Vol.4, No.5B, 52-56 (2013) Agricultural Sciences
Accumulation of microcystins in water and economic
fish in Phayao Lake, and fish ponds along the Ing
River tributary in Chiang Rai, Thailand
Niwooti Whangchai1*, Suthida Wanno1, Redel Gutierrez1,2, Korntip Kannika3,
Rattapoom Promna4, Norio Iwami5, Tomoaki Itayama6
1Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Sansai, Chiang Mai, Thailand;
*Corresponding Author: niwooti@hotmail.co.th
2College of Arts and Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines
3School of Agriculture and Natural Resources, Resources University of Phayao, Maeka, Mueang, Phayao, Thailand
4School of Energy and Environment, University of Phayao, Maeka, Mueang, Phayao, Thailand
5Graduate School of Science and Engineering, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo, Japan
6Graduate School of Engineering, Nagasaki University, 1-14 Bunkyou-machi, Nagasaki, Japan
Received 2013
This study determined the levels of microcystins
in water and fish from Phayao Lake, Phayao
Province and selected fish ponds along the Ing
River tributary in Chiang Rai Province. Samples
were collected monthly for 8 months (January to
August 2011 for Phayao Lake, and November
2008 to June 2009 for fish ponds) and were
analyzed by HPLC. The highest total micro-
cystin-LR levels in water and fish in Phayao
Lake were recorded in April 2010 at 2.60 ± 2.48
µg·L-1 and 0.20 ± 0.03 µg·kg-1 dry weight, re-
spectively. Microcystis aeruginosa Kütz were
the dominant species (271.6 ± 72.4 mm3/m3) in
the lake. Colony number of Microcystis spp
showed a positive correlation with soluble or-
thophosphate (r2 = 0.77). Similarly, Nile tilapia
ponds surveyed along the tributary in Chiang
Rai were cont aminated with microcystins as well.
The highest concen tratio n detec ted in water was
in March 2009 (0.58 ± 0.24 µg·L-1), whilst the
maximum concentration in fish was recorded in
April 2009 (2.68 ± 0.51 µg·kg-1 dry weight). Mi-
crocystis spp. dominated the pond waters and
was positively correlated with chlorophyll a
(r2=0.80) and soluble nitrate (r2=0.71). The high-
est concentration of the cyanobacteria was re-
corded in February 2009 at 4272.5 ± 62.3 mm3/m3.
Results showed that total microcystin-LR con-
centration in fish in Chiang Rai ponds were
higher than in Phayao Lake. This study sug-
gested the possible health risks associated w ith
the bioaccumulation of microcystins in fish (Nile
tilapia) cultivated in fish ponds along the tribu-
t ary in Chiang Rai and in Phayao Lake.
Keywords: Microcystins; Phayao Lake; Water; Nile
Thailand is one of the significant exporters of fish fil-
let in the United States, along with Taiwan, Mainland
China and Indonesia. Other key fillet markets include
Japan and Italy [1]. Tilapia is mainly derived from fish
farming in cages and ponds either through monoculture
or polyculture with other economic fishes such as hybrid
catfish. The consumption of Nile tilapia (Oreochromis
niloticus) in Thailand is very popular, especially in the
northern part where freshwater consumption rate was
reported to be up to 32 kg per person per year [2] The
Chiang Mai Aquacultural Cooperative (CMA Co-op)
also reported that demand for freshwater fish in Chiang
Mai amounts to 40,000 kg/day [3]. The largest producer
of tilapia (approximately 17.71 tons/day of fish products)
is in the Upper North, including the Phan District, Chiang
Rai Province, where tilapia production is primarily semi-
intensive based on natural foods derived from fertilizers
or animal manure [4,5]. The fish are generally raised
along with livestock, especially chicken and pigs, to re-
duce production costs. However, integrated fish farming
often face water quality problems caused by nutrients
(nitrogen and phosphorus) from animal wastes. Because
the volume of waste discharged into the fish pond is ex-
cessive, it results in a rapid growth of phytoplankton
(algal bloom) especially during summer, which mostly
often rendered the fish with musty off-flavor, or worst,
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N. Whangchai et al. / Agricultural Sciences 4 (201 3) 52-56 53
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/
contaminated with toxic microcystins.
Microcystin is a hepatoxin produced by blue-green
algae such as Anabaena spp., Oscillatoria spp., and Mi-
crocystis spp. with Microcystis aeruginosa, having the
most number of toxic species known. Microcystins are
cyclic peptides made from seven amino acids [6,7], con-
sidered as the most common and one of the most dan-
gerous groups of cyanotoxins found in water [8]. Micro-
cystins are toxic pollutants in the water that cause harm
to humans by inducing diseases of the digestive system.
These toxins affect the liver by inhibiting protein phos-
phatase and cause liver cancer in rats [9]. Magalhaes et
al. [10] reported the accumulation of microcystin in fish
used for human consumption, from the lake Jacarepagua
in Brazil.
Kwan Phayao is one the largest artificial lakes in
northern Thailand, located in the province of Phayao. It
covers an area of 2.3 km2, with a mean depth of 1.7 m
and located at an altitude of 380 meters above sea level.
It is situated at the southern tips of two mountains, Doi
San Klang and Doi Huai Nam Khao. The lake is fed by
the Ing River, which empties to the north. To the south
and west of the lakes are rice paddies, at the mouth of the
Ing River is marsh area, which is a significant residence
for water birds [11]. Kwan Phayao is the main source of
raw water for domestic, drinking and agricultural use of
Phayao residents. However, its water quality is deterio-
rating due to anthropogenic and farming activities, with
livestock effluent from the basin and sewage discharges
from Phayao City are being the major pollution sources.
These significant sources of nutrients, especially nitrogen
and phosphorus, contribute to the eutrophication of the
lake which leads to algal blooms and subsequently to
microcystin production, contaminating the water and
aquatic animals.
Therefore, the purpose of this research is to monitor
the levels of toxic microcystins and phytoplankton diver-
sity in Phayao Lake, Phayao province and in selected
aquaculture ponds in Chiang Rai province, Thailand.
2.1. Sample Collection
The present study was carried out in Phayao Lake due
to its geographical significance as a tourist destination in
Phayao province, and in selected fish ponds in the prov-
ince of Chiang Rai, along the Ing River tributary. The
period of sampling (8 months) was from January to Au-
gust 2011 (Phayao Lake) and from November 2008 to
June 2009 (Chang Rai fish ponds). Two-liter samples of
water were collected in polyethylene containers for physic-
chemical analysis and 1-L samples were collected for
microcystin analysis, from both lake and ponds. Samples
from Phayao Lake were collected from three fixed sam-
pling points as shown in Figure 1. Samples were pre-
served on ice, transported to the laboratory and stored at
Fish samples (Nile tilapia) were collected at the same
time as the water samples, with the aid of fishing net. A
total of 24 and 48 samples of Nile tilapia were sampled
from Phayao Lake and from 6 fishponds in Chiang Rai,
respectively during the period of study. Samples were
immediately kept in ice and transported to the laboratory.
Fish samples were weighed, filleted and stored in the
freezer at -20℃ until analysis.
Phytoplankton was sampled by filtration of pond water
(with a net of 10-µm mesh. Samples were concentrated
in a 30-mL bottle and preserved with Lugol’s solution.
2.2. Microcystin in Water
Microcystin in water was measured following a method
adapted from Prommana et al. [12] with modification.
Two-hundred-milliliter water sample was placed in a
500-mL beaker and boiled for 1 hour. The sample was
then filtered thru a 0.45 µm GF/C glass filter disc, and
the pH was adjusted to pH 7.0 using a solution of 0.01 M
NaOH or 0.01 M HCl. Microcystins (MCs) in the water
sample was extracted by solid phase extraction (SPE)
using a Strata-X 33 μm polymeric reversed phase (500
mg/6 mL) SPE cartridge. After conditioning the SPE
cartridge with 5 mL of 100% methanol followed by 10
mL of of Milli-Q water, the water sample was applied to
the cartridge at a flow-rate of 20 mL·min-1. After the
cartridge was washed with 20 mL of 10%methanol, res-
idue of MCs was eluted twice with 5 mL of 10% metha-
nol at a flow-rate of 5 mL· min-1.
The MC-containing fraction was evaporated to dryness.
This fraction was dissolved in 5 mL of 100% methanol
and was filtered through 0.45 µm Millipore filters.
One-hundred microliters of the methanolic extract was
then injected into the high performance liquid chromato-
graph (HPLC) for the detection and quantification of
Phayao Lake
inflow (Ing R iver)
outflow (Ing River)
collection points
Figure 1. Sketch map of Phayao Lake showing the sampling
N. Whangchai et al. / Agricultural Sciences 4 (201 3) 52-56
2.3. Microcystin Anal ysis in Fish
Deep-frozen fish fillets were thawed and then 10-g
samples were freeze-dried before extraction. One-gram
freeze dried sample was homogenized in a mortar and
then extracted three times with 10-mL 80% methanol,
mixed well with a vortex mixer for 10 minutes and was
left overnight. The extract was centrifuged at 5,000 rpm
for 10 minutes. The supernatant was transferred in a sep-
aratory funnel, 15 mL of hexane was added, the mixture
was shaken for 10 minutes and then the methanol frac-
tion was collected. This was repeated two more times
prior to HPLC analysis.
2.4. Water Quality and Nutrient Analysis
Temperature, pH, dissolved oxygen and turbidity were
measured in situ, using a multimeter (TOA DKK WQC-
22A Model, Japan). Alkalinity, hardness, total ammonia-
nitrogen, nitrate-nitrogen, nitrite-nitrogen, orthophos-
phate-phosphorus and chlorophyll a were determined in
the laboratory by standard methods [13].
2.5. Cyanobacterial Identification and
The identification of Microcystis colonies and other
cyanobacteria was carried out using a microscope.
Cyanobacterial cells were counted with a haemacytome-
ter and calculated as mm3/m3.
Microcystin-LR (MC-LR) was detected in 29% (7 out
of 24) of water sampled from Phayao Lake throughout the
survey period. The amount of total MC-LR in water
ranged from, “not detected” (ND) - 7.56 µg· L-1 with an
average concentration of 0.69+0.28 µg· L-1 (Figure 2).
On the other hand, Nile tilapia from the lake which were
collected at the same sampling date as with the contami-
nated water samples, were likewise found to be tainted
with toxic MC-LR, which ranged from ND - 0.26 µg· kg-1
dry weight. The average concentration in fish was 0.06 ±
0.02 µg· kg-1 dry weight. The highest total MC-LR con-
taminations in both water and Nile tilapia in Phayao Lake
were recorded in April 2010 (Figure 2).
Majority of water samples (67%) from the 6 fish ponds
in Chiang Rai province were contaminated with the
hepatoxin. The thirty-two MC-LR contaminated samples
of water had an average concentration of 0.22 ± 0.28
µg·L-1, where the highest concentration was detected in
March 2009 (Figure 3). Out of the 48 samples of Nile
tilapia, 23 were found positive for toxic MC-LR. Detected
concentrations ranged from ND - 5.91 µg· kg-1 dry weight
with an average of 1.22 + 0.48 µg·kg-1 dry weight. The
highest concentration recorded in fish was in April 2009
Figure 2. Concentration of MC-LR in lake water and fish
(Nile tilapia) sampled between January to August 2011.
Figure 3. Concentration of MC-LR in pond water and fish
(Nile tilapia) sampled between November 2008 and June
(Figure 3).
In Phayao Lake, Microcystis aeruginosa Kütz.
(271.6±72.4 mm3/m3) dominated the water. Other mi-
crocystin-producing cyanobacteria identified include,
Microcystis wesenbergii Kom. (120.8 ± 14.0 mm3/m3),
Anabaena spp. (1.0 ± 0.2 mm3/m3), Oscillatoria spp.
(74.0 ± 41.9 mm3/m3) and Cylindrospermopsis raci-
borskii (Wolosz.) Seenayya & Subba (5.5 ± 2.5 mm3/m3)
(Figure 4). Correlation analysis showed that Microcys-
tis species (Microcystis aeruginosa Kütz. and Micro-
cystis wesenbergii Kom.), had a positive relationship
with soluble orthophosphate (r2 = 0.77).
Likewise, Microcystis spp. dominated the Chiang
Rai ponds throughout the survey period. Anabaena spp.
and Oscillatoria spp. were also present in the ponds
(Figure 4). Biovolume of Microcystis spp. was posi-
tively correlated with chlorophyll a (r2=0.80) and sol-
uble nitrate (r2=0.71). Highest concentration of the
cyanobacteria was recorded in February 2009 at
4272.5±62.3 mm3/m3.
This study shows another compelling evidence of the
pervasive contamination of microcystins in eutrophic
surface waters and the bioaccumulation of these toxins in
fish tissues. The results, where 67% of water and 48%of
Nile tilapia samples from Chiang Rai freshwater ponds;
and 29% of water and Nile tilapia samples from Phayao
Lake, were contaminated with MC-LR, corroborate the
Copyright © 2013 SciRes. Openly accessible at http://www.scirp.org/journal/as/
N. Whangchai et al. / Agricultural Sciences 4 (201 3) 52-56 55
findings of previous studies conducted in freshwater
Scale bar = 10 µm
Figure 4. Microcystin-producing cyanobacteria identified in
Phayao Lake and in Chiang Rai ponds. (A) Oscillatoria spp. (B)
Microcystis wesenbergii Kom. (C) Cylindrospermopsis raci-
borskii (Wolosz.) Seenayya & Subba (D) Microcystis aerugi-
nosa Kütz. (E) Anabaena spp.
prawn and tilapia ponds in northern Thailand [12,14].
Due to the dominance of the genus Microcystis spp.,
(Microcystis aeruginosa Kütz. and Microcystis wesen-
bergii Kom) in Phayao Lake and in Chiang Rai ponds,
their presence in these surface waters could be impli-
cated for the production of the microcystins detected in
this study. This confirms the findings of Prommana [15]
that M. wesenbergii were found to be the dominant spe-
cies in Kwan Phayao reservoir (Phayao Lake). Similarly,
Peerapornpisal et al.[16] reported that Microcystis, which
include M. aeruginosa and M. wesenbergii, were the
main species found in water resources in Thailand whilst
Ruangrit et al. [14] reported that the dominant Microcys-
tis species in giant freshwater prawn ponds and in tilapia
ponds in northern Thailand were M. aeruginosa and M.
wesenbergii. Moreover, the presence of Microcystis spp.
in these bodies of water may be indicative of constant
nutrient enrichment, as shown by its established positive
correlation with soluble orthophosphate (r2 = 0.77) in
Phayao Lake, and with soluble nitrate (r2 = 0.71) in
Chiang Rai ponds. These species are known not to toler-
ate nutrient poor conditions in aquatic ecosystems [17].
Potential microcystin-producing genera such as Ana-
baena spp. and Oscillatoria spp. and Cylindrospermopsis
raciborskii were also identified in this study. However,
they were only present in small amounts and could not
be significant contributors to microcystin production in
the lake and ponds [12].
The mean level of MC-LR in Nile tilapia was higher
in ponds compared with that in the lake (Ta ble 1). De-
tected levels of MC-LR in the fish were below the rec-
ommended guideline value for a tolerable daily intake
(TDI) of microcystin at 0.04 μg kg-1 body weight day-1,
except for some fish pond samples which have exceeded
the allowed value. The apparent presence of microcystin
in Nile tilapia from both Phayao Lake and aquaculture
ponds in Chiang Rai could have a serious negative im-
plication on people that use the fish from these waters as
a source of meat. The toxins contained within fish tissues
may pose an alternative route of exposure to humans.
This is because there is sufficient published data that
implicate microcystins of bioaccumulation in fish tissues
As a planktivorous fish, Nile tilapia consumes cyano-
bacteria particularly Microcystis and other filamentous
species. Aside from getting contaminated with dissolve
microcystins from the water through the gills, direct in-
gestion of these toxic cyanobacteria, which through the
intestinal tract, could be another possible route of uptake
of microcystin. Zhao et al. [19] showed that microcystin
accumulation rates in muscle and liver tissues are
directly proportional to ingestion rates for Nile tilapia.
Furthermore, the contamination of fish with microcystins
also affects negatively the former’s growth and
productivity [20], thus pose problems as well in the
standpoint of aquaculture production and fish quality.
The extent of cyanobacterial contamination of Phayao
Lake is one significant issue in this study. The lake is the
main source of raw water for domestic, agricultural and
drinking purposes as well as a source of food and
livelihood for local fishermen. Although the MC-LR val-
ues detected were below the safety limits, the combined
risks from MC-LR contamination of water and fish pose
a possible health hazard to the residents and cannot be
overlooked. Similarly, aside from possible health risks to
humans, the presence of these toxins in Nile tilapia
production ponds could negatively affect fish growth and
productivity, and therefore, effective pond management
should be adopted to minimize these problems in
Table 1. Mean levels of Microcystin-LR in water and fish.
Source Water (µg·L-1) Fish (µg·kg-1 dw)
Phayao Lake 0.69+0.28 0.06+0.02
Fish ponds 0.22+0.09 1.22+0.48
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N. Whangchai et al. / Agricultural Sciences 4 (201 3) 52-56
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5. CONCLUSIONS [9] Zilberg, B. (1966) Gastroenteritis in Salisbury European
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The assessment on the levels of microcystins in water
and Nile tilapia in Phayao lake caused by the presence of
cyanobacteria i.e. Microcystis, due to nutrient enrichment
(eutrophication) should, therefore, be of great concern.
The communities normally use water from Phayao Lake
as raw water for household consumption, agricultural
and industrial use and the lake as source of food and
livelihood for local fishermen. The present study has also
suggested that Microcystis species and microcystins in
cultivation ponds in Chiang Rai, may pose a possible
hazard to aquatic organisms and to humans through the
food web. Consumers that feed on fish from these aqua-
culture ponds may be at the risk of continuous micro-
cystins poisoning. It is therefore recommended that fish
farmers should develop management strategies to control
cyanobacterial growth and minimize the incidence of
bioaccumulation of microcystin in cultured fish.
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