When sufficient resources are available, cyanobacteria may proliferate and bloom in reservoirs, lakes, rivers, estuaries, and coastal systems, where they may cause a multitude of water quality concerns, such as producing malodor, causing nocturnal oxygen deficiency leading to fish kills, disrupting the aquatic ecosystem, and posing a health hazard to wildlife, game, and humans, because of their potential to produce strong toxins (cyanotoxins) [1
]. Cyanobacteria can cause acute or chronic toxicity to animals and humans via different exposure routes, such as contaminated drinking water, fish or shell fish, through crops irrigated with cyanobacteria-infested waters, or through recreational exposure. Numerous animal poisonings associated with exposure to cyanobacteria have been reported by Hudnell et al., 2008 [3
]. The potency of the cyanobacterial toxins is underpinned by the death of 30 kg dogs exposed to anatoxins [4
] and microcystins [5
]. Three cows and ten calves died in northwest Queensland in 1997 and 148 people were hospitalized in Palm Island in 1979 after expoxure to cyanobacteria [6
]. In total, 458 suspected human illnesses and 175 animal deaths associated with cyanobacterial bloom events have been reported in the U.S.A. during 2007–2011 [9
Cyanotoxins are produced by several cyanobacterial species amongst others belonging to the genera Aphanizomenon
, and Oscillatoria
]. The most widespread and notorious class of cyanotoxins are the microcystins (MCs) that are known as non-ribosomal processed cyclic heptapeptides. The general structure is cyclo(-d
is (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid and X and Z are variable l
-amino acids on the 2 and 4 positions, which contribute mostly to the dozens of variants of MCs that have been detected. The amino acids on the 3 (d
-MeAsp) and 7 (d
-Glu6-Mdha) positions can also occur as demethylated variants. MCs are potent inhibitors of protein phosphatases, but the toxicity of different variants to mice varied substantially, where replacement of the hydrophobic leucine (L) in the first variable position with a hydrophilic amino acid (e.g., arginine, R) that dramatically reduces toxicity [11
Southern Vietnam, including the Mekong Delta, is a large area with lakes, ponds, rivers, primary canals, and reservoirs. It includes large systems such as the Mekong river, DongNai river, TriAn reservoir (323 km2
), DauTieng reservoir (264 km2
), and BinhThieng reservoir (192 hectare) and numerous smaller canals, streams and fish, shrimp, and duck ponds that are all vulnerable to point source pollution by sewage, ducks, and local fish farming. Consequently, these sites present a high risk for developing cyanobacterial blooms and as they are often in close vicinity to urban settlements, citizens might be at high risk of exposure to cyanobacterial toxins. In aquaculture in the Mekong Delta, cyanobacteria-infested water is commonly treated chemically, i.e., by chlorine or copper sulphate, which may then lead to high water concentrations of dissolved cyanotoxins from cell lysis [12
]. Hence, fish and other aquatic animals grown in the aquaculture ponds may contain cyanotoxins posing a potential risk to consumers (Figure 1
a,b). Surface water collected directly from the water bodies by local water supply stations in the Mekong Delta is generally treated by rock and sand filters in combination with chlorinated disinfection before supplying the water to the local communities [14
]. However, when cyanobacterial blooms occur in these water bodies, the presence of cyanotoxins in treated drinking water cannot be totally excluded. Local residents in the Mekong Delta use the surface water for daily bathing and washing (Figure 1
c,d). Hence, the presence of cyanobacteria in the water bodies mentioned above might infer a health risk to the local people. However, up-to-date information on cyanobacteria and cyanotoxins in southern Vietnam is very limited.
Studies on cyanobacteria in Vietnam have mainly focused on morphological taxonomy [15
]. Few later studies touched upon microcystins (MCs) producing cyanobacteria in natural lakes and reservoirs and showed the occurrence of microcystin variants MC-RR, MC-dmRR, MC-YR, MC-LA, MC-LY, and MC-WR in isolated cyanobacteria and field samples [21
]. A first report on MC accumulation in fish and bivalves was recently published by Pham et al. [25
], in which MCs concentrations varying from 0.06 to 3.15 µg MCs/g DW were determined in three fish and two bivalves collected in Dau Tieng Reservoir. The study areas and cyanobacterial samples in those studies are limited to a few water bodies, mainly large reservoirs. However, cyanobacteria blooms in small ponds and canals used for collecting drinking water or to cultivate fish or ducks may also pose a health risk to local people. We hypothesize that the cyanobacteria blooming in these small water bodies produce MCs and that MCs are also present in animals living in these water bodies. The aims of the current study were, therefore, (1) to determine the occurrence of cyanobacterial blooms and MCs; (2) to measure the MC content in isolated cyanobacteria strains; (3) to quantify the MC content in animals living in water bodies suffering from cyanobacteria; and (4) to assess health hazards caused by MC exposure to local people via estimated daily intake (EDI).
Eutrophication and hyper-eutrophication with the accumulation of high cyanobacterial biomass were observed in several water bodies (reservoirs, rivers and small ponds) in southern Vietnam. Eutrophication and cyanobacterial blooms in small ponds, where fish and/or ducks were cultivated, were more serious than those in reservoirs and rivers. Microcystis was the main potential toxin producer and the most common bloom-forming species in southern Vietnam. The MC concentrations ranged from <LOD to 11,039 µg/L or to 4033 µg/g DW in field samples. MCs were only found in isolated Microcystis strains and were <LOD in other isolates, including Anabaena, Anabaenopsis, and Planktothrix strains
MC-LR and MC-RR variants were most frequently found and the most abundant MC variants in MC-containing field samples. Three MC variants—MC-dmLR, MC-LW, and MC-LF—were recorded in Vietnam for the first time. MC-LR, MC-RR, and MC-LF significantly contributed to the total toxicity of MC-containing samples.
The MC content in fish was higher than in shrimp and snail. MC was mainly found in the visceral mass, liver, and gut, so consuming whole MC-containing fish and snails is not safe. It is strongly recommended that the whole viscera of fish and snails must be completely removed during food processing, especially when the animals are collected from water bodies with a high cyanobacteria biomass. The suckermouth catfish should be considered as an ornamental fish, while it is not a safe food source.
Cyanobacterial monitoring programs should be established to assess and minimize potential public health risks.
5. Materials and Methods
Seventeen water bodies including rivers, lakes, ponds, canals, and reservoirs in the vicinity of urban settlements throughout the Mekong basin, and in Southeast Vietnam (Figure 5
) were sampled once during the dry season (December–May/June) to assess blooms, potential eutrophication effects and resulting cyanobacteria and MCs.
At each collecting site, temperature, salinity, and pH were measured by pH/Cond 340i meter (WTW, Weilheim, Germany). Cyanobacterial-chlorophyll-a was measured with the bbe AlgaeTorch which is a lightweight instrument for the simultaneous quantification of the chlorophyll-a content of cyanobacteria and the total chlorophyll content of microalgae in water. (bbe Moldaenke GmbH, Schwentinental, Germany). Samples from sampling sites where Chl-a was higher than 200 µg/L were measured in a bucket after the dilution of collected scum material with tap water to maintain the advised measuring range for the AlgaeTorch. Cyanobacterial scum samples were also collected for isolation and filtration; sub-samples were preserved (in Lugol’s iodine) for microscopic analysis (dominant cyanobacterial species). In addition, 1 to 300 mL of surface water from water-bloom sites was filtered through GF/C filters. The filters and filtrates were stored at −20 °C upon MC analysis. Animals in infested water bodies with cyanobacterial bloom were collected to determine MCs in their tissues.
Samples for nutrient analysis were kept on ice and transported within 24 h to the laboratory of Water Quality, Institute for Environment and Resources where nutrients were analyzed colorimetrically with a spectrophotometer (DR/2010, Hach, Loveland, CO, USA) using the following APHA (2005) [59
] methods: Nitrate 4500NO3−
, ammonium 4500NH4+
, total nitrogen (TN) Kjeldahl 4500N, phosphate, and total phosphorus (TP) 4500P. The detection limits of the equipment for these parameters were 0.02 mg/L (nitrate), 0.04 mg/L (ammonium), 0.06 mg/L (TN Kjeldahl), and 0.05 mg/L for both TP and phosphate.
5.2. Strains Isolation
In the laboratory, single Microcystis
cells or colonies were picked out of the collected scum material by the micropipette-washing method [60
]. These isolates were grown in small glass tubes with a few mL modified WC medium (Woods Hole modified CHU10-medium) [61
] for several months at 25 °C, under a 14:10 h light/dark cycle at a light intensity of 70 µmol photon/m/s. When isolates reached a greenish appearance, they were transferred into 50 mL Erlenmeyer flasks and subsequently into 250 mL flasks. In total, there were 24 isolated strains (Table A1
and Table A2
, Appendix B
5.3. MC Analysis
The frozen filters stored at −20 °C were transferred to 8 mL glass tubes and dried for two hours in a freeze-drier (Alpha 1-2 LD, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Tissue and scum samples were also dried for several hours in the freeze-drier and 5 to 8 mg freeze-dried material was then transferred to 2 mL Eppendorf vials.
The filters, the tissue and scum samples were extracted three times at 60 °C in 2.5 and 0.5 mL 75% methanol and 25% Millipore water (v/v). The extracts were then dried in the Speedvac (Savant SPD121P, Thermo Scientific, Waltham, MA, USA) and subsequently reconstituted in 900 μL 100% methanol. The reconstituted samples were transferred to 2 mL Eppendorf vials with a cellulose-acetate filter (0.2 μm, Grace Davison Discovery Sciences, Deerfield, IL, USA) and centrifuged for 5 min at 16,000× g (VWR Galaxy 16DH, VWR International, Buffalo Grove, IL, USA). Filtrates were then transferred to amber glass vials for LC-MS/MS analysis. If needed, samples with high MC concentrations were diluted in methanol before re-analysis.
Concentrations of eight MC variants (dm-7-MC-RR, MC-RR, MC-YR, dm-7-MC-LR, MC-LR, MC-LY, MC-LW, and MC-LF) and nodularin (NOD) were determined by LC-MS/MS as described in [5
]. LC-MS/MS analysis was performed on an Agilent 1200 LC and an Agilent 6410A QQQ (Agilent Technologies, Santa Clara, CA, USA). The MCs were separated on an Agilent Eclipse 4.6 × 150 mm, 5-µm column. Hereto, a 10 µL sample was injected; the flow rate was 0.5 mL/min; the column temperature was 40 °C. Eluents were Millipore water with 0.1% formic acid (v
, Eluent A) and acetonitrile with 0.1% formic acid (v
, Eluent B) that were run using an elution program of 0–2 min 30% B, 6–12 min 90% B, with a linear increase of B between 2 and 6 min and a 5-min post run at 30% B. Detailed information on MS/MS settings for each MC can be found in [41
]; information on the recovery, repeatability, limit of detection, and limit of quantification of the analysis is given in [5
]. MCs were quantified against certified standards that were obtained from DHI LAB Products (Hørsholm, Denmark).