Microcystin kinetics (bioaccumulation and elimination) and biochemical responses in common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix) exposed to toxic cyanobacterial blooms.

Abstract

Two species of common edible fish, common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix), were exposed to a Microcystis spp.-dominated natural cyanobacterial water bloom for two months (concentrations of cyanobacterial toxin microcystin, 182-539 microg/g biomass dry wt). Toxins accumulated up to 1.4 to 29 ng/g fresh weight and 3.3 to 19 ng/g in the muscle of silver carp and common carp, respectively, as determined by enzyme-linked immunosorbent immunoassay. Concentrations an order of magnitude higher were detected in hepatopancreas (up to 226 ng/g in silver carp), with a peak after the initial four weeks. Calculated bioconcentration factors ranged from 0.6 to 1.7 for muscle and from 7.3 to 13.3 for hepatopancreas. Microcystins were completely eliminated within one to two weeks from both muscle and hepatopancreas after the transfer of fish with accumulated toxins to clean water. Mean estimated elimination half-lives ranged from 0.7 d in silver carp muscle to 8.4 d in common carp liver. The present study also showed significant modulations of several biochemical markers in hepatopancreas of fish exposed to cyanobacteria. Levels of glutathione and catalytic activities of glutathione S-transferase and glutathione reductase were induced in both species, indicating oxidative stress and enhanced detoxification processes. Calculation of hazard indexes using conservative U.S. Environmental Protection Agency methodology indicated rather low risks of microcystins accumulated in edible fish, but several uncertainties should be explored.

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2687
Environmental Toxicology and Chemistry, Vol. 26, No. 12, pp. 2687–2693, 2007
? 2007 SETAC
Printed in the USA
0730-7268/07 $12.00 ? .00
MICROCYSTIN KINETICS (BIOACCUMULATION AND ELIMINATION) AND
BIOCHEMICAL RESPONSES IN COMMON CARP (CYPRINUS CARPIO) AND SILVER
CARP (HYPOPHTHALMICHTHYS MOLITRIX) EXPOSED TO TOXIC
CYANOBACTERIAL BLOOMS
ONDRˇEJ ADAMOVSKY´,† RADOVAN KOPP,‡ KLA´RA HILSCHEROVA´,† PAVEL BABICA,† MIROSLAVA PALI´KOVA´,§
VERONIKA PASˇKOVA´,† STANISLAV NAVRA´TIL,§ BLAHOSLAV MARSˇA´LEK,† and LUDEˇK BLA´HA*†
†Centre for Cyanobacteria and Their Toxins (Institute of Botany, Czech Academy of Sciences and RECETOX, Masaryk University),
Kamenice 126/3, 625 00 Brno, Czech Republic
‡Department of Fishery and Hydrobiology, Mendel University of Agriculture and Forestry, Zeme ˇde ˇlska ´ 1, 613 00 Brno, Czech Republic
§University of Veterinary and Pharmaceutical Sciences, Palacke ´ho 1-3, 612 42 Brno, Czech Republic
(Received 19 March 2007; Accepted 12 July 2007)
Abstract—Two species of common edible fish, common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix),
were exposed to a Microcystis spp.–dominated natural cyanobacterial water bloom for two months (concentrations of cyanobacterial
toxin microcystin, 182–539 ?g/g biomass dry wt). Toxins accumulated up to 1.4 to 29 ng/g fresh weight and 3.3 to 19 ng/g in the
muscle of silver carp and common carp, respectively, as determined by enzyme-linked immunosorbent immunoassay.Concentrations
an order of magnitude higher were detected in hepatopancreas (up to 226 ng/g in silver carp), with a peak after the initial four
weeks. Calculated bioconcentration factors ranged from 0.6 to 1.7 for muscle and from 7.3 to 13.3 for hepatopancreas. Microcystins
were completely eliminated within one to two weeks from both muscle and hepatopancreas after the transfer of fish with accumulated
toxins to clean water. Mean estimated elimination half-lives ranged from 0.7 d in silver carp muscle to 8.4 d in common carp liver.
The present study also showed significant modulations of several biochemical markers in hepatopancreas of fish exposed to
cyanobacteria. Levels of glutathione and catalytic activities of glutathione S-transferase and glutathione reductase were induced in
both species, indicating oxidative stress and enhanced detoxification processes. Calculation of hazard indexes using conservative
U.S. Environmental Protection Agency methodology indicated rather low risks of microcystins accumulated in edible fish, but
several uncertainties should be explored.
Keywords—MicrocystinsBioaccumulationToxicokineticsBiomarkers
INTRODUCTION
Hepatotoxic microcystins (MCs) are a group of peptide
toxins produced by several species of freshwater cyanobac-
teria, such as Microcystis sp., Planktothrix sp., and so on [1].
Microcystins occurring as several structural variants are syn-
thesized nonribosomally during the growth phase and may
represent as much as 1% of the dry biomass. Although a por-
tion of produced MCs is present extracellularly, the majority
of MCs remain inside cyanobacteria, and toxins are released
only after cell death [1]. Microcystins are potent inhibitors of
serine/threonine protein phosphatase 1 and 2A [1], and they
tend to accumulate in liver. Hepatotoxicity, liver tumor pro-
motion, as well as other types of toxicity from MCs have been
intensively studied and documented [2,3]. The World Health
Organization suggested a limit for the tolerable daily intake
(TDI) of 0.04 ?g/kg body weight/d and corresponding pro-
visional guideline of 1 ?g/L for drinking waters for the most
often studied MC variant, MC-LR [1,4].
Although the human toxicity has been studied in detail, the
role of MCs in the aquatic environment remains questionable
[5,6]. Some reports have described levels of MCs in fish, their
metabolism, and also their toxicity [7–9], but detailed toxi-
cokinetics and critical evaluation of human health risks from
accumulated toxins remain to be resolved.
* To whom correspondence may be addressed
(blaha@recetox.muni.cz).
Published on the Web 7/24/2007.
An important mechanism of MC toxicity documented in
various laboratory animals [10], including fish [11], is oxi-
dative stress—that is, cell damage caused by the overproduc-
tion of reactive oxygen species. Oxidative stress causes de-
pletion of intracellular glutathione (GSH), lipid peroxidation,
and oxidative damage to other biomolecules [12]. Several bio-
markers of early toxic effects in fish after exposure to various
stressors, including MCs, have been suggested (e.g., modu-
lations of glutathione S-transferase [GST], glutathione reduc-
tase [GR], and glutathione peroxidase [GPx] [11–13]).
Major aims of the present study were to investigate kinetics
of accumulation and elimination of MCs in the tissues of two
cyprinid freshwater species, common carp (Cyprinus carpio)
and silver carp (Hypophthalmichthys molitrix). Both fish spe-
cies are among the most widespread fish in Europe and Asia,
and they often are cultured as important edible fish. In addition,
the present study examined profiles of biochemical markers in
hepatopancreas after cyanobacterial exposure and evaluated
the health risks of MCs accumulated in fish tissues.
MATERIALS AND METHODS
Experimental design
Experiments simulated the natural situation in the environ-
ment. Fish (C. carpio and H. molitrix; average age, two years)
were obtained from Pohor ˇelice Fisheries (Pohor ˇelice, Czech
Republic). Uptake and accumulation of MCs was studied in
the outdoor pond during two-month (nine-week) exposures of

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Environ. Toxicol. Chem. 26, 2007O. Adamovsky ´ et al.
Table 1. Kinetics of microcystin (MC) concentrations in the muscle and liver (ng MC/g tissue fresh wt) of common carp (Cyprinus carpio) and
silver carp (Hypophthalmichthys molitrix)a
Week
Water
MCsb
Biomass
MCsc
Silver carp
Fish weight (g)Muscle MCsLiver MCs
Common carp
Fish weight (g)Muscle MCsLiver MCs
Accumulation
0 22.7539 202 ? 46
(10)
319 ? 78
(10)
324 ? 78
(10)
0d
(3)
0d
(3)
125 ? 28
(10)
127 ? 42
(10)
128 ? 37
(10)
0d
(4)
0d
(4)
4 13.8 425
10.6 ? 9.9
(10)
5.2 ? 3.4
(7)
0.62/1.7
93.2 ? 50.7
(10)
124 ? 56
(7)
7.3/13.3
9.8 ? 6.4
(7)
7.3 ? 4.6
(7)
0.57/1.1
132 ? 59
(7)
68.7 ? 42
(7)
7.8/12.8
9 14.2182
BCFe(mean/maximum)
Elimination
0—
—
—
—
—
—
421 ? 92
(10)
380 ? 102
(10)
435 ? 86
(10)
0.9 ? 0.3
(5)
0d
(5)
0d
(5)
21.0 ? 14.8
(5)
9.3 ? 3.7
(5)
0.9 ? 0.8
(5)
46 ? 9
(10)
47 ? 16
(10)
40 ? 10
(10)
1.2 ? 0.3
(5)
0.2 ? 0.1
(5)
0d
(5)
17.2 ? 7.0
(5)
13.7 ? 2.7
(5)
2.3 ? 0.4
(5)
1
2
aValues represent the mean ? standard error, with the number of investigated fish given in parentheses.
bWater concentrations of total MCs (sum of MC-LR, -RR, and -YR; ?g/L).
cBiomass MCs concentrations (?g/g dry wt).
dLess than the limit of detection (liver, 0.31 ng/g fresh wt; muscle, 0.13 ng/g).
eBioconcentration factors (ratio between the mean/maximum tissue concentration and the average water concentration 17 ?g/L).
fish to a complex cyanobacterial bloom dominated by Micro-
cystis aeruginosa (45%), Microcystis ichthyoblabe(45%),and
Anabaena flos-aquae (5%). Kinetics of MC elimination (after
the transfer to clean water) was studied in fish that naturally
accumulated MCs in the pond with Microcystis spp. Fish were
not externally fed during experiments, and no mortalities were
recorded. Fish (n ? 3–10 individuals/treatment) were collect-
ed, weighed, and measured on weeks 4 and 9 (accumulation)
and on weeks 1, 2, 4, 6, and 8 (during elimination) (Table 1).
The tissue samples were immediately frozen and stored at
?80?C for analyses of MCs and biomarkers. Parameters of
water in the exposure/elimination experiments were as follows
(given for the accumulation and elimination experiments, re-
spectively; mean ? standard error): temperature, 18.9 ? 3.8
and 19.6 ? 1.3?C; dissolved oxygen, 18.2 ? 2.0 and 11.1 ?
3.2 mg/L; and pH, 9.4 ? 0.4 and 9.1 ? 0.2.
Toxin analyses by high-performance liquid
chromatography
Concentrations of MCs in the cyanobacterial biomass and
water (Table 1) were measured by high-performance liquid
chromatography (HPLC) as described by Lawton et al. [14]
with methods previously used in our laboratory [15]. Briefly,
extracts of lyophilized biomass (50% v/v methanol) or water
samples (MCs concentrated by solid-phase extraction using
SepPack C18 cartridges [Waters, Millford, MA, USA]) were
analyzed with a HPLC Agilent 1100 Series (Agilent Tech-
nologies, Waldbronn, Germany) on a Supelcosil ABZ? Plus
(length, 150 mm; inner diameter, 4.6 mm; film thickness, 5
?m; Supelco, Bellefonte, PA, USA) at 30?C. The binary gra-
dient of mobile phase (flow rate, 1 ml/min) consisted of H2O
plus 0.1% trifluoroacetic acid and acetonitrile plus 0.1% tri-
fluoroacetic acid (linear increase during 0–30 min from 20–
59% of acetonitrile). Chromatograms at 238 nm were recorded
with an Agilent 1100 Series photodiode-array detector, and
MCs were identified by the retention time and characteristic
absorption spectra (200–300 nm). Quantification was based on
external calibrations of three MC variants (MC-LR, -RR, and
-YR).
Tissue extractions
Tissue extractions were performed according to the method
described by Magalhaes et al. [16]. The frozen sample (0.4 g
fresh wt) was homogenized with methanol (3 ml), sonicated
in an ultrasonic bath for 30 min, and centrifuged at 4,000 g
for 10 min. Supernatant was collected and the pellet re-ex-
tracted three times using the same procedure. Obtained meth-
anol fractions were pooled and repeatedly extracted (three
times) with 1 ml of hexane to remove lipids (hexane layers
discarded). Methanol extract was evaporated at 50?C, and the
residue was dissolved in 1 ml of water and analyzed for MCs
using enzyme-linked immunosorbent immunoassay (ELISA).
Recovery of the method (?25%; data not shown) was not
considered during calculations to remain consistent withvalues
previously reported in the literature [16–21].
ELISA for MCs
Concentrations of MCs in the fish tissues were analyzed by
direct competitive ELISA according to the method described
by Zeck et al. [22] using a modification described previously
in detail [15]. Briefly, high-protein-binding, 96-well micro-
plates (Nunc, Wiesbaden, Germany) were incubated overnight
with the anti-mouse immunoglobulin (ICN MP Biomedicals,
Solon, OH, USA). After a wash, plates were incubated for 1
h with mouse monoclonal IgG MC10E7 developed against
MC-LR (5,000-fold dilution; ALEXIS, Lausen, Switzerland).
The reaction was based on the competition of MCs in the
sample with the conjugate of MC-LR–horseradish peroxidase
[22]. The activity of horseradish peroxidase was determined
using the 3,3?,5,5?-tetramethylbenzidine (absorbance, 420 nm;
reference, 660 nm) with a microplate reader (GENios Spectra
Fluor Plus; Tecan Group, Ma ¨nnedorf, Switzerland). Each sam-
ple was analyzed in three replicates and the results compared
with the 0.125 to 2 ?g/L calibration curve of MC-LR con-

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Microcystin toxicokinetics and biomarkers in fish
Environ. Toxicol. Chem. 26, 20072689
structed for each individual ELISA plate. Samples from both
exposed and control fish were analyzed, and no significant
nonspecific interferences of the tissue extracts with ELISA
were observed. The antibody used in the present study
(MC10E7) has been shown to have 100 and 96% cross-re-
activity with MC-LR and MC-RR, respectively [22]. Because
these two MC variants were dominant in the present study,
detected concentrations were considered to be a sum of MCs.
We cannot exclude that the ELISA also detected MC fragments
in fish tissues, such as glutathione-MC conjugates. This was
not studied in detail, however, our approach was comparable
with those in previous studies [16–21].
Biomarker analyses
Hepatopancreas samples (1 g) were homogenized on ice
with 1 ml of phosphate buffer saline (pH 7.2), and supernatant
was collected after centrifugation (5 min, 2,500 g, 4?C) and
stored at ?80?C before analyses. Protein concentrations were
determined according to the method of Lowry et al. [23] using
bovine serum albumin as a standard.
Concentration of glutathione was determined according to
the method described by Ellmann [24] using 5,5?-dithiobis-2-
nitrobenzoic acid as a substrate. Before analyses, the samples
were treated with trichloroacetic acid (25% w/v) and centri-
fuged (6,000 g, 10 min). Supernatant was mixed with 0.6 ?M
5,5?-dithiobis-2-nitrobenzoic acid in Tris-HCl/ethylenediami-
netetra-acetic acid (EDTA) buffer (0.5 M tris[hydroxymethyl]-
aminomethane–hydrochloric acid, 0.5 M Tris, and 12.5 mM
EDTA; pH 8.9) and incubated for 5 min at room temperature.
Absorbance was measured at 420/680 nm, and the concentra-
tions (nmol GSH/mg protein) were calculated from the cali-
bration of standard reduced GSH.
Glutathione S-transferase activity was measured spectro-
photometrically using 1 mM 1-chloro-2,4-dinitrobenzene and
2 mM GSH as substrates according to the method described
by Habig et al. [25]. Specific activity was expressed as nano-
moles of formed product per minute per milligram of protein.
Activity of GPx was determined from the rate of nicotin-
amide adenine dinucleotide phosphate (NADPH) oxidation,
recorded as the decrease in absorbance at 340 nm [26]. The
reaction mixtures contained 3 mM GSH, 1.2 mM butylhydro-
peroxide, 1 U of GR (1 U of GR reduces 1.0 mmol of oxidized
glutathione per minute at pH 7.6 at 25?C), and 0.15 mM
NADPH in 0.1 M potassium phosphate/1 mM EDTA buffer
(pH 7.0). Also, the activity of GR in fish was determined by
spectrophotometric measurement of NADPH oxidation in mi-
croplates [27]. The reaction mixtures contained 0.05 M po-
tassium phosphate/1 mM EDTA buffer (pH 7.0), 1 mM glu-
tathione-oxidized disodium salt, 0.1 mM NADPH, and the
tissue extract (0.25% v/v). Specific activities of both GPx and
GR were expressed as nanomoles of NADPH oxidized per
minute per milligram protein.
Statistical calculations
Significant differences were determined using Student’s t
test or analysis of variance followed by Dunnett’s post-hoc
tests. Data normality was checked with the Kolmogorov-Smir-
nov test, and homogeneity of variances was assessed with the
Levene’s test. The p values less than 0.05 were considered to
be statistically significant for all tests. Calculations were per-
formed using the Statistica for Windows? 7.0 softwarepackage
(StatSoft, Tulsa, OK, USA). Elimination kinetic curves and
MC half-lives were calculated using the one-phase exponential
decay equation incorporated in the GraphPad Prism 4 software
(GraphPad Software, San Diego, CA, USA).
RESULTS AND DISCUSSION
The present study describes toxicokinetics (accumulation
and elimination) of MCs in the tissues of common carp and
silver carp. Although several authors reported MC concentra-
tions in zooplankton, shellfish, or fish [28–32], the kinetics of
MC accumulation and elimination in fish have not been in-
vestigated in detail.
A summary of our results is given in Table 1 and in Figures
1 and 2. Microcystins accumulated in the muscle of common
carp and silver carp up to 9.8 and 10.6 ng/g fresh weight,
respectively. Concentrations approximately an order of mag-
nitude higher were determined in the hepatopancreas, which
is the target organ for MCs [33,34]. The muscle to liver con-
centration ratio in the present study (1:10) corresponded to
that in the previous study with Atlantic salmon [33], but a
higher ratio (1:20) was found in common carp compared with
that in the study by Li et al. [18].
Average MC concentrations in both studied species gen-
erally were comparable (Table 1), but slightly higher levels
were found in the liver of common carp in comparison to those
in the liver of silver carp (compare, e.g., week 4 of the ac-
cumulation experiment) (Table 1). This may be related to pos-
sible resistance of phytophagous silver carp to MCs in com-
parison with the benthophagous common carp (as also sug-
gested by Snyder et al. [19]). Calculated bioconcentration fac-
tors (BCFs; average and maximum tissue concentrations
divided by the average water concentration of 17 ?g/L) ranged
from 0.6 to 1.7 in the muscle and from 7.3 to 13.3 in the liver
of both species. To our knowledge, the BCFs for MCs in fish
were not previously reported, but our results generally cor-
respond to previously reported values for aquatic macrophytes
(MC BCF ?0.1–5.9 [35]). Higher BCFs (range, 12–22) were
reported for structurally related peptide cyanotoxin nodularin
in various zooplankton species [36].
Kinetics of MC accumulation in hepatopancreas seems to
be species-specific. In common carp, a peak in MC concen-
trations occurred after four weeks, followed by an apparent
decrease after nine weeks (a trend that is comparable to the
changes in muscle of both species) (Table 1). On the other
hand, continuous accumulation of MCs was recorded in he-
patopancreas of silver carp during the entire exposure period
(up to 124 ng/g fresh wt) (Table 1). Differences may be ex-
plained, for example, by phytoplanktivorous feeding of silver
carp, which actively ingests cyanobacterial cells, whereas only
passive MC intake can be expected in omnivorous and ben-
thophagous common carp [19].
The elimination experiment demonstrated that MC is rap-
idly removed from the tissues after the transfer of fish to clean
water (Table 1). In both species, calculated elimination half-
lives were shorter for muscle (0.7–2.8 d) than for liver (3.5–
8.4) (Fig. 1). To our knowledge, information regarding MC
depuration from the fish is rare [20,37,38]; however, studies
of MC elimination from some invertebrates also suggest fast
elimination of MCs. For example, a half-life of 8 d was re-
ported for freshwater snail [39], and half-lives from 3.0 to 4.8
d were observed in bivalves [40]. In contrast to the rapid
elimination observed in our manipulated experiments (Fig. 1),
slower MC removal from silver carp and Nile tilapia has been
reported in natural lakes (elevated MCs during the period 15–
40 d after the end of the accumulation period [20,37]).

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Environ. Toxicol. Chem. 26, 2007 O. Adamovsky ´ et al.
Fig. 1. Microcystin elimination from the tissues of common carp (Cyprinus carpio) (A and B) and silver carp (Hypophthalmichthys molitrix)
(C and D). Presented are individual tissue concentrations, elimination curves (solid lines) with 95% confidence intervals (dashed lines), and half-
lives in days (mean values with 95% confidence intervals in parentheses).
Taken together, bioaccumulation of MCs in the fish is a
dynamic process depending on both uptake and metaboliza-
tion/elimination [6]. Interspecies variability in MC metabolism
and elimination, however, as well as environmental factors
(e.g., temperature [40]) that may affect MC toxicokinetics will
require further research.
We also investigated a set of glutathione-related biomarkers
in the hepatopancreas of both fish species (Fig. 2). Activity of
GR (significantly elevated in a majority of experimental var-
iants, especially in common carp) was the most sensitive bio-
marker of cyanobacterial exposure (Fig. 2). On the other hand,
changes in GPx activity were less sensitive in our experiments.
Inductions of GST seem to correspond to detoxification of
MCs by GST-mediated conjugation with GSH [9,41,42]. El-
evated GSH concentrations and activities of the GR (the en-
zyme regenerating GSH from its oxidized form [13]) further
reveal increased demands for reduced GSH because of en-
hanced detoxification and/or oxidative stress induced by toxic
cyanobacteria [11,12,43]. Our present study, however, dem-
onstrates that biochemical adaptations are only temporary and
that prolonged exposures may result in signs of general tox-
icity—that is, suppression of GSH levels and inhibition of GR
activity (compare the four- and nine-week exposures for silver
carp, as shown in Fig. 2).
Apparent time-, species-, and MC variant–dependent var-
iability exists in biochemical responses of organisms to MCs
[44]. Inductions of GST are among the most often reported
responses [9,42] (present study), but other authors also have
reported rapid, 24-h inhibitions of GST in Corydoras paleatus
exposed to purified MC-RR [45]. Modulations of biomarkers
in the present study confirm an important role for oxidative
stress in the toxicity of complex cyanobacterial bloom, and it
also demonstrates that biochemical parameters (especially GR,
GST, and GSH) may serve as sensitive early markersofadverse
effects in fish. Direct interpretation of biomarker responses
remains complicated, however, and further research will be
needed to characterize both natural variability and temporal
changes in responses to toxicants.
It has been suggested that accumulated MCs in edible fish
may represent a risk to human health, and it has been dem-
onstrated that MCs are stable and not degraded by heat during
cooking [46]. We have calculated the hazard index (HI), a ratio
between the estimated daily intake (EDI) and chronic TDI,
based on our results using an U.S. Environmental Protection
Agency methodology [47]. To derive the EDI, we have con-
sidered a one-year exposure, 48 fish meals per year (100%
contaminated), ingestion rate of 132 g per serving of meat,
human body weight of 70 kg, and maximum concentration of
MCs in fish fillet observed in the present study (29.3 ng/g
fresh wt in silver carp). Using this worst-case scenario and
considering a chronic TDI (0.04 ?g/kg/d for MC-LR [1]), a
calculated HI of 0.19 indicates a nonsignificant risk from MCs
accumulated in fish meat (HI ? 1 [47]). Interestingly,relatively
high HIs, ranging from 2.35 to 3.66, which correspond to
realistically edible critical amounts of fish food (82–545 g/
serving) were reported previously by Magalhaes et al. [16].
Those authors, however, compared the single-day intake of
MCs with the chronic (i.e., year-round derived) TDI value,
which could overestimate the total risk. Another factor that
may affect total risk is relatively low recovery of MCs from
animal tissues (reported values range from 3% [48] to 25%
[present study]), which usually is not considered during cal-

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Microcystin toxicokinetics and biomarkers in fish
Environ. Toxicol. Chem. 26, 20072691
Fig. 2. Modulations of biochemical parameters in fish hepatopancreas after four and nine weeks of exposure to cyanobacterial biomass. Level
of glutathione (GSH; nmol/mg protein), activity of glutathione S-transferase (GST; nmol/min/mg protein), and activities of glutathione peroxidase
(GPx; nmol nicotinamide adenine dinucleotide phosphate [NADPH]/min/mg protein) and glutathione reductase (GR; nmol NADPH/min/mg
protein). Box includes the 25th to 75th percentiles, with the middle point representing the median and the whiskers showing the extremes. An
asterisk indicates a statistically significant difference from control (p ? 0.05, Student’s t test).

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Environ. Toxicol. Chem. 26, 2007 O. Adamovsky ´ et al.
culations but may lead to possible underestimation of EDI.
Taken together, MCs accumulated in edible fish tissues even-
tually may pose a risk to certain groups of people (e.g., fish-
ermen consuming large amounts of contaminated fish), but
uncertainties remain in both analytical approaches and risk
assessment calculations.
CONCLUSIONS
Our results demonstrate kinetics of MC accumulation and
elimination in two common Eurasian freshwater fish species,
common carp and silver carp. We found that in most cases,
maximum MC concentrations accumulated within the first four
weeks of exposure, and prolonged periods (nine weeks) re-
sulted in a less significant increase. Our results suggest rapid
elimination of MCs from the fish tissues (half-life in days),
but further research should focus on interspecies differences
in metabolization and natural factors affecting MC toxicoki-
netics. The role of oxidative stress and changes of detoxifi-
cation capacity in response of the fish on cyanobacterial ex-
posure was confirmed by modulations of several biochemical
parameters (e.g., GR, GSH, and GST in both species). Cal-
culation of hazard indexes using conservative U.S. Environ-
mental Protection Agency methodology indicates a rather low
risk of accumulated MCs in edible fish, but several uncertain-
ties should be explored.
Acknowledgement—This work was supported by the Ministry of Ed-
ucation of the Czech Republic (projects MSM 6215712402 and
IM6798593901) and by the National Agency for Agricultural Re-
search (NAZV/06/3233).
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