Glyphosate-based herbicides (GBHs), including brands such as Roundup^®, are the most used pesticides in the United States and for the world as a whole [1]. Commercial farmers spray GBHs in three main application contexts: (1) as a pre-planting herbicide, reducing competition for seedlings and young plants; (2) to reduce competition throughout the growing season on Roundup^® Ready crops, including most of the corn, sugar beets, soy, and canola, and smaller portions of zucchini, alfalfa, and other crops, grown in the US; and (3) to kill leafy vegetation before harvest and ease separation of non-vegetative, commercially important parts of crop plants, used extensively for sugar cane, wheat, oats, and legumes. Perhaps because of the timing of application, this last type of use seems to impact food supplies most greatly, since glyphosate residue concentrations measured in oat- and wheat-based foods are the highest among all the foods tested ^[2][3][2,3]. Yet these crops are not genetically modified to be herbicide-tolerant; though herbicide tolerant soybeans also contain high concentrations of glyphosate residue [4]. This results in continuous, low-concentration human exposure to residues through food [5] and, because of runoff, drift, and overspray, in drinking water and the environment for both humans and other animals that live in or near agricultural areas. In addition, GBHs are used extensively by homeowners to suppress vegetation around driveways, walkways and fence lines, and by public employees to suppress weeds in public spaces like sidewalks, parking lots, playgrounds, and schools. Therefore, during application and subsequent exposure, humans and other animals living in urban, suburban and rural environments can all be exposed sporadically to higher doses of GBHs (e.g., [6]). This supposition is borne out by examining glyphosate concentration in tissues (personal observation; ^{[7][8][9][10]}[7,8,9,10]), including its prevalence in human urine (reviewed in [11]), related to type of exposure (occupational vs. dietary; [12]) and type of diet (organic vs. conventional; [13]).

The marketing of Roundup^® focuses on its safety for animals based on two incorrect assumptions. Several different formulations are sold commercially, different formulations are sold in different countries, and those available to the public are slightly different from the formulations marketed for agriculture. To date, the active ingredient of all formulations is glyphosate, though secondary herbicides may also be included. The first argument for the safety of Roundup^® for animals is that glyphosate targets the shikimate pathway of amino acid synthesis, which is present in plants and microbes, but not in animals. Of course this does not preclude the possibility that glyphosate also interacts with other molecules to create toxic secondary effects. The second argument depends on experimental results showing that, when tested alone by Roundup^® manufacturers or scientists funded by them, glyphosate exhibited low toxicity to mammals. Regulatory agencies have never required testing of so-called inert ingredients within the various formulations, and tests with the whole GBH formulations are not required to get approval for marketing. Other ingredients within the various formulations, particularly the secondary herbicides and surfactants, have proven either to act synergistically, increasing the toxicity of glyphosate, or to be more toxic than glyphosate, e.g., ^{[14][15][16][17]}[25,26,27,28]. These effects are difficult to explore fully because the composition of each formulation is considered proprietary and therefore, herbicide manufacturers do not have to disclose the ingredients; and the composition of formulations can differ from country to country. Given the wide variety of animal species and biological systems known to be disrupted by GBH exposure, one or both of these arguments must be incorrect.

Thousands of peer-reviewed articles have been published that demonstrate toxic effects of glyphosate and/or GBH formulations on animals. A thorough review of all the toxic effects reported across all animal taxa studied is beyond the scope of any single review article, but particular types of GBH toxicity have been reviewed. Some particularly thorough reviews discuss health risks ^{[18][19][20][21]}[29,30,31,32] and ecotoxicology ^[22][33], while more focused reviews elaborate on the effects on water fleas ^[23][34], on bees ^[24][35] and honeybees ^[25][26][36,37], on fish ^[27][38], on amphibians ^[28][39], in South American agriculture ^[29][40], as studied in Brazil ^[30][41], in aquatic systems ^[31][32][42,43], and to offspring of exposed mothers ^[33][44]. Other reviews focus on particular outcomes of toxicity, including cancer and genotoxicity ^[34][35][36][45,46,47], pregnancy outcomes ^[37][38][39][48,49,50], mammalian nervous systems ^[40][51], and autism spectrum disorders ^{[41][42][43][44]}[52,53,54,55]. Since it is apparent that the effects of glyphosate and of GBH formulations often differ (reviewed in ^[17][45][28,56]), this distinction must be considered when possible.

Agricultural workers and others living in agricultural areas likely experience particularly high GBH exposure, e.g., ^[6][12][46][6,12,63]. While it is extremely difficult to study in detail, in human agricultural workers, herbicide exposure is implicated in cases of impaired kidney function ^[47][64], altered thyroid and reproductive hormone levels ^[48][65], and reduced sperm count ^[49][50][66,67], and increases time to pregnancy ^[51][68], chance of short gestation ^[52][69], preterm birth ^[53][70], and neurobehavioral birth defects in their children ^[54][71], perhaps partially by increasing the permeability of the blood–brain barrier and changing the metabolic activity of epithelial cells in the brain ^[55][72]. Workers in GBH factories experience increased likelihood of coronary artery disease ^[56][73]. Probability of advanced liver fibrosis in patients with fatty liver disease ^[57][74] is associated with GBH exposure, as are Parkinson’s Disease and parkinsonism ^[58][59][60][75,76,77], and autism spectrum disorders ^[61][62][78,79]. Experimentally, GBH exposure decreases human sperm motility ^[63][80]; transiently increases genotoxicity ^[64][81] via DNA lesions ^[65][82] and increases formation of micronuclei in peripheral white blood cells ^[66][83]; mimics estradiol in inducing the growth of cholangiocarcinoma cells ^[67][84]; induces genomic damage on human lymphocytes ^[68][85]; induces breast cancer cell growth through an estrogen receptor pathway ^[69][86]; and, at low doses, dysregulates gene expression in breast cancer cell lines, particularly in pathways related to cell cycle and DNA damage repair ^[70][87]. Consistent with cancer cell line studies, the development of non-Hodgkin lymphoma ^[36][71][72][47,88,89] and other cancers ^[73][90] have been correlated with GBH exposure. Human suicide attempts using GBH impair cardiac function ^[74][91], require attention to airways and cause renal damage ^[75][92], and may induce gastrointestinal symptoms and central nervous system complications ^[76][77][93,94] including hippocampal infarction ^[78][95].

2. Activity

Glyphosate affects the way many animals go about their day to day lives. Across studies about animal activity in a wide range of taxa, glyphosate affects activity levels, whether walking, crawling, swimming, or flying. When exposed to glyphosate, animals tend to be less active and do not move as far as unexposed animals. In the honeybee, Apis mellifera, an experiment conducted using 7 μg glyphosate/bee, 14 μg/bee, and 28 μg/bee found that exposure to higher concentrations caused the bees to have trouble reaching their hive ^[79][98]. Bees returning to the hive in a straight line or with five right-angle turns, 2 h after being fed sucrose solution containing 5 mg/L or 10 mg/L glyphosate, took more time to reach the hive in both sets of mazes ^[80][60]. A similar method was used by Balbuena et al. ^[81][99] to examine flight paths of bees after food-based glyphosate exposure at 2.5 mg/L, 5 mg/L, or 10 mg/L. Similarly, 2.5 mg/L glyphosate exposure strongly impacted how long it took bees to return home ^[25][36]. A different method was used to record total activity level over a 24 h period. Bees were more active than controls when exposed to Roundup^® at 1.2 mg/L or 6 mg/L, whereas exposure at 0.12 mg/L and 12 mg/L did not affect their activity levels, and exposure to 24 mg/L of Roundup^® decreased their activity ^[82][100]. While this is an unusual response pattern, the mechanism leading to this change in behavior is not known. If the change in activity results from the endocrine-disruptive properties of GBHs, which are known to be non-monotonic, this may not be surprising (e.g., ^[83][101]). Conversely, feeding 50- or 100-ng doses of glyphosate per bee, caused bees to sleep more ^[84][102]. Other studies did not identify changes in activity level after Roundup^® or glyphosate exposure. At concentrations of 0.5 mL/50 mL, 1 mL/50 mL, and 1.5 mL/50 mL, bees successfully returned to the hive ^[85][103]. However, these authors only considered whether the bees returned to the hive and not activity level or speed of return. Another study tested locomotion using a square box with four lights. One light was turned on at a time and when the bee was at the light it would turn off and the next light would turn on. This is an effective way to collect activity data because bees go toward lights. The data collected then were the duration of time it took for the bees to get to all the lights twice. When conducting this experiment with bees exposed to glyphosate at a concentration of 2.5 mg/L or 5 mg/L, they did not find a significant difference among the groups ^[86][104]. Walking animals also exhibit lower activity levels after exposure to glyphosate. In Swiss mice, the total distance traveled and velocity decrease when exposed to higher concentrations of GBHs given in oral gavages ^[87][88][105,106]. Exposure of mice to 50 mg/kg/day of a GBH had a large effect on their activity. The mice were less active and traveled a shorter distance than unexposed controls ^[89][107]. The Sprague Dawley rat also exhibited less locomotor activity when first injected with glyphosate at concentrations of 50, 100, or 150 mg glyphosate/kg. The rats traveled shorter distances, had less activity, and showed less stereotyped behavior in a dose-dependent pattern ^[90][108]. Another study showed less locomotor activity of Wistar rats injected with glyphosate at the lower concentration of 35 mg glyphosate/kg ^[91][109]. Similarly, arthropods also showed lower locomotor activity after GBH exposure. Arthropods tested include the small wolf spider, Pardosa milvina; the large wolf spider, Hogna helluo; and the ground beetle, Scarites quadriceps. All three arthropods showed a decrease in activity when they came in contact by touch with the herbicide ^[92][110]. The Madagascar hissing cockroach, Gromphadorhina portentosa, consumed up to 13.2 mg of glyphosate. Then, they were placed on a hamster wheel to see how long they could run for a maximum of 3 min. The results showed a significant decrease in time spent running on the wheel for cockroaches exposed to glyphosate compared to their unexposed conspecifics ^[93][111]. The fruit fly, Drosophila melanogaster, was also studied to see how different methods of feeding could affect their locomotor activity. The methods they used were putting the pesticide into agar-gelled feed (AM) and continuous liquid feeding (CLF), where flies ingested the pesticide in liquid, and how much they ingested could be quantified. Both methods decreased locomotor activity, though CLF yielded lower locomotor levels than when using the AM method ^[94][112]. The earthworm, Lumbricus terrestris, is also impacted when exposed to glyphosate. The GBH induced lower levels of activity when present throughout the soil ^[95][113]. In another earthworm, Octolasion cyaneum, there was very slight avoidance behavior towards the contaminated soil at the concentration of 249 μg, though this slight avoidance was not statistically significant ^[96][114]. Many aquatic organisms, including several types of fish, decrease activity and behave abnormally when exposed to GBH. Many studies using the zebrafish, Danio rerio, show that GBHs lower activity level at all stages, from larvae to adult fish. In a study that used both larvae and adults, glyphosate and Roundup^® at 0.01, 0.065, or 0.5 mg/L reduced swimming distance ^[97][115]. Glyphosate concentrations of 0.01, 0.5, or 5 mg/L also increased swimming activity levels in exposed larval and adult zebrafish during the day ^[98][116]. When exposed to glyphosate at 1000 μg/L, zebrafish larvae decreased swimming distance, number of rotations, mean velocity, and body mobility ^[99][117]. One study of larval zebrafish showed an increase in activity when exposed to GBH at the concentrations of 0.1 and 10 uM for a 7-day exposure period ^[100][118]. However, another study done on larval zebrafish showed an increase in activity levels when exposed to Roundup^® at 10⁶ to 10⁴ dilution but a decrease when exposed to GBH at 0.01 and 0.1 uM. Both exposure periods were 48 h at 5 days after fertilization ^[101][119]. The common carp, Cyprinus carpio, showed a decrease in activity level when exposed for 60 days versus an increase in activity level when exposed for only 12 h to GBH ^[102][103][120,121]. Collectively, these results show that in zebrafish and carp, the effect of GBH on activity depends on both concentration and duration of exposure. One clue to the variation might be that when glyphosate circulates in the water in the tanks, carp moved away from the part of the tank with contaminated water ^[103][121]. Under some conditions, changes in activity might reflect avoidance of contaminated water, while under other conditions might reflect an impairment in ability to behave normally. For example, when exposed to higher concentrations of glyphosate, the African catfish, Clarias gariepinus, showed loss of reflex, air gulping, and erratic swimming ^[104][122]. Two other studies that exposed catfish to glyphosate documented loss of equilibrium, increased startle responses, abnormal swimming, and restlessness ^[105][106][123,124]. When treated with glyphosate, the redbelly tilapia, Tilapia zilli, swam erratically and irregularly. After bursts of swimming, they became exhausted, more so at higher concentrations ^[107][125]. An additional study using the livebearer, Jenynsia multidentata, showed lower swimming activity levels when exposed to GBH ^[108][126]. The blue ridge two-lined salamander, Eurycea wildrae, exhibited lower burst distance swimming activity when exposed to GBH, and lower movement distance at higher temperatures ^[109][127]. Conversely, in a study using the hybrid fish surubim, a cross-breed of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum, fish showed higher swimming activity levels and increased ventilatory frequency when exposed to GBH ^[110][128]. Furthermore, the rainbow trout, Oncorhynchus mykiss, was moved from dark to light and back to dark conditions while exposed to different concentrations and formulations of GBH, including glyphosate alone. During the light period, fish have significantly lower activity levels compared to the dark periods, though during the dark periods, the fish swam a longer distance ^[111][129]. Other aquatic animals affected by glyphosate include the marsh frog, Pelophylax ridibundus. Marsh frog tadpoles exposed to water contaminated with 7.6 mg/L, 3.1 mg/L, and 0.7 mg/L glyphosate showed a decrease in activity level during the Gosner stage 25 ^[112][130]. In two different species of South American frogs, Boana faber and Leptodactylus latrans, tadpoles showed lethargy, convulsions, and rapid bursts of swimming when exposed to glyphosate at concentrations of 69, 161, 310, 550, and 1074.5 μg/L ^[113][131]. However, in the leopard treefrog, Boana pardalis, there was an insignificant decrease in activity levels when frogs were exposed to glyphosate ^[114][132]. The water flea, Daphnia magna, showed less swimming activity when exposed to GBH ^[115][133].

3. Foraging and Feeding Behavior

Exposure to glyphosate-based herbicides alters the feeding behavior of some organisms, while in other studies it has no effect. In many cases, GBH-laden food is avoided, while organisms pre-exposed to varying concentrations of GBHs for several hours or a few days before experimental trials only sometimes alter feeding behavior. Zebrafish larvae exposed throughout their first stage and tested 7 days after hatching exhibit altered feeding behavior. When zebrafish and their food, the rotifers, Brachionus calyciflorus and Lecane papuana, were exposed to GBH at concentrations of 0.8 mg/L, zebrafish decreased their food consumption. However, zebrafish pre-exposed to GBH consumed non-GBH food at normal rates. Since zebrafish rely on olfactory cues to find food, the authors suggest that changes in tastes or smells caused differential feeding on exposed rotifers ^[116][134]. Fruit flies consume less medium containing Roundup^® in a dose-dependent pattern ^[117][135]. Similarly, flies preferred an organic sucrose solution to a solution that contained Roundup^® Ready to Use, a GBH formulation with the active ingredients glyphosate and pelargonic acid. However, they did not show a preference for the solution with Roundup^® Super Concentrate, another GBH formulation containing the surfactant POEA and the active ingredient glyphosate, despite exposure to equal concentrations of glyphosate from both formulations. In the same study, flies given organic corn medium containing either Roundup^® Ready to Use or Roundup^® Super Concentrate at various concentrations later consumed more sucrose than those pre-fed with non-GBH medium. The authors attribute this to the flies consuming less of the GBH-contaminated medium, which prompted them to later consume more sucrose solution ^[118][136]. The spider, Alpaida veniliae, showed lower consumption rates when prey were exposed to GBH ^[119][137]. Effects of GBH exposure on honeybees are mixed. Newly emerged adult honeybees given food infused with glyphosate showed a decrease in food intake compared to the control ^[120][121][138,139]. However, a study conducted in the winter found honeybees consumed more food when it contained GBH at concentrations of 0.1, 1, and 10 μg/L. Species pre-exposed to glyphosate demonstrated varying effects. The pacu fish, Piaractus mesopotamicus, displayed decreased food consumption after they were exposed to glyphosate. Pacu were exposed chronically (10–15 days) to glyphosate at 0.2, 0.6 and 1.8 ppm. While the details differed among days, fish exposed to all concentrations exhibited decreased feeding on at least some days. Those exposed to 1.8 ppm also exhibited such a decrease ^[122][145]. Pre-exposed freshwater planarian, Girardia tigrina, exhibited decreased food consumption as the concentration of Roundup^® increased ^[123][146]. Another study tested how glyphosate and Roundup^® exposure influenced larvae of the damselfly, Coenagrion pulchellum. The larvae were exposed to 1 mg/L or 2 mg/L, both of which led to an increase in consumption of food compared to the control ^[124][147]. Predator cues did not affect food consumption when larvae of the damselfly, Enallagma cyathigerum, were pre-exposed to 2 mg/L of glyphosate for seven days ^[125][148]. Adult and spiderling Pardosa milvina environmentally exposed to GBH Buccaneer Plus ate more crickets than unexposed controls. The authors attribute this behavior to hyperactivity from exposure to Buccaneer Plus ^[126][149]. A similar study involving both P. milvina and another wolf spider, Tigrosa helluo, also showed that Buccaneer Plus altered predator efficiency. Environmental exposure via paper discs saturated with 12 mL/m² GBH placed in random locations around the testing apparatus mimicked fields exposed to GBH. T. helluo were allowed to prey on crickets, Acheta domesticus, and on P. milvina; P. milvina were observed preying on crickets. In the presence of the herbicide, T. helluo were able to capture prey faster than the control for both prey types. While exposed and unexposed P. milvina did not differ in timing of predation, they required more lunges to capture their prey ^[127][150]. Another wolf spider, Hogna cf. bivittata, displayed pest-specific effects from GBH exposure, as they captured caterpillars and ants with lower efficiency than the control, but not when preying on crickets ^[128][151], while a different spider, Pardosa agricola, and the ground beetle, Poecilus cupreus, showed no significant difference in prey capture rates ^[129][152]. The water flea, Daphnia pulex, reduced grazing by 40% after pre-exposure at glyphosate concentrations of 50 mg/L ^[130][153]. Three-keeled pond turtle, Mauremys reevesii, eggs were exposed to glyphosate concentrations of 0, 2, 20, 200, and 2000 mg/L, which led to an increase in foraging time in the hatchlings at the two highest concentrations ^[131][154]. While some studies indicate that GBH exposure alters feeding behavior, others do not. For instance, in a study involving two predators, the southern hawker dragonfly, Aeshna cyanae and smooth newt, Lissotriton vulgaris, the predatory activity of organisms exposed to GBH, chronically or acutely, was no different from those unexposed, suggesting that GBH had no effect on the foraging of the predators ^[132][133][155,156].

4. Anti-Predator Behavior

Glyphosate-based herbicides like Roundup^® have adverse effects on anti–predator capabilities of some organisms, but not others. For some organisms, exposure to GBHs led to a decrease in predator awareness. Zebrafish exposed to GBH were found to be in areas that put them at a higher risk of predation indicating loss in predator awareness compared to unexposed fish ^{[134][135][136][137][138]}[157,158,159,160,161]. In another fish, the common spiny loach, Lepidocephalichthys thermalis, exposure to Roundup^® (3 h and 15 days at 0.8 mg/L) led to an increase in activity in the presence of conspecific alarm cues (CC) ^[139][162]. Wood frog, Lithobates sylvaticus, tadpoles exposed to injured conspecific cues and Roundup^® did not change their activity, while tadpoles unexposed to Roundup^® decreased activity when exposed to cues from injured conspecifics, which indicates that glyphosate impairs the tadpole’s ability to respond to the threat of predation ^[140][163]. Gulf coast toad, Incilius nebulifer, tadpoles pre-exposed to Roundup^® and exogenous corticosterone (CORT) became more active in the presence of predator cues. Pre-exposure to the individual reagents and control all showed a decrease in activity ^[141][164]. Blue Ridge two-lined salamander showed synergistic effects of temperature and glyphosate on anti-predator behaviors. Use of refuge became less frequent in exposed salamanders at ambient temperatures (12 °C) and an interactive effect between elevated temperatures (23 °C) and glyphosate also had lower frequency of refuge use. Glyphosate led to a reduction in burst distance, speed and distance from a predator attack, unaffected by temperature ^[109][127]. When exposed to Roundup^®, damselfly larvae exhibited more activity in the presence of predator cues than the controls, which reduced their activity in the presence of predator cues. Exposed larvae walked more, faced their food and fed more often than the controls. However, the predator, the emperor dragonfly, Anax imperator, was not more effective at eating exposed larvae than the controls, despite the change in anti-predator behavior, perhaps because exposed larvae’s increased swimming speeds may counteract the reduced anti-predator behavior ^[142][165]. In damselfly, exposure to either Roundup^® or pure glyphosate at 1 or 2 mg/L induced slower escape speeds in the presence of predator cues. Roundup^® induced significantly slower escape speeds than glyphosate, indicating that “inert” ingredients affected their anti-predator capabilities ^[124][147]. Exposure to GBH decreased the amount of time wolf spiders spend ambulatory compared to controls in response to predator cues from beetles but not giant wolf spiders’ predator cues ^[143][166]. In other organisms, exposure to GBH causes little to no effects on anti-predator behavior. For instance, threat of predation from newts and dragonflies did not affect the anti-predation behavior of tadpoles of the agile frog, Rana dalmatina. Tadpoles exposed to varying levels of herbicide exhibited different behaviors, as the concentration increased, tadpoles decreased their activity around the predators. It was also shown that more tadpoles hid more often at the higher concentrations from the predators, more tadpoles hid from the dragonfly larvae than newts. Overall, the authors suggest that exposure to the herbicide did not significantly alter the tadpole’s anti-predator response ^[144][167]. Likewise, the anti-predator capabilities of marsh frog tadpoles were not affected by exposure to Roundup^® Power 2.0 ^[112][130].

5. Reproductive and Maternal Behavior

As an endocrine disruptor, glyphosate and GBHs particularly affect animal reproduction and reproductive behavior. Exposure to glyphosate can lead to a variety of negative effects on the reproductive systems of animals, including courtship, mating, fertility, and maternal behavior. Ait Bali et al. ^[88][106] found that the mice had difficulty conceiving and success rates rapidly declined when exposed to higher concentrations of glyphosate. The females who were not exposed to glyphosate had an 87% success rate for conceiving, females exposed to 250 mg/kg had a 60% success rate, and females exposed to 500 mg/kg had a 25% success rate. Similarly, fecundity rates and fertility rates of planaria decrease as the concentration of glyphosate increases ^[123][146]. In earthworms, L. terrestris and Aporrectodea caliginosa, and Japanese medaka, Oryzias latipes, it was found that fecundity and fertility rates were negatively impacted by GBH exposure ^[145][146][168,169]. The offspring of female Wistar rats exposed to GBH had lower rates of fertility as well ^[147][170]. Pinning behavior, considered crucial for the development of sexual competence in males, was diminished in both male and female offspring perinatally exposed to the highest dose of GBH. Both doses of GBH reduced female sexual behavior, as demonstrated by a decrease in the female’s receptiveness to the male’s sexual advances, measured by latency to the first lordosis, a postural change in females that indicates receptivity to mating, number of lordosis, and number of mounts without lordosis. Male sexual behavior, measured in latencies to the first mount, first intromission, first ejaculation, number of total mounts with or without intromissions, and number of ejaculations in 30 min, was unaffected. The study suggests that the prenatal and lactational exposure to GBH disrupted aromatase activity, leading to the impairment of sexual behavior in female offspring, including a precocious vaginal opening ^[148][171]. GBHs decrease masculinization of male mice exposed before puberty ^[149][150][172,173]. Both maternal exposure to glyphosate and exposure before puberty disturbed the masculinization process during the critical period of sexual hypothalamic differentiation. Sexual partner preference score, measured by (total time spent in estrous female area—total time spent in sexually active male area), increased and copulatory behavior was altered, with an increase in latency to first mount, first intromission, and mount after first ejaculation. In the same mice, exposure increased estradiol serum concentrations, but this did not lead to increased sexual arousal. However, the mice began puberty at a younger age, which may lead to an increase in sexual behaviors at a younger age ^[149][172]. Wolf spider males exhibit less courtship when exposed to glyphosate. Females were placed inside traps and 47.2% of the traps captured between one and four males. Traps with GBH on filter paper inside the trap captured fewer males than those treated with distilled water. Traps with GBH surrounding the opening also captured fewer males than those with only water on the filter paper ring. This suggests that the herbicide interferes with female pheromone production. In an olfactometer experiment, there was no significant difference in the choice of a corridor that the spider took regardless of the presence of a female or not, leading to belief that the spiders were not repulsed by GBH itself. The conclusion was made that the males had trouble in detecting and/or responding to the females pheromones ^[151][174]. Another study confirmed that exposure to glyphosate impaired sexual chemical communication between female and male wolf spiders, Pardosa agrestis, reducing the male spider’s ability to find their mate ^[152][175]. Male agrobiont spiders and beetles exhibit similar courtship behaviors and experience similar success rate and duration of mating regardless of whether the surface they were on contained GBH residues ^[129][152]. A similar study on wolf spiders and glyphosate showed no significant effect on courtship or sexual behavior in either sex of the spider ^[153][176]. This may be due to differences between species. While exposure levels from ^[153][176] are difficult to compare because of different experimental procedures (5.04 µg/cm² at 30.34%), they appear to be comparable among the other three studies (12 g/L ^[151][174], 14.4 g/L ^[129][152] and 15 mL/L ^[152][175]). Chronic sublethal exposure to glyphosate and another pesticide, thiacloprid, negatively affected colonies of the ant species, Cardiocondyla obscurior, decreasing the number of eggs and pupae when exposed to both pesticides simultaneously ^[154][177]. Specifically, queens’ reproductive performance decreased, possibly due to trade-offs between detoxification and reproduction. The density of endosymbionts in workers decreased, which could be responsible for the decrease in the queens’ reproductive performance. In addition, the pesticides had no effect on the sex ratio, but resulted in smaller colonies. The results highlight the importance of studying multiple stressors and the long-term effects of chronic exposure. Exposure to glyphosate and its commercial formulations can interfere with the reproductive fitness of fish by affecting their neural and endocrine systems. Exposure to glyphosate (0.5 mg/L in Roundup^®) decreases the sexual activity and sperm quality of male livebearers found in rice plantations in southern Brazil and northern Argentina ^[27][108][38,126]. Livebearers also experienced a reduction in copulation and mating success, thus decreasing sexual activity ^[155][178]. GBH exposure also negatively impacts mate attraction by changing territorial behavior, aggressiveness, and coloration of livebearers, zebrafish, and male Mozambique tilapia, Oreochromis mossambicus, all traits important in courtship behavior and mate attraction, which ultimately decreases reproduction. Territorial behavior is important because females lay their eggs within these territories, and those with more resources attract more females. Aggressiveness includes chasing and biting rival males to secure access to females. Finally, coloration indicates a male’s health and genetic quality, such that brighter and more colorful patterns attract females ^[155][156][178,179]. Adult zebrafish that are exposed to glyphosate in combination with warm temperatures showed significant malformities in offspring, which may ultimately negatively impact sexual development and behavior in later stages of life ^[157][180]. Conversely, GBH exposure did not significantly impact the fertility and reproductive potential of rainbow trout, since both the control and exposed fish had high fertility ^[111][129]. GBHs decreased ovary size and number of mature oocytes in fruit flies ^[158][181], which may account for GBH-induced reductions in fertility ^[159][61]. Several studies ^{[88][148][160][161]}[106,171,182,183] show that female rats and mice who were exposed to glyphosate while pregnant exhibited less maternal behavior, including decreased nursing, grooming of offspring, brooding, and reduced time spent in the nest compared to unexposed pregnant females. This negatively affects the offspring, interfering with their development and interactions with their environment. For example, offspring of exposed mothers had reduced locomotor function and mental health impairments. It was also found that maternal exposure to GBH had negative effects on maternal care of offspring, resulting in decreased body weight of rats at 75 and 90 days of age, with male offspring being more susceptible than females ^[148][171]. Another study ^[162][184] found that perinatal exposure to GBH reduced maternal care and aggressive behavior in rats, which may impair their ability to protect their offspring from predators. This was due to hormonal deregulations that decreased maternal reflexes and motivation. The time and number of pups retrieved decreased with a high dose of GBH, and maternal grooming and nesting was also observed to decrease. Maternal grooming and nesting are important for the pups’ development of endocrine and emotional responses to stress, and the lack of such grooming or nesting by the mother can alter the pup’s endocrine development and its response to stress later in life. Maternal behavior of Wistar rats exposed to two different concentrations of GBH during pregnancy and lactation was not affected, nor did it impact water and food intake of mothers or their body weight, gestational length, or litter size. There were also no visible external malformations in the pups or any effect on their body weight due to GBH intake by mothers. These findings suggest that exposure to GBH during pregnancy and lactation did not have any significant adverse effects on maternal behavior of rats ^[163][185]. No significant changes were observed in maternal behavior of Wistar rats between the experimental and control groups ^[161][183]. These conflicting results may be attributed to the lower doses of GBH in both Gallegos ^[163][185] and de Oliveira ^[161][183] and the different formulations that were used. Additionally, there were no observed behavioral changes in Japanese medaka fish despite induced altered expression in reproductive related genes ^[146][169]. Nikbakhtzadeh and Fuentes ^[164][186] found that exposure to glyphosate was lethal to eggs, larvae and pupae, prolonged larval development, and delayed pupation of the mosquito, Culex quinquefasciatus. Female mosquitoes avoided ovipositing in glyphosate-contaminated water ^[164][186], but glyphosate at 5 mg/L from Roundup^® Super Concentrate had no effect on where female field crickets, Gryllus lineaticeps, chose to lay their eggs ^[165][187].

6. Learning, Memory, and Cognition

Learning involves the acquisition of new information, while memory is the ability to retain that information and apply it in future situations. Studies that focus on visual and olfactory learning tasks indicate that some sensory learning systems are extremely susceptible to GBH exposure, while in other situations they may not be affected at all. In the mosquito, Aedes aegypti, for example, habituated less to a visual stimulus after exposure to a dose only 5% of the lethal dose, and almost completely lost habituation at higher but still field-relevant concentrations ^[166][188]. The effect of GBH on honeybee sensory learning is more complicated. In two-color discrimination associative learning, whether the association was between neutral stimuli and electric shock ^[82][100] or between a sucrose reward and an aversive solution ^[167][189], GBH exposure did not affect visual learning. However, in a 10-color discrimination scenario, which is a realistic foraging situation for honeybees, GBH-exposed bees failed to learn during the second half of training, resulting in significantly worse performance than unexposed control bumblebees, Bombus terrestris ^[167][189]. The same authors found no effect on 10-odor discrimination. Glyphosate exposure did impair olfactory learning in 9-day-old young adult honeybees (but not at 5 or 14 days, ^[120][138]) and adult honeybees ^[79][86][98,104] in some two-choice associative learning situations, but not in another ^[168][190]. Apparently, difficult sensory learning tasks are more likely to be damaged by GBH exposure than simple ones. Even in paradigms in which sensory learning is not impaired by GBH exposure, memory often is. Though Hernandez et al. ^[168][190] found no effect on learning, or on memory overall, exposure did shorten memory retention from long-term to medium-term sensory memory. Helander et al. ^[167][189] found that sensory memory was significantly and strongly impaired in the same situations as learning and 10-color discrimination but not 2-color or 10-odor situations. Importantly, this was true whether bees were exposed to GBH before or after learning acquisition. Similarly, Herbert et al. ^[86][104] and Luo et al. ^[79][98] identified deficiencies in short-term and medium-term olfactory memory in exposed honeybees. A study of farmers in Uganda identified that visual memory is also impaired by pesticide exposure in humans, as are language memory, perceptual motor function, complex attention, and processing speed. Specifically, glyphosate exposure is associated with impaired visual memory, as measured by the Benton visual retention test ^[169][191]. Spatial learning ability has been assessed based on maze completion in rats and turtles, and homeward flight paths in honeybees. In an open field experiment, honeybees exposed to GBH took longer to accomplish homeward flight, and were less likely to transition from an indirect flight path on their first trial to a direct path on the second ^[81][99]. Similarly, turtles exposed to GBH took longer to complete a cross maze, and those exposed to high concentration took longer than low concentration ^[131][154]. Rat spatial learning was examined using the Morris Water Maze test, where they use visual cues outside of the water to find a submerged platform in opaque water. Those rats exposed to GBH took longer to find the correct quadrant and the platform during the second half of the learning phase, regardless of the exposure concentration ^[170][192]. GBH exposure impaired spatial memory even more strongly than spatial learning. In honeybees, those exposed to GBH at 25% or 50% of the ED₅₀ (ED₅₀ = 10 mg/L in sucrose) concentration took 6 or 10 times longer to complete a simple maze 2 h after training than unexposed controls. After 24 h, the results were only slightly less pronounced. In addition, while control bees required no course corrections, exposed bees did in a dose-dependent manner. Differences between exposed and unexposed bees were even greater in a complex maze, both in terms of completion time and course corrections ^[80][60], again indicating that more complex types of learning and memory decrease more than simple ones. Rodents’ spatial memory was also impaired, including rats tested in the water maze test mentioned above ^[170][192] (but not ^[171][193]). In addition, chronic GBH exposure reduces short term spatial memory in a y-maze among young mice exposed through maternal dosing, prenatally, and through lactation ^[88][106] (but not those exposed only during gestation ^[172][194]) and chronically exposed adult mice ^[173][195]. Mice and rats explore and spend more time with an unfamiliar (novel) object than one they have spent time interacting with in the past. However, mice exposed to GBH fail to discriminate between novel and familiar objects. In adults, chronic and subchronic exposure significantly reduce discrimination. This effect most prominently impacts short-term (6-h) memory ^[89][107]; and is dose-dependent when young mice are exposed through maternal dosing ^[88][106]. These results are similar in rats, both in terms of an increase in variance among exposed females and overall novel object recognition impairment in males ^[174][196] or both sexes ^[170][192]. In contrast, Del Castilo et al. ^[171][193] did not observe a decrease in novel object recognition in 3-month-old mice exposed to GBH since pregnancy; the difference may be attributed to lower doses. Consistent GBH exposure is detrimental to aversive stimulus-avoidance memory in mice and in fish. In both taxa, electric shocks are applied when the animal enters a dark area of their arena during training. Short-term memory is measured as the latency to enter the dark area 2 h later; long-term memory is tested 24 h after training. Mice exposed to 500 mg/kg, whether as adults ^[173][195] or through maternal dosing ^[88][106] exhibited shorter latency to enter the dark area after 24 h, whether dosing was chronic, subchronic or acute. Maternally dosed and chronically dosed adults’ short-term avoidance memory was also impaired at this dose. A lower dose of 250 mg/kg impaired avoidance memory after acute and subchronic dosing in adults (short-term memory), subchronic and chronic dosing in adults (long-term memory), and maternal dosing (long-term only). In zebrafish ^[97][115] and a livebearer fish ^[108][126], long-term aversive stimulus memory is impaired by GBH exposure. These are also among the few learning and memory papers that directly compare exposure to different formulations. Consistent with results from a wide variety of taxa comparing the effects of glyphosate to those of formulated GBHs on many different behavioral, physiological, morphological, and genetic endpoints (reviewed in ^[17][45][28,56]), Bridi ^[97][115] found that Roundup^® exposure affected memory more than exposure to glyphosate alone, and Sanchez et al. ^[108][126] compared two different Roundup^® formulations with somewhat different results. Most information about how GBH exposure affects humans is based on case studies resulting from accidental acute exposure or intentional exposure during suicide attempts. Most of these case studies indicate that short-term and/or verbal memory loss occurs. The exception is Wang et al. ^[58][75], who report a case in which the patient exhibited parkinsonian syndrome, but without short-term memory loss. Other cases do report short-term memory loss, often beginning quickly (hours to days after exposure) ^{[78][175][176]}[95,197,198]. In some cases, memory loss lasted for many months ^[78][95] or years ^[177][199] through the end of the study. In other cases, dramatic improvements were observed ^[175][176][197,198]. Types of memory affected include word recall ^[175][197], confusion, verbal memory, general memory and delayed memory ^[78][95], and both retrograde and anteriograde amnesia ^[78][176][95,198]. While there is some variation among these case studies, both in how patients were assessed and the memory impairments reported, the overall pattern indicates that short-term and language memory are most often affected.

7. Social Behaviors

Studies on a variety of animal species have assessed the effects of pesticides and herbicides on social behavior. The scope of these investigations includes effects on anxiety-related and depressive-like behavior, aggression, autism spectrum disorders (ASD), and more. In critical developing stages of the brain such as the prenatal, postnatal, and adolescent periods, exposure may be even more detrimental, having possible impacts not just on higher cognitive functioning like in learning and memory, but also on social and emotional behavior as well as the development of ASD ^[87][178][105,200]. Generally, herbicides like glyphosate have been reported to affect motor and emotional functioning in addition to sociability in several non-target animals. Though neurotoxicity of glyphosate on humans is less frequently studied, studies have made associations between glyphosate and neuropathology like ASD and Parkinson’s disease. Herbicides like glyphosate have been shown to cause neurotoxic effects linked to changes in mood such as anxiety-related and depressive-like behavior. In rodents, the open field (OF) test is commonly used to assess locomotor activity and emotional reactivity to new environments, where a tendency to stay in the peripheral areas in the OF arena, termed thigmotaxis, and thus, less time spent in the center, indicates anxiety-like behavior ^[89][107]. Elevated plus maze (EPM) tests have also similarly been used to assess anxiety in rodents. Employing these methods, studies on GBH exposure in mice have reported decreased locomotor activity ^[89][179][107,201], indicating effects on nervous system function, and increased anxiogenic behavior ^{[87][88][89][180][181]}[105,106,107,202,203]. Ait Bali et al. ^{[87][180][181]}[105,202,203] have shown that these effects are dose-dependent and occurred after subchronic (6 weeks) and chronic (12 weeks) GBH exposure, but not acute. Another study by Bicca et al. ^[182][204] assessed subchronic exposure of Zamba^® GBH at 50 mg/kg on rodents and explored the potential therapeutic effects of the flavonoid, quercetin. The results showed increased anxiety in the EPM test with fewer open arm entries and less time spent in the open arm. These effects were largely recovered by quercetin. Conversely, some studies did not find anxiolytic behavioral effects of glyphosate in the OF test ^{[171][179][183]}[193,201,205], and Joaquim et al. ^[179][201] noted reduced exploratory behavior of only male mice during the EPM test. Exposure to GBH during critical development stages of the brain may have important effects on emotional behavior later in life. De Castro Vieira Carneiro et al. ^[172][194] reported that mice between postnatal day (PND) 25–28 that were exposed to 0.3 mg/kg/day of GBH during gestation crossed lines more frequently in OF tests, suggesting hyperactivity, and exhibited increased marble burying behavior which may be indicative of anxiety. In another study, rats were exposed to either 0.65 or 1.3 g/L of GBH during gestation and lactation. Females at PND 45 that were exposed to the highest concentration of GBH crossed fewer squares in an OF test, indicating decreased locomotor activity. These effects were also observed in 90-day-old male and female rats exposed to either concentration of GBH. The authors state their findings are positively correlated to an increase in emotional response in adulthood ^[163][185]. Glyphosate also affects locomotor activity and anxiety in other non-target organisms such as zebrafish. A study assessing the potential effects of global warming on glyphosate toxicity found that zebrafish exposed to glyphosate at increasing temperatures spent more time at the bottom of the tank and had more erratic movements suggesting increased anxiety ^[184][206]. They also found that glyphosate exposure at increasing temperatures caused disruptions in the zebrafish’s circadian rhythm, where they spent less time swimming during the light portion of the cycle and more in the dark portion. Ivantsova et al. ^[100][118] compared the effects of glyphosate and AMPA (glyphosate’s main metabolite) as well as a mixture of the two on zebrafish larvae. While glyphosate, but not AMPA or the mixture, induced hyperactivity in zebrafish, there were no observed effects on anxiety with any of the treatments. In livebearers, unlike what has generally been seen in rodents, unexposed fish spend more time in the peripheral areas in an OF test than the central area where there is increased predatory susceptibility, indicating natural anxiety behavior ^[108][126]. Two formulations of GBH, Roundup^® Original and Roundup^® Transorb, increased the amount of time fish spent in the central area, while a third formulation, Roundup^® WG, did not. The authors suggest that this could be due to a depressive-like state coinciding with reduced alertness. Similarly, Lanzarin et al. ^[138][161] found that zebrafish embryos exposed to the highest tested concentration of GBH did not exhibit evasion behavior when introduced to an aversive stimulus compared to the unexposed embryos. This decreased perception of fear could be due to adverse effects on CNS development, specifically in the habenula region of the brain which plays a role in aversive response control. While it is unclear whether the effects of GBH on evasion behavior are caused by a depressive-like state, other studies have also linked glyphosate exposure to depression-like behavior. Ait Bali et al. ^[87][180][105,202] reported that subchronic and chronic, but not acute, exposure to GBH not only induced anxiety in mice but also depressive behavior, where mice subjected to a tail suspension test and splash test showed a dose-dependent increase in immobility time and decrease in grooming time, respectively. These results were in agreement with Joaquim et al. ^[179][201], who also reported increased immobility in the tail suspension test with both male and female mice acutely exposed to GBH. Mice exposed to GBH also spent more time immobile in a forced swim test and this, like the anxiety effects previously discussed, also improved with quercetin therapy ^[182][204]. Rats exposed to 0.36% glyphosate in water from gestational day 5 until PND 60 demonstrated prolonged immobility time and decreased time climbing in a forced swim test, indicative of depressive-like behavior, though no effects on anhedonia-like behavior were seen ^[185][207]. Glyphosate affects honeybees’ ability to carry out social activities that rely on functions such as directional flight ^[81][99], appetite ^[86][104], associative learning, and circadian rhythms ^[82][100]. Decreased social interaction was also observed in rodents exposed to GBH during gestation ^[161][186][183,208] and also throughout the lifespan from pregnancy until adulthood ^[171][193]. Impaired social behavior was also found in livebearers exposed to Roundup^® for 96 h ^[108][126], as demonstrated by a preference for the side of the aquarium with fewer fish. De Oliviera et al. ^[161][183] found that maternal exposure to GBH reduced the number of ultrasonic vocalizations emitted by pups, which is an early social communicative deficit. Additionally, the pups exhibited increased latency to reach the GBH-treated dam’s shavings, signifying a defect in olfactory discrimination which is important for social behavior development. In a three-chamber sociability test, GBH-treated mice spent less time and made less visits to another conspecific and spent more time with an inanimate object, indicating adverse effects on adult mice social skills ^[88][106]. While many studies have reported negative effects of GBH on social behavior, a few studies ^[138][172][161,194] did not identify such effects. Changes in aggression also occur in some animals exposed to glyphosate. In Sanchez et al.’s ^[108][126] study, livebearers were tested for aggressive behavior by observing their proximity to their own reflection in a mirror, which indicated preference for an “opponent”. All GBH-treated fish spent more time in proximity to the mirror and thus demonstrated more aggressiveness than non-treated fish. In contrast, Bridi et al. ^[97][115] reported that glyphosate impaired aggressive behavior in zebrafish, utilizing a similar methodology. Pinning behavior is an important assessment of play fighting behavior in rodents where the goal of the rat is to wrestle the opponent onto its back and stand over it ^[148][171]. Pinning behavior was impaired in both male and female offspring of dams exposed to the highest GBH dose of 150 mg/kg/day from day 15 of gestation to PND 7. Since pinning behavior is critical to the development of male rat sexual competence, this could affect sexual behavior in adulthood. Over the past few decades, there has been a rapid increase in the prevalence of autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by difficulties in social communication and unusually limited and repetitive behaviors and interests ^[187][209]. With the simultaneous increase in global herbicide and pesticide use, many studies assessing the association between pesticides and neurodevelopmental disorders like ASD have shown a strong relationship ^{[178][188][189][190]}[200,210,211,212]. Del Castilo et al. ^[171][193] found that both male and female 3-month-old mice exposed to GBH since pregnancy exhibit more repetitive marble burying, a behavior used to assess stereotyped behavior in mouse models of autism. Other studies also support these findings ^[161][172][183,194], reporting an increase in repetitive/stereotyped behavior in rodents exposed to GBH during gestation and throughout the lifespan ^[171][193]. In addition, in a novel object recognition test, mice demonstrated cognitive deficits after maternal glyphosate exposure, suggesting ASD-like cognitive impairment ^[186][208]. Some case studies have assessed the effects of pesticides and herbicides on neurological disorders like ASD and Parkinson’s disease in humans. For example, a study by von Ehrenstein et al. ^[62][79] examined birth data between 1998–2010 from the Central Valley of California, a major agricultural location. The risk of developing ASD correlates with exposure to glyphosate and other herbicides such as chlorpyrifos, diazinon, malathion, avermectin, and permethrin. This risk increases following prenatal exposure to ambient pesticides within 2000 m of their mother’s residence during pregnancy. Exposure during the first year of life can further increase the risk of ASD with intellectual disability comorbidity. Other studies have linked glyphosate exposure, through occupational exposure or accidental ingestion, to Parkinsonian syndrome ^{[58][177][191]}[75,199,213]. A case study by Zheng et al. ^[192][214] detailed the events of a previously healthy 58-year-old woman following acute glyphosate exposure, where the patient developed Parkinsonian syndrome that completely resolved after treatment with ATP, pralidoxime iodide, and scopolamine hydrobromide.