Arch Environ Contam Toxicol (2014) 66:379–389
DOI 10.1007/s00244-013-9993-2
Variation in Cyanobacterial Hepatotoxin (Microcystin) Content
of Water Samples and Two Species of Fishes Collected
from a Shallow Lake in Algeria
Amina Amrani • Hichem Nasri • Amina Azzouz
Yacine Kadi • Noureddine Bouaı̈cha
Received: 15 October 2013 / Accepted: 30 December 2013 / Published online: 21 January 2014
Ó Springer Science+Business Media New York 2014
Abstract Microcystins (MCs) produced from cyanobac-
teria can accumulate in freshwater fish tissues. In this study,
variations in these toxins content were examined monthly in
water samples and two species of fish in Lake Oubeira,
Algeria, from April 2010 to March 2011. During the study
period, MCs were analyzed using protein phosphatase type
2A (PP2A) inhibition assay. In lake water, total (dissolved
and intracellular toxins) MC concentrations by PP2A ranged
from 0.028 to 13.4 lg equivalent MC-LR/l, with a peak in
September 2010. MC-LR was the dominant variant (90 % of
the total) in water samples, followed by MC-YR and MC-
(H4)YR. The highest MC concentration in the omnivorous
common carp (Cyprinus carpio) was found in the order
intestine [ hepatopancreas [ muscle; however, in the car-
nivorous European eel (Anguilla anguilla) the order was
liver [ intestine [ muscle. Highest MC concentrations in
the intestine tissue of the common carp were found between
August and November 2010 where high MC concentrations
were detected in water samples, whereas high levels of MCs
in the liver of the European eel were found later between
January and February 2011. During the entire period of
study, the World Health Organization (WHO) lifetime limit
A. Amrani  H. Nasri  A. Azzouz
Laboratoire Biodiversité et Pollution des Écosystèmes, Institut
des Sciences de la Nature et de la Vie, Université d’El Tarf,
36 000 El Tarf, Algérie
Y. Kadi
Laboratoire Central de Pathologie, Hôpital El Taref,
36 000 El Tarf, Algérie
N. Bouaı̈cha (&)
Laboratoire Ecologie, Systématique et Evolution, UMR
8079-Université Paris-Sud/CNRS/AgroParisTech, Université
Paris-Sud, Bâtiment 362, 91405 Orsay Cedex, France
e-mail: noureddine.bouaicha@u-psud.fr
•
for tolerable daily intake was exceeded only in common carp
muscle.
When meteorological conditions and nutrient supply are
favorable, cyanobacteria flourish in lakes and rivers where
they can produce blooms and thus represent an emerging
human and environmental health concern (for review see
O’Neil et al. 2012). Maximum growth rates are attained by
most cyanobacteria at temperatures [25 °C. For example,
within the North African basin, in Egypt (Mohamed et al.
2003), in Morocco (Oudra et al. 2001), in Tunisia (El Herry
et al. 2008), and in Algeria (Nasri et al. 2004, 2007, 2008)
cyanobacterial blooms have been observed in several sur-
face freshwaters during the warmest months particularly in
summer and early autumn. The occurrence of these harmful
microorganisms in aquatic ecosystems is often accompa-
nied by a production of a variety of cyanotoxins, which are
generally classified into four categories, i.e., hepatotoxins,
neurotoxins, irritant toxins, and cytotoxins, depending on
their mode of action (Chorus and Bartram 1999). The
presence of such toxins has been reported throughout the
world, and it appears that liver-toxic microcystins (MCs)
are more commonly found in 40–75 % of cyanobacterial
blooms (Chorus and Bartram 1999).
In marine ecosystem, human poisoning through phyco-
toxins highlights that biomagnifications of these toxins in
the aquatic food chain may cause illness in humans when
biota from higher trophic levels are consumed and that this
risk must be assessed in freshwater ecosystems for cy-
anotoxins as well. Freshwater aquacultures are the fastest
growing animal food sector in the world, and cultured fish
supply now provides [13 % of the animal protein intake
for the human population (WHO 2007). Therefore,
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380
aquaculture farming in some Mediterranean countries has
grown to offset the increased demand. Therefore, this
intensive aquaculture commonly results in eutrophic con-
ditions and the occurrence of toxic cyanobacterial blooms
(Magalhaes et al. 2001). Several studies have been reported
that cyanobacterial toxins are known to bioaccumulate in
common aquatic vertebrates and invertebrates, including
zooplankton, mollusks, and crustaceans, as well as fishes,
which pose a potential risk to both animal and human heath
if such aquatic animals are consumed (see review by Ib-
elings and Chorus 2007; Ettoumi et al. 2011). Although no
case of human poisoning by this route has been reported in
the literature, this eventuality must not be ignored. Indeed,
a study by Chen et al. (2009) reported that MCs were
identified for the first time in the serum of a chronically
exposed human population (fishermen at Lake Chaohu,
China) together with indication of hepatocellular damage.
Freshwater fish comprise only a minor proportion of fish
products in some countries. So far, only a few studies have
measured MC content in feral fishes in Brazil (Magalhaes
et al. 2001), Egypt (Mohamed et al. 2003), Portugal (Va-
sconcelos 1999), China (Xie et al. 2005), Mexico (Berry
et al. 2011) and the United States (Schmidt et al. 2013) in
only a few species. Such studies are still lacking in Algeria,
where people commonly consume freshwater fish regard-
less of the danger of MCs. Therefore, the main goal of this
study was to evaluate the presence of MCs in water sam-
ples and different tissues (hepatopancreas, liver, intestine,
and muscle) of two fish species, i.e., the common carp
Cyprinus carpio (Linnée 1758) and the European eel
Anguilla anguilla (Linnée 1758) captured in Lake Oubeira,
looking to associate the levels of MCs found in water with
those observed in fish tissues.
Materials and Methods
Site Description and Raw Water and Fish Sample
Collection
Lake Oubeira is a shallow, polymictic waterbody, located
in northeastern Algeria (36°530 N and 008°230 E), home to
El Kala National Park, and has an average elevation of
25 m above sea level. It is the first largest freshwater lake
in Algeria and has an estimated surface area of 2,200 ha
and a maximum depth of *4 m. In 1984, the lake was
included as a wetland under the Ramsar Convention
[Ramsar Convention Official Website (www.ramsar.org)]
because it is considered an important natural reserve for
migratory birds and wildfowl species. Fishing and irriga-
tion are some of the activities practiced in and around its
waters. Cyanobacterial blooms with the dominance of
Microcystis spp., [90 % of the algal biomass, have been
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Arch Environ Contam Toxicol (2014) 66:379–389
previously reported in this lake (Nasri et al. 2004, 2008).
The high concentration of MCs was detected in autumn
2004 at 29 mg equivalent MC-LR/l (Nasri et al. 2004). In
October 2005, the death of two species of freshwater turtle
was observed in this lake during a Microcystis spp. bloom
(Nasri et al. 2008). The trophic status of the lake and the
increase in cyanobacterial bloom occurrences has led
authorities to cease using this lake as a source for the
production of drinking water. However, it is still used for
irrigation and aquaculture particularly for two fish species:
the common carp C. carpio (Linnée 1758) and the Euro-
pean eel A. anguilla (Linnée 1758). The common carp is
omnivorous, and its diet consists of insects, larvae, mol-
lusks, and various aquatic plants and algae. However, the
European eel is carnivorous and a carrion feeder, and its
diet is highly biased toward fishes, water snails, worms,
aquatic insects, and different and varied waste, such as fish
corpses.
During the period of study (April 2010 to March 2011),
raw water and fish samples were collected monthly from
the same site in Lake Oubeira. For MC analysis in raw
water, 1 l of lake water taken from the surface at the same
site at 10 m from the shore was filtered through glass mi-
crofiber filters (GF/C; Whatman, VWR, Fontenay Sous
Bois, France) to separate MCs present in both the water
(dissolved or extracellular toxins) and the cyanobacterial
cells (intracellular toxins) using a vacuum pump. The filters
were air-dried, packed in aluminum foil, and stored at
-20 °C until further processing and analysis. Filtered
water samples (filtrates) were maintained in glass bottles at
-20 °C until concentration and analysis. Every water
sample was taken in triplicate. For assessment of MC
bioaccumulation, species of locally consumed fish,
including predominantly the common carp, C. carpio, and
the European eel, A. anguillan were obtained monthly from
small commercial catches of fisherman fishing within Lake
Oubeira during water-quality sampling. During the 1-year
sampling period, the body weight of the common carp and
the European eel varied between 250 and 1,400 g and 400
and 1,050 g, respectively. However, at each date of sam-
pling, every fish species was taken by triplicate with a
similar body weight. After capture, fishes were ice-cooled
and transported to the laboratory where they were imme-
diately dissected, and, subsequently, hepatopancreas, liver,
intestine, and 100 g of muscle for each fish were separated.
Tissues were cut into small portions and transferred to
50-ml tubes, lyophilized, and stored at -20 °C for sub-
sequent extraction and analysis.
MC Extraction from Water and Fish Samples
For the determination of free dissolved MCs (extracellular
toxins), filtrates (500 ml in triplicate) were applied to C-18
Arch Environ Contam Toxicol (2014) 66:379–389
solid-phase extraction cartridge (LiChrolut RP-18, 500 mg;
Merck, France) previously washed with 5 ml of methanol
[high-performance liquid chromatography (HPLC) grade]
and further conditioned with 5 ml of Milli-Q water (Milli-
Q Plus ultrapure water purification system; Millipore,
France). Toxins were eluted using 3 ml of methanol
(HPLC grade) containing 0.5 % trifluoro acetic acid. The
eluate was evaporated to dryness at 40 °C under decreased
pressure, suspended in 1 ml of 50 % (v/v) aqueous meth-
anol, and stored at -20 °C until further analysis by phos-
phatase type 2A (PP2A) inhibition assay. For the
determination of cellular MCs (intracellular toxins), air-
dried filters were placed in glass tubes, covered with 5 ml
of 75 % (v/v) aqueous methanol, and sonicated for 5 min
in an ultrasonic bath (Ultrasonic 300). The suspension was
centrifuged at 10,000 g for 5 min; the supernatant was
retained; and the filters were re-extracted two times as
previously, described. Combined supernatants were evap-
orated to dryness at 40 °C under decreased pressure, sus-
pended in 1 ml of 50 % (v/v) aqueous methanol, and stored
at -20 °C before analysis by PP2A inhibition assay.
For MC extraction from fish tissues, 100 mg dry weight
(dw) of each lyophilized tissue (hepatopancreas, intestine,
and muscle for the common carpe; liver, intestine, and
muscle for the European eel) were homogenized with 1 ml
of 75 % (v/v) aqueous methanol using a Teflon pestle.
Homogenates were introduced for 5 min in an ultrasonic
bath (Ultrasonic 300) followed by centrifugation at
10,000 g for 10 min. The supernatant was retained and the
residue re-extracted two times as previously described.
Combined supernatants were stored at -20 °C before
analysis by PP2A inhibition assay.
Analysis of Microcystins by PP2A Inhibition Assay
MC concentrations in the water and in each fish tissue
sample were determined using PP2A inhibition assay
according to Bouaı̈cha et al. (2002). Briefly, the PP2A
activity was determined by measuring the coloration
associated with the formation at 37 °C of p-nitrophenol (p-
NP) from the substrate p-nitrophenyl phosphate (p-NPP)
using a microtiter plate reader (CERES UV900 Hdi). The
p-NPP (80 mM) was dissolved in the Tris–HCl buffer
containing 40 mM Tris–HCl, 34 mM MgCl2, 4 mM eth-
ylene diamine tetraacetic acid, and 4 mM dithiothreitol, pH
8.3. The PP2A was diluted in the Tris–HCl buffer and
supplemented with bovine serum albumin (BSA) at
0.5 mg/ml to a concentration of 200 mU/ml. The assay was
then performed by adding 100 ll of the sample solution
prepared in the Tris–HCl buffer to 50 ll of the enzyme
solution in a 96-well microtiter plate. After incubating at
37 °C for 5 min, 50 ll of substrate (p-NPP) was added.
The rate of p-NP production was measured after 1 h at
381
405 nm. The ‘‘toxin equivalent’’ concentration (MC-LR
equivalent/l or g dw) in each sample was determined from
the linear part of the calibration curve (20–1,000 pg/ml
with pure MC-LR; Novakits, France) produced in each
assay, which was between approximately 25 and 75 %
inhibition of PP2A. The test sample will, therefore, be
diluted or concentrated so that the inhibition value will be
in the linear portion of the standard curve. The limit of
quantification for PP2A was 5 ng/l of water samples or
20 ng/g dw of tissue samples. For water and fish samples,
analysis was performed on three different water and fish
samples and each analysis was replicated three times. The
total MC content in the lake water was obtained by the
addition of cellular (intracellular) and dissolved (extracel-
lular) MC concentrations.
Identification of Microcystin Variants by Liquid
Chromatography–Tandem Mass Spectrometry
Liquid chromatography coupled to tandem mass spec-
trometry (LC–MS/MS) analysis was performed in the
Laboratory PESSAC, INRA de Grignon, France. The
electrospray ionization mass spectrometry in positive mode
experiment for confirming the identities of some MC-LR
variants in lake water, harvested on September 2010 where
the highest MC concentrations were observed with the
PP2A assay, was performed using a Waters Acquity Ul-
traperformance LC separations module coupled to a
Quattro Ultima triple-quadrupole mass spectrometer
(Waters, France). The sample was chromatographed on an
Acquity ULPC BEH C18 column (100 9 2.1 mm i.d.,
1.7 lm) with a linear gradient (300 ll/min) of water (sol-
vent A) and acetonitrile (solvent B) each containing 0.1 %
acetic acid as the mobile phase. The gradient consisted of
0 min at 95 % A, then to 45 % A at 7.50 min, to 100 % B
at 8.50 min (1-min hold), followed by a return to 95 % A at
9.60 min with a 2-min hold to equilibrate the column. The
MS/MS was run in precursor/product ion mode as descri-
bed in Mekebri et al. (2009). Precursor-ion scanning (m/z
500–1200) for m/z 135 was performed with collision
energy at 90 eV.
Histopathology of Fish Hepatopancreas and Liver
Tissues
Three specimens of each species C. carpio and A. anguilla
with a mean body weight of 800 g were obtained from
small commercial catches of fisherman fishing within Lake
Oubeira in October 2010. Two control carps (C. carpio)
with a mean body weight of 20 g were purchased from a
local fish hatchery. The carps were kept at a water tem-
perature of 15 °C in dechlorinated tap water under con-
tinuous aeration and fed with pellet food at a rate of 1 %
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382 Arch Environ Contam Toxicol (2014) 66:379–389

Fig. 1 Distributions and 14000 2000

variations of extracellular
Extracellular toxin 1800
(broken line) and intracellular

Microcystin-LR equivalent (ng/l)

Microcystin-LR equivalent (ng/l)

12000 Intracellular toxin
(unbroken line) MCs in surface 1600
water collected monthly in Lake
10000

(Extracellular toxin)
Oubeira from April 2010 to 1400

(Intracellular toxin)
March 2011. Data were
1200
expressed as the mean value and 8000
SDs of three replicate 1000
determinations
6000
800

4000 600

400
2000
200

0 0

Date of sampling

body weight/day for 6 months. However, breeding of the sampling period from April 2010 to March 2011, the total
European eel is not well controlled in our laboratory; MC concentrations, corresponding to the addition of the
therefore, we do not have a control for this species. After intracellular and the extracellular fractions, ranged between
dissection, hepatopancreas and liver tissues from captured 0.028 and 13.4 lg equivalent MC-LR/l. The percentage of
and control fishes were quickly removed and rinsed in tap MC levels found in the extracellular fraction ranged from
water for subsequent histopathological analysis. Liver and 67 to 99 % for all sampling dates except in June and July
hepatopancreas tissues were fixed in 5–10 % neutral buf- where it ranged from 1.6 to 32 %, respectively. Therefore,
fered formalin, dehydrated through a graded series of eth- in the current study, the dissolved fraction contributed a
anol, cleared in xylene, embedded in paraffin, sectioned at a high percentage to total MC concentrations in this lake.
thickness of 5 lm, and stained with hematoxylin (H) and
eosin (E) for histological examination. Subsequently, liver Microcystin Variant Identification by LC–MS/MS
and hepatopancreas tissues were examined microscopically
(Leica DM 1000 LED, France), and their histological The LC–MS/MS tandem mass spectra of the water lake
abnormalities were recorded and photographed. fraction containing dissolved toxins and collected on Sep-
tember 2010, where the highest MC concentration was
observed with the PP2A assay, are shown in Figure 2.
Results These spectra showed the presence of three peaks (6.096,
6.294, and 6.371 min) giving a fragment ion at m/z 135, a
Seasonal Variation of Microcystin Concentrations typical fragment for MCs attributed to a [C9H11O]? frag-
in the Lake Water ment of the Adda residue. The parent ion [M?H]? of the
more abundant peak at 6.371 is m/z: 995.7. The parent ions
The MC concentrations in water (extracellular toxins) and [M?H]? of the two minor peaks at 6.096 and 6.294 min
algal samples (intracellular toxins) were analyzed by the are m/z: 1049.5 and 1045.7, respectively. To establish the
PP2A inhibition assay, and their seasonal variation is structural identities of these peaks, collision-induced dis-
shown in Figure 1. Measurable MC levels were detected in sociation spectra of the [M?H]? ions were generated. The
dissolved and particulate fractions in all of the examined product ions’ spectra of the peaks at 6.294 and 6.371 min
samples, with concentrations ranging from 18 to 11,800 summarize some of the important fragment ions present in
and from [10 to 1,600 ng/l, respectively. High concen- the MS/MS spectra of MC-YR (1017.5 and 603.5) and
trations of cell-bound MCs were found in 2010 in early MC-LR (967.9 and 553.4), respectively. However, the peak
autumn (September to October) with a maximum value of at 6.096 is not very intense, and except the fragment ion at
1.6 lg/l. Extracellular MC concentrations in all cases, m/z 135, no further characteristic fragment ions were
except June and July, are several orders of magnitude observed. In the literature, the only MC variant with a
greater than those of intracellular MC concentrations with a [M?H]? ion at m/z: 1049.5 described until now is MC-
peak in September of 11.8 lg/l. During the 12-month homotyrosineR (MC-(H4)YR) (Namikoshi et al. 1992).

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Arch Environ Contam Toxicol (2014) 66:379–389
Fig. 2 LC–MS/MS precursor ion spectra of the water sample fraction containing dissolved toxins from Lake Oubeira harvested on September
2010 exhibit parent ions [M?H]? of MCYST-LR (m/z: 995.5), MCYST-YR (m/z: 1045.5), and MCYST-(H4)YR (m/z: 1049.5)
Therefore, according to retention time and the parent ion
[M?H]?, this minor compound should be MC-(H4)YR.
Thus, the water sample form Lake Oubeira collected on
September 2010 presents three variants of MCs: MC-LR,
MC-YR, and MC-(H4)YR.
Variation of MC Contents in Various Organs of Fish
MCs measured in different common carp tissue samples
collected between April 2010 and March 2011 ranged from
343 to 771 ng equivalent MC-LR/g DW in hepatopancreas
tissue, from 371 to 3,059 ng equivalent MC-LR/g DW in
intestine, and from 329 to 680 ng equivalent MC-LR/g DW
in muscle (Fig. 3). The highest MC concentration was
found in the intestine (3,059 ng equivalent MC-LR/g DW),
followed by the hepatopancreas (771 ng equivalent MC-
LR/g DW) and the muscle (680 ng equivalent MC-LR/g
DW) (Fig. 3). MC levels in different European eel tissue
samples ranged from 86 to 333 ng equivalent MC-LR/g
DW in liver, from 66 to 233 ng equivalent MC-LR/g DW
in intestine, and from 54 to 67 ng equivalent MC-LR/g DW
in muscle (Fig. 4). The highest MC concentration was
found in the liver (333 ng equivalent MC-LR/g DW), fol-
lowed by the intestine (233 ng equivalent MC-LR/l) and
the muscle (67 ng equivalent MC-LR/g DW) (Fig. 4).
383
During the sampling period, the mean of MC concen-
trations in intestine, liver, and muscle tissues was highest in
the omnivorous fish C. carpio and lowest in the carnivo-
rous fish A. anguilla. The highest MC concentration in the
common carp was found in the order intestine [ hepato-
pancreas [ muscle; however, in the European eel the order
was liver [ intestine [ muscle. Except in April 2010, the
highest MC concentrations in the intestine tissue of the
omnivorous fish C. carpio were found between August and
November 2010 (Fig. 3) where high MC concentrations
were detected in water samples of Lake Oubeira (Fig. 1).
The high MCs concentration detected in the intestine of the
common carp in April 2010 can be the result of an earlier
bloom of cyanobacteria that occurred in Lake Oubeira.
However, for the carnivorous fish A. Anguilla, the highest
MC concentrations in liver tissue were found later between
January and February 2011 (Fig. 4).
Histopathology of Common Carp Hepatopancreas
and European Eel Liver
No fish mortalities were observed during the period of
study, and the appearance of the examined organisms was
macroscopically normal. However, during the period Sep-
tember to October 2010, when MC levels were greater, we
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384 Arch Environ Contam Toxicol (2014) 66:379–389

Fig. 3 Distributions and 4500 14000

variations of total MC Hepatopancreas
concentrations in raw water 4000 Intestine 12000
(broken line) and their

Microcystin-LR equivalent (ng/g DW)

Microcystin-LR equivalent (ng/l)

accumulation in the Muscle
3500
hepatopancreas (open bars), Total MCs
10000
intestine (gray bars), and
3000
muscle (black bars) of the
common carp (C. carpio) 8000
captured monthly in Lake 2500
Oubeira from April 2010 to
March 2011. Data are expressed 2000 6000
as the mean value and SDs of
three different fishes replicated 1500
three times 4000
1000

2000
500

0 0

Date of sampling

Fig. 4 Distributions and 500 14000

variations of total MC Liver
concentrations in raw water 450
Intestine
(broken line) and their 12000
Microcystin-LR equivalent (ng/g DW)

Muscle
accumulation in the liver (open

Microcystin-LR equivalent (ng/l)

400
bars), intestine (gray bars), and Total MCs
muscle (black bars) of the 350 10000
European eel (A. anguilla)
captured monthly in Lake 300
Oubeira from April 2010 to 8000
March 2011. Data are expressed 250
as the mean value and SDs of
6000
three different fishes replicated 200
three times
150 4000

100
2000
50

0 0

Date of sampling

noticed that when we cutting hepatopancreas (common
carp) and liver (European eel) tissues into small portions
before lyophilization, the liver appeared to be a firm tissue;
however, the hepatopancreas appeared to be crumbled.
Microscopic examination of the H&E-stained hepatopan-
creas section of the common carp control showed no
histopathological degeneration (Fig. 5a). However, micro-
scopic examination of the H&E-stained hepatopancreas
sections of the common carp collected from Lake Oubeira
in October 2010 showed the presence of several abnor-
malities characterized by liver steatosis with large, fat
nodules at the periphery of hepatocytes (Fig. 5b), dilatation
of the lumen of hepatic sinusoid (Fig. 5c), and the presence
of siderotic macrophages (Fig. 5d), an inflammatory cell
type indicating intrahepatic hemorrhage. Microscopic
examination of the HE-staining liver section of the Euro-
pean eel collected during the same period and at the same
site showed the presence of several abnormalities

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Arch Environ Contam Toxicol (2014) 66:379–389
a b
Fig. 5 Histopathological changes in H&E-stained hepatopancreas
section of common carp (C. carpio) and in H&E-stained liver section
of European eel (A. anguilla) captured in Lake Oubeira during
October 2010. a Hepatopancreas section of the common carp control
with no histopathological degeneration (original magnification 940).
b Hepatocytes with lipid nodules (arrows) (original magnification
920). c Dilatation (arrows) of the lumen of the hepatic sinusoid
characterized by hydropic degenerations, focal necrosis,
and haemosiderin deposits (Fig. 5e). Haemosiderin is an
iron-stockage complex that it is most commonly found in
macrophages and is especially abundant after hemorrhage.
Potential Risks of MCs in Lake Oubeira
To avoid potential health risk, the World Health Organiza-
tion (WHO) established the tolerable daily intake (TDI) for
MCs over the lifetime of 40 ng/kg body weight/d for MC-
LR equivalents (WHO 2008). In this study, a coefficient of 5
was used to convert dry weight to fresh weight (fw) of
muscle tissues of the common carp and the European eel
(Dong et al. 2012). The estimated daily intake (EDI) of MCs
for an adult weighing 60 kg ingesting 100 g of edible organs
(muscle) of these fish is shown in Fig. 6. Compared with the
TDI, i.e., 40 ng/kg body weight/day, muscle tissues of the
common carp captured in Lake Oubeira between April 2010
and March 2011 were not deemed safe for consumption
(Fig. 6) due to their having concentrations of MCs 2.7–5.7
times greater than the TDI. However, muscle tissues of
European eel captured during the same period and at the
same site never exceeded the TDI. Ibelings and Chorus
(2007) used the seasonal WHO guideline value of 0.4 lg/kg
body weight/day to estimate a seasonal (daily exposure for
several weeks during the cyanobacterial season) tolerable
385
c
d e
(original magnification 910). d Presence of siderotic macrophages
(arrow), an inflammatory cell type indicating intrahepatic hemorrhage
(original magnification 910). e Normal parenchyma (black arrow),
hydropic degeneration (white arrowhead), focal necrosis (black
arrowhead), and haemosiderin deposits (broken arrow) (original
magnification 940)
intake value for MC-LR of 300 lg/kg fw of food for adults
and 40 lg/kg fw of food for children with 100 g fw of
contaminated fish being consumed per day. Using the
guidelines of Ibelings and Chorus, none of the muscle tissue
samples of the common carp and the European eel in our
study would have exceeded the seasonal value for adults.
However, for children only muscle tissue samples of the
common carp would have exceeded the seasonal value. In a
recent study, Mulvenna et al. (2012) assigned an acceptable
daily limit for MCs in fish of 39 lg/kg fw for adults (aged
C17 years) and 24 lg/kg fw for children (age 2–16 years).
A factor of 319 g fw of fish consumed/d for children and
377 g fw/day for adults was used to generate the guideline
values. Using these guidelines, all muscle tissue samples of
the common carp are above the limit for children and adults;
however, none muscle tissue samples of the European eel are
above the limit for children and for adults.
Discussion
In this study, although no dense bloom of cyanobacteria
was observed during the 12-month sampling period from
April 2010 to March 2011, total MC concentrations ranged
between 0.028 and 13.4 lg equivalent MC-LR/l. Com-
pared with the total toxin concentration, the proportion of
123
386
Fig. 6 EDI of MCs by a person
consuming 100 g (fresh weight)
Ingested MCs (ng equiv. MC-LR /kg bw/day)
of muscle of common carp C.
carpio (open bars) and
European eel A. anguilla (black
bars) captured monthly in Lake
Oubeira from April 2010 to
March 2011. The horizontal
broken line indicates the
maximum lifetime TDI for
humans (40 ng/kg/day)
proposed by WHO (Chorus and
Bartram 1999)
extracellular MCs was generally greater ([65 %) than
intracellular MCs, except in June and July 2010, when the
proportion of dissolved toxins were 1.6 and 32 %,
respectively. These high percentages of dissolved MCs in
the water of Lake Oubeira could be the result of their
release from cyanobacterial cells during the senescence and
decomposition periods of an earlier bloom. Indeed, several
results reported that toxic cyanobacterial blooms occurred
throughout the year in this lake (Nasri et al. 2004, 2008).
The LC–MS/MS tandem mass spectra of the dissolved
toxins in the water lake harvested on September 2010,
where the highest MC concentrations were observed with
the PP2A assay, showed the presence of a major MC-LR
variant (90 % of the total) and two minor variants MC-YR
(6 %) and MC-(H4)YR (4 %). As described in our earlier
studies (Nasri et al. 2004, 2007), the profile of MC variants
in Lake Oubeira usually consists of MC-LR as the domi-
nant variant in water samples followed by two or three
hydrophobic variants, such as MC-RR, MC-YR, D-Asp3-
MC-LR, and, for the first time in this study, MC-(H4)YR.
MCs present in this lake can therefore enter aquatic
organisms, especially commercial fish species, by being
absorbed through the gills when they filter contaminated
water or through the diet (see Ettoumi et al. 2011). Once
accumulated in fish, these toxins can affect liver tissue and
cause cell damage and death through inhibition of protein
phosphatases (reviewed in Malbrouck and Kestemont
2006). In the present study, the indication of hepatocellular
and liver damage of the common carp and the European
eel, respectively, consistent with MC poisoning, was indi-
cated both by the accumulation of these toxins and by the
presence of several abnormalities in these tissues. These
123
Arch Environ Contam Toxicol (2014) 66:379–389
Common carp
240
European eel
200
160
120
80
40
Date of sampling
abnormalities, observed after histopathology examination
of the hepatopancreas of the common carp, are character-
ized by hepatopancreas steatosis, dilatation of the lumen of
hepatic sinusoid, and the presence of siderotic macro-
phages, inflammatory cells indicating intrahepatic hemor-
rhage. In liver of European eel, haemosiderin deposits,
which are most commonly found in macrophages and are
especially abundant after hemorrhage, were observed.
Atencio et al. (2008) reported that pathological lesions,
including macrovesicular steatosis, were observed in the
liver of Tinca tinca after exposition by oral route to
cyanobacterial cells. In addition, toxicity of MCs is medi-
ated through the active transport of these toxins into
hepatocytes by the bile acid organic anion transport system
followed by inhibition of eukaryotic serine/threonine pro-
tein phosphatases 1 and 2A (Eriksson et al. 1990), thus
leading to hyperphosphorylation of proteins associated
with the cytoskeleton in hepatocytes (Toivola et al. 1997).
Therefore, the rapid loss of the sinusoidal architecture and
attachment to one another leads to accumulation of blood
in the liver, and death most often results from hemorrhagic
shock in fish (Jiang et al. 2011). Our histopathological
observations, especially dilatation of the lumen of hepatic
sinusoid and the presence of intrahepatic hemorrhage, seem
to be in agreement with previous studies concerning
cyanobacterial hepatotoxicity in fish (Dong et al. 2012;
Ernst et al. 2006; Fischer and Dietrich 2000). In a previous
study, we reported for the first time the death of two
freshwater terrapin species, E. orbicularis and M. leprosa,
in Lake Oubeira during October 2005, with liver crumbling
evident after examination of the fresh carcass of M. leprosa
(Nasri et al. 2008). Recently, Mitsoura et al. (2013)
Arch Environ Contam Toxicol (2014) 66:379–389
observed histopathological changes, characterized by the
presence of lipid droplets and sporadic hemorrhagic
symptoms in the liver of the common carp caught from
Lake Karla (Greece) with significant MCs concentrations
(0.75–3.9 lg/l).
It has been well documented that the liver is the target
organ for MCs accumulation in fish (Ernst et al. 2006;
Papadimitriou et al. 2012). This may be attributable to the
high density of organic anionic transporters on the surface
of hepatocytes (Fischer et al. 2005). However, numerous
studies have proven that high concentration of these toxins
could be also accumulated in different organs of fish, such
as viscera, kidney, and muscle, whereas muscle usually
presents the lowest MC content (Fischer and Dietrich 2000;
Mohamed et al. 2003; Schmidt et al. 2013; Zhang et al.
2009). Tricarico et al. (2008) reported that crayfish (Pro-
cambarus clarkii) collected in Massaciuccoli Lake (Italy)
accumulated cyanotoxins in all of the organs analyzed. The
highest concentration of MCs was found gradually in
intestine > hepatopancreas = stomach > abdominal mus-
cle. These results are in reasonable agreement with the
results reported here for the common carp; however, in the
European eel the highest MC concentrations was found in
the order liver [ intestine [ muscle. In fact, differential
accumulation of MCs in fish tissues of different species has
been noted by several investigators (Berry et al. 2011;
Palı́ková et al. 2011; Papadimitriou et al. 2012). Recently,
Schmidt et al. (2013) reported that of 129 fish belonging to
5 different species taken from a eutrophic lake in Ohio
(Grand Lake St. Marys, USA) tested for MCs, only black
crappie (Pomoxis nigromaculatus) and common carp (C.
carpio) tested positive for MC-LR. In Lake Oubeira, MC
concentrations in the common carp tended to be greater
when MC concentrations in water were also high during
cyanobacterial development in autumn. However, in the
European eel the high MC concentration was observed
later in winter where MC concentrations in water were low.
This variability is likely attributable to the differences in
diet between the two species sampled. The results from the
literature show that MCs are taken into fish from both
water and bloom material, both by way of the gills and the
intestine followed by their distribution and accumulation in
different organs (Cazenave et al. 2003). Therefore, in the
current study, the highest MC concentrations in fish
intestine were observed in the common carp with a peak at
1933.3 ng equivalent MC-LR/g DW on November 11,
2010, compared with 108.4 ng equivalent MC-LR/g DW
on October 13, 2010, for the European eel (Figs. 3, 4), in
autumn when MC concentrations in water were also
extremely high. This suggests that the omnivorous fish
(common carp) takes up MCs from both water and
cyanobacterial cells more rapidly than the carnivorous fish
(European eel). However, the highest levels of MCs in liver
387
tissues of the carnivorous fish (European eel) were
observed later in winter when total MC concentrations in
the lake water were lowest (Fig. 4). This suggests that in
the carnivorous fish, MC accumulation could have occur-
red through food web transfer, but this remains unknown
without diet toxicity data. Nevertheless, several studies
have been reported that MCs can be transferred to greater
trophic levels, i.e., from zooplankton and mollusks to
carnivorous fish species, in natural and aquaculture systems
(Mohamed et al. 2003; Smith et al. 2008). Smith et al.
(2008) reported that [80 % of noncovalently bound MC in
zooplankton Bosmina fed to sunfish Lepomis gibbosus was
directly transferred to the sunfish, indicating that free or
conjugated MCs can travel efficiently up the aquatic food
web. Thus, the variations of MC concentrations in different
tissues of the two fish species captured in this study are in
agreement with those reported in the literature. Xie et al.
(2005) reported that MC concentration in liver was highest
in carnivorous fish, followed by omnivorous fish, and was
lowest in phytoplanktivorous and herbivorous fish. In
contrast, gut MC concentration generally showed a
reversed pattern with concentrations being highest in
phytoplanktivorous fish followed by omnivorous and car-
nivorous fish. In contrast, Zhang et al. (2009) found that
MC concentration in muscle was highest in omnivorous
fish, followed by phytoplanktivorous fish, and was lowest
in carnivorous fish.
Until now, more attention has been paid to human
uptake of MCs through drinking water (WHO 1998) than to
bioaccumulation of MCs in aquatic animals in natural
waters. Fishes, at the top of the aquatic food chain, are
likely to be most affected by exposure to toxic cyanobac-
teria, and so their consumption may pose great risk to
humans. During the entire period of this study, the WHO
lifetime limit for TDI was exceeded only in common carp
muscle. However, if we use the guidelines established by
Ibelings and Chorus (2007) (described previously in
Potential Risks of MCs in Lake Oubeira), none of the
muscle tissue samples of the common carp and the Euro-
pean eel during this same period would have exceeded the
seasonal value for adults. However, for children only
muscle tissue samples of the common carp would have
exceeded the seasonal value. The present results are in
agreement with several studies reported in the literature.
For example, Garcia et al. (2010) reported that the highest
concentration of MCs occurring in crab tissue was 105 lg
MCs/kg fw representing an EDI, based on human body
weight and amount of crab tissue consumed, of 0.46 lg
MCs/kg body weight/d, a value [10 times the WHO TDI
guideline. Peng et al. (2010) indicated that most of the
aquatic products from three large Chinese lakes seem to be
unsafe for human consumption due to MC accumulation
with EDI values 2–148 times greater than the lifetime
123
388
WHO TDI guideline. A previous study showed that MC
accumulation in muscle of crucian carp and common carp,
harvested from Lake Taihu in China, varied from 0.93 to
3.55 times TDI values (Zhang et al. 2009). Recently,
Schmidt et al. (2013) reported that of 129 samples of 5 fish
species taken from Grand Lake St. Marys (Ohio, USA),
only two samples that tested positive for MC—black
crappie (P. nigromaculatus) and common carp (C. car-
pio)—would have exceeded the seasonal value for children
or the lifetime value for adults.
This current study was designed to evaluate only free
MCs in tissues of the captured two fish species. However,
MCs are known to bind covalently to protein phosphatases
or other cysteine-containing peptides [e.g., reduced gluta-
thione (GSH)]; therefore, conventional methods, such as
organic extraction, only extract the unbound portions of the
toxin from the aquatic tissues, which will lead to a false
determination of total toxin concentrations (Dionisio Pires
et al. 2004; Ibelings et al. 2005; Nasri et al. 2008; Ott and
Carmichael 2006). It is estimated from the oxidation of the
bound MCs in tissues that as much as 60–90 % of the total
MCs contained within in the organism’s tissues are cova-
lently bound (Ibelings et al. 2005; Lance et al. 2010; Nasri
et al. 2008). Smith et al. (2008, 2010) reported that protease
degradation of protein phosphatase enzyme–MC complex
releases peptides that have similar toxicity to the GSH
conjugate. Nevertheless, free MCs have generally been
accepted to represent the total risk to organisms and
humans due to the scarce data regarding the bioavailability
of the covalently bound portion of MCs. Therefore, studies
on the release of the active form of MCs from the cova-
lently bound portion during gut passage must be performed
to estimate their contribution to the total MC content of an
aquatic organism.
Conclusion
These results demonstrate the year-round presence of MCs
in water and feral fishes from Lake Oubeira. Our observed
MC concentrations in fish, especially the common carp (C.
carpio), fall within the range of concentrations reported in
other studies from around the world, thus confirming that
accumulation of MC in this omnivorous fish is of global
concern. Therefore, we recommend that MC content in
fishery products of this lake, particularly the common carp,
be strictly monitored seasonally to avoid human intoxica-
tion and that the local authorities warn the public to be on
the alert for poisoning by contaminated fish.
Acknowledgments This research was supported by a fund from the
Agence Universitaire de la Francophonie (Grant No. 6313PS002). We
thank Ghislaine Delarue, INRA de Versailles (France), for LC–MS/
123
Arch Environ Contam Toxicol (2014) 66:379–389
MS analysis. We also thank Hind Kherfi, Laboratoire Central de
Pathologie, Hôpital El Taref, Algérie, for assistance with histopa-
thological observations. We also thank anonymous reviewers for their
comments and suggestions on the manuscript.
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