005; Mafra et al., 2010;u et al., 2007). In this study, a two-compartment model was ini-ially assumed to quantitatively predict the uptake and depurationynamics of PSTs, based on the fact that the DG would be the pri-ary site of absorption, storage and then release of the toxins to

ther visceral tissues, as was evident from previous field studies.owever, during the experimental period, the transfer of PSTs fromG to the other tissues was not observed, except for the kidneyhere very low toxin concentrations were detected. In this case,e were left with the one-compartment model, which, instead

f having a toxin flow from a rapidly detoxifying compartment to a compartment 2 characterized by slow detoxification, hashe foremost amount of the toxins in the octopuses remaining inhe DG. Therefore, toxin depuration was better described with anxponential decay model from a single compartment. This modelesulted from the negligible toxin transfer coefficients from diges-ive gland to kidney resulting in negligible toxin concentrationsetected in kidney. The use of this model is only valid because there

s a tissue accounting for the greatest majority of the toxin content,hich in this case is the digestive gland. Similarly, Li et al. (2005)

uggested a one-compartment model after calculating negligibleransfer coefficients of PSTs between hepatopancreas and other tis-ues of the green-lipped mussel (Perna viridis). Silvert and Cembella1995) also recognized that the extreme degree of simplification,.e. one-compartment model, is often the only appropriate modelo use. Although Blanco et al. (1997) compared the two models forST detoxification kinetics in mussels showing marginally better

esults with the two-compartment model, the anatomical distribu-ion of the toxins was not examined, and the second compartmentas hypothesized from the model output results. After exposing

ysters and mussels to domoic acid-producing diatoms, Mafra et al.

logy 146 (2014) 205– 211

(2010) observed that a two-compartment model resulted in com-parable fits, but with higher degree of uncertainty.

Predictions inferred from the use of first-order kinetic mod-els can become useful in understanding the movement of toxinsthroughout the various links of marine food webs, especially incephalopods, because they play a key ecological role linking lowerto higher trophic levels of the coastal marine food webs. Octopusesshowed low depuration and excretion rates, allowing the DG toaccumulate PSTs at considerably high levels for longer periods oftime. Thus, O. vulgaris is a potential vector of PSTs to higher levelsof marine food webs.

This study points out the need to identify the mechanismsproviding the octopuses with the ability to metabolize and detox-ify HAB-toxins. Despite of the remarkably high levels of toxinsdetected no apparent harm neither signs of behavioral changeswere observed. Coastal octopuses have probably evolved to acquireadditional mechanisms that enable them to tolerate the HAB-toxins.

Acknowledgments

The Portuguese Foundation for Science and Technology (FCT)supported this study (in part) through project grant PTDC/BIA-BEC/103266/2008, Programa Ciência 2007 to R. Rosa and ProgramaCiência 2008 to PR Costa. We also appreciate the valuable com-ments given by the two anonymous reviewers.

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