Microcystin Accumulation in Nile Tilapia, <i>Oreochromis niloticus</i> and Giant Freshwater Prawns, <i>Macrobrachium rosenbergii</i> in Green Water System Cultivation

doi:10.4236/ijg.2013.45B010

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International Journal of Geosciences, 2013, 4, 60-63

http://dx.doi.org/10.4236/ijg.2013.45B010 Published Online September 2013 (http://www.scirp.org/journal/ijg)

Micr o c ystin Accumulation in Nile Ti lapia, Oreochromis

niloticus and Giant Freshwater P r awns, Macrobrachium

rosenbergii in Green Water Sys tem Cultivation

Khomsan Ruangrit1, Yuwadee Peerapornpisal1, Jeeraporn Pekkoh1, Niwooti Whangchai2*

1Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand

2Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiang Mai, Thailand

Email: *[email protected]

Received July 2013

ABSTRACT

Phytoplankton including blue-green algal or cyanobacterial blooms frequently occurred in aquaculture ponds. Some

cyanobacteria produced cyanotoxins that may accumulate in the food web and eventually in the aquacult ure products. I n

this study, accumulatation of microcystins in Nile tilapia (Oreochromis niloticus) and giant freshwater prawn (Macro-

brachium rosenbergii) cultured in green water system was investigated. Nile tilapia was cultured in green water sy stem

and fish food; green water system with Microcystis aeruginosa Kützing and fish food and green water system with M.

aeruginosa. Giant freshwater prawn was cultured: in green water systems with and without toxic M. aeruginosa. Mi-

crocystins of 8.32 ± 0.76 and 9.35 ± 1.45 µg·kg−1 d.w. were detected in fish cultured in green water system with M.

aeruginosa and fish food and in green water system with M. aeruginosa, respectively. Microcystins of 14.42 ± 1.63

µg·kg−1 was found in prawn samples. It implied that aquaculture products were likely to be contaminated with micro-

cystins. This finding is useful for aquaculture in terms of food safety.

Keywords: Microcystis aeruginosa Kützing; Microcystins; Aquaculture; Green Wa t e r System

1. Introduction

Thailand is the fourth ranking Nile tilapia (Oreochromis

niloticus) producer in the world since 2000. Its produc-

tion has increased almost exponentially [1]. It is con-

sumed and exported to other countries. Nile tilapia is

mostly raised in earthen ponds using manures and other

recyclable wastes, as low cost commercial pellet feeds

are not necessary for growing tilapia and the traditional

cultivation, nutrient-enriched water, “green water”, pro-

duced by the addition of animal manure or fertilizer is

sufficient to achieve a marketable fish [2] as well as the

prawns [3]. There are many aquaculture ponds through-

out Thailand where giant freshwater prawns, Macrobra-

chium rosenbergii, are cultured. In fact, the prawns can

be grown in all freshwater bodies [3]. They are commer-

cially important because they are widely used for human

consumption. Domestic consumption was 70 % of total

production [1]. Green water systems can cause eutrophi-

cation of surface water, resulting in increased occurrence

of toxic cyanobacterial bloom, especially Microcystis [3].

The occurrence can create a significant water quality

problem, including their ability to produce toxins, name-

ly microcystins (MCs). The toxins accumulate in aquatic

organisms and are transferred to higher trophic levels. It

involves the risk for human exposure through the con-

sumption of contaminated aquatic organisms [4-6]. In

Thailand, cyanobacterial genera with known toxin- pro-

ducing taxa occurred in many reservoirs in all regions.

Microcystis aeruginosa is the most frequently blooms

[7-9]. Ruangrit et al. [10] found high amount of M. aeru-

ginosa in prawn pond and microcystins were detected in

prawn. Therefore, it is needed to clarify whether MCs are

able to accumulate in aquatic organism cultured with

traditional method. The data would be useful for food

safety aspect and public health to avoid the damaging

effect of cyanobacteria and their toxins.

2. Materials and Methods

2.1. Culturing of Nile Tilapia and Giant

Freshwater Prawn

Nile tilapia about 5 cm in size and giant freshwater

prawn about 5 - 7 cm in size were obtained from the Fa-

culty of Fisheries Technology and Aquatic Resources,

Maejo University, Chiang Mai, Thailand. The fish were

cultured in 3 cement ponds, 1.5 m × 1.5 m and the depth

of 0.50 m containing green water 0.30 m deep, 30 fish in

*Corresponding a uthor.

K. RUANGRIT ET AL.

each pond. Feeding treatments were; 1) Green water sys-

tem (Tr. 1). 2) Green water system with 18 - 30 × 106

cells·L−1 M. aeruginosa from natural pond and combined

with commercial pellet feed (Tr. 2). 3) Green water sys-

tem with 18 – 30 × 106 cells·L−1 M. ae ruginosa (Tr. 3).

The prawns were cultured in two cement ponds of

similar size, 30 prawns were in the pen (0.45 m × 0.45 m

with water depth of 0.30 m) made of blue net (mezh size

2 mm) attached to the pond, 30 prawns were outside the

pen. Feeding treatments were: Green water system (Tr. 1)

and green water system with M. aeruginosa combined

with commercial pellet feed (Tr. 2).

Completely randomize design (CRD) with duplicate

treatments were carried out. Both fish and prawn were

cultured for 2 months. Water samples were collected

every two weeks to determine the amount of M. aerugi-

nosa, phytoplankton and microcystins.

2.2. Identification and Enumeration of M.

aeruginosa and Phytoplankton

Morphological classification of Microcystis spp. and phy-

toplankton were done under compound microscope (Olym-

pus model CH30RF200) using related texts such as

Komárek and Komáková-Legnerová [12] and Hindak [13].

Cells of M. aeruginosa we re counted on a haem acytometer .

2.3. Analysis of Microcystins

2.3.1. Extraction of Microcystins

Microcystins were extracted after Kankaanpää et al. [4]

with modification. Fish and prawn tissues were dissected

and freeze-dried at −20˚C for 24 - 72 hours before ex-

traction and ELISA analysis. One mL of 100% methanol

was added into 2 g fish and prawn tissues for extraction

overnight. The extracts were centrifuged at 12,000 rpm

for 30 min and the supernatants were concentrated to 150

µl with a heat blo ck at 50 ˚C, overnight and centrifuged at

12,000 rpm for 30 min before ELISA analysis.

2.3.2. Microcystin Analysis by ELISA Assay

ELISA Microcystin Plate Kit (Catalog No. EP022), EN-

VIROLOGIX INC was used and performed in accor-

dance with the manufacturer’s instructions. A standard

curve was constructed using three calibrations 0.16, 0.5

and 2.5 µg·L−1 supplied with the kit. The absorbance at

450 nm was measured with a microplate reader (Spectra

MR, DYNEX Technologies). The microcystin concentra-

tion in each extract was expressed as MC-LR equivalent.

3. Results and Discussion

3.1. Identification and Enumeration of M.

aeruginosa and Phytoplankton in Fish Ponds

Dominant species of phytoplankton excluding M. aeru-

ginosa were found to belong to 5 divisions i.e. Divisions

Chlorophyta, Cyanophyta, Euglenophyta, Bacillariophyta

and Pyrrhophyta. Dominant species were Chlorophyta

(green algae) such as Scencedesmus spp . and Pediastrum

spp. The species composition was similar in each treat-

ment except Tr. 2, Microcystis wesenbergii was found as

dominant species.

Tr. 1 had lowest amounts of phytoplankton. Whereas

the highest amounts of phytoplankton were found in Tr.

3 and Tr. 2, respectively (Figures 1 and 2) .

3.2. Microcystin Contents in Fish Samples

High microcystin contents in fish samples were detected.

Microcystin contents of 8.32 ± 0.76 and 9.35 ± 1.45

µg·kg−1 d.w. were found in the fish of Tr. 2 and Tr. 3,

respectively. Whereas microcystins in the corresponding

Tr. 2 and T r. 3 pond s w ere 2 0.08 ± 0 .24 and 1 9.52 ± 0.49

µg·L−1, respectively.

Microcystins were released from dead Microcystis

cells causing high content of microcystin in the water.

However, microcystin in the fish was obtained from in-

gestio n of Microcystis cells by the fish [14,15].

Figure 1. Amounts of phytoplankton and M. aeruginosa in

each treatment (Replicate I fish pond).

Figure 2. Amounts of phytoplankton and M. aeruginosa in

each treatment (Replicate II fish pond).

K. RUANGRIT ET AL.

3.3. Identification and Enumeration of M.

aeruginosa and Phytoplankton in Prawn

Ponds

Dominant phytoplankton species excluding M. aerugi-

nosa in the prawn pond were diatoms such as Cyclotella

spp. and Acthanthidium spp. The amounts of M. aerugi-

nosa and other phyto plankton a re s hown in Figure 3.

3.4. Microcystin Contents in Prawn Samples

Microcystin detected in the second pond (at the end of

cultivation) was 21.19 ± 0.31 µg·L−1 and in the prawn

tissue was 14.42 ± 1.63 µg·kg−1 d.w.

This study was conducted in a short period. Dominant

species of phytoplankton excluding M. aeruginosa were

similar in each treatment because similar green water

was used. Both fish and prawn cultivation in Tr. 1 had

lowest amounts of phytoplankton. Tr. 2 and Tr. 3 had

higher amounts because not only biomass of M. aerugi-

nosa was added but also phytoplankton associated with

M. aeruginosa. Fish samples in Tr. 2 and Tr. 3 which

contained high amounts of M. aeruginosa also contained

high amounts of MCs.

Effect of giant freshwater prawn consumption beha-

vior on the microcystin accumulation in two types of

cultivation i.e. inside and outside the pens, in the same

pond was not different. Microcystins can interact with

humic and fulvic substances, suspended particulate mat-

ter or sediments. Prawns are bottom dweller and con-

sume feed which falls to the bottom of the pond. If

prawns are forced to avoid feeding at the bottom by pen,

toxin accumulation could be lower. Unfortunately, the

experiment had to condu ct in cement pond which had no

sediment. It was shown that MC contents from both

types of cultivation were comparable.

MC content in the prawn tissue was higher than that in

the fish tissue. It might be possible that prawns came into

Figure 3. Amounts of phytoplankton and M. aeruginosa in

each treatment (prawn pond).

contact with microcysin in sediment which were released

by dead Microcystis cells at the bottom of the pond.

Moreover, a number of studies have demonstrated that

MCs can be excreted quickly by fish. Soares [16] showed

that 48% of the total MCs ingested by Tilapia rendalli

were eliminated with feces during a 30-day experiment.

4. Conclusion

Tilapia and prawn aquaculture prefer nutrient-enriched

water (green water), produced by the addition of animal

manure or fertilizer, for supporting the growth of tilapia

and prawn. Pond management is essential for a produc-

tive aquaculture farm. In this sense, adequate nutrient

levels will allow the right biomass and structure of phy-

toplankton. An excessive supply of nutrients will result

in an over-enrichment that eventually will promote algal

blooms. Additionally, nutrients in excess will alter phy-

toplankton composition with a resulting change of do-

minant species; such changes imply the substitution of

larger species for smaller ones, particularly cyanobacte-

ria.

5. Acknowledgements

The authors thank the Graduated School, Environmental

Sciences Section and the Conservation and Utilization of

Biodiversity Project, Biology Department, Faculty of

Science, Chiang Mai University for providing a research

grant.

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