Foodweb transfer, accumulation, and depuration of microcystins, a cyanobacterial toxin, in pumpkinseed sunfish (Lepomis gibbosus)

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doi:10.1016/j.to

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Toxicon 48 (2006) 580–589

www.elsevier.com/locate/toxicon

Foodweb transfer, accumulation, and depuration ofmicrocystins, a cyanobacterial toxin, in pumpkinseed sunfish

(Lepomis gibbosus)

Juliette L. Smitha,�, James F. Haneyb

aFaculty of Environmental and Forest Biology, State University of New York, Syracuse, College of Environmental Science and Forestry,

1 Forestry Drive, Syracuse, NY 13210, USAbZoology Department, University of New Hampshire, Durham, NH 03824, USA

Received 16 November 2005; received in revised form 5 July 2006; accepted 6 July 2006

Available online 11 July 2006

Abstract

Zooplankton accumulate microcystins (MC), a potent cyanobacteria toxin, and therefore may act as vectors of the toxin

up the aquatic food web; however this transfer has not yet been quantified. In addition there is a lack of information

regarding fish’s ability to metabolize MC when administered a low dose over a longer period of time. We monitored MC

concentrations in three levels of an aquatic food web: phytoplankton, zooplankton, and sunfish (Lepomis gibbosus).

Bosmina appeared to be both a major accumulator of MC in zooplankton and the major vector of MC to sunfish.

In an accumulation experiment, sunfish were brought into the laboratory and fed MC-rich zooplankton pellets

(50 ngMCkg�1d�1) for 9 days. Zooplankton directly transferred MC to sunfish, resulting in liver and muscle tissue

accumulation. However, after 6 days of accumulation fish significantly decreased concentrations in their liver and muscle

tissue, indicating the induction of a detoxification and excretion pathway. Sunfish retained MC in their liver and muscle

tissue, showing no significant changes in toxin concentration over 2 weeks of fasted depuration.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Microcystin; Accumulation; Depuration; Detoxification; Vector; Microcystis; Sunfish

1. Introduction

Microcystins (MC) are a common cyanobacterialtoxin that generally accumulates in the liver orhepatopancreas of an exposed organism, and bindsto a nucleophilic site on protein phosphatases PP1and PP2A (Robinson et al., 1991; MacKintoshet al., 1995; Craig et al., 1996), possibly resulting in

e front matter r 2006 Elsevier Ltd. All rights reserved

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ng author. Tel.: +1 315 470 4847;

6934.

ss: [email protected] (J.L. Smith).

hepatocyte degradation and fatal liver hemorrha-ging (Fischer and Dietrich, 2000a; Zimba et al.,2001). The toxin is well known for its inhibitoryeffect on PP1 and PP2A in mammals (Xu et al.,2000), fish (Fischer and Dietrich, 2000a; Fischeret al., 2000), amphibians (Fischer and Dietrich,2000b), and zooplankton (DeMott and Dhawale,1995). The toxin has also been found to accumulateand have histopathological effects in muscle, gill,and kidney tissue of multiple fish species (Rodgeret al., 1994; Carbis et al., 1996; Kotak et al., 1996;Fischer and Dietrich, 2000a).

.

ARTICLE IN PRESSJ.L. Smith, J.F. Haney / Toxicon 48 (2006) 580–589 581

Aquatic organisms can be exposed to MC via theconsumption of toxic cyanobacteria (Li et al., 2004;Xie et al., 2004) or aquatic organisms that hadpreviously accumulated MC in their tissues; how-ever, negligible amounts enter the system throughthe gills or epithelium (Tencalla et al., 1994; Kentet al., 1996). MC have been found to accumulate inspecies of zooplankton (Watanabe et al., 1992;Thostrup and Christoffersen, 1999), freshwatermussels (Eriksson et al., 1989; Amorim andVasconcelos, 1999), crayfish (Liras et al., 1998;Vasconcelos et al., 2001), marine biota (Andersenet al., 1993), and multiple fish species (Vasconcelos,1999; Fischer and Dietrich, 2000a; Magalhaes et al.,2003; Ibelings et al., 2005). A few studies haveimplicated invertebrates as a vector of the toxin tohigher trophic organisms (Andersen et al., 1993;Kent et al., 1996; Babcock-Jackson et al., 2002;Ibelings et al., 2005); however, the direct link hasnot been observed under controlled laboratoryconditions.

We combined a field monitoring study and alaboratory accumulation experiment to investigatethe direct transfer of MC from zooplankton tosunfish. MC was administered at a sub-lethal dose,50 ng kg�1 d�1, over 9 days, to investigate the abilityof fish to accumulate and metabolize the toxin whenorally exposed under more ‘‘natural’’ conditions.Most studies have administered MC to fish in single,acute doses (4500 mg kg�1) via intraperitonealinjection or gavaging with whole cyanobacteriacells. Although these techniques are useful todetermine pathological effects, neither the dosenor the route of administration represents naturalconditions. Furthermore, intraperitoneal injectioncauses the toxin to bypass presystemic hepaticelimination and therefore be delivered to otherorgans in an excessive and unnatural manner(Carbis et al., 1996). We also conducted a depura-tion experiment to determine loss of MC from liverand muscle tissue when the toxic source is removed.

2. Methods

2.1. Field study collections

We sampled sunfish, zooplankton, and phyto-plankton from Barbadoes Pond (Madbury, NH)every 4 days from 4 July to 30 August 2000.Barbadoes Pond (5.80 ha, maximum depth of14.6m) was selected for study due to its relatively

high concentrations of MC within its major foodweb compartments.

Nine juvenile pumpkinseed sunfish (Lepomis

gibbosus) were collected and fish age (scale), forklength and wet weight were recorded. Liver tissuewas harvested and the nine livers were pooled intogroups of three to provide enough mass for toxinanalysis. Samples were frozen at �401C until laterMC analysis.

A 50-mm mesh, 30-cm diameter plankton net wasused to collect net plankton. Zooplankton andphytoplankton fractions were separated by provid-ing a light and dark separatory apparatus, relyingon zooplankton phototaxis. On each sample daytriplicate zooplankton and phytoplankton sampleswere (1) preserved with 4% formalin/sucrose forenumeration and (2) frozen at �401C for MCanalysis.

2.2. Accumulation experiment

Mass quantities of zooplankton, ca. 1 kg freshweight, were collected from Barbadoes Pond on 26June 2001 using a 375-mm mesh, 0.5-m diameterplankton net. The large mesh size sampled primarilylarge zooplankton, excluding most phytoplankton.Samples were frozen and a razor blade was used toremove a thin layer of phytoplankton coloniesthat had formed above the dense zooplanktonmass. The zooplankton mass consisted mostly ofDaphnia catawba, 76.573.6%, and Diaphanosoma,16.473.8%, with Bosmina, and copepods makingup the remainder, 7.171.3%. Zooplankton werelyophilized to dryness, 24 h, and stored below 20 1Cuntil used to create food pellets. Fresh pellets wereproduced daily by adding 0.583 g of lyophilizedzooplankton to 10mL of 3% Bactose Agar.

Sunfish were collected from Barbadoes Pond on 9July and 30 September 2001 for duplicate trials;fish averaged 1.5770.12 g wet weight (ww) and1.8670.10 g ww, respectively. Individual fish wereacclimated for 3 weeks in 1-L glass bottles, contain-ing 1L of aerated, MC-free well water. Fish werefasted during the acclimation period in an attempt todecrease liver toxicity and variability betweenindividual fish. During both the acclimation periodand experiment, 90% of the tank water was changedevery 48h and fish were exposed to a 13:11 hlight:dark cycle. During the 9-day experiment, fishconsumed 4pellets d�1, totaling 77.2 pgMCd�1.Each pellet contained 19.3 pgMC and averaged11.770.3mg wet weight. After 9 days, fish consumed

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a total of 694.8 pgMC, equal to a sub-lethal dose of0.5mgMCkg�1 body weight. Fish livers and skeletalmuscle tissue (taken from above the dorsal line) werecollected from three randomly selected fish on days0, 2, 4, 6, and 9. Fish samples were frozen at �40 1Cuntil extraction.

2.3. Depuration experiment

Sunfish were collected from Barbadoes Pond on 5and 30 July 2001 for duplicate trials; fish averaged1.7670.07 and 1.8670.08 g ww, respectively. In-dividual fish were immediately placed in 1-L glassbottles, commencing the experiment. Fish werestarved during the experiment. Fish liver, muscletissue, and tank water (including solid waste) wereharvested from three fish for 9 consecutive days andthen on days 11 and 14. Fish and water sampleswere frozen at �40 1C until MC analysis.

2.4. MC analysis

In preparation for MC analysis, all samples (netphytoplankton, net zooplankton, fish liver andmuscle, food pellets, and tank water) were frozenand thawed three times to rupture cells. Addition-ally, fish tissue was homogenized with a hand-held,electric mortar and pestle (Kontes). All sampleswere extracted for 24 h in 80% methanol at roomtemperature. Phosphate buffer solution (PBS) wasadded to bring the methanol concentration to 10%.Extracted samples were then filtered through a 0.2-mm, 13-mm syringe filter (PTFE, Filter Media,Whatman). Fish liver and muscle extract waslyophilized to dryness and resuspended in Milli-Qwater to reach 7- and 9-fold concentrations,respectively. Likewise, tank water was concentrated30-fold. All net zooplankton and phytoplanktonwere diluted 25- and 50-fold, respectively, withMilli-Q water to place them within the range ofELISA detection limits.

ELISA was performed as described by theEnviroLogix Inc. kit (Portland, ME). MC-LRstandards included in the kit, 160, 500, and1600 pgml�1, were diluted to increase the range ofsensitivity to 25–1600 pgml�1. All results arereported as MC-LR equivalents. Optical densitywas read via Bio-tek Instruments Inc. EL800microplate reader and toxin concentrations werederived from a cubic log–log standard curve (KCJunior software). A dual wavelength, at 630 nm, was

run to remove interference from bubbles and plateirregularity.

2.5. Statistical analysis

Data from the all three studies were analyzedusing one-way ANOVA (repeated measures), Tu-key’s post hoc test, t-test, and regression analysis(SYSTAT 9.0). Regression residuals were plottedand values were log transformed if heteroscedacitywas observed. Percent composition data from theenumeration of net zooplankton and phytoplanktonin vertical tows were arcsine transformed before anyregression analyses were performed. Significancelevels were set at pp0.05.

3. Results

3.1. Field study

MC were detected in sunfish livers, zooplankton,and net phytoplankton throughout the field study inBarbadoes Pond; 4 July–28 August 2000 (Fig. 1(A)and (B)). Sunfish liver MC was not correlated withzooplankton or net phytoplankton MC. Net phy-toplankton MC was positively correlated withzooplankton MC with a 4 day lag (r2 ¼ 0.33,p ¼ 0.02).

MC concentrations in the net zooplankton,per unit biomass, were positively correlated withthe percent composition of Bosmina (r2 ¼ 0.21,p ¼ 0.05), and negatively correlated with the densityof copepods (r2 ¼ 0.28, p ¼ 0.04). Zooplanktontoxicity did not correlate with the abundanceof any other zooplankton genera (i.e., Daphnia,Ceriodaphnia, Diaphanosoma) or group (i.e., cope-pods, cladocera).

Concentrations of MC in the net phytoplankton,per unit biomass, were positively correlated with thedensity and percent composition of Microcystis sp.(log–log transformed, r2 ¼ 0.28, p ¼ 0.03). Thetoxicity of net phytoplankton was not correlatedwith other genera of cyanobacteria present inBarbadoes Pond, including Anabaena and Coelo-

sphaerium.The concentration of MC in sunfish liver tissue

remained relatively constant over the samplingperiod (p40.05), averaging 3.870.2 ng g�1 ww.MC concentrations in sunfish liver were notcorrelated with fish age, fork length, or wetweight of the fish. Liver concentrations weresignificantly correlated with density of Bosmina

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6000

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9000Net Zooplankton MCNet Phytoplankton MC

Mean = 3.82 ± 0.15 ng g-1ww

(A)

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Fig. 1. Microcystin concentrations in net zooplankton and phytoplankton (450 mm) of Barbadoes Pond 2000 (A). Microcystin

concentrations in the liver tissue of sunfish (Lepomis gibbosus) (B). No significant differences were detected in liver concentrations between

sampling dates (p ¼ 0.70). Values are mean71SE.

J.L. Smith, J.F. Haney / Toxicon 48 (2006) 580–589 583

(Fig. 2(A), r2 ¼ 0.28, p ¼ 0.03) and net Microcystis

colonies (Fig. 2(B), log–log transformed, r2 ¼ 0.24,p ¼ 0.04). Microcystis and Bosmina densities werenot correlated. No correlations were detectedbetween liver concentrations and any other genera(Daphnia, Ceriodaphnia, Diaphanosoma, Anabaena,Coelosphaerium) or large taxonomic group ofplankton studied (copepods, calanoid, cyclopoid,cladocera, cyanobacteria).

3.2. Accumulation experiment

The dosage and exposure time used in theaccumulation experiment did not result in sunfish

mortality. Fish continued to consume toxic zoo-plankton pellets at the same rate throughout theexperimental period (4 pellets d�1). ELISA detectedMC in all fish liver and muscle tissue samplesproduced by the accumulation experiment. Despitethree weeks of acclimation in MC-free water,sunfish retained detectable levels in their liver andmuscle tissue, from then on regarded as the baselevel on day 0 of the experiment.

In both trials, MC rapidly accumulated in theliver tissue of sunfish and then decreased to levelsbelow the initial concentration by day 9, despitethe continuous supply of newly delivered toxin(Fig. 3(A) and (C)). The first trial, July 2001,

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Bosmina Density (L-1)

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p = 0.04

Adj R2 = 0.24

p = 0.03Adj R2 = 0.28

(A)

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Fig. 2. Relationship between the concentration of microcystin in sunfish liver (Lepomis gibbosus) and the density of Bosmina, Barbadoes

Pond 2000 (A). Relationship between the log density of Microcystis colonies and log microcystin concentrations in sunfish liver tissue (B).

Dashed lines represent 95% confidence intervals.

0 9

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Fig. 3. Microcystin concentrations in sunfish (Lepomis gibbosus) liver and muscle tissue in the first trial (July 2001) of the accumulation

experiment (A and B) and the second trial (September 2001) (C and D). Bars with a letter in common do not differ significantly (Tukey’s

post hoc, pp0.05) (A). Fish were fed toxic zooplankton throughout the experiment.

J.L. Smith, J.F. Haney / Toxicon 48 (2006) 580–589584

showed significant differences between samplingdays; however, no significance was detected withinthe second trial, conducted with fish collected in lateSeptember 2001. In the first trial, initial average

liver MC, 2.571.0 ng g�1 ww, significantly in-creased to 9.970.1 ng g�1 ww after 2 days of feedingupon toxic zooplankton, and 11.270.2 ng g�1 wwon day 4. After 6 days of feeding, liver MC became

ARTICLE IN PRESSJ.L. Smith, J.F. Haney / Toxicon 48 (2006) 580–589 585

variable and then significantly decreased on day 9 to1.470.2 ng g�1 ww. In the second trial, liver MCsimilarly decreased by day 9 to 3.270.2 ng g�1 ww.Overall, average liver MC for the first and secondtrials, 6.171.2 and 7.271.8 ng g�1 ww, respectively,were not significantly different.

The overall average muscle MC for trials 1 and 2was 0.06370.03 and 0.17770.04 ng g�1 ww, respec-tively. Muscle MC concentrations significantlydecreased on days 6 and 9 (Fig. 3(B)), followingthe general trend seen in the liver tissue. Muscle MCin trial 2; however, stayed relatively constant overtime (Fig. 3(B)). Liver and muscle MC were notcorrelated.

3.3. Depuration experiment

ELISA detected MC in all fish, muscle, and tankwater samples once samples were concentrated asdescribed in the methods. MC concentrations didnot change significantly (ANOVA, p40.05) in liver,muscle, or tank water (water data not shown) overthe sample period (Fig. 4(A)–(D)). Total MCconcentrations in the tank water, summed for theentire sampling period, were 53.6 and 51.1 pgmL�1,from trials 1 and 2, respectively. Average dailyconcentration in the tank water were 5.970.9 and5.770.8 pgmL�1, respectively. Liver, muscle, andtank water MC were not correlated in the depura-tion experiment.

0 4 9 11 14

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012345678

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765321

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Fig. 4. Microcystin concentrations in sunfish (Lepomis gibbosus) liver

(A and B) and second trial (C and D) (July 2001). Fish were fasted

concentrations were detected between sample days (ANOVA, p40.05)

4. Discussion

4.1. Transfer vectors of MC

We found evidence for the direct transfer of MCfrom zooplankton to sunfish and the subsequentaccumulation of toxin in the liver tissue throughboth a field study and laboratory experiment. In thefield study, Barbadoes Pond (Madbury, NH),Bosmina appeared to be a major vector of MC tosunfish through July and August 2000. The con-centration of hepatotoxin in the zooplankton(450 mm) increased significantly as Bosmina becamemore abundant in the water column, suggestingBosmina was a major accumulator in BarbadoesPond. Furthermore, fish appear to have consumedtoxic Bosmina, and accumulate MC, when thesmall cladoceran was a more abundant prey item(Fig. 2(A)). As Bosmina densities declined throughthe summer, fish may have switched to another,more plentiful prey species, accounting for thedecrease in fish liver concentrations. Unlike theaccumulation of MC found in Bosmina, overallzooplankton MC decreased as copepods becamemore dominant. Zooplankton MC was not corre-lated with the abundance of any other zooplanktongenera or large taxonomic group studied. Copepodsare able to discriminate between toxic and non-toxic cyanobacteria, allowing them to avoid tox-icosis (Fulton and Paerl, 1987), while Bosmina and

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765321

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and muscle tissue in the first trial of the depuration experiment

throughout the experiment. No significant differences in toxin

.

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Daphnia ingest toxic cyanobacteria leading to MCaccumulation (Watanabe et al., 1992; Thostrup andChristoffersen, 1999). Four days were necessary forpeaks in phytoplankton toxicity to transfer tozooplankton in Barbadoes Pond (Fig. 1(A)).

MC concentration in sunfish liver was alsopositively correlated with the density of Microcystis

colonies in the net phytoplankton of BarbadoesPond, 450 mm (Fig. 2(B)). Large colonies may beingested accidentally when they are in high abun-dance. Gavaging fish with Microcystis led to MCintoxication and hepatocyte necrosis in commoncarp and rainbow trout, showing that cyanobacteriacan be digested, and MC absorbed through the gut,under laboratory conditions (Tencalla et al., 1994;Carbis et al., 1996; Fischer et al., 2000).

In the accumulation experiment, sunfish consumedMC-rich zooplankton. After just 2 days of feeding,the concentration of MC significantly increased insunfish livers (Fig. 2(A)). The hepatotoxin continuedto accumulate in liver tissue through 4 days offeeding. The dose, 50ngMCkg�1 d�1, was not lethaland represented natural levels. Pellets consisted of76.573.6% Daphnia, with Bosmina, Diaphanosoma,and copepods making up the remaining ca. 24%.From body length measurements and the use of alength–weight regression equation (Burns, 1969), wecalculated that fish consumed approximately100 daphnids d�1 during the experiment. This iscomparable to natural conditions, where smallyellow perch ingested as many as 213 daphnids d�1

in mid August (Wu and Culver, 1994).MC appears to be absorbed through the GI tract

of fish in greater proportion when the toxin isadministered through a vector, zooplankton, ratherthan through toxic cyanobacteria directly. Ourstudy shows that 80% of the administered MCin zooplankton was absorbed and accumulated inthe liver tissue, in a non-covalent form, within24 h of exposure (Fig. 3). Tencalla et al. (1994)estimated that rainbow trout only absorbed 10% ofthe MC from toxic Microcystis aeruginosa cellsin 24 h, and Bury et al. (1998) found evidence foronly 0.3% transfer when fish were gavaged withpurified MC-LR. The efficient transfer from zoo-plankton to fish liver observed in our study mayreflect utilization of the total MC load, boundand unbound, in the diet. If MC is released fromprotein phosphatases during digestion by fish, thecovalently bound MC in the zooplankton willbe readily available for absorption by the GItract. Future research needs to be conducted to

determine if covalently bound MC is transferable upthe food web.

4.2. Metabolism of MC

Due to the extraction method used (80% MeOH)and detection with ELISA, this work reports MCvalues that are an integration of numerous forms ofMC, including variants and detoxification conju-gates. ELISA (EnviroLogix) has good crossreactiv-ity with many variants of MC (Carmichael and An,1999) and MC that has been biotransformed(detoxified) at the Mdha residue (N-methyldehy-droalanine) with glutathione, cysteine–glycine, orcysteine (Metcalf et al., 2002). However, becauseMC-LR, the most potent MC variant with an LD50

of 50 mg kg�1 in mice, was used as the standard forthis assay, results are reported as MC-LR equiva-lents (Kotak et al., 1995).

The potential of MC to covalently bind in animaltissue (Williams et al., 1997a, b) raises someimportant, but largely unexplored questions aboutthe total MC burden that accumulates in organismsin aquatic food webs. It is important to point outthat our results do not report the total tissue load ofMC. Instead, we measured the MC that was notcovalently bound to the target molecules, proteinphosphatase 1 and 2A. As in similar studies thathave measured methanol-extracted MC using ELI-SA (Amorim and Vasconcelos, 1999; Thostrup andChristoffersen, 1999, Zimba et al., 2001, Magalhaeset al., 2003), strictly speaking, we are reporting thefraction of MC that was extractable with MeOH.Williams et al. (1997a, b) reported that only ca. 13%of the total MC load in fish liver was extractable viaMeOH method, 24 h after exposure. The remainingtoxin was said to be irreversibly, covalently boundto protein phosphatase 1 and 2A, inhibiting proteinphosphatase activity.

In the accumulation experiment, sunfish, fromBarbadoes Pond, were fed toxic zooplanktonpellets for 9 consecutive days. Our experiment wasdesigned to represent natural conditions, and there-fore is unique in that (1) fish began the accumula-tion experiments with a base level of MC in theirtissues; (2) a vector, zooplankton, was used asthe source of toxin; and (3) a sub-lethal dose,50 ngMCkg�1 d�1, was repeatedly delivered. Over-all, the concentration of MC in fish liver and muscletissue increased with 4 days of feeding, becamevariable on day 6, and decreased by day 9 to aconcentration below the initial base level (Fig. 3).

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To date, this is the only laboratory study toshow that fish accumulate and then significantlydecrease the concentration of free MC in their livertissue despite a constant influx of toxin fromzooplankton.

Sunfish retained relatively constant levels of MCin their liver and muscle tissue through 3-weeks offasted acclimation, and then accumulated 80% ofthe administered toxin in the liver tissue after 2 daysof feeding (Figs. 3 and 4), indicating that proteinphosphatases 1 and 2A (PP1 and PP2A) may havebeen near saturation with MC upon collection fromBarbadoes Pond. Otherwise, the majority of newlydelivered toxin would have preferably bound(covalently) to PP1 and PP2A within hours ofabsorption (Robinson et al., 1991; MacKintoshet al., 1995; Craig et al., 1996) and been undetect-able by the assay methods used.

MC concentrations continued to increase insunfish liver and muscle tissue until day 6 whenconcentrations became variable, suggesting enzymeinduction (i.e., the switching on of a MC detoxifica-tion pathway), and elimination via bile excretion.By day 9 of the accumulation experiment, fish liverand muscle tissue successfully exported the majorityof MC and/or detoxification products in theirtissues to concentrations below base levels (Fig. 3).MC detoxification, via its conjugation to glu-tathione, has been identified in fish, zooplankton,and aquatic plants (Pflugmacher et al., 1998, 2001;Wiegand et al., 1999). Detoxification products andthe parent toxin have been detected in urine, fecesand bile of fish and mussels (Sahin et al., 1996;Williams et al., 1997a; Amorim and Vasconcelos,1999). It seems unlikely that the sudden decrease inliver MC on day 6 was due to delayed covalentbinding of MC to protein phosphatases 1 and 2A.Irreversible, covalent binding, instead, has beenobserved to occur within hours, rather than days(Robinson et al., 1991; MacKintosh et al., 1995,Craig et al., 1996).

Our results from the accumulation experimentagree with Xie et al. (2004), where the concentrationof non-covalently bound MC (MC-LR and MC-RRcombined) increased in the liver tissue of silver carpand then significantly decreased despite a constantinflux of toxin from Microcystis viridis cells. It is ofinterest that the onset of toxin elimination wasfaster in sunfish (6 days) as compared to silver carp(40–60 days). The time needed for enzyme inductionand toxin elimination may have been dictated by (1)the fasting period, (2) the frequency and duration of

toxin exposure, and/or, (3) species life history.Fasted juvenile goldfish were more severely andmore rapidly affected by MC-LR when intraper-itoneally injected (Malbrouck et al., 2004). Theoverall pattern of accumulation and depuration;however, was indistinguishable between fed andfasted goldfish, indicating that our study mayexemplify an elevated, but parallel response toMC. Secondly, the fish used in our study wereexposed to high levels of MC in the year priorto the experiment (Fig. 1); however, Xie et al.(2004) utilized silver carp from a fishery, sug-gesting they were reared in a MC-free environment.Pre-exposure to MC may have decreased thesensitivity of our fish to the detrimental effects ofMC and allowed for early enzyme induction insunfish.

The same overall pattern of liver accumulationwas seen in the second trial of the accumulationexperiment; however, significant changes in liverMC concentrations were not detected over time(Fig. 3(C)). Instead, MC concentrations were muchmore variable between fish. Fish collected for thesecond trial, in September 2001, had a greaterbase level of MC in their liver and muscle tissueupon the commencement of the accumulationexperiment than did fish collected in July 2001(Fig. 3). September fish were most likely morevariable due to individual differences in toxinexposure, diet, and metabolism rates over thesummer months.

4.3. Sequestration of MC

The liver and muscle tissue of sunfish retainedrelatively constant levels of MC once fish wereremoved from Barbadoes Pond and were held in aMC-free environment for 2 weeks (Fig. 4). Theseresults contradict other depuration studies in whichsilver carp (Xie et al., 2004) and two species ofmussels (Vasconcelos, 1995; Yokoyama and Park,2003) successfully depurated MC in less than 15days. Because our fish were starved during thedepuration period, unlike the other studies, it can beinferred that the discrepancy is likely due to fasting.Therefore, our results more likely represent winterconditions when fish may not be actively feeding butwill retain MC in their tissue until spring. Yokoya-ma and Park (2003) showed that decreased tem-peratures, such as in autumn and winter, also led toslowed depuration rates for the freshwater bivalve,Unio douglasiae.

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Acknowledgements

We thank Profs. M. Ikawa and J. Sasner for theirideas and laboratory support. Much gratitude alsogoes out to Prof. K. Schulz from SUNY-College ofEnvironmental Science and Forestry for her com-ments on the manuscript. Support for this work wasprovided through the University of New HampshireAgricultural Experiment Station, Hatch Grant#205.

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