Ecotoxicity of glyphosate-based herbicide (GBH) to aquatic plants worldwide.
Glyphosate-based herbicides (GBHs) are chemicals developed to control unwanted plants such as weeds or algae. These chemicals act on EPSPS enzyme that blocks the production of tyrosine, phenylalanine, and tryptophan amino acids causing plant death. This biochemical pathway exists only in plant organisms. Despite the target use, GBHs have been related to toxic effects on nonplant organisms, such as invertebrates, fishes, amphibians, reptiles, birds, and mammals, including humans. This chapter is focused on ecotoxicological effects of GBHs on aquatic environment, showing a perspective of studies since this kind of product was developed until nowadays, an analysis of how many studies for each taxonomic group. Furthermore, we analyzed specifically the toxic effect of GBHs on each taxon, and finally, we discuss future perspectives and suggestions for a better regulation and application for this chemical.
- water quality
- weed control
Herbicides are chemical compounds used mostly to control weed (i.e., uncultivated) plants in agriculture and forestry and also for algae control [1, 2]. Herbicide formulations are designed to affect mainly plants, affecting specific plant biochemical pathways. However, it is common that this kind of pesticides affects nontarget organisms such animals, including aquatic organisms [3, 4].
The most used herbicide worldwide is glyphosate-based herbicide (GBH), such as Roundup® from Monsanto, and its usage has been increased  mainly due to the development of transgenic glyphosate-resistant crops . Glyphosate (N-(phosphonomethyl) glycine (CAS no. 1071-83-6)) is a weak organic acid with a molecular weight of 169.09 M and has a half-life of 7–142 days in water and 76–240 in soil [6, 7]. Glyphosate has high solubility in water (10,000–15,700 mg L−1 at 25°C), and it readily dissolves and disperses in an aquatic environment.
Glyphosate affects a specific plant biochemical pathway, inhibiting the action of the enzyme 3-enolpyruvylshikimic acid 5-phosphate synthase (EPSPS) that is necessary for biosynthesis of amino acids such as phenylalanine, tyrosine, and tryptophan  (Figure 1). Animals do not have this biochemical pathway, and hypothetically, they would be safe from glyphosate. However, the use of glyphosate requires that some other compounds as surfactants are added to the commercial formulation to increase adhesion to the leaf surface and absorbance by plants, trespassing the waxy cuticle . There are a variety of surfactants, but the most common used on glyphosate-based formulations has been polyethoxylated amine (POEA). This surfactant is known to be more toxic to animals then glyphosate itself [6, 9].
As mentioned above, glyphosate
In terrestrial animals, glyphosate reaches these organisms through direct application and contaminated food consumption. However, application of GBH in an aquatic environment is not so common when compared to terrestrial environments. Despite this, GBH can reach the aquatic environment through many ways. It can be applied directly on water bodies for algae control, although the opposite effect can be found, with proliferation of some species of algae due to the increase of phosphorus levels . GBH can also reach the aquatic environment through leaching, run-off, and contaminated food source .
As mentioned, glyphosate has high solubility in an aquatic environment. Some studies say that 50% of glyphosate in natural waters dissipates by water flow and decomposition in a few days to 2 weeks [19, 20, 21]. Despite that, glyphosate binds to soil particles and solid surfaces , which makes its dissipation difficult. The by-products of glyphosate decomposition are sarcosine and aminomethylphosphonic acid (AMPA). The first one is known to be nontoxic  and the second one less or equally toxic for aquatic organisms than glyphosate [24, 25]. This substance has also a great solubility and dissipates in water in 7–14 days. POEA in natural environments degrades by microbial decomposition in 14 weeks and its half-life is estimated in 21–42 days .
Considering that glyphosate
2. Studies about glyphosate-based herbicides on the aquatic environment
One of the first studies that evaluated the effects of glyphosate and GBH in aquatic environments was performed by Folmar et al. . According to Thomson’s ISI WoS (Institute for Scientific Information, Web of Science) database, using keywords as “glyphosate,” and “aquatic environment,” since 1979 to the present day, 233 papers have been published that evaluated the toxicological effects of glyphosate in aquatic environments (Figure 2). These papers addressed the toxic effects of glyphosate on various types of organisms. The invertebrate group was the most studied, with 52 published articles (21.3%), followed by fish with 51 (20.9%), amphibians 40 (16.4%), plant 31 (12.7%), and aquatic environment 30 (12.3%). The other groups were present in 40 published articles (16.4%) (Figure 3). For the investigated period and database, there were no papers which have evaluated the toxicological effects of glyphosate in aquatic mammals and birds. This scarcity of studies demonstrates the lack of knowledge on the risk of exposure of these groups in aquatic environments contaminated by glyphosate.
2.1 Aquatic plants
Glyphosate in the aquatic environment causes the death of the macrophyte community, which serves as a microhabitat for zooplanktonic, phytoplanktonic, and periphytic communities, and this leads to top-down control of planktonic organisms, affecting refuge and feeding to fish , triggering a chain effect. Studies have evaluated the effects of glyphosate on aquatic lentils (
|Species||Group||Chemical||Glyphosate concentration (μg L−1)||Effect||Reference|
|Nostocaceae||Gly. (acid)||0.1–8.8 mM||Increases growth|||
|Phormidiaceae||Gly. (acid)||0.005–0.048 mM||Increases growth|||
|Chlorellaceae||Gly. (acid)||293,000||Chlorophyll fluorescence/decreases PP|||
|Hydrocharitaceae||DCMU Gly. (acid)||11,600||Decreases chlorophyll fluorescence|||
|Gly. (acid) Roundup®||46,900||Increases growth|||
|Leptolyngbyaceae||Gly. (acid)||0.003–0.02 mM||Increases growth|||
|Onagraceae||Gly. (acid)||4000 and 108,000||Bioaccumulation|||
|Microcystaceae||Gly. (acid)||3–37||Increases growth and toxin production|||
|Gly. (acid)||15,000||Increases growth and toxin production|||
|Haloragaceae||Gly. (acid) Roundup®||840||Decreases root|||
|Gly. (n.c.)||220||Chlorophyll fluorescence|||
|Nostocaceae||Gly. (acid)||>50 mM||Increases growth|||
|Chlorophyceae||Gly. (acid)||200,000||Chlorophyll fluorescence/decreases primary productivity (PP)|||
|Nostocaceae||Gly. (acid)||0.005–0.02 mM||Increases growth|||
Dörr  studied the effect of glyphosate on the growth and production of secondary metabolites by toxigenic strains of the cyanobacteria
The effects of herbicides on nontarget aquatic plants are emerging as a major conservation issue in aquatic biodiversity .
Another important community in aquatic ecosystems that is also affected by the use of glyphosate is the periphyton. In terms of primary production, the periphyton has a photosynthetic contribution 77% higher than that of phytoplankton . Among the most common and potentially toxic outcrossing cyanobacteria,
The exposure to GBH reduced 78% of the primary productivity of phytoplankton when used at low concentrations (0.125 mg L−1)  and at high concentrations (3.8 mg L−1) , causing a disturbance in the trophic levels. In freshwater systems, glyphosate at high levels stimulated eutrophication by increasing total phosphorus and favoring the growth of cyanobacteria on the periphyton, which altered the typology of the study ecosystem that was a mesocosm .
Species-based differences in sensitivity to GBH exposure may lead to decreased richness and abundance of ecosystem species . Even though herbicides are thought to kill terrestrial plants, it can have an even more devastating effect in water, due to the imbalance that causes mortality of algae and aquatic plants. This causes an increase in decomposing organic matter in the water, which will reduce the concentrations of dissolved oxygen in the system and increase the stress of aquatic communities . Thus, algae and aquatic plants are considered as nontarget organisms that are sensitive to the effects of glyphosate, and the damage to the balance of the aquatic environment is of concern. The damage of glyphosate on the aquatic plant community ranges from the death of the plant itself to the reduction of environmental heterogeneity promoted by the local plants. Consequently, this leads to the death of other aquatic species, causing an imbalance in the ecosystem.
2.2 Aquatic invertebrates
One of the pioneer studies of the effects of GBH on invertebrate organisms was carried out by Tsui and Chu  that studied the effects of this chemical on
|Species||Chemical||Exposure time (h)||LC50 (μg L−1)||Reference|
|Eskoba®, Panzer Gold®, Roundup Ultramax®, Sulfosato Touchdown®||48||250–16,770|||
|Roundup®, POEAE, Glyphosate acid||96||18,000 (9400–32,000)|||
|Rodeo®, X-77 Spreader®, ChemTrol®||48||1,216,000 (996,000–1,566,000)|||
|Eskoba®, Panzer Gold®, Roundup Ultramax®, Sulfosato Touchdown®||48||2670–15,430|||
|Roundup®, POEAE, Glyphosate acid||48||3000 (2600–3400)|||
|Eskoba®, Sulfosato Touchdown®||48||1620–31,410|||
|Rodeo®, X-77 Spreader®, ChemTrol®||48||218,000 (150,000–287,000)|||
|Roundup®, POEAE, Glyphosate acid||48||62,000 (40,000–98,000)|||
|Roundup®, POEAE, Glyphosate acid||96||43,000 (28,000–66,000)|||
|Rodeo®, X-77 Spreader®, ChemTrol®||96||720,000 (399,000–1,076,000)|||
|Rodeo®, X-77 Spreader®, ChemTrol®||96||1,177,000 (941,000–1,415,000)|||
|Eskoba®, Sulfosato Touchdown®||48||1220–1,282,000|||
|Roundup®||24||18.3 ± 12.9|||
Specifically about microinvertebrates (<35 μm), these organisms persist within resting eggs (or egg banks) in lake sediments . They represent a major source of regenerative potential in lake ecosystems near agricultural areas, and play a key role in influencing the active population and community dynamics, seasonal succession, biogeographic patterns, and the evolution of populations [36, 37]. Despite the widely accepted importance of resting egg banks in the ecology of aquatic micro-invertebrates’ communities, recently, experimental studies have demonstrated that the extensive and inappropriate use of commercial GBH, associated with agricultural activities, may impair the hatching of resting eggs in the sediment of lakes [38, 39]. Gutierrez and collaborators  indicated that the GBHs (Sulfosato Touchdown®) affect the hatching dynamics of micro-invertebrates, and selectively alter the species richness and abundance of community hatched from lake sediment. Portinho and associates  extended these findings and indicated that commercial herbicides as Roundup® (a.i. glyphosate) separate or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) have the potential to suppress emergences of micro-invertebrates from resting egg banks from lake sediments.
The environmental implication of this scenario suggests that changes in micro-invertebrates’ structure and composition induced by herbicides will occur, causing not only negative impacts on the process of recolonization from resting egg banks but also shifts in community composition. Recent attempts to develop guidelines for protecting aquatic organisms have focused on emergence from resting egg banks within the context of an ecological community , with potential implications for studies related to environmental risk to, and integrity assessment of, aquatic ecosystems.
Fish species are particularly vulnerable to GBH and their susceptibility depends on the commercial formulation, fish species, fish developmental stages, and exposure conditions, such as concentrations, exposure time, and route of exposure. Furthermore, gender-specific response of fish to GBH has been indicated in guppy
In general, the surfactant and the commercial formulation showed higher toxicity to fish when compared to active ingredient (glyphosate pure) and their metabolite (AMPA). The 50% lethal concentration (i.e., LC50) of GBHs for fish has high variability, ranging from 1000 to 9750 μg L−1 [6, 41]. Chandrasekera and Weeratunga  found a LC50 of 976 μg L−1 for 48 h of exposure in fries of
Glyphosate and formulation compounds can be taken by fish via gills and digestive tract through ingestion of contaminated food or water [6, 45]. Once inside the organisms, glyphosate is absorbed and distributed to the whole body through blood circuit, reaching several tissues. GBHs can affect fishes in different ways, affecting many organs and as well molecular levels. In liver, vacuolization process was reported in hepatocytes and nuclear pyknoses; in kidney, studies report Bowman capsule dilatation and accumulation of hyaline drops in tubular cells; and in gills, glyphosate causes hyperplasia, lamellar fusion and aneurism [46, 47, 48, 49, 50]. Besides that, Langiano and Martinez  showed activation of the stress axis, with increased blood glucose levels. Souza-Filho and collaborators  also showed genotoxic effects in fish cells. Concerning to enzymes, Sandrini and collaborators  showed that glyphosate impairs acetylcholinesterase activity in synapses, preventing detaching of acetylcholine from receptors, impairing electric transmission by neurons. This can impair muscle contraction and information transmittance. GBH in sub-lethal levels can also impair fish feeding behavior as shown by Giaquinto and collaborators . Also, a recent
OMIC technologies, such as proteomics, transcriptomics, and metabolomics, have been applied to investigate the molecular mechanisms and toxicity of GBHs on fish. For example, proteomics-based methods (two-dimensional gel electrophoresis associated with mass spectrometry and bioinformatics) were used to complement the knowledge about the ecotoxicity of GBH on
The herpetofauna is composed of reptiles and amphibians, and due to the low mobility, physiological requirements, and habitat specificity, this group has become ideal models for environmental conservation studies . Amphibians are sensitive to exposure to contaminants and are considered good bioindicators in monitoring water quality . Characteristics such as permeable skin, reproduction, and larval stages dependent on the aquatic environment make anuran amphibians highly vulnerable to pesticide contamination . Evidence suggests that anuran species decline is related to the intensive use of pesticides [58, 59, 60].
The decline of amphibian populations is related to the increase of environmental pollutants, the influence of climate change, habitat fragmentation, exposure to ultraviolet radiation, and human-induced environmental changes [61, 62]. Contamination of water bodies next to agricultural areas generally increases during the rainy season, that is, widely used to breed by most species of amphibians, and many species use temporary ponds and small streams adjacent to agricultural areas as part of their life cycle, harming the reproductive period and larval development [57, 58, 63]. During the rainy season, the agrochemical present in the soil are susceptible to be transported down the soil profiles and/or surfaces/underground water bodies and consequently affect the amphibian population  and other environmental (a) biotic elements [6, 64].
Herbicides may delay or inhibit the metamorphosis of amphibians directly impacting their reproduction . According to Walker and collaborators , the main routes of herbicide absorption in anuran amphibians are through contaminated food ingestion and skin absorption of pollutants dissolved or suspended in water. After absorption, the substance is transported to different compartments of the body through blood. The effect of herbicides on tadpoles is less known when compared to adult amphibians, since the larvae of the anurans are less visible, and unlike adults, they do not have vocalization. Tadpoles of various species have not yet been described, which makes it even more difficult to study these organisms in depth .
The reduction in larval survival due to exposure to glyphosate was observed by Simioni and collaborators , Figueiredo and Rodrigues , and Costa and collaborators  in larvae of
|Species||Chemical||Exposure time (h)||LC50 mg a.i./L||Reference|
Reptiles are extremely sensitive to herbicide formulations and may exhibit changes in their behavior after exposure of these xenobiotics . This group is fairly uniform and exposure to GBHs may affect its energy storage process [75, 76]. Schaumburg and collaborators  found that exposure to sublethal concentrations of glyphosate during the embryonic phase of
Currently in the Neotropical region, about 40 studies relate the indiscriminate use of herbicides based on glyphosate with the risk to biodiversity of herpetofauna. Schiesari and collaborators  reported that some species of amphibians, including tadpoles and adults and some reptiles are sensitive to exposure to formulations based on glyphosate. Exposure to sublethal concentrations of glyphosate is sufficient to cause irreversible damage to the DNA of amphibians and reptiles, so the use of GBH should be controlled in arable areas avoiding the decline of species that make up the herpetofauna group.
2.5 Aquatic birds
Glyphosate when used in recommended rates is considered not bioaccumulative and of low toxicity in birds . However, the present acquaintance is not enough to make affirmation about low toxicity risk and low exposure of birds to herbicide considering the possible complex process behind the movement and accumulation of glyphosate, additives, and waste in the environment. Moreover, even the few available studies [82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96] have found direct and indirect effects of glyphosate on bird species (Figure 4). Among those, only five studies along years 1994 and 2017 on Google Scholar database have analyzed effects on aquatic bird species. Direct effects have been analyzed on male ducks (
Indirect effects have been found in wetlands where the glyphosate is used to control the increase of
The direct effect of glyphosate on aquatic plants and macroalgae  can also affect aquatic birds once they make up the varied and plentiful diet of many of those birds. Changes in physiological, histological, and behavioral levels and lethal cases have been documented in fishes due to use of glyphosate [87, 88]. In this way, piscivorous birds can also be suffering indirect effects. In fact, all aquatic birds’ food chain can be affected by glyphosate once effects on invertebrates [81, 87, 88], amphibians , and reptiles  have already been confirmed.
Birds are very similar in their physiology and anatomy. Then, studies that have tested direct and indirect effects of glyphosate on nonaquatic birds can be also considered here. In Japanese quails (
Therefore, the controlled and scaled use of glyphosate in large areas is necessary to contribute to conservation of environmental heterogeneity and biological diversity avoiding the plausible effects on bird communities [83, 84, 85, 94]. To know what plants are important to bird diet and to promote techniques that do not eliminate all of those plants from the place are important activities before glyphosate application . More studies that aim to analyze the bird contamination by herbicides are also necessary . Long-term studies that encourage collaborative work between ecologist, toxicologist, and chemist are more pertinent .
2.6 Aquatic mammals
For the best of our knowledge, GBH or glyphosate only was not tested in aquatic mammals. Searching on Web of Science website for the terms “Glyphosate AND mammal AND aquatic,” there is no study reported to date. Despite that, mammals in general are considered less sensible to GBH damages than other groups due to reduced contact with the environment of mammals when compared to other groups as fishes, amphibians, or aquatic invertebrates . The main way that GBH or the active ingredient glyphosate reaches mammals’ bodies is through the digestive tract. However, it seems to be poorly absorbed and is excreted essentially nonmetabolized . Essentially, mammals that were tested were rats, mice, and dogs , tested through injection or ingestion. Some studies report glyphosate in humans in medical case studies. Reported direct effects of GBH on mammals are described as a “wide range of clinical manifestations” such as skin and throat irritation, hypotension, or death  and include heart arrhythmias and atrioventricular block, cardiac electrophysiological changes and conduction blocks , pregnancy problems , disrupt transcriptional expression of the steroidogenic acute regulatory protein in testicle  and aromatase activity, alter mRNA levels, and interact with enzymes . Indirect effects on mammals can be due to reduction of vegetation and animals that are a source of food such as invertebrates  and fishes. Although these mentioned studies were conducted in nonaquatic mammals, it is expected that aquatic mammals have similar or even more accentuated effect, since they have intense contact with water, and if it is contaminated, the exposure will be higher.
3. Regulations and perspectives
Despite the fact that GBHs were developed to control weeds, acting specifically in a restrict plan biochemical pathway, several studies demonstrated that there are many side effects on nontarget organisms in all great groups as reported extensively here. Looking to control these side effects, governments for many countries around the world established limits for usage and concentrations in water bodies. The USA, for example, allows 700 μg L−1 in water bodies, while Canada allows 280 μg L−1 in drink water. The Brazilian law is a little more restrictive, allowing 65 μg L−1 in water bodies class 2 that is used for crop and recreation of first degree (direct contact) . However, we could check here that these maximum concentrations allowed are not safe for biodiversity conservation. Considering the Brazilian law, the more restrictive in American countries, populations of yellowtail tetra fish (
However, even with all those regulations, it is not being obeyed, since there is a large range of glyphosate and its metabolite (e.g., AMPA) concentrations in hydroresources [6, 64]. Therefore, another way of action for environment safety is preserving marginal forests of rivers, surveillance, and environment education. Another sustainable way to achieve this goal is changing the crop production matrix from large scale, that is, conventional-based production model to a smaller integrative-/organic-based production system, with controlled or restrictive usage of pesticides and other agrochemicals.
We are thankful to FAPEG (#201710267001261) for financial support.
Conflict of interest
The authors declare that there is no conflict of interest.