An invasive mussel is in trouble: How do glyphosate, 2,4-D and its mixture affect Limnoperna fortuneiʹs survival?
Graphical abstract
Introduction
An anthropogenic factor of environmental change is defined as any modification driven by human activities affecting ecosystems either directly or indirectly. In aquatic ecosystems, contamination and the presence of invasive species have been major anthropogenic factors over the last 50 years (Millennium Ecosystem Assessment 2005). The fact that these factors act simultaneously emphasizes the importance of studying their joint effects to prevent unpleasant "ecological surprises” (Townsend et al. 2008).
The golden mussel Limnoperna fortunei (Dunker) stands out among freshwater alien species in South America (Boltovskoy 2015). This mytilid bivalve is characterized by showing heteromyarian shell morphology, byssal attachment to solid substrata, and gregariousness. It occurs naturally in China, Thailand, Korea, Laos, Cambodia, Vietnam, and Indonesia (Ricciardi 1998) and was accidentally introduced into South America through ship ballast waters about three decades ago (Pastorino et al. 1993). The species has been reported to reach high densities in natural environments (150,000 ind. m–2) as well as in invaded areas (Darrigran and Pastorino 2004). Golden mussels are able to colonize soft-water habitats, heated waters, and organically-enriched waters (Morton 1977; Deaton et al. 1989; Darrigran and Pastorino 1995). Due to their high filtering capacity, they act as ecosystem engineers (Sylvester et al. 2005) that can alter the abundance and composition of plankton (Cataldo and Boltovskoy 2000), modify trophic interactions and food availability of benthic and pelagic species, increase the depth of the photic zone, thus favoring macrophytic growth (Boltovskoy et al. 2009), and affect oxygen availability and rates of sedimentation and nutrient recycling (Karatayev et al. 2007).
Agrochemicals used in industrial agriculture are considered contaminants in both terrestrial and aquatic ecosystems (Schwarzenbach et al. 2010; Banin and Kafkafi 2015). Glyphosate (N-phosphonomethyl glycine) is a nonselective, broad-spectrum and post-emergence herbicide extensively used worldwide. It inhibits the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme involved in the shikimic acid pathway. This causes the cessation of aromatic amino acid biosynthesis, followed by reduced protein synthesis, growth, and premature cellular death (Lydon and Duke 1988).Currently, the appearance of glyphosate-resistant weeds has led to the combined use of glyphosate and other herbicides (Metzler et al. 2011), such as 2,4-D (2,4-Dichlorophenoxyacetic acid) (Singh and Singh, 2016). The 2,4-D is an auxin-type, systemic herbicide inducing overgrowth of vascular cambium in dicots, widely used for post-emergence control of annual and perennial weeds (Raghavan et al. 2004).
Both glyphosate and 2,4-D are water-soluble and their residues in crop fields can be transferred to surrounding aquatic environments by surface runoff (Kloeppel et al. 1997). Glyphosate contributes to the acceleration of eutrophication in freshwater systems (Pérez et al. 2007; Vera et al. 2010, 2012), while 2,4-D modifies microbial richness, functional diversity and activity (Aguayo et al. 2014). Moreover, the commercial 2,4-D-based formulation AsiMax 50® has been reported to induce structural changes in phyto- and picoplankton communities both in turbid and clear freshwater systems (Lozano et al. 2019). There is little information on the environmental impact of mixtures of glyphosate and 2,4-D. Lozano et al. (2018) observed a concentration-dependent effect of a glyphosate and 2,4-D mixture on phytoplankton, with a significant decrease in abundance and important changes in primary production (Lozano et al. 2020). According to Lozano et al. (unpublished data), the effect of the glyphosate and 2,4-D mixture is likely to vary according to the functional properties of freshwater, as evidenced by significant differences in dissolved oxygen concentration between mesotrophic and eutrophic systems. These authors observed that dissolved oxygen concentration showed a sinusoidal dynamic, with decreasing and increasing values of herbicide concentration in water after application of a single dose of glyphosate, 2,4-D or their mixture.
There are some physical and chemical drivers that decrease the fitness of L. fortunei. For example, low-level sublethal chlorination was observed to weaken byssal threads (Rajagopal et al. 1998). As a result, mussels became detached from the substrate and although this increased mortality, it did not reach 100% (Cataldo et al. 2003). L. fortunei showed no significant mortality at glyphosate concentrations of up to 40 mg/L (Di Fiori et al. 2012) and the interaction between mussels and this herbicide had strong synergistic effects on the structure and function of freshwater ecosystems (Pizarro et al. 2015; Gattás et al. 2016; De Stéfano et al. 2018).
Freshwater systems in the Pampa plain, the main agricultural region of Argentina, are increasingly contaminated with agrochemicals (Pérez et al. 2017). In this region, the presence of glyphosate has been detected in shallow lakes of Buenos Aires Province (Castro Berman et al. 2018) but little is known about the occurrence of 2,4-D. Notably, although these freshwater systems are in the vicinity of the entry site of L. fortunei, they have not been invaded. In an attempt to identify possible reasons for its absence, in the present study we conducted different experiments.
The fact that glyphosate, 2,4-D and their mixture change the dissolved oxygen dynamics in water led us to question if these changes affect musselʹs survival and if there are differences when herbicides are applied either combined or separately. In an effort to answer these questions, we carried out two successive manipulative experiments at different scales: first, a 22-d indoor microcosm experiment under controlled conditions with and without air supply to analyze a possible impact of changes in dissolved oxygen on the survival of mussels exposed to glyphosate, 2,4-D and their mixture; and then, an 18-d outdoor mesocosm experiment without air supply to compare the effect of both herbicides and their mixture on musselʹs survival. We tested the following hypotheses: 1. Changes in dissolved oxygen in water induced by glyphosate, 2,4-D and their mixture cause a significant mortality in L. fortunei; 2. The impact on musselʹs survival differs if these herbicides are used separately or in combination. 3. The mixture of herbicides acts additively on musselʹs survival.
Section snippets
Collection of Limnoperna fortunei mussels
We selected mussels with mean shell length of 19.7 ± 0.9 mm, which were manually collected on the banks of the Rio de la Plata estuary, near Buenos Aires City, Argentina (34° 32’ 15’’ S, 58° 26’ 30’’ W) and carried immediately to the laboratory. They were carefully separated from the substrate and acclimated in aquaria with dechlorinated water for one week under constant temperature (24 ± 1°C), natural photoperiod, and continuous aeration. During this period, mussels were fed a daily diet of
Indoor Experiment
The effect of air supply on daily DO fluctuation in the presence of L. fortunei is shown in Fig. 1. I-CA revealed no significant temporal fluctuations, with a mean DO value of 8.6 ± 0.2 mg/L throughout the study (Fig. 1a). In the absence of air supply, DO dropped significantly in I-C (Fig. 1b) compared with I-CA from days 1 to 7 (GLMM p<0.05), reaching a minimum of 4.7 ± 0.3 mg/L on day 3. A significant increase was observed from day 12 onward (GLMM p<0.05), rising to a maximum of 13.2 ± 1.7
Discussion
Our results indicated that glyphosate and 2,4-D had a negative effect on Limnoperna fortunei survival providing new insight into the harmful impact of herbicides on non-target organisms. In particular, this tended to be higher in scenarios with glyphosate applied alone or mixed with 2,4-D, supporting previous evidence of its toxic effect on animals (Gill et al. 2018). Moreover, DO fluctuations in water seemed to play a key role in the survival of mussels. Changes in DO due to the combination of
Conclusions
Our work demonstrated that the herbicides glyphosate and 2,4-D, both in pure form, had a negative effect on the golden mussel Limnoperna fortunei survival providing new insight into the harmful impact of herbicides on non-target organisms. Herbicides produce significant changes in DO dynamics, being more pronounced when glyphosate is present. These disturbances of DO in water mediated by the herbicides in conjunction with the presence of mussels, lead system to hypoxia. Fluctuations in DO due
Author contributions
Cecilia E. Miranda: Conceptualization, Methodology, Investigation, Software, Formal analysis, Writing- Original draft preparation. Carlos D. Clauser: Methodology, Investigation. Verónica Lozano: Methodology, Investigation, Visualization. Daniel H. Cataldo: Conceptualization, Software, Methodology, Formal analysis. Haydée N. Pizarro: Conceptualization, Supervision, Writing- Original draft preparation, Funding acquisition.
Funding
This work was supported by PICT 2014 1586, UBACyT 20020130100248BA and PIP 11220130100399 all under H. Pizarro direction.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Dr. Maria Solange Vera and Dr. Carmen Sabio y García for field assistance.
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