Dean G. Thompson

Ecological Risk Assessment for Aquatic Organisms from Over-Water Uses of Glyphosate

Although the herbicide glyphosate is most widely used in agriculture, some is used for the control of emergent aquatic weeds in ditches, wetlands, and margins of water bodies, largely as the formulation Rodeo. This article presents an ecological risk assessment (ERA) of glyphosate and some of the recommended surfactants as used in or near aquatic systems. Glyphosate does not bioaccumulate, biomagnify, or persist in a biologically available form in the environment. Its mechanism of action is specific to plants and it is relatively nontoxic to animals. As a commercial product, glyphosate may be formulated with surfactants that increased efficacy but, in some cases, are more toxic to aquatic organisms than the parent material. For this risk assessment, three model exposure scenarios--static or low-flow systems such as ponds, flowing waters such as streams, and systems subjected to tidal flows such as estuaries--were chosen and application rates from 1 to 8 kg glyphosate/ha were modeled. Additional measured exposure data from several field studies were also used. As acute exposures are most likely to occur, acute toxicity data were used as effect measures for the purposes of risk assessment. Toxicity data were obtained from the literature and characterized using probabilistic techniques. Risk assessments based on estimated and measured concentrations of glyphosate that would result from its use for the control of undesirable plants in wetlands and over-water situations showed that the risk to aquatic organisms is negligible or small at application rates less than 4 kg/ha and only slightly greater at application rates of 8 kg/ha. Less is known about the environmental fate and toxicology of the surfactants commonly used in combination with the Rodeo formulation of glyphosate. The surfactants used for this purpose were judged not to be persistent nor bioaccumulative in the environment. Distributional analysis of measured deposition concentrations of LI 700, suggest that this surfactant presents an insignificant acute risk to aquatic organisms. Assuming similar applications rates, significant ecological effects would not be expected from the use of some other surfactants such as Induce or X-77. Risks from the use of glyphosate +MON 0818 (Roundup) were slightly greater than those from glyphosate and surfactants such as LI 700; however, in over-water uses, risks were still considered small. Similar small risks were observed for measured concentrations of glyphosate in surface waters resulting from aerial application of Vision (a formulation equivalent to Roundup) to forestry areas in Canada. Concentrations measured after ground application presented a greater risk, but the data were sparse and the assessment is more uncertain.

Azadirachtin: An Effective Systemic Insecticide for Control of Agrilus planipennis (Coleoptera: Buprestidae)

ABSTRACT The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive pest discovered in North America in 2002, is now well established and threatens ash (Fraxinus spp.) trees throughout the continent. Experiments were conducted to 1) examine the efficacy of an alternative natural pesticide, azadirachtin, to control emerald ash borer, and 2) determine foliar uptake and dissipation patterns after systemic injections of azadirachtin into trunks of small (2.2 cm diameter at breast height [dbh]), uninfested green ash trees. We found no evidence of mortality of adult beetles. In contrast, fewer larvae completed their development at dose levels ≥1.7 mg (AI)/cm dbh and development ceased beyond the second instar at dose levels ≤13.6 mg (AI)/cm dbh. Substantial concentrations (11.2 µg/g dry mass [SD = 7.55]) of azadirachtin were present in leaves within 7 d of treatment. After rapid initial uptake, concentrations in leaves declined logarithmically during the 55 d after injection. A similar pattern was observed in a separate experiment that examined the uptake and translocation of azadirachtin in larger green ash trees (22 cm dbh) treated with 250 mg (AI)/cm dbh with the EcoJect injection system. In another experiment, recently infested plantation green ash trees treated with doses ≥40 mg (AI)/cm dbh had significant reductions in adult emergence ≈1 yr postinjection. Given the inhibition of larval development, reduction of adult emergence, and the occurrence of foliar residues at biologically active concentrations, we conclude that azadirachtin is effective in protecting ash trees from emerald ash borer.

Laboratory and field exposure of two species of juvenile amphibians to a glyphosate-based herbicide and Batrachochytrium dendrobatidis

Herbicides are commonly used in agriculture and silviculture to reduce interspecific competition among plants and thereby enhance crop growth, quality, and volume. Internationally, formulations of glyphosate-based herbicides are the most widely used herbicides in both these sectors. A large amount of work has focused on the effects of these herbicides on amphibians. Several laboratory and mesocosm studies have demonstrated that various formulations of glyphosate herbicides can be acutely toxic to larval and juvenile amphibians at concentrations at the upper end of environmental realism. However, to date there has been little work done investigating such effects in natural systems, limited work on juvenile amphibians, and only a few studies have investigated interactions with other stressors. We conducted a 16 day field experiment in which juveniles of two amphibian species (Lithobates clamitans and Lithobates pipiens) were exposed to the herbicide Roundup WeatherMax™ at four application rates (0, 2.16, 4.32 and 8.64 kg a.e./ha) to investigate effects on survival, liver somatic index (LSI), body condition, and incidence of disease caused by Batrachochytrium dendrobatidis (Bd). In a separate 16 day laboratory experiment, we exposed juvenile L. clamitans to both the herbicide and Bd. Results of our studies showed that this particular herbicide formulation had no effect on juvenile survival, LSI, body condition, or disease incidence, nor was there an interaction between exposure to herbicide and exposure to the disease in tests which closely mimic real world exposure scenarios. These experiments suggest that Roundup WeatherMax as typically used in agriculture is unlikely to cause significant deleterious effects on juvenile amphibians under real world exposure conditions.

Community-level disruptions among zooplankton of pond mesocosms treated with a neem (azadirachtin) insecticide

A natural, plant-derived insecticide, neem, is being evaluated as an alternative insect pest control product for forestry in Canada. As part of the process to investigate the environmental safety of neem-based insecticides, a mesocosm experiment was conducted to assess the effects of neem on natural zooplankton communities. Replicate (n=5), shallow (<1 m) forest pond enclosures were treated with Neemix 4.5, at concentrations of 0.035 (the expected environmental concentration), 0.18, 0.70, and 1.75 mg/l active ingredient, azadirachtin. Zooplankton communities were quantitatively sampled over a 4-month experimental period in treated and control enclosures, and water samples were collected to track azadirachtin concentrations. Concentrations in water declined linearly with estimated DT(50) values of 25-29 days. Trends in abundance over time among populations of cladocerans, copepods, and rotifers were found to differ significantly among treatments. At the two highest test concentrations, adverse effects were obvious with significant reductions in several cladoceran species, and near elimination of the three major copepod species present. More subtle effects at the two lowest test concentrations were determined by comparing the community structure of enclosures across treatment levels and over time through an analytical process based on the multivariate statistical software, PRIMER. Significant effects on community structure were detected at both of these lower concentrations, including the expected environmental concentration of 0.035 mg/l azadirachtin. Differential responses among species (some increases, some decreases) caused detectable disruptions in community structure among zooplankton of treated enclosures. Perturbations to zooplankton communities were sufficient to cause measurable differences in system-level metabolism (midday dissolved oxygen concentrations) at all but the lowest test concentration.

Exposure of juvenile green frogs (Lithobates clamitans) in littoral enclosures to a glyphosate-based herbicide.

The majority of studies on the toxicity of glyphosate-based herbicides to amphibians have focused on larval life stages exposed in aqueous media. However, adult and juvenile amphibians may also be exposed directly or indirectly to herbicides. The potential for such exposures is of particular interest in the littoral zone surrounding wetlands as this is preferred habitat for many amphibian species. Moreover, it may be argued that potential herbicide effects on juvenile or adult amphibians could have comparatively greater influence on overall recruitment, reproductive potential and thus stability of local populations than effects on larvae. In this experiment, juvenile green frogs (Lithobates clamitans) were exposed to two concentrations (2.16 and 4.27 kg a.e./ha) of a glyphosate-based herbicide formulation (VisionMax®), which were based on typical application scenarios in Canadian forestry. The experimental design employed frogs inhabiting in situ enclosures established at the edge of small naturalized wetlands that were split in half using an impermeable plastic barrier. When analyzed using nominal target application rates, exposure to the glyphosate-based herbicide had no significant effect on survival, body condition, liver somatic index or the observed rate of Batrachochytrium dendrobatidis infection. However, there were marginal trends in both ANOVA analysis and post-hoc regressions regarding B. dendrobatidis infection rates and liver somatic index in relation to measured exposure estimates. Results from this study highlight the importance of field research and the need to include multiple endpoints when examining potential effects of a contaminant on non-target organisms.

A silviculture application of the glyphosate‐based herbicide VisionMAX to wetlands has limited direct effects on amphibian larvae

Herbicides are commonly used in agriculture and silviculture to reduce interspecific competition among plants and thereby enhance crop growth, quality, and volume. Internationally, glyphosate-based herbicides are the most widely used herbicides in both of these sectors. Laboratory and mesocosm studies have demonstrated that some formulations are toxic to amphibian larvae below concentrations that approximate predicted maximal or worst-case exposure scenarios. However, field studies have not found evidence of toxicity at these concentrations. The authors conducted a replicated field experiment involving 10 naturalized wetlands split in half with an impermeable plastic barrier to assess the direct toxicity of a glyphosate formulation commonly used in silviculture (VisionMAX™). The herbicide formulation was applied directly to the surface of one side of each wetland at one of two target aqueous exposure rates (highu2009=u20092,880, lowu2009=u2009550u2009µg acid equivalents [a.e.]/L), and the other side was left as an untreated control. The survival and growth of green frog larvae (Lithobates clamitans) were assessed for two years following herbicide treatment. The herbicide did not have a negative impact on survival or growth of L. clamitans larvae at either treatment level. In fact, mean larval abundance was typically greater in the treated sides than in control sides within the year of herbicide application. These results indicate that typical silviculture use of VisionMAX poses negligible risk to larval amphibians, likely because the combined effects of sorption and degradation in natural wetlands limit the exposure magnitude and duration.

The direct and indirect effects of a glyphosate‐based herbicide and nutrients on Chironomidae (Diptera) emerging from small wetlands

Laboratory and mesocosm experiments have demonstrated that some glyphosate-based herbicides can have negative effects on benthic invertebrate species. Although these herbicides are among the most widely used in agriculture, there have been few multiple-stressor, natural system-based investigations of the impacts of glyphosate-based herbicides in combination with fertilizers on the emergence patterns of chironomids from wetlands. Using a replicated, split-wetland experiment, the authors examined the effects of 2 nominal concentrations (2.88u2009mg acid equivalents/L and 0.21u2009mg acid equivalents/L) of the glyphosate herbicide Roundup WeatherMax, alone or in combination with nutrient additions, on the emergence of Chironomidae (Diptera) before and after herbicide-induced damage to macrophytes. There were no direct effects of treatment on the structure of the Chironomidae community or on the overall emergence rates. However, after macrophyte cover declined as a result of herbicide application, there were statistically significant increases in emergence in all but the highest herbicide treatment, which had also received no nutrients. There was a negative relationship between chironomid abundance and macrophyte cover on the treated sides of wetlands. Fertilizer application did not appear to compound the effects of the herbicide treatments. Although direct toxicity of Roundup WeatherMax was not apparent, the authors observed longer-term impacts, suggesting that the indirect effects of this herbicide deserve more consideration when assessing the ecological risk of using herbicides in proximity to wetlands.

The response of amphibian larvae to exposure to a glyphosate-based herbicide (Roundup WeatherMax) and nutrient enrichment in an ecosystem experiment

Herbicides and fertilizers are widely used throughout the world and pose a threat to aquatic ecosystems. Using a replicated, whole ecosystem experiment in which 24 small wetlands were split in half with an impermeable barrier we tested whether exposure to a glyphosate-based herbicide, Roundup WeatherMax™, alone or in combination with nutrient enrichment has an effect on the survival, growth or development of amphibians. The herbicide was applied at one of two concentrations (low=210 μg a.e./L, high=2880 μg a.e./L) alone and in combination with nutrient enrichment to one side of wetlands and the other was left as an untreated control. Each treatment was replicated with six wetlands, and the experiment was repeated over two years. In the high glyphosate and nutrient enrichment treatment the survival of wood frog (Lithobates sylvaticus) larvae was lower in enclosures placed in situ on the treated sides than the control sides of wetlands. However, these results were not replicated in the second year of study and they were not observed in free swimming wood frog larvae in the wetlands. In all treatments, wood frog larvae on the treated sides of wetlands were slightly larger (<10%) than those on the control side, but no effect on development was observed. The most dramatic finding was that the abundance of green frog larvae (Lithobates clamitans) was higher on the treated sides than the control sides of wetlands in the herbicide and nutrient treatments during the second year of the study. The results observed in this field study indicate that caution is necessary when extrapolating results from artificial systems to predict effects in natural systems. In this experiment, the lack of toxicity to amphibian larvae was probably due to the fact the pH of the wetlands was relatively low and the presence of sediments and organic surfaces which would have mitigated the exposure duration.

Field Evaluation of Triclopyr Ester Toxicity to Fish

Two field experiments were conducted to assess the predictions of laboratory time-toxicity tests regarding lethal effects of triclopyr butoxyethyl ester (TBEE) on fish in standing and flowing water bodies. Large lake enclosures were treated with TBEE by backpack sprayer at concentrations of 0.25–7.6 mg/L, expressed as acid equivalents. Median dissipation times for TBEE in lake water ranged from 4–8 d.Effects of the treatments on survival and growth of caged rainbow trout were measured. All trout died by 3 d at initial concentrations of 0.69–7.6 mg/L. There was 43% mortality of rainbow trout in the enclosure treated at 0.45 mg/L, and no mortality in the 0.25 mg/L enclosure or the controls. The treatments at the two lower concentrations had significant adverse effects on the growth rates of surviving trout. TBEE was applied to sections of a forest headwater stream at nominal concentrations of 0.8 mg/L and 2.7 mg/L. These concentrations represent maximum-expected environmental concentrations in 50- and 15-cm deep bodies of water, respectively, when directly oversprayed at an application rate of 3.84 kg/ha. Concentrations of TBEE were rapidly dissipated (as much as 70% decline within 55 m) and exposure periods at concentrations above 0.1 mg/L varied from 25 min in the low-concentration area to 55 min in the high-concentration area. The applications of TBEE at both initial test concentrations did not result in any mortality of resident brook trout. There were no significant effects of the herbicide treatments on the growth of 1+ and 2+ brook trout, but there were indications that the growth of 0+ trout was reduced as a result of exposure to TBEE in the stream. These results were in general agreement with the predictions of laboratory time-toxicity tests.

Analytical study of azadirachtin and 3-tigloylazadirachtol residues in foliage and phloem of hardwood tree species by liquid chromatography-electrospray mass spectrometry.

A rapid and sensitive LC-ESI-MS method has been developed and validated for the quantitation of azadirachtin and 3-tigloylazadirachtol in deciduous tree matrices. The method involves automated extraction and simultaneous cleanup using an accelerated solvent technique with the matrix dispersed in solid phase over a layer of primary-secondary amine silica. The limits of quantification were 0.02 mg/kg for all matrices with the exception of Norway maple foliage (0.05 mg/kg). Validation at three levels (0.02, 0.1, and 1 mg/kg), demonstrated satisfactory recoveries (71-103%) with relative standard deviation <20%. Two in-source fragment ions were used for confirmation at levels above 0.1 mg/kg. Over a period of several months, quality control analyses showed the technique to be robust and effective in tracking the fate of these natural botanical insecticides following systemic injection into various tree species for control of invasive insect pest species such as the emerald ash borer and Asian longhorned beetle.