Effects of Glyphosate and Their Metabolite AMPA on Aquatic Organisms

Glyphosate (N-(phosphonomethyl)glycine) is a herbicide used to kill broadleaf weeds and grass, developed in the early 1970s. The widely occurring degradation product aminomethylphosphonic acid (AMPA) is a result of glyphosate and amino-polyphosphonate degradation. The massive use of the parent compounds leads to the ubiquity of AMPA in the environment, and particularly in water. Considering this, it can be assumed that glyphosate and its major metabolites could pose a potential risk to aquatic organisms. This review summarises current knowledge about residual glyphosate and their major metabolite AMPA in the aquatic environment, including status and toxic effects in aquatic organisms, mainly fish, are reviewed. Based on the above, we identify major gaps in the current knowledge and some directions for future research knowledge about the effects of worldwide use of herbicide glyphosate and its major metabolite AMPA. The toxic effect of glyphosate and their major metabolite AMPA has mainly influenced growth, early development, oxidative stress biomarkers, antioxidant enzymes, haematological, biochemical plasma indices, caused histopathological changes in the aquatic organism.

concentrations in the environment, they may be present as complex mixtures. The metabolites may be as toxic as their parental compound or even higher. Therefore, the presence of these substances raises significant ecotoxicological concerns [e.g., [24][25][26].
Due to repeated application of pesticides arise to physical and chemical changes in water properties that are reflected in the modification of the cellular and biochemical biology of aquatic communities, leading to significant changes in tissues, physiology, and behaviour [27][28]. Therefore, it may affect the daily or seasonal rhythm and reproduction ability of aquatic organisms. Environmental stress from xenobiotics may cause losing of habitats and consequently losing freshwater biodiversity [29][30], which imply that the use of pesticides, despite their advantage in controlling pests, diseases, fungi, etc. has adversely impacted their ubiquity in the environment [e.g., [16][17][31][32].
As far as is known, in the literature is several studies and reports about an occurrence or toxic effects of different type of pesticides and their metabolites; nevertheless, their global extents and spatial extent of exposure remain largely unknown [2,33]. Considering this information, we decided to write a review to summarise the toxic effects of often using herbicide glyphosate and their metabolite AMPA on aquatic organisms.

Glyphosate (N-(phosphonomethyl)glycine)
Glyphosate (GLY) is belonging to the phosphonoamino acid class of pesticides. Glyphosate is an acid that can be associated with different counter cations to form salts [15]. This herbicide is a crop desiccant, broad-spectrum, non-selective, post-emergency herbicide that affects all annual and multiannual plants and aquatic weed control in ponds, lakes, canals, etc. [34][35].
Unlike GLY, whose small molecule consists of a linear chain with weak bonds, the molecules of other herbicides are usually arranged in aromatic circular structures. This difference reduces the persistence of glyphosate in the environment [36]. For higher water solubility, GLY is formulated as potassium salts or isopropylamine salts, and a surfactant, poly-oxyethylene amine (POEA), is added to enhance the efficacy of the herbicide. Another formulation, Rodeo, contains the isopropylamine salt (IPA) of GLY without the surfactant and is primarily used for controlling aquatic weeds [35,37] or Roundup Transorb, which contain a mix of 15% POEA and additional surfactants [38]. The Roundup includes 48% of active agent IPA [34] or potassium salts in the range 167-480 g . l -1 depends on the type of area where the Roundup is applied [39].

Environmental fate
Even though solid bond to the soil amount of GLY which leach or runoff to surfaceor ground-waters is low [40], spray drift from the ground and aerial applications of glyphosate may enter to aquatic ecosystems [41]. Hight application rates, rainfall, and a flow route that does not include transportation of GLY through the soil from watersheds comprise the most risk for offsite transport of GLY [9]. For example, the United States Environmental protection agency [15] reports predicted GLY concentration from direct applications into a standard pond in 103.8-221.5 μg/L for daily peak, 101.8-217.5 μg/L for 21-day average, and 98.4-210 μg/L for 60-day average. In water bodies, the glyphosatebased herbicide is usually detectable as glyphosate acid equivalent at a range level from 0.01 mg/L to 0.7 mg/L and reaching the worst case for surface waters of 1.7 mg/L [42][43][44]. Coupe et al. [9] reported concentration of GLY for Mississippi, Iowa, and France ranged from 0.03 to 73 μg/L, 0.02-1.6 μg/L, and 1.9-4.7 μg/L, approximately.
This herbicide is unique for its high efficiency, transformation on major metabolite AMPA due to microbial degradation [16,40], and physiochemical properties: water solubility 11.6 g/L at 25°C, low lipophilicity LogP <-3.2 at 20°C, dissociation constant of 2.3 at 25°C [40]. Under aerobic conditions, the half-life of GLY ranges from 1.8 to 109 days in soil and 14-518 days in water-sediment systems; however, in anaerobic water-sediment systems ranges from 199 to 208 days [15]. Nevertheless, according to published data halflife of GLY ranges from 7 to 14 days [40].
Owing to their high-water solubility and extensive usage in the environment (especially in shallow water systems), GLY contamination has emerged as an urgent issue. Therefore the exposure of non-target aquatic organisms to these herbicides is a concern of ecotoxicologists [16,37].

Acute toxicity
It has been already mentioned that initial testing of GLY did not fully demonstrate its toxic effects, and therefore the amount for use was not over-regulated. U.S. EPA divided toxicity of GLY into slightly toxicity with concentration 10 -100 mg/L and almost non-toxicity with higher concentration > 100 mg/L to fish species with acute LC50 values from > 10 to > 1000 mg/L [15]. The lethal concentration for fish is in the range 0.295 to 645 mg/L (Table 1), for amphibians is in the range 6.5 to 115 mg/L ( Table 2) and for invertebrate species from 35 to 461.54 mg/L ( Table 3).

Toxic effects
2.2.1 Fish GLY toxicity has been studying in recent years on various kinds of aquatic organisms. Exposure to GLY may cause several changes in fish (Table 4), such as haematologic changes, biochemical processes in tissues [38], genotoxicity [52,58], histopathological damage, immunotoxicity [48,59], or cardiotoxicity [60]. There are just several data about the chronic effects of glyphosate on non-target organisms. For example, [74] studied chronic exposure to glyphosate with a concentration of 1 μg/L on rainbow trout for 10 months. No significant changes in reproduction, metabolism, nor even oxidative response were observed. However, occasional impacts on immune response have occurred. Other chronic effects were studied with different concentrations of glyphosate (0.2, 0.8, 4 and 16 mg/L) in Oreochromis niloticus for 80 days [75]. It was evaluated that glyphosate exposure reduced antioxidative ability, disturbed liver metabolism, promoted inflammation and suppressed immunity.

Mussels
Glyphosate caused changes in hemolymph [76], changes in the reproduction system and 50% inhibition of cholinesterase activity [77] in mussels (Table 5). Table 5. Toxic effects of glyphosate and its commercial product on mussels.

Invertebrate species
Exposure to GLY may cause several changes in invertebrate species (Table 6), such as biochemical processes in tissues, development, or behaviour.

AMPA (aminomethylphosphonic acid)
AMPA belongs to the aminomethylenephosphonates chemical group. It is the primary metabolite of GLY degradation process with a significant measured concentration in the environment. Additional sources of AMPA originate from organic phosphonates using in water treatment [86], from the degradation of phosphonic acids used in Europe in detergent and industrial boilers and cooling (EDTMA, DTMP, ATMP. HDTMP) [15,86]. Because of phosphonate and amine functional groups, AMPA will form metal complexes with Ca 2+ , Mg 2+ , Mn 2+ , and Zn 2+ . AMPA is sorb firmly to soil [87].

Environmental fate
AMPA has a lower water solubility and longer soil half-life than glyphosate. Presence of AMPA in freshwater, sediment, and suspended particulate is commonly measured in significant quantities [10,88], and even more frequently (67.5%) than glyphosate (17.5%) [15,[89][90]. The Water Framework Directive [91] provides a procedure to set Environmental Quality Standards for AMPA at level 450 mg/L. Coupe et al. [9] reported concentration of AMPA in freshwater environments for Mississippi and Iowa ranged 2.6 μg/L, 0.02-5.7 μg/L. In France, AMPA was detectable with the highest concentration at level 44 μg/L. AMPA, like glyphosate, also degrade in water and soil but significantly slower. Because its adsorption to particulates is possibly stronger is lower penetrability to cell membranes. The concentration of AMPA in the sediment can fluctuate depending on its degradation rate relative to GLY [92].

Acute toxicity
AMPA toxicity has been already studied in recent years on various kinds of organisms. Although [52] observed no acute toxic effect of AMPA on fish species, other studies showed acute toxicity values from 27 to 452 mg/L (Table 7).

Toxic effects
Although AMPA has been studied less than glyphosate, Reddy et al. [96] pointed on affecting chlorophyll biosynthesis, which leads to plant growth reduction. That means that AMPA can also be translocated to diverse plant tissue. AMPA is also known as a phytotoxin, which can amplify the indirect effects of glyphosate on physiological processes. On the other hand, due to its chemical similarity, AMPA can compete with glycine in biological sites and pathways, affecting chlorophyll biosynthesis and thus the photosynthetic process [97]. Plants treated with AMPA showed a decreased glycine, serine, and glutamate [98]. There is almost no data for chronic effects and exposure to AMPA for aquatic organisms. The chronic toxicity of AMPA to Pimephales promelas and Daphnia magna was studied by Levine et al. [86]. Evaluating NOEC for P. promelas was determined 12 mg/L, and no-observed-effect concentration for D. magna was 15 mg/L.

Conclusion
GLY and AMPA et environmental relevant concentrations usually do not cause direct lethality. However, glyphosate as a separate compound or as a component of commercial products used in agriculture and its primary metabolite AMPA may have adverse effects on non-target aquatic organisms. GLY mainly affected oxidative stress, antioxidant enzymes, blood parameters and cause several histopathologic changes in gills, liver and kidneys, and not least genotoxicity, immunotoxicity and cardiotoxicity in fish; oxidative stress, antioxidant enzymes, and haemocyte parameters in mussels. In comparison to AMPA, in literature is gaps in knowledge about its toxicity on aquatic organisms. AMPA may cause genotoxicity, immunotoxicity in fish, adverse changes in haemolymph parameters, affected mussels' antioxidant enzymes, and developmental delay and survival of tadpoles.
There are also concerns about potential bioconcentration effects and breeding in organisms of these compounds. Considering the increasing consumption of herbicides and their repeated application worldwide, we assume that the presence of GLY and AMPA in the aquatic environment requires stricter control and further studies of the potentially toxic effects of these substances on the non-target organism. Further needs to be found bioindicators for polluted aquatic environments of GLY and AMPA.
Author Contributions: Conceptualization, data curation, writing -original draft preparation, N.T.; writing -review and editing A.S.; supervision J.V. All authors have read and agreed to the published version of the manuscript.