Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes
Introduction
The non-selective, post-emergence herbicide glyphosate (N-(phosphonomethyl)glycine) is one of the most widely used pesticides. It is utilized for weed control, i.e. in agriculture, forestry, urban areas and even aquaculture. It is usually formulated as the isopropylamine salt and in case of the most common formulation Roundup Ultra combined with the surfactant polyoxyethylene amine (POEA) (Giesy et al., 2000). Due to cultivation of genetically modified glyphosate-resistant crops, the use of glyphosate clearly increased during the last years, raising again concerns regarding the potential environmental impact of this herbicide (Kolpin et al., 2006).
Due to its high adsorption tendency in soil (Kd values up to 900 L kg−1) and its fast degradation by microorganisms, glyphosate is generally regarded as having low potential to contaminate surface waters or groundwater (Borggaard and Gimsing, 2007, de Jonge et al., 2001, Vereecken, 2005). However, leaching of glyphosate and its degradation product AMPA (amino-methylphosphonic acid) up to 1 m depth has been observed in laboratory and field studies, suggesting a potential risk for the aquatic environment (Kjaer et al., 2005, Landry et al., 2005, Stadlbauer, 2005). Moreover, glyphosate and AMPA are present in surface waters worldwide in considerable concentrations, e.g. up to 2.2 μg L−1 in US rivers (Feng et al., 1990, Kolpin et al., 2006). The limit value for single pesticides in groundwater in Europe is 0.1 μg L−1 and for the sum of pesticides 0.5 μg L−1 (CEC, 1991). Because of the high water solubility of glyphosate (10 g L−1) and its increasing occurrence in the aquatic environment, the herbicide and its formulations have a growing relevance for aquatic ecotoxicology. The toxicity of technical-grade glyphosate to aquatic invertebrates is known to be only minor (LC50 values of >55 mg L−1 glyphosate, WHO, 1994). However, an increased toxicity of glyphosate formulations like Roundup Ultra has been reported and related to its surfactants, e.g. POEA (Bringolf et al., 2007, Giesy et al., 2000, Tsui and Chu, 2003, Tsui and Chu, 2004). Lower toxicity thresholds (LC/IC50 < 2 mg a.i. L−1) were determined for the amphipod Hyalella azteca, the copepod Acartia tonsa and the algae Sceletonema costatum, respectively (Tsui and Chu, 2003, Tsui and Chu, 2004).
The blackworm Lumbriculus variegatus (Muller), a typical sediment dwelling invertebrate, was selected to study the impact of glyphosate and Roundup Ultra. The oligochaete L. variegatus, native to European and North American river and lake sediments, feeds on decomposing plant material and microorganisms. It shows moderate sensitivity to xenobiotics (Drewes, 1997). Scientific studies conducted with L. variegatus focused on the bioaccumulation and depuration of organic substances and metals in the worm, as well as on the biological impact of the xenobiotics, using acute toxicity, the locomotor and burial behavior or the feeding rate as toxicological endpoints (Landrum et al., 2004, Leppanen and Kukkonen, 2004, Maenpaa and Kukkonen, 2006). Only few studies have been conducted to gain more insight in the enzymatic and physiological response of the organism when exposed to anthropogenic pollutants. Activity of the cytochrome P-450 enzymes seems to be limited (Ankley and Collyard, 1995). Enzymes as delta-aminolevulinic acid dehydratase (ALA-D) or inhibition of cholinesterase (ChE) activity, as biomarker of lead or organophosphorous pesticides exposure, respectively, have been recognised to be useful (Aisemberg et al., 2005, Kristoff et al., 2006). Also glutathione S-transferase (GST) and catalase (CAT) reacted to an exposure of the organism to the herbicide paraquat or to dissolved organic matter (Wiegand et al., 2007), as well as to atrazine and contaminated sediments (Contardo-Jara and Wiegand, 2008). Physiological endpoints proved to be reliable indicators for exposure and for negative sub-lethal impact of pollutants on the organism, especially activity changes of the biotransformation system enzymes, modifying chemical substances, or the enzymes involved in the defence against oxidative stress, preventing cell damage of proteins, lipids, and DNA. To our knowledge, this is among the first studies assessing superoxide dismutase (SOD) activity in L. variegatus as biomarker for oxidative stress.
The aim of this study was to investigate if a sediment living organism, such as L. variegatus is negatively influenced by an exposition to pure glyphosate and the formulation Roundup Ultra in surface waters. Therefore we studied (1) the bioaccumulation in the worm tissue and (2) the activity changes of the biotransformation enzymes soluble GST (sGST) and membrane bound GST (mGST), as well as of the antioxidant enzymes SOD and CAT in response to increasing concentrations of glyphosate and Roundup Ultra in water.
Section snippets
Rearing of the organisms
The culture of L. variegatus, originating from NOAA/Great Lakes Environmental Research Laboratory, Ann Arbor, Michigan, USA, reproduces exclusively asexual through fragmentation (Lesiuk and Drewes, 1999) ensuring a genetically homogenous worm population. The worms were reared according to Leppanen and Kukkonen (1998) in 5 L glass tanks containing artificial tank water reconstituted from Milli-Q-grade water (100 mg L−1 Instant Ocean sea salt, 200 mg L−1 CaCl2, 103 mg L−1 NaHCO3) at 20 ± 1 °C and a
Bioaccumulation study
An average dry weight (dw) of 12.6 ± 0.5% and a lipid content of 1.6 ± 0.3% of fresh weight were determined for L. variegatus.
The glyphosate concentration in the test medium was constant over the 4-day test period. The degradation product AMPA (amino-methylphosphonic acid) was not detectable.
With increasing glyphosate concentrations, the total amount of glyphosate accumulated in L. variegatus per gram fresh body mass increased, in both, the pure glyphosate and the Roundup Ultra treatments (Fig. 2).
Discussion
In this work we studied the bioaccumulation potential and physiological effect of pure glyphosate and the formulation Roundup Ultra in an oligochaete. L. variegatus was shown to be suitable for determination of both parameters, the bioaccumulation in animal tissue and changed enzyme activity of the biotransformation system, e.g. GST and of the antioxidant defense, e.g. SOD and CAT.
Conclusion
This study shows that glyphosate accumulates in L. variegatus, despite the hydrophilic character of the herbicide. The accumulated amounts of glyphosate and the added surfactants in Roundup Ultra cause an elevation of the biotransformation enzyme sGST at non-toxic concentrations. The accumulation and the enzymatic response of the worms were clearly higher in the animals exposed to Roundup Ultra, indicating that the formulation Roundup Ultra is of more ecotoxicological relevance than the
Acknowledgements
We would like to thank the German Research Foundation (Deutsche Forschungsgemeinschaft – DFG) for providing a PhD grant for V. Contardo-Jara and E. Klingelmann within the graduate school 780/II “Perspectives on Urban Ecology II”. We are indebted to the Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry (JKI), for providing laboratory facilities enable to the handling of 14C. The authors thank S. Chakrabati and J. Krüger for helpful technical assistance and S.
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Both authors contributed equally to the experimental work and the manuscript.