Elsevier

Harmful Algae

Volume 27, July 2013, Pages 88-97
Harmful Algae

Accumulation and detoxification dynamic of cyanotoxins in the freshwater shrimp Palaemonetes argentinus

https://doi.org/10.1016/j.hal.2013.05.007Get rights and content

Highlights

  • MCLR and Nod accumulated in the shrimp P. argentinus.

  • MCLR accumulated is removed after exposure ends (74% after 3 days).

  • GST and GR seems to be involved in MCLR removal after exposure ends.

  • First report of Nod in natural fresh water in South America.

  • Low levels of Nod caused biochemical disturbances in P. argentinus.

Abstract

The uptake and accumulation of microcystin-LR (MC-LR) in the shrimp Palaemonetes argentinus was investigated using both laboratory and field assays. Shrimps were exposed in aquarium during 1, 2, 3 and 7 days to 1, 10 and 50 μg L−1 MCLR. Accumulation (0.7 ± 0.2 μg MC-LR g−1) was observed after three days exposures to 50 μg L−1 toxin. Then, shrimps were relocated in fresh water (free of MCLR) to verify the detoxification dynamic, showing a drop to 0.18 ± 0.01 μg MCLR g−1 after three days. The activity of glutathione-S-transferase, measured in the microsomal fraction (mGST), was significantly increased during the exposure period, with further increment during the detoxification period. Furthermore, cytosolic GST (sGST) and glutathione reductase (GR) increased their activities during detoxification, while inhibition was observed for catalase (CAT) with no significant changes for glutathione peroxidase (GPx). Current results suggest that GSH conjugation could be an important MC detoxification mechanism in P. argentinus and that MCLR induce oxidative stress in this shrimp.

Field exposures were carried out in San Roque Reservoir (Córdoba, Argentina) after a cyanobacteria bloom. Nodularin (Nod) presence was measured for the first time in this waterbody (0.24 ± 0.04 μg L−1), being the first report of Nod in South America freshwaters. Nod was also detected in Palaemonetes argentinus (0.16 ± 0.03 μg g−1) after three weeks of exposure in this reservoir, with the concomitant activation of mGST, sGST and CAT.

Although internal doses of Nod were low throughout the exposure, they were enough to cause biochemical disturbances in Palaemonetes argentinus.

Further laboratory studies on Nod accumulation and antioxidant responses in Palaemonetes argentinus are necessary to fully understand these field results. P. argentinus should be considered a potential vector for transferring cyanotoxins to higher trophic levels in aquatic environments.

Introduction

Serious eutrophication accompanied with the presence of massive cyanobacterial blooms and cyanotoxins have been documented in many inland waters worldwide (Gurbuz et al., 2009). Among cyanotoxins, microcystins (MCs) and nodularins (Nod) are considered the most dangerous groups, mainly because both are potent hepatotoxins (for a review see: Zurawell et al., 2005, Wiegand and Pflugmacher, 2005) and tumor promoters (Nishiwaki-Matsushina et al., 1991, Nishiwaki-Matsushina et al., 1992). MCs are constituted by seven amino acid (one characteristic, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid-ADDA, and 2 variable), while Nod is formed by five amino acid (preserving the characteristic amino acid ADDA; Sivonen et al., 1989).

In natural environments, MCs were found in a wide range of aquatic biota such as fish (Mohamed et al., 2003, Cazenave et al., 2005, Deblois et al., 2008, Chen et al., 2009, Amé et al., 2010), shrimps (Chen and Xie, 2005, Pflugmacher et al., 2005), gastropods (Chen et al., 2005, Zhang et al., 2012), bivalves (Gerard et al., 2009), and aquatic plants (Mitrovic et al., 2005). Cyanotoxins were present in diverse organs but also in the muscle and other edible parts of studied animals. On the other hand, the accumulation of Nod has been observed in flounders, mussels, clams (Sipiä et al., 2002) and eiders (Sipiä et al., 2008).

To our knowledge, there are not reports on the presence of cyanotoxins in Palaemonetes argentinus, which is omnivorous, feeding throughout the water column, preying small components of the plankton and benthos. P. argentinus is one of the most widely distributed decapods in the littoral region of Argentina, Paraguay, Uruguay and southern Brazil, inhabiting freshwater ponds and lakes (Morrone and Lopreto, 1995). Despite its important role in the food chain, little is known about the dynamics of accumulation–detoxification of cyanotoxins in this shrimp.

In according to Ito et al. (2002), the toxicity of MC depends on the balance between accumulation and metabolism. MC conjugation with glutathione (GSH) is catalyzed by glutathione-S-transferases (GST) in different aquatic organisms such as plant, mollusk, crustacean and fish. This conjugation is generally considered the primarily route for MC detoxification in aquatic organisms, by forming more polar compounds, facilitating the excretion (Pflugmacher et al., 1998, Beattie et al., 2003). However, upon uptake, absorption and distribution, it can inhibit in the liver the key regulatory enzymes: serine-threonine protein phosphatases (PPs) 1 and 2A (Yoshizawa et al., 1990, Honkanen et al., 1994, Annila et al., 1996). Furthermore, some evidences suggest a close connection between cellular hyperphosphorylation state and oxidative stress generation induced by the exposure to MCs (Amado and Monserrat, 2010). When oxidative stress takes place in the cell, the antioxidant system is activated. This system comprises enzymatic antioxidant defense such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) (Amado and Monserrat, 2010).

The main goal of this work was to examine the uptake, detoxification and accumulation of MCLR in shrimps, experimentally exposed to MCLR dissolved in water. Additionally, we aimed to verify laboratory results obtained by evaluating the accumulation of cyanotoxins in shrimps exposed in their natural habitat after a cyanobacteria bloom. The antioxidant enzymatic activities (CAT, GR, GPx and GST) were also measured to evidence the oxidative stress induced by cyanotoxin exposure.

Section snippets

Standard cyanotoxins

MCs and Nod used in different assays and measurements were provided by Sigma–Aldrich (Sigma–Aldrich Argentina S.A.).

Animals

Freshwater shrimps (31.6 ± 6.2 mm, 0.15 ± 0.03 g), used for laboratory exposures, were obtained from free MCs natural water (San Antonio River, Córdoba, Argentina) and then acclimated to controlled aquarium conditions during 20 days (water reconstituted from deionized water with 100 mg L−1 sea salt, 200 mg L−1 CaCl2, 103 mg L−1 NaHCO3, light/dark cycle of 12 h:12 h, temperature 21 ± 1 °C). Shrimps

Test of solvent efficiency for microcystin extraction from P. argentinus

Our current results reveal that the best solvent to extract MCLR from Palaemonetes argentinus was methanol 70%:TFA 0.1% (recovery = 112 ± 7% of MCLR spiked). On the other hand, extraction using BuOH:MeOH:H2O (1:4:15) resulted in recoveries below 10–20%.

The extraction solvents used for the extraction of MCs from animal tissues vary greatly in the literature (Barco et al., 2005, Smith and Boyer, 2009). The solvent selected for Palaemonetes argentinus was successfully used also in the mussel (Mytilus

Laboratory exposure

Exposure to cyanotoxins represents a health risk to aquatic organisms, wild life, domestic animals, and humans upon drinking or ingesting these compounds (Malbrouck and Kestemont, 2006). Although these toxins are rarely ingested by human in quantity high enough to reach a lethal acute dose, chronic toxic effects from exposure through food need to be considered, especially if there is long-term frequent exposure (Magalhães et al., 2001). Palaemonetes argentinus in all life cycle stages were

Conclusions

The present study confirms the bioaccumulation of MCLR in Palaemonetes argentinus. However, MCLR was partially eliminated from the shrimp after transferring to free-MC medium (74% after 3 days). Increased GST activity suggests that GSH conjugation can be an important MC detoxification mechanism also in P. argentinus. However, during laboratory exposure, it seems that GST in not mainly involved in the elimination of MCLR. Alternative mechanisms, like MXR, could be responsible for MCLR removal

Acknowledgements

This work was supported by grants from the Agencia Nacional de Promoción Científica y Técnica (FONCyT-PICT/2007 1209 and 1225), Secretaría de Ciencia y Técnica (SECyT) and CONICET (National Research Council, Argentina).[SS]

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