Commercially formulated glyphosate can kill non‐target pollinator bees under laboratory conditions

The use of glyphosate‐based herbicides in agroecosystems has increased over the past few years because of the advent of genetically modified glyphosate‐resistant crops and resistant weeds. This is alarming because of potential damaging effects on non‐target organisms. In sub‐Saharan Africa, for example Ghana, many rural farmers have not received training in the use of glyphosate‐based herbicides, thus tend to apply higher than recommended concentrations on farms. Therefore, this study investigated the effect of glyphosate‐based herbicides on beneficial insects under laboratory conditions, using Apis mellifera L. (Hymenoptera: Apidae, Apini) and Hypotrigona ruspolii (Magretti) (Hymenoptera: Apidae, Meliponini) as models. The bees were put in contact for 24 h with the recommended concentration of Sunphosate 360 SL, a glyphosate‐based herbicide, 2× the recommended concentration, or distilled water as control. The effect of the herbicide on the bees was compared to the effect of a lambda‐cyhalothrin insecticide. Generally, more bees died after contact with plants freshly sprayed with the herbicide than on herbicide‐treated filter paper. In both cases, more bees died after contact with the higher concentration of the herbicide. These findings suggest that beneficial insects, specifically A. mellifera and H. ruspolii, may get killed if they are sprayed upon or come into contact with plants that have been freshly sprayed with (more than) the recommended concentration of glyphosate‐based herbicides. Therefore, it is important to restrict access and use of such herbicides to trained personnel who will comply with spraying guidelines, that is, recommended concentrations and timing of spray. Spraying at a time when insects are flying about may be detrimental to beneficial insects such as pollinator bees, parasitoids, and predators.     

Can aerial herbicide application control Grey Willow (Salix cinerea L.) and stimulate native plant recovery in New Zealand wetlands?

Grey Willow (Salix cinerea L.) poses a significant threat to wetland ecosystems in New Zealand. To manage the ecological impacts and to control further spread, cost‐effective large‐scale control methods are needed. We investigated the response of Grey Willow and dominant wetland plant groups to the aerial boom‐spray application of glyphosate at 9 L/ha and triclopyr (amine) at 18 L/ha at three New Zealand wetlands. We found glyphosate substantially reduced the dominance of tall (>2 m) Grey Willow with commensurate increases in the dominance of most native plant groups. Triclopyr (amine) application resulted in poor Grey Willow control, was not associated with increased native plant group dominance, and some native plant groups declined where triclopyr (amine) was applied. We conclude that the aerial application of glyphosate is an effective large‐scale Grey Willow control tool and could be used to initiate the restoration of native plant communities in wetlands dominated by Grey Willow. But, evidence of Grey Willow recovery after control suggests that increases in native plant dominance will be reversed as Grey Willow re‐establish. Further research is needed to determine how to maintain and enhance native plant dominance after control, and to determine how to manage Grey Willow in fen areas where the Grey Willow canopy is discontinuous and nontarget herbicide impacts can occur. The aerial boom‐spray application of triclopyr (amine) for large‐scale Grey Willow control should be discontinued as it does not provide effective control and results in negative ecological outcomes.     

Seed screening of three pine species for glyphosate sensitivity for forest restoration

Austrian black pine (Pinus nigra J.F. Arnold), Scots pine (Pinus sylvestris L.), and maritime pine (Pinus pinaster Aiton) are commonly used in restoration practices in the Mediterranean base including Turkey. Direct seeding can secure desired level of tree establishment and survival on degraded sites wherever, favorable safe sites are scattered throughout the landscape. Glyphosate is a commonly used herbicide for weed control in forest, nursery and restoration sites worldwide, due to its broad-spectrum efficacy and considerably less toxicity on the environment compared to other chemicals. The present study screened the glyphosate phytotoxicity of three dominant pine species in Turkey during a seed germination trial, after seeds had been presoaked in 15 different dose-glyphosate solutions varying between 0 and 5%, v:v. Glyphosate applications damaged seed germination speed more than cumulative germination rate. As a systemic herbicide, glyphosate appeared highly phytotoxic to Austrian black pine. Scots pine showed an intermediate glyphosate phytotoxicity. Maritime pine was the least sensitive species, tolerating glyphosate at ≤1% doses. In conclusion, glyphosate may be used at low doses on restoration sites seeded with maritime pine while it is not recommended to be used on degraded sites seeded with Austrian black pine and Scots pine.     

Responses of invertebrates to herbicide in Salix cinerea invaded wetlands: Restoration implications

The use of herbicides to control weeds, particularly large invasions, has now become an essential management tool in many ecological restoration projects. The herbicide glyphosate is routinely used to control the invasive weed, Grey Willow (Salix cinerea), within New Zealand wetlands. However, little is known about the effects of glyphosate on invertebrates. We determine the short‐term effects of glyphosate on the abundance and composition of the nontarget canopy invertebrate community in wetlands invaded by Grey Willow in New Zealand. Initially, the application of glyphosate and a surfactant showed no detectable effect on the canopy invertebrates examined in this study. However, 27 days after herbicide application, significant Grey Willow canopy loss caused dramatic decreases in the abundance of invertebrates in the glyphosate‐treated plots compared with the unsprayed plots. Invertebrates appeared to be sensitive to changes in vegetation structure, such as canopy loss. These results agree with previous studies that have shown that the negative impacts of glyphosate on invertebrate communities are related to indirect effects via habitat modification as the herbicide‐treated vegetation dies. From a terrestrial invertebrate perspective, this study suggests that the use of glyphosate herbicide is suitable for the control of invasive weeds within wetland restoration projects as it appears to have negligible impact on the canopy invertebrate assemblage.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 × 10⁻⁸. Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.     

Investigations into the movement of glyphosate from treated to adjacent untreated plants

Transfer of glyphosate from treated to adjacent untreated plants was investigated under glasshouse conditions using wheat and Agropyron repens. When glyphosate was used at concentrations characteristic of conventional field application rates, and where shoot contact was prevented, no symptoms were observed on untreated plants. When there was shoot contact, and when glyphosate was used at 2 kg a. i./ha (10 g a. i./litre), phytotoxic effects were observed on untreated plants. At higher concentrations of glyphosate (90 or 180 g a. i./litre), typical of selective applications with ropewick or roller applicators, evidence of root transfer of herbicide was found. In pot experiments these phytotoxic effects were variable, due, perhaps, to variable amounts of root contact. Confining the roots, by growing the plants in tubes, increased the level of phytotoxicity.     

Effects of genetically modified maize events expressing Cry34Ab1, Cry35Ab1, Cry1F,and CP4 EPSPS proteins on arthropod complex food webs

Four genetically modified (GM) maize (Zea mays L.) hybrids (coleopteran resistant, coleopteran and lepidopteran resistant, lepidopteran resistant and herbicide tolerant, coleopteran and herbicide tolerant) and its non‐GM control maize stands were tested to compare the functional diversity of arthropods and to determine whether genetic modifications alter the structure of arthropods food webs. A total number of 399,239 arthropod individuals were used for analyses. The trophic groups’ number and the links between them indicated that neither the higher magnitude of Bt toxins (included resistance against insect, and against both insects and glyphosate) nor the extra glyphosate treatment changed the structure of food webs. However, differences in the average trophic links/trophic groups were detected between GM and non‐GM food webs for herbivore groups and plants. Also, differences in characteristic path lengths between GM and non‐GM food webs for herbivores were observed. Food webs parameterized based on 2‐year in‐field assessments, and their properties can be considered a useful and simple tool to evaluate the effects of Bt toxins on non‐target organisms.     

Evaluation of glyphosate application in regulating the reproduction of riparian black locust (Robinia pseudoacacia L.) after clear-cutting,and the possibility of leaching into soil

Black locust (Robinia pseudoacacia L.)—an invasive alien species in riparian forests—is becoming more prevalent in many rivers of eastern Japan. Riparian black locust forests are typically cut down to maintain river-flow capacity. However, such forests often reproduce rapidly by stump sprouting and root suckering and regenerate by germination. Thus, more effective riparian forest management approaches are required. To regulate the reproduction of black locust forests after clear-cutting, we examined the regrowth-inhibiting effects of glyphosate herbicide application to stumps, in accordance with current river management protocol (i.e., winter logging operation). Further, we investigated the concentrations of glyphosate leaching into the soil at a depth of 30 cm in a riparian area of the Tenryu River. Our results showed that glyphosate application to stumps completely inhibited stump sprouting but not root suckering or seedling germination. The glyphosate concentration leaching into the soil reached a maximum (2.6 ± 0.7 mg kg^?1, mean ± standard error) on day 1 after the application, and subsequently declined to below the detection limit on day 2. Thus, the rapid degradation of glyphosate was confirmed, despite the fact that the herbicide leached into the soil after application to the stumps. The glyphosate application has limited effectiveness against root suckering and germination of riparian black locust forests after clear-cutting in winter, in accordance with the current river management protocol.     

Evaluating Tolerance of Herbicide and Transplantation by Cane (a Native Bamboo) for Canebrake Restoration

Canebrakes (bamboo grasslands dominated by Arundinaria spp.) were once a widespread ecosystem across the Southeastern United States, and many species of wildlife depended upon them. Early settlers replaced this system with subsistence agriculture and today few canebrakes remain. The restoration of canebrakes is critical to the recovery of several wildlife species; however, restoration is complicated because (1) seed is uncommon and often predated, (2) competition from hardwood species, including the exotic Chinese privet (Ligustrum sinesnse), often prevent cane establishment, and (3) cane depends on disturbance regimes that have been disrupted in the Southeast. We investigated the tolerance of Switch cane (Arundinaria tecta) to four commonly used herbicides that are effective at controlling privet and other hardwoods: hexazinone (Velpar‐L), glyphosate (Razor Pro), triclopyr (Garlon 3A), and imazapyr (Chopper). We also investigated the possibility of transplanting cane culms, and the factors affecting successful transplant. Cane tolerated hexazinone and triclopyr but was damaged or killed by glyphosate and imazapyr. Although many measures of weather and cane condition were not predictors of transplant success, the Keetch–Byram drought index was a strong predictor, and is available through most state forestry offices. Selective herbicides and deliberately timed transplantation may be important canebrake restoration tools.     

Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)

Silver catfish (Rhamdia quelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.     

Effects of glyphosate [N-(phosphonomethyl)glycine] on photosynthetic pigments, stomatal response and photosynthetic electron transport in Medicago sativa and Trifolium pratense

Stomatal response, chlorophyll and carotenoid content, and photosynthetic electron transport were investigated in Medicago sativa L. (cv. Aragón) and Trifolium pratense L. (cv. Violeta) after plants were exposed to various glyphosate [ N -(phophonomethyl)glycine] concentrations. The herbicide decreased the content of photosynthetic pigments when applied at concentrations of 0.15, 1.5 and 15 m M , showing a greater effect on chlorophyll pigments than on carotenoids. The chlorophyll a / b ratio increased as a consequence of a major effect on chlorophyll b. Glyphosate treatment (15 m M ) led to a closure of stomates after seven days in both species. The results suggest that glyphosate acts as an electron transport inhibitor, because glyphosate pre-incubated chloroplasts, isolated from control plants, showed reduced photosystem II and photosystem I activities. In general, clover was more sensitive to glyphosate than glyphosate, lucerne.     

The effect of varying rates of glyphosate and an organosilicone surfactant on the control of gorse

The herbicidal effect of glyphosate applied to gorse (Ulex europaeus L.) was improved by the addition of increasing amounts (0.5–20 g/litre) of Silwet L-77, an organosilicone surfactant. Increasing the rate of herbicide also enhanced control. There was a highly significant interaction between surfactant rate and herbicide dosage; as the amount of Silwet L-77 was increased the rate of glyphosate could be reduced without loss of herbicide efficacy. However, without any added organosilicone surfactant, glyphosate did not provide more than 73% control of gorse at any rate up to 6.5 kg a.i./ha. With the addition of Silwet L-77, complete mortality of all plants could be achieved with 2.2 kg glyphosate/ ha.     

Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plants

A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-¹⁴C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production.The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.     

The influence of form of deposit on the phytotoxicity of MCPA, paraquat and glyphosate applied as individual drops

MCPA, paraquat and glyphosate were applied as individual drops (200–400 μm) to pot-grown plants of radish (Raphanus sativus) or wild oat (Avena fatua), using concentrations appropriate to very low volume applications of these herbicides. For a given dose per plant, herbicide activity was unaffected by concentration of MCPA or paraquat but was enhanced as the concentration of glyphosate was increased. The activity of all three herbicides on both species was affected by variation of the site of application but not by drop size. On radish the greatest activity resulted when paraquat was applied to the cotyledons, glyphosate to the interveinal areas of true leaves and MCPA to the veins of true leaves. This is discussed in relation to herbicide mobility and local toxicity following applications at high concentration.     

Glyphosate-Tolerant Crops: Genes and Enzymes

This review focuses on the genes for the enzymes 5-enolpyruvyl-3-phosphoshikimlc acid synthase (EPSPS) and the glyphosate oxidoreductase (GOX). These genes have been used to genetically engineer plants that are resistant to the herbicide glyphosate. Overproduction of glyphosate-insensitive.EPSPS in transgenic crops has been used to overcome the deleterious effuts of this herbicide. The introduction into plants of GOX also confers glyphosate tolerance to plants and augments the tolerance of transgenic plants already expressing a glyphosate tolerant EPSPS. These genes also provide a method for selecting transformed plant tissue using the glyphosate tolerance as the selectable marker in the presence of inhibitory concentrations of glypllosate. Glyphosate tolerant transgenic plants of beet, corn, cotton, lettuce, poplar, potato, rapeseed. soybean, tobacco, tomato, and wheat have already been field tested and are entering agriculture.     

Absorption,translocation, and metabolism of 14C-thifensulfuron in soybean (Glycine max), spurred anoda (Anoda cristata), and velvetleaf (Abutilon theophrasti)

The absorption, translocation, and metabolism of thifensulfuron-methyl {methyl 3-[[[[(4-methoxy)-6-methyl-1,3,5-triazin-2-yl]-amino]-carbonyl] amino]sulfonyl]-2-thiophenecarboxylate} were investigated in tolerant Essex soybean [Glycine max (L.) Merr.], moderately tolerant Vance soybean, and spurred anoda [Anoda cristata (L.) Schlecht.], and susceptible velvetleaf (Abutilon theophrasti Medic.). Radiolabeled (thiophene-2-¹⁴C) thifensulfuron-methyl was absorbed readily by young seedlings of all species following a foliar spray with the herbicide. Spot-applied ¹⁴C-thifensulfuron-methyl was absorbed by the treated leaf of all species. Absorption of thifensulfuron-methyl was limited when excised stems of all species were dipped into the herbicide solution for 2 h. Translocation of absorbed thifensulfuron-methyl to other plant parts was limited in all species, regardless of the method of its application. Root exudation of leaf-applied thifensulfuron-methyl was observed in all species and it was higher in seedlings of spurred anoda and velvetleaf. The two soybean cultivars metabolized 62–70% of absorbed thifensulfuron-methyl at 3 days after treatment with spot-applied ¹⁴C-thifensulfuron. Velvetleaf and tolerant spurred anoda metabolized about 50% of the absorbed herbicide. The major metabolite formed in all species appeared to be deesterified thifensulfuron acid. Differential metabolism seems to be a contributing factor in the selectivity of thifensulfuron-methyl between the two soybean cultivars and velvetleaf. The metabolic basis for the moderate tolerance of spurred anoda to thifensulfuron-methyl is not understood at the present time.Plant Pathology, Physiology, and Weed Science Department, Contribution no. 628.     

Development of native forest species of the Atlantic forest in soil contaminated with hormonal herbicides

Abstract

Hormonal herbicides, used in pastures, can suffer drift and reach forests. The sensitivity and potential phytoremediation of native species to herbicide residues should be evaluated. The objective of this study is to evaluate the initial development of native Atlantic Forest tree species in soil contaminated with hormonal herbicides. The experiment was conducted in a greenhouse in a 4 x 8 factorial scheme. The first factor had the control and the herbicide Tordon^® in three doses (0.166, 0.333 and 0.666?L ha^?1) and the second consisted of the forest species Anadenanthera colubrina (Vell.), Cassia ferruginea (Schrad.) Schrad. ex DC., Dalbergia villosa (Benth.) Benth., Machaerium nyctitans (Vell.) Benth., Machaerium opacum Vogel, Piptadenia gonoacantha (Mart.) JF Macbr., Senegalia polyphylla (DC.) Britton and Rose, Senna macranthera (DC Collad.) HS Irwin and Barnaby. The emergence, height, survival, emergence speed index, intoxication, root volume, stem diameter, root and shoot dry mass, leaf area and leaf numbers of the forest species were evaluated. The A. colubrina, D. villosa and M. opacum initial development was reduced by the herbicides 2.4-D plus picloram residues. S. macranthera and P. gonoacantha are tolerant to this mixture and, therefore, show potential for phytoremediation of degraded areas containing residues of these compounds.     

An Experimental Investigation into the Response of Some New Zealand Sand Dune Species to Salt Spray

The tolerance to salt spray of 29 species, mainly from New Zealandsand dunes, was investigated. Plants were grown in water culturein a glasshouse and subjected to overhead salt spraying at intervals.Growth rates in many species were reduced by salt spray buta significant decrease occurred only in six native herbs. However,many species showed sensitivity in leaf necrosis. Tolerant speciesincluded Scirpoides nodosa, Elymus farctus and Desmoschoenusspiralis. Ammophila arenaria, tolerant of spray as an adult,was less so when younger. There was little correlation between tolerance to salt sprayand tolerance to root salinity. Some species were tolerant toboth, e.g. S. nodosa and E. farctus, and some intolerant toboth, e.g. Wahlenbergia congesta. One species, Lupinus arboreus,was glycophytic in respect to root salt but tolerant of aerialsalt. Other species, such as Senecio elegans L. and Austrofestucalittoralis, were intolerant of salt spray but tolerant of mediumroot salinities. For some species salt spray tolerance correlated well with fielddistribution, e.g. D. spiralis and Bromus diandrus. However,some species present in semi-fixed dunes close to the sea havemuch lower tolerance than would be expected from their fieldsituation, e.g. W. congesta. This apparent inconsistency couldbe explained by the ameliorating high rainfall on the West Coast,or protection by ridges. One environmental variable alone, suchas salt spray, could not explain the field distribution formany species. Salt spray, growth rate, live leaf area, New Zealand, dune species, root salinity     

Absorption and efflux of glyphosate by cell suspensions

The absorption and efflux of [¹⁴C]-glyphosate (N-[phosphonomethyl]glycine) was studied in maize (Zea mays L. cv. Aussie) and soybean (Glycine max L. Merr. cv. Maple Arrow) cell suspensions. Glyphosate absorption was complex: at low external herbicide concentrations (3-250 M) there was evidence for a single active uptake system with an apparent Km of 31 M and Vmax of 11 nmol g^-1 fr. wt. 2 h^-1. The system was inhibited by carbonylcyanide m-chlorophenyl hydrazone (CCCP), orthovanadate, diethylstilbestrol (DES), phosphate, and phosphonoformic acid (PFA) suggesting the glyphosate carrier to be a phosphate transporter energized by the plant plasmalemma ATPase. At higher external glyphosate concentrations the operation of this carrier was masked as passive diffusion became the dominant absorption mechanism. Any non-specific binding of glyphosate to the cell surface during absorption was low (0.02-0.02 nmol g^-1 fr. wt). Efflux kinetics of [¹⁴C]-glyphosate suggests the herbicide to be located in the cells in three kinetically distinct compartments: after 24 h uptake of radiolabelled herbicide, 71% of absorbed glyphosate was found in the slow compartment (t1/2 162 h), 19% in the medium (t1/2 185 min) and 10% in the fast (t1/2 27 min). The implications of these results in relation to the delivery of glyphosate to its subcellular target site and subsequent phytotoxicity are discussed.Keywords: Zea mays, Glycine max, glyphosate (N-[phosphonomethyl]glycine), absorption, compartmentation.