Evaluation of biochemical markers in the golden mussel Limnoperna fortunei exposed to glyphosate acid in outdoor microcosms

Highlights

• Impact of glyphosate acid on L. fortunei was assessed employing outdoor microcosms.

• The exposition to glyphosate acid altered biochemical processes in L. fortunei.

• TBARS increase and SOD and CES decrease indicate that glyphosate had adverse effects.

• Multibiomarker approach provides knowledge of the impact of glyphosate on L. fortunei.

Abstract

In this study, the impact of technical grade glyphosate acid on Limnoperna fortunei was assessed employing outdoor microcosms treated with nominal glyphosate concentrations of 1, 3 and 6 mg L⁻¹. At the end of the experiment (26 days), catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), acetylcholinesterase (AChE), carboxylesterases (CES) and alkaline phosphatase (ALP) activities, and lipid peroxidation levels were analyzed. GST and ALP activities and lipid peroxidation levels showed a significant increase with respect to controls in the mussels exposed to glyphosate (up to 90, 500 and 69 percent, respectively). CES and SOD activities showed a significant decrease in glyphosate exposed bivalves with respect to controls (up to 48 and 37 percent, respectively). CAT and AChE did not show differences between exposed and no exposed bivalves. The increase in lipid peroxidation levels and the decrease in SOD and CES activities observed in L. fortunei indicate that glyphosate had adverse effects on the metabolism of this bivalve. The results of the present study also indicate that a “multibiomarker approach” provides a more precise knowledge of the impact of glyphosate on L. fortunei.

Introduction

Worldwide, the intensive agricultural technologies are associated with the use of agrochemicals (Carvalho, 2006). These compounds can reach water bodies in a variety of ways, leading to a gradual increase in global water pollution (Nitschke and Schüssler, 1998). In Argentina, the continued use of agrochemicals plus the expansion of the agricultural frontier have augmented the areas where these compounds are applied. Given these facts, both agrochemical usage and their concentration in the environment have increased in recent years.

Glyphosate (N-phosphonomethyl glycine) is a broad-spectrum post-emergent systemic organophosphonate herbicide. Transgenic glyphosate-resistant soybean is one of the transgenic crops most used in the world and represents almost 52 percent of the total surface area of land cultivated with genetically modified organisms (James, 2010). Argentina is the second largest producer of soybean in the world. In this country, soybean crops occupy about 50 percent area out of the total cropped area, corresponding more than 99 percent to transgenic soybean (Aizen et al., 2009, Bonny, 2011, FAOSTAT, 2011). The scheme of application of glyphosate-based herbicides commonly used in these crops usually involves three sequential applications every 25–30 days (Blum et al., 2008, Strandberg and Bruus Pedersen, 2002).

Glyphosate can enter water bodies bordering fumigated lands through surface runoff, spray drift or direct overspray applications (World Health Organization, 2005). Information on glyphosate concentration levels in water bodies is more available from Europe and North American countries than from Argentina. Glyphosate levels exceeding the fresh water aquatic biota standard (65 μg L⁻¹) (CCME, 2007) and/or the drinking water limit (USEPA standard=0.7 mg L⁻¹ and European standard=0.1 μg L⁻¹) have been reported for a number of water bodies located in the following countries: France (165 μg L⁻¹), Canada (5.1–13.832 mg L⁻¹) and USA (1.2–1.7 mg L⁻¹) (CCME, 2007, CCME, 2012, IFEN, 2006, Miller et al., 2010, Page et al., 2006, World Helath Organization, 2005).

Though the consumption of glyphosate in Argentina reached 200 million liters in 2010 (CASAFE, 2011), few studies have determined glyphosate concentration levels in local water bodies. Peruzzo et al. (2008) have found levels of glyphosate of 0.1–0.7 mg L⁻¹ in water bodies located near agricultural plantations of the Pampa Region (Buenos Aires, Argentina). These values depended on the time elapsed between glyphosate application and rainfall. On the other hand, Sobrero et al. (2007) have considered that the concentration of glyphosate in argentine water bodies would cover a range of 0.1–80 mg L⁻¹. The lowest concentrations would correspond to water bodies located near to fumigated crop fields whereas the highest would correspond to water bodies that have received accidental spills or intentional dumping (washing of containers and machinery).

Glyphosate is highly soluble in water (12 g L⁻¹ at 25 °C) and its half-life can vary from a few days to 60 days, depending on the conditions in the environment (Vereecken, 2005). Glyphosate primary mechanism of action is by inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the production of aromatic amino acids in plants and microorganisms.

Although glyphosate has been considered a low-toxicity compound for animals, several studies have reported adverse effects on terrestrial and aquatic organisms. In particular, glyphosate can alter the survival, growth and development of several aquatic species, such as microalgae, protozoa, crustaceans and bivalves (Bringolf et al., 2007, Conners and Black, 2004, Fuentes et al., 2011, Mottier et al., 2013, Relyea, 2005, Tsui and Chu, 2003). Glyphosate can also alter several metabolic parameters that have been proposed as biomarkers of exposure. These biomarkers include parameters related to oxidation of biomolecules, and to changes in the levels of antioxidant, detoxificant and general metabolism enzymes (Romero et al., 2011).

Alkaline phosphatase (ALP) activity is considered an indicator of the general metabolic state of the organisms. Van Dyk and Pletschke (2011) have suggested its use for the detection of organochlorine, carbamate and organophosphate pesticides, and alterations in ALP activity have been detected in fish exposed to glyphosate (Jiraungkoorskul et al., 2003, Neškovic et al., 1996). Alterations in the detoxification systems such as increases in the activity of glutathione-S-transferase (GST), and in the expression of cytochrome P450 gene 4 family (CYP4) have also been reported in oligochaetes, amphibians and cladocerans (Contardo-Jara et al., 2009b, Lajmanovich et al., 2011, Le et al., 2010).

Several herbicides increase reactive oxygen species (ROS) levels, triggering lipid damage and antioxidant responses in the cell (Agrawal and Sharma, 2010). These antioxidant responses include increases in the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, and glutathione reductase, among others. There are evidences that glyphosate or its commercial formulations are able to increase oxidative damage and antioxidant defences in oligochaetes, amphibians, fish, and microalgae (Contardo-Jara et al., 2009b, Costa et al., 2008, Lushchak et al., 2009, Modesto and Martinez, 2010, Romero et al., 2011). These facts indicate the participation of oxidative stress processes in the toxic mechanism of action of glyphosate in these species. Furthermore, different authors have analyzed the effects of commercial formulations of glyphosate on biomarkers usually proposed for organophosphate pesticides, such as acetylcholinesterase (AChE) and carboxylesterases (CES). These studies have shown inhibition of AChE activity in the silver cat fish Rhamdia quelen (Glusczak et al., 2007), and inhibition of AChE and CES activities in Rhinella arenarum tadpoles (Lajmanovich et al., 2011).

Bivalves are sessile filter-feeders organisms that have been established as suitable bioindicators for monitoring water quality in water bodies subjected to potentially pollution risk (i.e. Mussel Watch Program, Sericano et al., 1995). The ability of bivalves to bioaccumulate toxic compounds determines their participation in the transfer of environmental contaminants to higher trophic levels (Hunt et al., 2003). The golden mussel Limnoperna fortunei (Mytilidae, Bivalvia) is an invasive bivalve of China and Southeast Asia origin (Darrigran and Ezcurra, 2000, Ricciardi, 1998). The presence of L. fortunei in the Río de la Plata basin, Argentina, was registered for the first time in 1991 (Pastorino et al., 1993). Since then, L. fortunei has expanded to other areas in the country, due to the characteristics of its reproductive cycle and its high adaptability to different environmental conditions such as high load of organic matter (Darrigran and Ezcurra, 2000). The great phenotipic plasticity, high fecundity and byssal attachment to different substrates that characterizes this species allow L. fortunei to colonize new environments, with the consequence of a wide spatial and temporal distribution of the species. Thus, the specimens of L. fortunei can be collected in great quantity in different seasons of the year. All this, together with its ability to adapt to captivity makes L. fortunei a very useful tool for biomonitoring polluted water bodies and a suitable model organism for the study of the impact of pesticides and other contaminants on biota.

In the present study, specimens of L. fortunei were exposed to environmentally relevant concentrations of glyphosate in microcosms simulating a representative ecological environment of the Pampa Region of Argentina. The experimental design aimed to recreate the impact of a time period of exposure to glyphosate (26 days) similar to the elapsed time between two applications of the pesticide in an agricultural scheme. Our hypothesis was that the exposition of L. fortunei to glyphosate in conditions that mimicked its natural environment may affect biochemical parameters related to oxidation of biomolecules and to changes in the levels of antioxidant, detoxificant and general metabolism enzymes. The aims were (1) to analyze the impact of glyphosate on TBARS levels, and SOD, CAT, AChE, CES, GST and ALP activities in L. fortunei, and (2) to assess the suitability of these parameters as biomarkers of glyphosate exposure in this mussel.