Seasonal Dynamics of the Hepatotoxic
Microcystins in Various Organs of Four
Freshwater Bivalves from the Large Eutrophic
Lake Taihu of Subtropical China and the Risk
to Human Consumption
Jun Chen, Ping Xie
Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater
Ecology and Biotechnology of China, Institute of Hydrobiology, Chinese Academy of Sciences,
Wuhan 430072, People’s Republic of China
Received 23 May 2005; revised 6 July 2005; accepted 7 July 2005
ABSTRACT: So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of
bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern
and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral
mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii,
Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003–Sep. 2004 in Lake Taihu with
toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the
organs were done by LC–MS and HPLC. The major toxins were present in the hepatopancreas (45.5–
55.4%), followed by visceral mass with substantial amount of gonad (27.6–35.5%), whereas gill and foot
were the least (1.8–5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass,
gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 g/g DW, respectively. There were rather
good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r ¼
0.75–0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the
hepatopancreas and the 13C (r ¼ 0.919) or 15N (r ¼ 0.878) of the foot, indicating that the different MC
content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of
MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were
above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four
bivalves were 8–23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk
of consuming bivalves in Lake Taihu. # 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 572–584, 2005.
Keywords: microcystin accumulation; four bivalves; organ distribution; seasonal dynamics; human consumption; Lake Taihu
INTRODUCTION
Correspondence to: P. Xie; e-mail: [email protected]
Contract grant sponsor: Ministry of Science and Technology of PRC.
Contract grant number: 2002AA601011 (863 project).
Contract grant sponsor: Chinese Academy of Sciences.
Contract grant number: KSCX2-SW-129.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/tox.20146
C
2005 Wiley Periodicals, Inc.
572
The occurrence of toxic cyanobacterial blooms in eutrophic
lakes, reservoirs, and recreational waters has become a
worldwide problem (Paerl et al., 2001). Among cyanotoxins, the hepatotoxic microcystins (MCs) are considered
to be one of the most dangerous groups, which are known
to be potent hepatotoxin (Carmichael, 1994; Dawson,
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
573
Fig. 1. Photos of four bivalves collected from Meiliang Bay of Lake Taihu. A: Anodonta
woodiana; B: Lamprotula leai; C: Hyriopsis cumingii; D: Cristaria plicata.
1998) and tumor promoter (Nishiwaki-Matsushima et al.,
1991, 1992). Exposure to MCs represents a health risk to
animals (Falconer and Choice, 1992; Carmichael, 1994,
1997; Falconer et al., 1994; Falconer, 1998) and humans
(Yu, 1989, 1995; Azevedo et al., 2002). No case of human
deaths caused by oral consumption of cyanobacteria toxins
has yet been documented, because humans are not likely to
drink directly from water containing water blooms with
high enough levels of cyanotoxins that can lead to acute
lethal exposure. However, chronic toxic effects from exposure through food need to be considered, especially if there
is long-term frequent exposure.
In natural waters, MCs are found to accumulate in a
wide range of aquatic animals such as fish (Magalhàes
et al., 2001; Mohamed et al., 2003; Xie et al., 2005),
shrimps (Chen and Xie, 2005), gastropods (Zurawell et al.,
1999; Chen et al., 2005), and bivalves (Watanabe et al.,
1997; Williams et al., 1997; Yokoyama and Park, 2002).
The toxins are present not only in the viscera but also in the
edible muscle/foot. This means that oral consumption on
animals containing MCs is risky to human health.
There have been extensive studies on MC bioaccumulation in bivalves both in the laboratory (Eriksson et al.,
1989; Lindholm et al., 1989; Vasconcelos, 1995; Amorim
and Vasconcelos, 1999; Yokoyama and Park, 2003) and
in the field (Prepas et al., 1997; Watanabe et al., 1997;
Williams et al., 1997; Vanderploeg et al., 2001; Yokoyama
and Park, 2002). However, these studies almost focus on
MC accumulation in the hepatopancreas or the whole body
with very limited information on the MC distribution in different organs of bivalves: only one measurement for Unio
douglasiae from Lake Suwa (Watanabe et al., 1997), and
for Anodonta cygnea and Mytilus galloprovincitalis in laboratory experiments (Eriksson et al., 1989; Vasconcelos,
1995). Moreover, all the above-mentioned field studies are
limited to temperate climate, lacking information from subtropical waters.
In China, freshwater bivalves are commercially important because they are widely used for human consumption.
They are not only cultured in ponds but also abundantly
present in freshwater lakes. However, during the past decades, eutrophication in Chinese lakes has progressed rapidly, resulting in frequent outbreak of toxic cyanobacterial
blooms in many large lakes such as Lakes Taihu and
Caohu, where production of freshwater bivalves are an
important industry. Therefore it is quite likely that oral consumption of these bivalves exposed to high MC levels
could lead to chronic human intoxication. However, up to
now, there have been no studies on MC accumulation in
bivalves from Chinese waters.
The present research was conducted on four freshwater
bivalves, Anodonta woodiana, Hyriopsis cumingii, Cristaria
plicata, and Lamprotula leai (Fig. 1), in the large shallow,
eutrophic subtropical Lake Taihu, where heavy toxic cyanobacterial blooms occur regularly in the warm seasons of
every year. The purposes of this study are mainly to examine
the distribution patterns and the seasonal dynamics of three
common MCs (MC-LR, -YR and -RR) in various organs
(hepatopancreas, intestine, visceral mass, gill, foot, and rest)
of the bivalves and to discuss the possible mechanisms
underlying these patterns with comments on the potential risk
to human health when these bivalves are consumed.
574 CHEN AND XIE
TABLE I. Body length (BL), body weight (BW), d13C and d15N of the feet (in August) of the four bivalves
collected from Meiliang Bay of Lake Taihua
Species
A. woodiana
L. leai
H. cumingii
C. plicata
a
BL (mm)
BW (g)
13C
15N
Natural Range
130 6 8.4
100 6 6.5
190 6 12.4
240 6 13.5
77.4 6 13.9
59.6 6 7.3
89.3 6 12.5
114.9 6 27.8
29.84
27.01
29.21
24.7
4.48
9.81
5.87
12.6
China, USSR, Japan, and Korea
China and Vietnam
Endemic to China
China, USSR, Japan, and Korea
Geographic distributions of these species are also listed (Liu, 1979). Data of 13C and 15N (%) are kindly provided by Dr. Jun Xu.
MATERIALS AND METHODS
Taihu Lake (30850 –32880 N and 119880 –1218550 E) is located in the east part of China. It is the third largest
freshwater lakes in China, and has a surface area of
2428 km2, a mean water depth of 1.9 m and a maximum
depth of about 3.5 m. During the past decades, the lake
has witnessed a steady increase in eutrophication, characteristic of a regular occurrence of cyanobacterial surface
blooms in the warm seasons of each year (Pu et al.,
1998a,b). Meiliang Bay (water surface area 135 km2), a
part of Lake Taihu, accommodates municipal and industry wastewater from Wuxi City, but also acts as principal water source for the city. Meiliang Bay is the most
eutrophic part of the lake, characteristic of extremely
dense accumulation of toxic Microcystis blooms by wind
in the summer. In this area, A. woodiana, H. cumingii,
C. plicata, and L. leai are four commercially important
freshwater bivalves (Table I).
The bivalves were collected monthly from surface sediment of Meiliang Bay during October 2003 and September
2004. The collected animals were immediately frozen at
–208C, and then dissected into six parts, intestine (including
intestinal walls), hepatopancreas, visceral mass (excluding
hepatopancreas and intestines, thus mainly composed of
gonad), gill, foot, and rest in the laboratory. The collected
organs were frozen at –808C prior to MC analysis. We
pooled, respectively, all intestine, hepatopancreas, visceral
Fig. 2. A comparison of the chromatograms (monitored at 238 nm) of the standard MCLR, -YR and -RR (5.00 g/ml1), the extracts of intestine, hepatopancreas, and visceral
mass of Lamprotula leai collected from Lake Taihu in August 2004. (Note on HPLC analysis: a gradient starting at 50% (v/v) aqueous methanol with 0.05% trifluoroacetyl (TFA)
was increased to 70% (v/v) aqueous methanol with 0.05% TFA in 25 min at a flow rate of
1 ml/min).
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
Fig. 3. ESI LC/MS analysis of microcystins in the hepatopancreas of Hyriopsis cumingii
(August 2004).
575
576 CHEN AND XIE
Fig. 3. (Continued.)
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
577
Fig. 3. (Continued.)
mass, gill, foot, and rest of five dissected animals. Thus,
each value represents an average amount of MCs in the
organs of five individuals.
During the study period, individuals of H. cumingii and
C. plicata were also hung in the surface layer of the lake
water using bags tied to a rope. In August and September,
Fig. 4. ESI LC/MS analysis of the foot of Cristaria plicata (September 2004).
578 CHEN AND XIE
Fig. 5. The seasonal changes of MC-LR, -YR, and -RR concentrations (ng/g1 DW) in (a)
hepatopancreas, (b) intestine, (c) viscera mass, (d) rest, (e) gill, and (f) foot of the freshwater bivalve Anodonta woodiana.
samples of these two bivalves were collected from both surface water and bottom sediment to look for possible difference in MC content in various organs of the bivalves between the two habitats.
Extraction and analysis of the MCs in the organs (0.5 g
lyophilized sample for each organ) of the study animals
basically followed the method of Chen and Xie (2005). The
toxin-containing fraction was subjected to a HPLC equipped
with an ODS column (Cosmosil 5C18-AR, 4.6 150 mm,
Nacalai, Japan) and a SPD-10A UV–vis spectrophotometer
set at 238 nm. MC concentrations were determined by comparing the peak areas of the test samples with those of the
standards available (MC-LR, MC-YR, and MC-RR, Wako
Pure Chemical Industries, Japan). The limit of detection
and the limit of quantification for the MCs were 0.02 and
0.07 g/g1, respectively.
Qualitative analysis of MCs was performed using a
Finnigan LC–ESI–MS system. MS tuning and optimization
were achieved by infusing MC-RR and monitoring the
[M þ H]þ ion at m/z 1038. MS conditions were as follows:
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
579
TABLE II. A comparison of MC contents (ng/g1) in various organs of two bivalves
collected from both bottom sediment and surface layer in the Meiliang Bay
of Lake Taihu in August and Septembera
August
Hyriopsis cumingii
Intestine
Hepatopancreas
Visceral mass
Gill
Foot
Rest
13C of foot
15N of foot
Cristaria plicata
Intestine
Hepatopancreas
Visceral mass
Gill
Foot
Rest
13C of foot
15N of foot
September
Surface Sample
Bottom Sample
Surface Sample
Bottom Sample
1277
3722.1
293.7
180.5
523.2
252.7
29.32
6.43
1129.5
3995.7
265.9
230.8
584.2
486.8
29.21
5.87
781.6
1880.2
758.3
106.5
98.7
130.9
701.3
1789.8
803.2
86.5
143.7
169.9
5285.9
5997.3
158.2
138.8
256.1
199.7
24.5
12.94
5505.8
6508.7
189.9
168.2
218
216.4
24.7
12.6
1646.3
390.8
35.4
60.5
74.8
78.1
1736.9
355.5
52.4
36
70.6
86.7
C and 15N data (%) of foot of both bivalves in August are kindly provided by Dr. Jun Xu.
a 13
ESI spray voltage 4.54 kV, sheath gas flow rate 30 unit,
auxiliary gas flow rate 0 unit, capillary voltage 45.67 V,
capillary temperature 2308C, and multiplier voltage
801.62 V. Data acquisition was in the positive ionization
centroid mode with full mass mode at a mass range between
800 and 1500.
Stable carbon and nitrogen isotope ratios in the feet of
the bivalves were analyzed with Delta Plus (Finnigan) continuous flow isotope ratio mass spectrometer directly
coupled to an EA1110 elemental analyzer (Carlo Erba) for
combustion (see Xu et al., 2005 in detail).
RESULTS
The chromatograms of the MC-LR, -YR, and -RR standards,
and the extracts of intestine, hepatopancreas, and visceral
mass of L. leai are compared in Figure 2, indicating that the
toxins were taken up by the mussel. The presence of MCs in
the hepatopancreas of H. cumingii was also confirmed by ESI
LC/MS measurements (Fig. 3). Based on total ion chromatogram, mass chromatograms monitored at m/z 1,038, and the
presence of [MþH]þ ion at m/z 1,038, it is confirmed that
peak A was derived from MC-RR. Similarly, peak B was
derived from MC-LR, as the peak was detected by monitoring with m/z 995, and the mass chromatogram showed [M þ
H]þ ion at m/z 995. Since MC-YR in the hepatopancreas was
rare, the [M þ H]þ ion monitored at m/z 1,045 was not as
well as MC-RR and MC-LR. In addition, ESI LC/MS
revealed that a GSH conjugate of MC-LR was present in the
foot of C. plicata (m/z 1302.10) (Fig. 4).
The monthly changes in MC contents of all mussels
were studied, and the representative was showed in Figure 5.
During the study period, there were great temporal variations in MC contents in various organs of all bivalves. The
hepatopancreas and intestine of all bivalves showed remarkably high peaks in July or August, whereas the highest
MC peaks in the feet appeared in different months (July for
A. woodiana, August for H. cumingii, October for C. plicata, and May for L. leai, respectively). The hepatopancreas of C. plicata had the highest MC level (38.48 g/g1
DW); the maximum MC contents in intestine and foot
observed for H. cumingi were 20.65 and 0.58 g/g1 DW,
respectively, whereas the maximum MC contents in visceral mass, gill, and rest observed for L. leai were 1.70,
0.64 and 0.61 g/g1 DW, respectively. MC contents
in various organs of Both H. cumingii and C. plicata and
13C and 15N values of their feet had little difference
between the individuals from the surface layer and those
from the bottom sediment (Table II), indicating that the
bivalves on the sediments were probably also filtering
cyanobacteria from the water column.
Distribution studies of the toxin in the bivalve organs
revealed that the major part of the toxins was present in the
hepatopancreas (35.5% for A. woodiana, 43.4% for
H. cumingii, 41.2% for C. plicata, and 47.4% for L. leai),
followed by visceral mass (25.7% for A. woodiana, 21.6%
for H. cumingii, 27.2% for C. plicata, and 34% for L. leai),
580 CHEN AND XIE
TABLE III. Dry weight of the different bivalve organs as a percentage of total
weight, and MC contents and percentage of toxins present in the different
organs of four freshwater bivalves collected from Meiliang Bay of
Lake Taihu during Oct. 2003 and Sep. 2004
Toxins (%)
Species/Tissue
A. woodiana
Intestine
Hepatopancreas
Visceral massa
Gills
Foot
Rest
H. cumingii
Intestine
Hepatopancreas
Visceral massa
Gills
Foot
Rest
C. plicata
Intestine
Hepatopancreas
Visceral massa
Gills
Foot
Rest
L. leai
Intestine
Hepatopancreas
Visceral massa
Gills
Foot
Rest
a
Dry Weight (%)
MCs (g/g)
Mean (Min.-Max.)
Including
Intestine
Excluding
Intestine
2.2
5.1
40.2
9.5
12.1
31.1
2.17 (0.42–7.88)
1.54 (0.15–5.96)
0.14 (0.007–0.31)
0.08 (0.034–0.12)
0.072 (0.024–0.19)
0.066 (0.035–0.11)
22.1
35.5
25.7
3.5
4.0
9.4
45.5
32.9
4.4
5.1
12.0
21.7
43.4
21.6
1.4
3.2
8.7
55.4
27.6
1.8
4.1
11.1
3.7
8.3
45.3
9.1
10.0
23.6
3.83 (0.035–20.65)
3.42 (0.076–12.50)
0.31 (0.052–0.80)
0.10 (0.022–0.23)
0.21 (0.015–0.58)
0.24 (0.052–0.49)
5.7
2.9
58.0
6.6
7.0
19.8
1.68 (0.069–5.64)
5.79 (0.16–38.48)
0.19 (0.017–0.71)
0.093 (0.035–0.22)
0.11 (0.033–0.23)
0.096 (0.037–0.22)
23.5
41.2
27.2
1.5
1.9
4.7
53.9
35.5
2.0
2.5
6.1
1.6
6.1
45.2
7.8
9.9
29.5
0.93 (0.18–2.53)
4.25 (0.38–13.23)
0.41 (0.037–1.70)
0.22 (0.004–0.64)
0.19 (0.021–0.52)
0.17 (0.043–0.61)
2.8
47.4
34
3.1
3.4
9.2
48.8
35.0
3.2
3.5
9.5
Excluding hepatopancreas and intestines.
whereas gills, foot, and the rest of the tissues had altogether
less than 16.9% of the total toxin. If intestines are excluded,
up to 55.4% and 35.5% of the toxin burden were allocated
in the hepatopancreas and visceral mass, respectively
(Table III).
There were significantly positive correlation between
MCs in the intestines and those in the hepatopancreas (for
Anodonta woodiana, r ¼ 0.9715, p < 0.05; for Hyriopsis
cumingii, r ¼ 0.9647, p < 0.05; for Cristaria plicata, r ¼
0.7529, p < 0.05; for Lamprotula leai, r ¼ 0.8723, p <
0.05). Also, well correlation between MCs in the intestines
and those in the visceral mass was only found for Lamprotula leai (r ¼ 0.8634, p < 0.05), whereas there was no correlation between MCs in the intestines and those in the feet
of the mussels.
There appeared to be positive correlations between the
maximum MC content in the hepatopancreas and the 13C
(r ¼ 0.919, p ¼ 0.08) or 15N (r ¼ 0.878, p ¼ 0.122) of the
foot, indicating that the different MC content in the hepatopancreas of the four bivalves might be due to different food
ingestion.
DISCUSSION
This is the first study to examine the relationship of MC
contents between intestine and hepatopancreas of bivalves,
and we found a rather good positive correlation (r ¼ 0.75–
0.97, p < 0.05) for four bivalves. Yokoyama and Park
(2002) report that in Lake Suwa, the MC content in the hepatopancreas of Unio douglasiae was linearly correlated
with intracellular MCs in phytoplankton, expressed as g/L
or g/g (r ¼ 0.85, p < 0.05). Zurawell et al. (1999) reported
that the concentrations of MC-LR in the tissue (whole body
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
excluding shell) of three gastropods were positively correlated with toxin in the phytoplankton of the water column
based on log-log transformed data in seven Canadian lakes
(r ¼ 0.37–0.50, p < 0.05). In the eutrophic Lake Chaohu of
China, there was a rough positive correlation between MC
of digestive tracts and MC of hepatopancreas of the snail
Bellamya aeruginosa (r ¼ 0.64, p ¼ 0.17) (Chen et al.,
2005), but no correlation between MCs in the stomach and
those in the hepatopancreas of two shrimps (for Palaemon
modestus: r ¼ 0.48, p ¼ 0.41; for Macrobrachium nipponensis: r ¼ 0.159, p ¼ 0.764) (Chen and Xie, 2005).
These results may reflect a difference in feeding mode
among the mollusks, and the filter-feeding bivalves apparently show the most significant correlation between MC
contents of hepatopancreas and those of their food items.
Similarly, in Sepetiba Bay of Brazil, a significant correlation was observed between MC concentration in seston
samples and in the muscle of Tilapia rendalli, a phytoplanktivorous fish (r ¼ 0.96, p < 0.05) (Magalhàes et al.,
2003).
In the present study, if intestines are excluded, the hepatopancreas of the four bivalves from Lake Taihu accumulated 45.5–55.4% of total MCs although the dry weight was
only 2.9–8.3% of the total body, indicating that hepatopancreas is the target organ of MCs, and visceral mass (mainly
gonad) retained 27.6–35.5% of total MCs, suggesting that
the gonad might be the second important target organ of
MCs. In a laboratory experiment, after the bivalve Anodonta cygnea was fed with toxic Oscillatoria aghardii, the
hepatopancreas accumulated 40% of the total MC in spite
of a small proportion (3.5%) in dry weight (Eriksson et al.,
1989). In Lake Chaohu, gonads of two shrimps (Palaemon
modestus and Macrobrachium nipponensis), the crayfish
Procambarus clarkia, and the snail (Bellamya aeruginosa)
are also found to be the second target organ of MCs (Chen
and Xie, 2005; Chen et al., 2005). A substantial amount of
MCs (1.19 g/g DW) was also found in the gonad of the
bivalve Unio douglasiae from Lake Suwa, Japan (Watanabe
et al., 1997). In the present study, MC content in gill and
foot of the four bivalves from Lake Taihu was relatively
low, contrary to the results of U. douglasiae from Lake
Suwa (Watanabe et al., 1997), but similar to the experimental result of A. cygnea (Eriksson et al., 1989). MC levels in
bivalves from Lake Taihu are within the range of the reported values from literatures (Table IV).
In the present study, the bivalves accumulated high
amount of MC in the hepatopancreas (e.g. up to 38.5 g/g
for C. plicata) in summer, but declined to low MC level in
other seasons, indicating that bivalves are quite tolerant to
MCs and are able to depurate these cyanotoxins efficiently.
When Mytilus galloprovincitalis were fed with toxic
M. aeruginosa strain for 16 days, their mortality was less
than 1% in spite of accumulation of up to 10.5 g MC/g
DW (whole body) (Vasconcelos, 1995). Eriksson et al.
(1989) also showed experimentally high accumulation of
581
MCs (up to 70 g/g DW) (whole body). Yokoyama and
Park (2003) reported extremely high accumulation of MCLR (130–250 g/g DW) in the hepatopancreas of U. douglasiae in laboratory experiment. Pires et al. (2004) reported that when zebra mussel (Dreissena polymorpha) was
fed with toxic Microcystis, they accumulated up to 10.8 g
MC-LR/g DW1 in the whole body. Rapid depuration rates
of MCs by bivalves are reported from the aforementioned
experimental studies: high MC contents decline sharply to
rather low level within 2–3 weeks.
In the present study, a glutathione (GSH) conjugate of
MC-LR was detected in the foot sample of C. plicata. This
conjugate is considered to be the first step in the detoxication of MC-LR in various aquatic animals, and its presence
has been confirmed experimentally (in vitro) in the freshwater mussel Dreissena polymorpha (Pflugmacher et al.,
1998), the marine mussel Mytilus edulis (Sipiä et al., 2001),
zebra fish (Danio rerio) (Weigand et al., 1999), and brine
shrimp (Artemia salina) (Beattie et al., 2003). Quantitative
evaluations of such conjugates among different animals are
needed in our future study, to clarify divergence in detoxication mechanisms.
WHO proposed a provisional tolerable daily intake
(TDI) of 0.04 g/kg1 bw per day for MC-LR (Chorus and
Bartram, 1999). We estimated for the bivalves the critical
amount (gram wet weight) that is necessary to ingest to
reach the TDI for MC. A coefficient of 5 was used to convert dry weight to wet weight, and since i.p. LD50 in mice
for MC-RR and -YR is about 5 times and 2.5 times higher
than that for MC-LR, respectively (Gupta et al., 2003),
coefficients of 0.2 and 0.4 were used to convert MC-RR
and -YR into MC-LR equivalent, respectively. Considering
an adult of 60 kg, who ingests on the average 300 g of
bivalve foot a day, 15 of the 28 analyzed foot samples
(54%) were above this limit. For instance, in May 2004, the
concentration of MC-LReq in Lamprotula leai foot sample
reached 0.058 g/g1 WW, representing an estimated daily
intake of 0.29 g/kg1 of body weight. This is 7.25 times
the TDI value suggested by WHO. During the study period,
the mean daily intakes from feet of A. woodiana, H. cumingii, C. plicata, and L. leai were estimated to be 0.043,
0.109, 0.051, and 0.107 g MC-LReq/kg1 bw, respectively, and the maximum daily intakes were 0.13, 0.196,
0.115 and 0.29 g MC-LReq/kg1 bw of MC-LReq,
respectively. On the other hand, bivalves are traditional
eaten as a whole in some Chinese foods (usually the
bivalves are put in clean water for a couple of days to
empty out the gut contents before cooking), and during the
study period, the overall mean MC-content for these four
bivalves were 0.064, 0.188, 0.096, and 0.131 g MC-LReq/
g1 WW, respectively. This indicates that the daily intakes
from these four bivalves reached, respectively, 0.32, 0.94,
0.48, and 0.66 g MC-LReq/kg1 bw (8, 23.5, 12, and 16.5
times the TDI value suggested by WHO) when the bivalves
are eaten as a whole. Since MCs are not broken down by
b
a
MC
MC
Hepatotoxin
MC-LR
MC-LR
MC
Whole
Whole
Whole
Whole
Digestive tract
Whole
Hepatopancreas
Hepatopancreas
Hepatopancreas,
intestine, visceral
mass, gill,
foot, and rest
Hepatopancreas
Hepatopancreas
Hepatopancreas, intestine,
visceral mass, gill,
foot, and rest
Hepatopancreas
Whole
Hepatopancreas, intestine,
visceral mass, gill,
foot, and rest
Hepatopancreas, intestine,
visceral mass, gill,
foot and rest
Hepatopancreas
Hepatopancreas
Gill and muscle
Gonad
Gut
Hepatopancreas
Whole
MC-LR
MC-RR, -LR
MC-RR, -LR
MC-RR, -LR
MC-RR, -LR
MC-RR, -LR
Hepatotoxin
MC-RR, -LR, -YR
MC-RR, -LR
MC-LR
MC-RR, -LR, -YR
MC-RR, -LR
MC-RR, -LR
MC-RR, -LR, -YR
MC-RR
MC-RR, -LR
MC-RR,
-LR, -YR
MC
MC
MC
MC-LR
MC
Whole
Whole
Whole
Hepatopancreas
Whole
MC
Toxin
Whole
Organ
Remieux oxidation
GC/MS
PPase assay
Remieux oxidation
GC/MS
ELISA
HPLC-UV
HPLC-UV
ELISA
63.4 g/g WW
HPLC-MS
HPLC-PDA
HPLC-UV, LC-MS
HPLC-MS
HPLC-PDA
HPLC-UV, LC-MS
HPLC-PDA
HPLC-MS
HPLC-UV, PLC-MS
0.21 g/g WW
12.6 g/g DW
5.96, 7.88, 0.31,
0.12, 0.19,
0.11 g/g DW
0 g/g WW
297 g/g DW
38.48, 5.64, 0.71, 0.22,
0.23, 0.22 g/g DW
0 g/g DW
16.3 g/g DW
212.50, 20.65,
0.80, 0.23, 0.58,
0.49 g/g DW
313.23, 2.53, 1.70,
0.64, 0.52,
0.61 g/g DW
250 6 40 g/g DW
2.7 g/g WW
2.0 g/g WW
1.19 g/g WW
1.31 g/g WW
420 g/g DW
0.1 g/g DW
HPLC-PDA
HPLC-MS
HPLC-MS
HPLC-MS
HPLC-MS
HPLC-PDA
ELISA
HPLC-UV, LC-MS
HPLC
HPLC
HPLC
PPase assay
30 g/g DW
70 g/g DW
136 g/g DW
1.35 g/g DW
1.49 g/g DW
10.5 g/g DW
27.6 g/g DW
16 g/g DW
0.204 g/g WW
336.9 g/g WW
PPase assay
Analysis Method
0.022 g/g WW
Concentrationa
Maximum concentration of toxin in the literature expressed as micrograms of toxin per gram dry weight (DW) or wet weight (WW).
Sample of field experiment.
Macoma balthica
Unio douglasiae
Lamprotula leai
Corbicula sandai
Dreissena polymorpha
Hyriopsis cumingii
Cristaria plicata
Anodonta woodiana
Anodonta grandis simpsoniana
Anodonta cygnea
Mytilus galloprovincialis
Mytilus edulis
Organism
TABLE IV. A comparison of microcystin contents in bivalves reported both in literatures and in the present study
Laboratory experiment
Lake Suwa, Japan
Lake Suwa, Japan
Lake Suwa, Japan
Lake Suwa, Japan
Lake Suwa, Japan
Northern Baltic Sea
Lake Taihu, China
Lake Biwa, Japanb
Laboratory experiment
Lake Taihu, China
Lake Suwa, Japan
Lake Suwa, Japan
Lake Taihu, China
Laboratory experiment
Laboratory experiment
Laboratory experiment
Lake Driedmeat,
AB, Canadab
Lake Suwa, Japan
Lake Suwa, Japan
Lake Taihu, China
Northern Baltic Sea
Laboratory experiment
Laboratory experiment
Laboratory experiment
Campbell River,
BC, Canada
Campbell River,
BC, Canada
Laboratory experiment
Laboratory experiment
Location
Yokoyama et al, 2003
Watanabe et al., 1997
Watanabe et al., 1997
Watanabe et al., 1997
Watanabe et al., 1997
Yokoyama et al., 2002
Sipiä et al., 2002
Present study
Ozawa et al., 2003
Pires et al., 2004
Present study
Watanabe et al., 1997
Yokoyama and Park, 2002
Present study
Watanabe et al., 1997
Yokoyama and Park, 2002
Present study
Sipiä et al., 2001
Vasconcelos, 1995
Vasconcelos, 1995
Amorim and
Vasconcelos, 1999
Lindholm et al., 1989
Eriksson et al., 1989
Eriksson et al., 1989
Prepas et al., 1997
Williams et al., 1997
Williams et al., 1997
Williams et al., 1997
Williams et al., 1997
Reference
SEASONAL DYNAMICS OF MICROCYSTINS IN BIVALVES
cooking (they are chemically heat stable) (Harada et al.,
1996), consumption of bivalve is clearly a high risk to
human health. Because MCs do not readily undergo proteolytic or hydrolytic attack (Dawson, 1998), these toxins can
enter the food chain and finally end up in human food.
Therefore, the long-term impact of MCs to the aquatic
organism and public health cannot be overlooked. It is also
needed in our future studies to evaluate the potential harmful effects of MCs on human health by multiple exposure
routes through aquatic food, drinking water, and swimming
in lakes with toxic cyanobacterial blooms.
We express our deep thanks to Dr. Jun Xu for his kind supply
of the stable isotope data. Thanks are also due to Dr. Longgen
Guo and Mr. Rongle Fang and Dawen Zhang for their tremendous
help in the field collection of the bivalves.
REFERENCES
Amorim A, Vasconcelos M. 1999. Dynamics of microcystins in
the mussel Mytilus galloprovincialis. Toxicon 37:1041–1052.
Azevedo SMFO, Carmichael WW, Jochimsen EM, Rinehart KL,
Lau S, Shaw GR, Eaglesham GK. 2002. Human intoxication by
microcystins during renal dialysis treatment in Caruaru-Brazil.
Toxicology 181–182:441–446.
Beattie KA, Ressler J, Wiegand C, Krause E, Codd GA, Steinberg
CEW, Pflugmacher S. 2003. Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp
Artemia salina. Aquat Toxicol 62:219–226.
Carmichael WW. 1994. The toxins of Cyanobacteria. Sci Am
270:78–86.
Carmichael WW. 1997. The cyanotoxins. In: Callow JA, editor.
Advances in Botanical Research, Vol. 27. London: Academic
Press. p 211–256.
Chen J, Xie P. 2005. Tissue distributions and seasonal dynamics
of the hepatotoxic microcystins-LR and -RR in two freshwater
shrimps, Palaemon modestus and Macrobrachium nipponensis,
from a large shallow, eutrophic lake of the subtropical China.
Toxicon 45:615–625.
Chen J, Xie P, Guo LG, Zheng L, Ni LY. 2005. Tissue distributions and seasonal dynamics of the hepatotoxic microcystins
-LR and -RR in a freshwater snail (Bellamya aeruginosa) from
a large shallow, eutrophic lake of the subtropical China. Environ Pollut 134:423–430.
Chorus I, Bartram J. 1999. Toxic Cyanobacteria in Water. A
Guide to Public Health Consequences, Monitoring and Management. London: E & FN Spon. p 416.
Dawson RM. 1998. The toxicology of microcystins. Toxicon 36:
953–962.
Eriksson JE, Meriluoto JAO, Lindholm T. 1989. Accumulation of
a peptide toxin from the cyanobacterium Oscillatoria agardhii
in the freshwater mussel Anodonta cygnea. Hydrobiology 183:
211–216.
Falconer IR. 1998. Algal toxins and human health. In: The Handbook of Environmental Chemistry, Vol. 5, Part C: Quality and
583
Treatment of Drinking Water II (Hrubec J, editor). Berlin:
Springer. p 53–82.
Falconer IR, Choice A. 1992. Toxicity of edible mussels (Mytilus
edulis) growing naturally in an estuary during a water bloom of
the blue-green alga Nodularia spumigena. Environ Toxicol
Water Qual 7:119–123.
Falconer IR, Burch MD, Steffensen DA, Choice M, Coverdale
OA. 1994. Toxicity of the blue-green alga (cyanobacterium)
Microcystis aeruginosa in drinking water to growing pigs, as
an animal model for human injury and risk assessment. Environ
Toxicol Water Qual 9:131–139.
Gupta N, Pant SC, Vijayaraghavan R, Lakshmana Rao PV. 2003.
Comparative toxicity evaluation of cyanobacterial cyclic peptide toxin microcystin variants (LR, RR, YR) in mice. Toxicology 188:285–296.
Harada KI, Tsuji K, Watanabe MF, Kondo F. 1996. Stability of
microcystins from cyanobacteria. III. Effect of pH and temperature. Phycologia 35:83–88.
Lindholm T, Eriksson JE, Meriluoto JAO. 1989. Toxic cyanobacteria and water quality problems—examples from a eutrophic
lake on Åland, South West Finland. Water Res 23:481–486.
Liu YY. 1979. Fauna Sinica of Economic Animals (Freshwater
Mollusca). Beijing, China: Science Press (in Chinese).
Magalhàes FV, Soares RM, Azvedo SMFO. 2001. Microcystin
contamination in fish from Jacarepaguá Lagoon (Rio de Janeiro,
Brazil): ecological implication and human health risk. Toxicon
39:1077–1085.
Magalhàes VF, Marinho MM, Domingos P, Oliveira AC, Costa
SM, Azevedo LO, Azevedo SMFO. 2003. Microcystins (cyanobacteria hepatotoxins) bioaccumulation in fish and crustaceans
from Sepetiba Bay (Brasil, RJ). Toxicon 42:289–295.
Mohamed ZA, Carmichael WW, Hussein AA. 2003. Estimation of
microcystins in the freshwater fish Oreochromis niloticus in an
Egyptian fish farm containing a Microcystis bloom. Environ
Toxicol 18:137–141.
Nishiwaki-Matsushima R, Nishiwaki S, Ohta T, Yoszawa S, Suganuma M, Harada K, Watanabe MF, Fujiki H. 1991. Structurefunction relationships of microcystins, liver-tumor promoters,
in interaction with protein phosphatase. Jpn J Cancer Res 82:
993–996.
Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, Suganuma M,
Yoszawa S, Kohyama K, Ishikaawa T, Carmichael WW, Fujiki
H. 1992. Liver tumor promotion by the cyanobacterial cyclic
peptide toxin microcystins-LR. J Cancer Res Clin Oncol 118:
420–424.
Ozawa K, Yokoyama A, Ishikawa K, Kumagai M, Watanabe MF,
Park HD. 2003. Accumulation and depuration of microcystin
produced by the cyanobacterium Microcystis in a freshwater
snail. Limnology 4:131–138.
Paerl HW, Fulton RS, Moisander PH, Dyble J. 2001. Harmful
freshwater algal blooms, with an emphasis on cyanobacteria. Sci
World J 1:76–113.
Pflugmacher S, Wiegand C, Oberemm A, Beattie KA, Krause E,
Codd GA, Steinberg CEW. 1998. Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication. Biochim
Biophys Acta 1425:527–533.
584 CHEN AND XIE
Pires LMD, Karlsson KM, Meriluoto JAO, Kardinaal E, Visser
PM, Siewertsen K, Donk EV, Ibelings BW. 2004. Assimilation
and depuration of microcystin–LR by the zebra mussel, Dreissena polymorpha. Aquat Toxicol 69:385–396.
Prepas EE, Kotak BB, Campbell LM, Evans JC, Hrudey SE,
Holmes CFB. 1997. Accumulation and elimination of cyanobacterial hepatotoxins by the freshwater clam Anodonta grandis
simpsoniana. Can J Fis Aquat Sci 54:41–46.
Pu PM, Hu WP, Wang GX, Zhang SZ, Hu CG, Yan JS. 1998a.
The new strategy for improving the aqua-ecological environmental in Taihu Lake Basin, China. How can we solve the
problem of lack of qualified water and deterioration of environment and natural resources in Taihu Lake basin. J Lake Sci 10:
47–58.
Pu PM, Hu WP, Yan JS, Wang GX, Hu CG. 1998b. A physicoecological engineering experiment for water treatment in a
hypertrophic lake in China. Ecol Eng 10:79–90.
Sipiä VO, Kankaanpää HT, Lahti K, Carmichael WW, Meriluoto
JAO. 2001. Detection of nodularin in flounders and cod from
the Baltic Sea. Environ Toxicol 16:121–126.
Vanderploeg HA, Liebig JR, Carmichael WW, Agy MA, Johengen TH, Fahnenstiel GL, Nalepa TF. 2001. Zebra mussel
(Dreissena polymorpha) selective filtration promoted toxic
Microcystis blooms in Saginaw Bay (Lake Huron) and Lake
Erie. Can J Fish Aquat Sci 58:1208–1221.
Vasconcelos VM. 1995. Uptake and depuration of the heptapeptide toxin microcystin-LR in Mytilus galloprovincialis. Aquat
Toxicol 32:227–237.
Watanabe MF, Park HD, Kondo F, Harada K, Hayashi H, Okino
T. 1997. Identification and estimation of microcystins in freshwater mussels. Nat Toxins 5:31–35.
Wiegand C, Pflugmacher S, Oberemm A, Meems N, Beattie KA,
Steinberg CEW, Codd GA. 1999. Uptake and effects of microcystin-LR on detoxication enzymes of early life stages of the
zebra fish (Danio rerio). Environ Toxicol 14:89–95.
Williams DE, Dawe SC, Kent ML, Andersen RJ, Craig M,
Holmes CFB. 1997. Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus edulis, and in vivo
evidence for covalently bound microcystins in mussel tissues.
Toxicon 35:1617–1625.
Xie LQ, Xie P, Guo LG, Li L, Yuichi M, Park HD. 2005. Organ
distribution and bioaccumulation of microcystins in freshwater
fishes with different trophic levels from the eutrophic Lake
Chaohu, China. Environ Toxicol 20:293–300.
Xu J, Xie P, Zhang M, Yang H. 2005. Variation in stable isotope
signatures of seston and a zooplanktivorous fish in a eutrophic
Chinese lake. Hydrobiologia 541:215–220.
Yokoyama A, Park HD. 2002. Mechanism and prediction for contamination of freshwater bivalve (Unionidae) with the cyanobacterial toxin microcystin in the hypereutrophic Lake Suwa,
Japan. Environ Toxicol 17:424–433.
Yokoyama A, Park HD. 2003. Depuration kinetics and persistence
of the cyanobacterial toxin microcystin-LR in the freshwater
bivalve Unio douglsiae. Environ Toxicol 18:61–67.
Yu SZ. 1989. Drinking water and primary liver cancer. In: Tang
ZY, Wu MC, Xia SS, editors. Primary Liver Cancer. New
York: China Academic Publishers. p 30–37.
Yu SZ. 1995. Primary prevention of hepatocellular carcinoma.
J Gastroenterol Hepatol 10:674–682.
Zurawell RW, Kotak BG, Prepas EE. 1999. Influence of lake trophic status on the occurrence of microcystin-LR in the tissue of
pulmonate snails. Freshwater Biol 42:707–718.