Subchronic oral toxicity of microcystin in common carp (Cyprinus

carpio L.) exposed to Microcystis under laboratory conditions

Xiao-Yu Lib, Ik-Kyo Chungc, Jung-In Kimd, Jin-Ae Leea,*

aSchool of Environmental Science and Engineering, Inje University, Kimhae 621-749, South KoreabSchool of Life Science, Henan Normal University, XinXiang 453002, People’s Republic of China

cDepartment of Marine Science, Pusan National University, Busan 609-735, South KoreadSchool of Food and Life Science, Biohealth Products Research Center, Inje Univeristy, Kimhae 621-749, South Korea

Received 25 March 2004; accepted 17 June 2004

Abstract

The subchronic oral toxicity of microcystin in common carp (Cyprinus carpio L.) was investigated in this study. The fish

(mean body weight of 322G36 g, nZ10) were orally exposed to Microcystis by feeding with bloom scum at a dose of 50 mg

microcystins/kg body weight under laboratory conditions for 28 days. Growth assay results showed that microcystin could

completely inhibit the growth of carp, but failed to change the fish hepatosomatic index. Ultrastructural examination by electron

microscope revealed severe damage in hepatocytes derived from the treated fish. Serum biochemical assays with commercial

kits indicated that alanine aminotransferase and aspartate aminotransferase activities were significantly increased as compared

to control levels, but g-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase activities remained unchanged.

Protein phosphatase inhibition assay revealed that the microcystin concentrations were 261.0G108.3 ng microcystin-LR

equivalent/g fresh weight in hepatopancreas and 38.3G12.3 ng microcystin-LR equivalent/g fresh weight in muscle. The latter

is above the limit recommended by the World Health Organization for human consumption. Therefore, we recommend that a

warning system be instituted for announcing the occurrence of microcystin-producing water bloom and the possible risk of

human intoxication.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: Microcystis bloom; Common carp; Subchronic toxicity of microcystin; Serum enzyme; Bioaccumulation

1. Introduction

Microcystis bloom occurs frequently in bodies of

freshwater all over the world (Codd et al., 1999). The

bloom not only deceases water quality, but also increases

the risk of toxicity to both animals and humans, because

many Microcystis species (mainly Microcystis aeruginosa)

can produce highly toxic microcystin (MCYST) (Toranzo

et al., 1990; Jochimsen et al., 1998; Falconer, 1999;

0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2004.06.010

* Corresponding author. Tel.: C82-55-320-3248; fax: C82-55-

334-7902.

E-mail address: envjal@ijnc.inje.ac.kr (J.-A. Lee).

Zimba et al., 2001). MCYST is a cyclic peptide hepatotoxin,

and acute dosage by intraperitoneal (i.p.) injection can kill

animals (Kotak et al., 1996; Gupta et al., 2003). Toxicolo-

gically, it appears that MCYST causes damage when it is

taken up by hepatocytes and inhibits serine/threonine

protein phosphatases 1 and 2A (Eriksson et al., 1990;

Yoshizawa et al., 1990; Runnegar et al., 1993). The resulting

imbalance in protein phosphorylation disrupts liver cytos-

keleton, which leads to massive hepatic haemorrhages that

cause death in rodents (Dawson, 1998).

MCYST-induced liver damage has been correlated with

the activity of certain plasma enzymes, such as g-glutamyl

transferase (GGT), alanine aminotransferase (ALT), aspar-

tate aminotransferase (AST), alkaline phosphatase (ALP)

Toxicon 44 (2004) 821–827

www.elsevier.com/locate/toxicon

X.-Y. Li et al. / Toxicon 44 (2004) 821–827822

and lactate dehydrogenase (LDH) (Falconer et al., 1994;

Fischer and Dietrich, 2000; Malbrouck et al., 2003). The

bioaccumulation of MCYST in fish tissues has been

reported (Eriksson et al., 1986; Magalhaes et al., 2001,

2003; Mohamed et al., 2003). This could lead to the transfer

of the toxin to human upon consumption of contaminated

fish, thereby affecting human health. Until now, there have

been no studies on subchronic oral exposure of carp to

microcystin under laboratory conditions.

The Naktong river is the second largest river in South

Korea, and it is an important resource for agriculture,

industrial development, and drinking water. Since 1993,

heavy cyanobacterial blooms have been observed in this

river, and a predominant species in the bloom material was

identified as toxic Microcystis aeruginosa (Lee et al., 1997).

Thus, there is much concern about using the Naktong river

as a source of drinking water and the possible effects of this

water on human health.

Common carp is a dominant species in the Naktong river

and often captured by the local fishermen for food. Because

the carp is omnivore, Microcystis can comprise a significant

portion of its diet, which might lead to MCYST accumu-

lation in its tissues during the bloom periods. Therefore, it is

important to evaluate MCYST toxicity to fish as well as

the toxic impact of Microcystis bloom on both fish and

human health.

This study aimed to determine the subchronic toxicity of

MCYST in common carp by examining ultrastructural

alternations in hepatocytes and changes in the activity of

various plasma enzymes. We also verified the presence of

MCYST in carp tissues, to evaluate the potential risks for

human health if MCYST-contaminated carp from the

Naktong river are consumed.

2. Materials and methods

2.1. Chemicals

MC-LR, -RR and -YR (purityO95%) were purchased

from Wako (Wako Chemical Co., Japan). Protein phospha-

tase 2A (PP 2A) and a commercial PP assay kit was obtained

from Promega (Promega, USA). Bovine serum albumin

(BSA), glutaraldehyde, EGTA, EDTA, and all other

reagents used were of the highest purity available and

were purchased from Sigma (Sigma, USA). Serum enzyme

assay kits were obtained commercially from ASAN (ASAN,

Korea).

2.2. Bloom scum collection and HPLC analysis

Microcystis bloom scum was collected from the Naktong

river using a phytoplankton net (25-mm diameter mesh) and

stored at K70 8C until used for fish feeding. Microcystis and

other associated algal species were identified as described

by Lee et al. (1997) and counted using a hemocytometer.

Extraction of MCYST from bloom scum and quantitative

HPLC analysis of MCYST levels within the scum was

performed according to method of Harada et al. (1988), with

minor modifications.

2.3. Fish

Common carp (mean body weight 322G36 g) was

kindly provided by Jinhae Inland Fisheries Research

Institute, National Fishery Research and Development

Institute, Korea. The fish were divided into two 400-L

tanks containing drinking water (one tank received treat-

ment and one tank served as a control). The fish were fed

adlibitum with commercial carp food at a rate of 1.5% of

body weight. Water temperature was maintained at 20G1 8C and dissolved oxygen values were maintained between

7.1 and 7.6 mg lK1. Fish were exposed to a 12-h light/12-h

dark photoperiod, and the tank water was changed weekly.

2.4. Exposure and sampling

After acclimatization for 2 weeks, fish in the treatment

tank (nZ10) were exposed orally to Microcystis by feeding

with bloom scum. The scum was mixed in the commercial

carp food powder and made into small sticky pellets. The

pellets were put into tank, and they fell down on the bottom

of tank for fish to take. It was ensured that fish should eat up

all pellets within 1 h. A low dose of 50 mg toxin/kg body wt,

about one tenth of the oral LD50 of MCYST in carp, was

adopted for subchronic exposure. According to this

exposure dose and the toxin concentration of bloom scum

(determined from HPLC analysis), we determined the

quantity of bloom scum that was necessary to add to carp

food. Control fish (nZ10) was fed only the commercial

food. The body length and weight of fish were measured

prior to exposure and fish were tagged for individual

recognition. Sampling was conducted after 4 weeks of

exposure. At sampling, the body length and weight of fish

were measured and blood was collected. Fish were then

killed by a blow to the head, the body cavity was opened,

and the liver weight was determined. The hepatosomatic

index (HSI) was obtained by expressing the liver weight as a

percentage of body weight. Liver and muscle samples from

all treated fish and several controls were taken for MYCST

analysis. Other liver samples from treatment and control fish

were obtained for transmission electron microscope (TEM)

analysis.

2.5. TEM

Liver samples were prefixed in a 2.5% glutaraldehyde

solution (in 0.1 M Na-cacodylate buffer, pH 7.4) for 2 h at

4 8C and postfixed in cold 1% osmium tetroxide (in 0.1 M

Na-cacodylate buffer, pH 7.2) for 2 h at 4 8C. Ultra-thin

sections were sliced with glass knives (LKB, ultra-

microtome, Nova, Sweden). The sections were examined

Table 1

Methods used for plasma enzyme assays performed with kits (ASAN, Korea)

Enzyme Method Reference

Alanine aminotransferase (ALT) Reitman–Frankel method Reitman and Frankel (1957)

Aspartate aminotransferase (AST) Reitman–Frankel method Reitman and Frankel (1957)

g-glutamyl transferase (GGT) 5-Aminosalicylic acid method Persijn and van der Slik (1976)

Lactate dehydrogenase (LDH) Wroblewski–LaDue method Wroblewski and LaDue (1955)

Alkaline phosphatase (ALP) Kind–King method Kind and King (1954)

X.-Y. Li et al. / Toxicon 44 (2004) 821–827 823

with an electron microscope (JEOL, JEM 1200EX-II,

Japan).

2.6. Blood collection and serum enzyme assay

At sampling, blood was collected from fish heart with a

syringe. Serum was recovered by centrifugation at 3000g for

10 min at 4 8C, and stored at K70 8C until needed. All

analyses were conducted using commercially available

serum enzyme assay kits (ASAN, Korea) based on

colorimetric reaction (Table 1).

2.7. Determination of MCYST concentration in liver

and muscle

MCYST was extracted from fish liver and muscle using

the method described by Malbrouck et al. (2003). MCYST

concentrations in fish tissues were analyzed by a highly

sensitive radioisotope protein phosphatase assay (Jones and

Orr, 1994). [32P]-phosphorylase-A was prepared by the

method of Cohen et al. (1988). Inhibition of protein

phosphatase was expressed as MCYST concentration

(MC-LR equivalent).

2.8. Statistical analysis

Results were expressed as the meanGSD. The two-tailed

Student’s t-test for sample equal variance was used to

compare the growth rate, hepatosomatic index and serum

enzyme activity changes between the treated and control

samples. Differences were considered to be statistically

significant at P!0.05.

Fig. 1. Growth rate of common carp during a period of 4 weeks of

oral exposure to Microcystis bloom scum (P!0.01). The exposure

dosage was 50 mg microcystin/kg body weight. Data are expressed

as the meanGSD (nZ10).

3. Results

3.1. Characteristics of Microcystis bloom scum

Microscopic examination revealed that cyanobacterial

bloom in the Naktong river was dominated by Microcystis

aeruginosa (Relative abundance, 91.3%). HPLC analysis

showed that three kinds of microcystins (MC-RR, -LR and

-YR) were identified in bloom material, of which MC-RR

was the dominant variant (Relative concentration, 73.7%)

and the total concentration of toxin in bloom scum was

357.3G26.8 mg/g dry weight.

3.2. Effect of MCYST on fish growth

Although the body color of treated fish became darker

than that of control fish, no obvious differences were

observed in appetite, swimming activity or other behavior of

control versus treated fish. No fish died during the course of

the experiment. Examination of the intestinal contents

revealed that most bloom scum could be digested by carp

(data not shown). Growth assay results showed that

the growth rate of the treated fish was significantly lower

than that of control fish (Fig. 1), while no significant

difference in the hepatosomatic index of fish was found in

the two groups of carp (Fig. 2).

3.3. Ultrastructural observations

TEM analysis revealed a number of ultrastructural

alterations in hepatocytes from MCYST-treated fish.

When hepatocytes from control and treated fish were

examined by electron microscope under low magnification

(2000!), sections derived from control fish had intact

plasmalemma and distinct cell junction (Fig. 3A). However,

hepatocytes from the treated fish had lost the intact

plasmalemma as well as the cell junction between

cells (Fig. 3B). Under higher magnification (5000!),

Fig. 2. Hepatosomatic index of common carp after 4 weeks of oral

exposure to Microcystis bloom scum (PO0.05). The exposure

dosage was 50 mg microcystin/kg body weight. Data are expressed

as the meanGSD (nZ10).

X.-Y. Li et al. / Toxicon 44 (2004) 821–827824

a widespread swelling of the hepatocytic endomembrane

system (mainly consisting of endoplasmic reticulum (ER),

mitochondria and Golgi body) was observed in the treated

hepatocytes as compared with controls (Fig. 3C and D).

Dilation of cisternae of the rough ER and ER transformation

into concentric membrane whorls were recognized in the

treated hepatocytes under higher magnification levels

(10,000!) (Fig. 3E and F). Compared with normal

mitochondria (Fig. 3G), it appeared that the injured

hepatocytes exhibited numerous electron-lucent, mem-

brane-bound vacuoles (Fig. 3H) that originated from

damaged mitochondria. Extensive vacuolization of the

cytoplasm was evident in necrotic hepatocytes (Fig. 3D

and F). Some MCYST-damaged hepatocytes with lipid

droplets were also found in the sections derived from the

treated fish (Fig. 3I).

3.4. Serum biochemistry

Serum biochemical analysis indicated that ALT and AST

activities were significantly higher in the treated fish as

compared with controls, but GGT and LDH activities

remained unchanged (Table 2). There was a tendency for

ALP activity to be higher in treated fish than in control fish,

but this difference was not statistically significant.

3.5. MCYST concentration in carp liver and muscle

The protein phosphatase inhibition assay showed

MCYST concentrations in the hepatopancreas and muscle

of carp were 261.0G108.3 ng MC-LR equivalent/g fresh

weight and 38.3G12.3 ng MC-LR equivalent/g fresh

weight, respectively (Fig. 4). MCYST concentration in

muscle was higher than the tolerable daily intake (TDI) level

of 0.04 mg MC-LR/kg body weight per day recommended

by the World Health Organization (WHO) if a person of a

60-kg body weight ingests 100-g of fish muscle per day.

4. Discussion

Although there are several studies describing the oral

toxicity of MCYST in mammals after application of a

sublethal dose over a long period of time (Falconer et al.,

1994; Heinze, 1999; Ueno et al., 1999; Orr et al., 2003), only

one report is available about oral exposure to MCYST in

carp. In that study, fish were gavaged with a single sublethal

bolus dose of toxic Microcystis aeruginosa for an acute test

(Fischer and Dietrich, 2000). Oral exposure studies are

needed to assess the potential risk of fish to such toxins,

particularly studies in which fish receive subchronic

exposure by feeding them toxic algae, which represents a

natural route of exposure. In this study, the growth of carp

was found to be inhibited by MCYST. This inhibition was

assumed to result from severe liver damage due to the toxin,

which was verified by serum biochemical changes and TEM

observations. Ultrastructural examination revealed wide-

spread swelling and vacuolization of the hepatocytic

endomembrane system, with this severe injury possibly

causing hepatocytic necrosis. These findings are consistent

with those of Rabergh et al. (1991) and Kotak et al. (1996),

who tested the effects of MC-LR on carp and rainbow trout,

respectively. These results are also similar to our previous

observation in carp hepatocytes exposed to MC-LR, in

which the swollen ER and mitochondria were found in the

toxin-treated hepatocytes (Li et al., 2001). Therefore, it

appears likely that MCYST intoxication kills fish by hepatic

necrosis, rather than by the massive hepatic hemorrhaging

that is frequently reported in mammals (Falconer et al.,

1994). To our knowledge, this report is the first to determine

oral toxicity of MCYST in carp by feeding fish bloom scum

under laboratory conditions over an extended period of time.

This is also the first study to verify toxin bioaccumulation in

carp tissues after exposure.

Algal cells are thought to be lysed in the fish gut after

ingestion, and digestion releases intercellular MCYST into

the intestinal lumen of fish (Falconer, 1993). Following

intestinal absorption, the toxin is taken up into hepatocytes

via a carrier-mediated transport system, and it then inhibited

the activity of serine/threonine protein phosphatases 1 and

2A (Runnegar et al., 1993). This inhibition could disturb the

cellular phosphorylation balance and cause hyperphosphor-

ylation of a variety of functional proteins, which leads to

apoptosis and/or necrosis of hepatocytes (Dawson, 1998).

Our TEM results are consistent with this mechanism.

Hepatic injury also could be observed macroscopically

when the treated fish were sampled and dissected. There-

fore, Fish kills by Microcystis bloom can be assumed to

result from extensive liver damage.

Since hepatic physiological and functional alterations

can be revealed by early changes in plasma

Fig. 3. Ultrastructural effects of MCYST on hepatocytes from carp exposed orally to Microcystis at a dose of 50 mg microcystin/kg body weight

for 28 days. (A) Control hepatocytes with intact plasmalemma and cell junction. (B) Hepatocytes from MCYST-treated carp. Note that these

hepatocytes have no visible plasmalemma and cell junction. (C) Ultrastructure of normal hepatocytes with stacks of rough ER. (D) Hepatocytes

from the treated fish showing a swollen endomembrane system. (E) The swollen RER in hepatocyte from toxin-treated fish. (F) Concentric

membrane whorls originating from the toxin-damaged ER (arrow). (G) Mitochondria of hepatocyte from control fish. (H) Vacuoles originating

from the toxin-damaged mitochondria. (I) MCYST-damaged hepatocytes with lipid droplets (arrow). RER, rough endoplasmic reticulum;

M, mitochondria; G, Golgi body; V, vacuole; N, nuclei.

X.-Y. Li et al. / Toxicon 44 (2004) 821–827 825

aminotransferase activity when an animal receives an

external shock, serum enzymes such as ALT, AST, and

GGT have been used as pathological and toxicological

indicators in clinical examination and toxicity research.

Table 2

Serum enzyme activities of common carp after 4 weeks of oral exposure

weight

ALT AST

Control 12.8G6.6 65.4G16.8

Treatment 37.4G27.3 91.1G26.3

Significance P!0.05 P!0.05

All analyses were conducted using commercially available serum enzyme

The International Unit (U lK1) was adopted as the enzyme activity value, e

meanGSD (nZ10).

Rabergh et al. (1991) reported that statistically significant

increases in activity of three liver enzymes, ALT, AST, and

LDH, were found in the plasma of common carp injected

with MC-LR at a dose of 150 mg/kg body weight.

to Microcystis bloom scum at a dose of 50 mg microcystin/kg body

GGT LDH (W-U) ALP

42.5G21.7 1305.5G706.1 12.1G7.3

33.1G30.3 1383.1G647.8 18.5G16.9

PO0.05 PO0.05 PO0.05

assay kits (ASAN, Korea) based on colorimetric reaction (Table 1).

xcept for LDH (W-U, Wroblewski Unit). Data are expressed as the

Fig. 4. Concentrations of MCYST in liver and muscle of common

carp after 4 weeks of oral exposure to Microcystis bloom scum. The

exposure dosage was 50 mg microcystin/kg body weight. MCYST

concentration was determined by a protein phosphatase assay and

expressed as MC-LR equivalents. Data are represented as the

meanGSD (nZ10).

X.-Y. Li et al. / Toxicon 44 (2004) 821–827826

Our results are consistent with this report, except that LDH

activity remained unchanged at the termination of our test.

Falconer et al. (1983) reported that there was a statistically

significant increase in GGT activity in people drinking from

a contaminated reservoir during the period of Microcystis

bloom, as compared to a local population with different

water supplies. An increase of GGT activity was also

observed in MCYST toxicity studies with pigs and rodents

(Fawell et al., 1993; Falconer et al., 1994). In contrast, no

changes in GGT activity were observed in our study.

Metabolic difference between species may explain this

discordance.

According to Falconer (1993), MCYST is taken up by a

bile-acid transporter in intestinal and liver cells. The toxin is

partially excreted through the feces and the rest accumulates

in liver, muscle and other tissues. Bioaccumulation of

MCYST in liver and tissue was observed in both mammals

(Falconer et al., 1994; Orr et al., 2003) and fish (Magalhaes

et al., 2001, 2003; Malbrouck et al., 2003; Mohamed et al.,

2003). Thus, transfer of MCYST to humans through

consumption of contaminated fish is a potential threat to

human health. Based on recommendations of the WHO, the

maximum allowable concentration for MCYST in drinking

water was established as 1 mg lK1 (Falconer et al., 1994). A

TDI value of 0.04 mg/kg body weight day was also set as a

provisional value by the WHO (Kuiper-Goodman et al.,

1999). Based on this limit, the MCYST concentration

observed in carp muscle in this study (38.3G12.3 ng/g fresh

weight) would result in a higher than acceptable TDI value

if a person of 60 kg body weight consumes 100-g of fish

muscle per day. For this reason, we recommend that fish

taken from the Naktong river during Microcystis blooms

should be tested for MCYST prior to consumption, and that

a warning system be instituted to alert local populations

when toxin-producing water blooms occur, as these may

represent a risk to human health.

Acknowledgements

We would like to thank Drs Yong-Chul Cho and

Eon-Jong Kang (Jinhae Inland Fisheries Research Institute,

National Fishery Research and Development Institute,

Korea) for their kind gift of common carp. This work was

supported by APEC (Asia Pacific Economics Cooperation)

Postdoctoral Fellowship in Science and Engineering

(Granted No. AP-01) funded by KOSEF (Korea Science

and Engineering Foundation) and KOSEF research project

in Science and Engineering (Granted No. R04-2003-000-

10130-0) in 2003.

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