Rethinking the term "glyphosate effect" through the evaluation of different glyphosate-based herbicide effects over aquatic microbial communities.

Glyphosate-based herbicides (GBH) -the most widely used herbicides in agriculture worldwide-are frequently generalized by the name of "glyphosate". However, GBH encompass a variety of glyphosate salts as active ingredient and different adjuvants, which differ between products. These herbicides reach water bodies and produce diverse impacts over aquatic communities. Yet, the risk assessment assays required for the approval focus mostly on active ingredients. Herein, we compared the effect of five different GBH as well as of monoisopropylamine salt of glyphosate (GIPA) on aquatic microbial communities from natural shallow lakes that were mixed and allowed to evolve in an outdoor pond. We performed an 8-day long assay under indoor control conditions to evaluate the effects of exposure on the structure of nano-plus microphytoplankton (net phytoplankton, with sizes between 2 and 20 µm and >20 µm, respectively) and picoplankton (size ranging between 0.2 and 2 µm) communities through microscopy and flow cytometry, respectively. Significantly different effects were evident on the structure of microbial communities dependent on the GBH, even with herbicides sharing similar active ingredients. Each GBH evoked increases of different magnitude in bacterioplankton abundance. Furthermore, GIPA and a formulation decreased the abundance of a phycocyanin-rich (PC-rich) picocyanobacteria (Pcy) cytometric population and GIPA further altered Pcy composition. Also, two GBH increased net phytoplankton total abundance and, unlike the tested GBH, no apparent effect of GIPA was detected on this community structure. These results demonstrate that GBH effects on aquatic microbial communities should not be summarized as "glyphosate" effects considering that the formulations have effects beyond those exerted by the active ingredients alone. This work intends to alert on the lack of real knowledge regarding the consequences of the variety of GBH on natural aquatic ecosystems. Indeed, the wide use of the term "glyphosate effect" should be thoroughly rethought.

An invasive mussel is in trouble: How do glyphosate, 2,4-D and its mixture affect Limnoperna fortunei's survival?

Contamination and biological invasions are important factors that alter the functioning of freshwater systems. We carried out two experiments involving daily measurements of dissolved oxygen (DO) and Limnoperna fortunei mortality: 1) the impact of DO on mussel survival mediated by glyphosate, 2,4-D and their mixture was analysed in a 22-day indoor experiment (IE) under controlled conditions using microcosms with L. fortunei, with and without air supply; and 2) the effect of each herbicide and their mixture on mussel accumulated mortality was compared in a 18-day outdoor experiment (OE) using mesocosms without air supply, with and without L. fortunei. Results showed that glyphosate, alone or mixed affected L. fortunei survival both directly and indirectly. In IE we observed direct toxicity of glyphosate in treatments with air supply, with accumulated mortality of 20.0% for glyphosate and 10.0% for the mixture. In OE, L. fortunei deepened the changes in the patterns of DO fluctuations driven by the herbicides, which led to hypoxia in the system. The accumulated mortality was 46.7, 8.6 and 48.2% for glyphosate, 2,4-D and the mixture, respectively. This study contributes to the understanding of the mechanisms that control the invasion of L. fortunei in freshwater systems influenced by agrochemicals.

Invasive species versus pollutants: Potential of Limnoperna fortunei to degrade glyphosate-based commercial formulations.

The intensive use of glyphosate in industrial agriculture may lead to freshwater contamination, encouraging studies of its toxic effect on non-target aquatic organisms. Glyphosate-based commercial formulations contain adjuvants, making them even more toxic than the active ingredient (a.i.) itself. The golden mussel Limnoperna fortunei is a freshwater invasive species which has been found to increase glyphosate dissipation in water and to accelerate eutrophication. The aim of this study is to evaluate the capability of L. fortunei to reduce the concentration of glyphosate in two commercial formulations, Roundup Max® and Glifosato Atanor®. Results were compared with the decay of the a.i. alone and in presence of mussels. Evasive response and toxicity tests were performed in a first set of trials to analyze the response of L. fortunei exposed to Roundup Max® and Glifosato Atanor®. Subsequently, we conducted a 21-day degradation experiment in 2.6-L microcosms applying the following treatments: 6 mg L-1 of technical-grade glyphosate (G), Glifosato Atanor® (A), Roundup Max® (R), 20 mussels in dechlorinated tap water (M), and the combination of mussels and herbicide either in the technical-grade (MG) or formulated form (MA and MR) (all by triplicate). Samples were collected at days 0, 1, 7, 14 and 21. No significant differences in glyphosate decay were found between treatments with mussels (MG: 2.03 ± 0.40 mg L-1; MA: 1.60 ± 0.32 mg L-1; MR: 1.81 ± 0.21 mg L-1), between glyphosate as a.i. and the commercial formulations, and between the commercial formulations, suggesting that the adjuvants did not affect the degrading potential of L. fortunei. In addition to the acceleration of glyphosate dissipation in water, there was an increase in the concentration of dissolved nutrients in water (N-NH4+ and P-PO43-) even higher than that caused by the filtering activity of the mussels, probably resulting from stress or from the degradation of glyphosate and adjuvants. We believe that a larger bioavailability of these nutrients due to glyphosate metabolization mediated by mussels would accelerate eutrophication processes in natural water bodies. The approach used here, where L. fortunei was exposed to two commercial formulations actually used in agricultural practices, sheds light on the potential impact of glyphosate decay on water bodies invaded by this species.

New findings on the effect of glyphosate on autotrophic and heterotrophic picoplankton structure: A microcosm approach.

Massive use of glyphosate-based herbicides in agricultural activities has led to the appearance of this herbicide in freshwater systems, which represents a potential threat to these systems and their communities. These herbicides can affect autotrophic and heterotrophic picoplankton abundance. However, little is known about glyphosate impact on the whole structure of these assemblages. Herein, we used an 8-day long microcosm approach under indoor controlled conditions to analyze changes in the structure of picoplankton exposed to a single pulse of glyphosate. The analyzed picoplankton correspond to two outdoor ponds with contrasting states: "clear" (chlorophyll-a = 3.48 µg L-1± 1.15; nephelometric turbidity, NTU = 1) and "turbid" (chlorophyll-a = 105.96 µg L-1 ± 15.3; NTU = 48). We evaluated herbicide impact on different picoplankton cytometric populations and further explored changes in bacterial dominant operational taxonomic units (OTUs) fingerprinting. We observed that glyphosate induced a drastic decrease in the abundance of phycocyanin-rich picocyanobacteria. Particularly, in the turbid system this effect resulted in an 85 % decrease in the abundance of the whole autotrophic picoplankton. Glyphosate also changed the structure of the heterotrophic fraction by means of changing bacterial dominant OTUs fingerprinting patterns in both systems and by shifting the relative abundances of cytometric groups in the clear scenario. These results demonstrate that upon glyphosate exposure picoplanktonic fractions face not only the already reported changes in abundance, but also alterations in the composition of cytometric groups and of bacterial dominant operational taxonomic units. This research provides suitable and still little explored tools to analyze agrochemical effects on picoplanktonic communities.

Impact of interaction between Limnoperna fortunei and Roundup Max® on freshwater phytoplankton: An in situ approach in Salto Grande reservoir (Argentina).

The joint impact of the glyphosate-based commercial formulation Roundup Max® and the invasive mussel Limnoperna fortunei on phytoplankton and water quality was assessed in Salto Grande reservoir, a scenario were both stressors coexist. We performed an in situ mesocosm approach, through a 7-day experiment using 400-L enclosures. The following treatments were applied by triplicate: addition of 250 mussels (M); addition of 5 mg L-1 of active ingredient (a.i.) in Roundup Max® (R); addition of 250 mussels and 5 mg L-1 of a.i. in Roundup Max® (MR), and controls, without any addition (C). R showed higher total phosphorus (TP) and ammonium nitrogen (NNH4+) concentrations due to the herbicide input, and a significant increase in algal abundance, biovolume and chlorophyll a levels (Chl-a). In M mussels grazed on phytoplankton, which resulted in subsequent phosphates (SRP) release. A decrease in species diversity was observed in R and M with respect to C. In MR, there were higher TP and NNH4+ concentrations, a decrease in biovolume, an antagonistic effect on Chl-a and a synergistic effect on phytoplankton abundance. Species diversity and evenness showed a significant decrease due to the explosive growth of a small and opportunistic Chlorophyta, Spermatozopsis exsultans. The dominance of this species may be due to negative selectivity for S. exsultans and/or release of potential competitors by L. fortunei, and to the input of nutrients by Roundup Max® and/or removal of competitors by its toxicity.

Effect of glyphosate acid on biochemical markers of periphyton exposed in outdoor mesocosms in the presence and absence of the mussel Limnoperna fortunei.

Glyphosate is currently the most widely used herbicide in agricultural production. It generally enters aquatic ecosystems through surface water runoff and aerial drift. We evaluated the effect of glyphosate acid on biochemical parameters of periphyton exposed to concentrations of 1, 3, and 6 mg/L in outdoor mesocosms in the presence and absence of the mussel Limnoperna fortunei. Periphyton ash-free dry weight, chlorophyll a content, carotene/chlorophyll a ratio, lipid peroxidation levels, and superoxide dismutase and catalase activities were determined at days 0, 1, 7, 14, and 26 of the experimental period. Ash-free dry weight was similar between control and glyphosate-treated periphyton in the absence of L. fortunei. The latter had significantly lower carotene to chlorophyll a ratios and enzyme activities, and higher lipid peroxidation levels and chlorophyll a content than the former. These results show an adverse effect of glyphosate on the metabolism of periphyton community organisms, possibly inducing oxidative stress. On the contrary, no differences were observed in any of these variables between control and glyphosate-treated periphyton in the presence of L. fortunei. Mussels probably attenuated the herbicide effects by contributing to glyphosate dissipation. The results also demonstrate that biochemical markers provide useful information that may warn of herbicide impact on periphyton communities. Environ Toxicol Chem 2017;36:1775-1784. © 2016 SETAC.

Impact of multiple anthropogenic stressors on freshwater: how do glyphosate and the invasive mussel Limnoperna fortunei affect microbial communities and water quality?

The study of the joint effect of multiple anthropogenic stressors is important because the emerging consequences are often unpredictable on the basis of knowledge of single effects. We explored the joint impact of glyphosate and the invasive golden mussel Limnoperna fortunei on freshwater phytoplankton, bacterioplankton and periphyton, and on the physical and chemical properties of the water. We manipulated both stressors simultaneously in a 25-day experiment using outdoor mesocosms; we assayed technical-grade glyphosate acid at four concentrations: 0, 1, 3 and 6 mg gly L(−1) under scenarios with and without mussels. The addition of the glyphosate significantly increased total phosphorus according to the concentration used; the high clearance rate of L. fortunei significantly decreased phytoplanktonic abundance leading to low values of turbidity. The mussel significantly stimulated the development of filamentous green algae (metaphyton). Interestingly, the combined effect revealed that L. fortunei accelerated the dissipation of glyphosate, which showed a 4-fold decrease in its half-life; this promoted the rapid bioavailability of glyphosate-derived phosphorus in the water. The interaction had a synergistic effect on soluble reactive phosphorus concentrations and was directly dependent on the concentration of glyphosate. A synergistic effect was also observed on bacterioplankton, water turbidity and metaphyton, thus inducing enhanced and rapid eutrophication. The ability of mussels to reduce glyphosate in water may be valued as positive, but our results allow us to predict that the invasion of Limnoperna fortunei in natural freshwater systems contaminated by glyphosate will accelerate the negative impact of the herbicide associated with eutrophication.

Comparative effects of technical-grade and a commercial formulation of glyphosate on the pigment content of periphytic algae.

We investigated the potentially different effects of one of the most commonly used glyphosate formulations in Argentina, Glifosato Atanor(®), and the technical-grade glyphosate on the pigment content, as biomass indicators of the algal fraction in a freshwater periphytic community. A laboratory bioassay was carried out in 250-ml beakers. Two treatments were used: technical-grade glyphosate acid and Glifosato Atanor(®) (isopropylamine salt of glyphosate 48 % w/v), which were at a concentration of 3 mg active ingredient per liter. Treatments and the control (without herbicide) were replicated in triplicate. The concentrations of chlorophyll a and b and carotenes were determined at 0, 2, 6, 10, 24, 48, 96 and 192 h after herbicide addition. A significant increase in pigment content was observed for both herbicides after a 2-day exposure. Moreover, the formulation had little or no effect compared to the active ingredient, suggesting that the additives of Glifosato Atanor(®) may not enhance glyphosate toxicity.

Impact of the invasive mussel Limnoperna fortunei on glyphosate concentration in water.

The use of glyphosate has increased dramatically during the past years around the world. Microbial communities are altered when glyphosate reaches water bodies. The freshwater golden mussel Limnoperna fortunei is an invasive species that has rapidly dispersed since it was introduced in Argentina two decades ago. Mussels alter aquatic conditions through their filtrating activity by increasing water clarity and nutrient recycling. We aim to evaluate the potential capacity of the golden mussel to reduce glyphosate concentration in water, in laboratory conditions. Firstly, the evasive response of mussels to glyphosate (10, 20, and 40 mg l⁻¹) was evaluated and a toxicity test was carried out for these concentrations. A three-week experiment was then performed to assess glyphosate variation under mussel presence for two mussel sizes. Finally, mussels' role on glyphosate concentration was evaluated considering different mussel parts (living organisms and empty shells) through another three-week experiment. Laboratory experiments were performed in triplicate using 2-l microcosms. An initial glyphosate concentration between 16 and 19 mg l⁻¹ was used, and when mussels or valvae were added, 20 organisms per aquaria were used. Samples were obtained at days 0, 1, 2, 4, 8, 14, and 21. Glyphosate decreased by 40% under large mussel presence in both experiments, and was reduced by 25% in empty shell treatments. We believe that part of the herbicide that disappears from the water column is adsorbed in valvae surface, while another proportion is being mineralized by microbial communities in shells' biofilm. The mechanisms by which living mussels increase glyphosate dissipation would be degradation, possibly mediated by bacteria associated to mussel's metabolism. Glyphosate half-life depended on mussel and valvae presence and varied with mussel size. L. fortunei presence (either alive or as empty valvae) alters glyphosate concentration in water. We provide preliminary observations from laboratory experiments, with strong potential ecological consequences, about two stressors that could be acting jointly on the environment.

Direct and indirect effects of the glyphosate formulation Glifosato Atanor® on freshwater microbial communities.

Glyphosate-based formulations are among the most widely used herbicides in the world. The effect of the formulation Glifosato Atanor(®) on freshwater microbial communities (phytoplankton, bacterioplankton, periphyton and zooplankton) was assessed through a manipulative experiment using six small outdoor microcosms of small volume. Three of the microcosms were added with 3.5 mg l(-1) of glyphosate whereas the other three were left as controls without the herbicide. The treated microcosms showed a significant increase in total phosphorus, not fully explained by the glyphosate present in the Glifosato Atanor(®). Therefore, part of the phosphorus should have come from the surfactants of the formulation. The results showed significant direct and indirect effects of Glifosato Atanor(®) on the microbial communities. A single application of the herbicide caused a fast increase both in the abundance of bacterioplankton and planktonic picocyanobacteria and in chlorophyll a concentration in the water column. Although metabolic alterations related to oxidative stress were induced in the periphyton community, the herbicide favored its development, with a large contribution of filamentous algae typical of nutrient-rich systems, with shallow and calm waters. An indirect effect of the herbicide on the zooplankton was observed due to the increase in the abundance of the rotifer Lecane spp. as a consequence of the improved food availability given by picocyanobacteria and bacteria. The formulation affected directly a fraction of copepods as a target. It was concluded that the Glifosato Atanor(®) accelerates the deterioration of the water quality, especially when considering small-volume water systems.

New evidences of Roundup (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems.

Argentina is the second largest world producer of soybeans (after the USA) and along with the increase in planted surface and production in the country, glyphosate consumption has grown in the same way. We investigated the effects of Roundup (glyphosate formulation) on the periphyton colonization. The experiment was carried out over 42 days in ten outdoor mesocosms of different typology: "clear" waters with aquatic macrophytes and/or metaphyton and "turbid" waters with great occurrence of phytoplankton or suspended inorganic matter. The herbicide was added at 8 mg L(-1) of the active ingredient (glyphosate) in five mesocosms while five were left as controls (without Roundup addition). The estimate of the dissipation rate (k) of glyphosate showed a half-life value of 4.2 days. Total phosphorus significantly increased in treated mesocosms due to Roundup degradation what favored eutrophication process. Roundup produced a clear delay in periphytic colonization in treated mesocosms and values of the periphytic mass variables (dry weight, ash-free dry weight and chlorophyll a) were always higher in control mesocosms. Despite the mortality of algae, mainly diatoms, cyanobacteria was favored in treated mesocosms. It was observed that glyphosate produced a long term shift in the typology of mesocosms, "clear" turning to "turbid", which is consistent with the regional trend in shallow lakes in the Pampa plain of Argentina. Based on our findings it is clear that agricultural practices that involve the use of herbicides such as Roundup affect non-target organisms and the water quality, modifying the structure and functionality of freshwater ecosystems.