Glyphosate-based herbicides are the most widely used herbicides in the world, including in Canadian forestry. In general, glyphosate-based herbicides are considered relatively non-toxic to wildlife species due, in part, to rapid breakdown of the chemical in the environment. However, recent work has shown that glyphosate can persist for at least one year after application at low concentrations leading to concern over the persistence of trace levels in the environment. Using two independent studies we characterize the short- (18 days) and long-term (1 year) persistence of glyphosate in vegetation which are commonly, but differentially, browsed by WhiteiTailed Deer (Odocoileus virginianus), Moose (Alces alces), and Black Bear (Ursus americanus), or used as traditional medicines by Indigenous people and compare the residues to exposure thresholds. In the short-term study, glyphosate concentrations within the application block exceeded the general and maximum residue level (MRL) for fresh fruit set by Health Canada (0.1 ppm) for up to 18 days after application. In the long-term study, glyphosate concentrations were above the MRL one week after application and below the MRL one month and one year after application. Under the assumptions that all vegetation contained glyphosate at the highest observed concentration, animals only consume vegetation from herbicide treated areas, and animals consume the physiological maximum level of vegetation daily, Moose, White-Tailed Deer, and Black Bears could exceed the Acceptable Daily Intake for glyphosate (0.3 mg/kg/day) for up to 18 days after application. Subsequently, given dissipation of residues in vegetative matrices as observed in this study, we consider it highly unlikely that the species considered herein could consume enough vegetation throughout their lives to pose a risk to their health. Overall, our two independent studies demonstrate that trace levels of glyphosate persist in vegetation for up to one year after application, however, observed concentrations are unlikely to pose risk to wildlife. We caution that operational practices as typically imposed in Canadian forestry are very important and effective in minimizing risk.

Exploratory analysis of field-derived data highlighted a number of important statistical and experimental design considerations for aquatic mesocosm studies. Abundance data for numerically dominant zooplankton taxa were characterized by distinct heterogeneity of variance (coefficient of variation range 10 to 173%) proportional to the mean and related to both time of observation (0 to 77 days postapplication) and taxonomic level of investigation. Log-transformation did not obviate violations in parametric statistical assumptions, necessitating the use of procedures robust to or not assuming variance homogeneity. Using appropriate RM-ANOVA techniques and multiple comparison tests, an overall LOEC value of 1.0 ppm was estimated for effects on numerical abundance. Log-transformation served to linearize the inherently curvelinear concentration-response relationship. Threshold and temporal effects on the concentration-response relationship resulted in significant lack of fit for the linear model in many cases. Statistically significant, adequately fitting models were observed in 21 of 63 cases, with 16 of these meeting the minimum criteria (Fregression/F(0.05;1,12) > 4) for predictive value of the model. A nonlinear regression technique using an exponential decline model wherein both a and b parameters were considered as linear functions of time provided good fit statistics for 5/8 taxa investigated. Examination of residuals provided a posteriori evidence that the nonlinear model adequately addressed the three-dimensional nature of the problem. EC50 values estimated using linear and nonlinear regression procedures differed markedly, ranging from 0.004 to 0.045 and 0.06 to 1.26, respectively. Potential improvements to experimental design and data analysis, including disproportionate replication and three-dimensional methods of data analysis, were identified in relation to regulatory assessment of aquatic mesocosm impact data.

The direct effects of large-scale disturbances are readily studied because their effects are often apparent and result in large changes to ecosystems. Direct effects can cascade through the ecosystem, leading to indirect effects that are often subtle and difficult to detect. Managing anthropogenic disturbances, such as chemical contamination, requires an understanding of both direct and indirect effects to predict, measure, and characterize the impact. Using a replicated whole-ecosystem experiment and path analyses (assesses the effects of a set of variables on a specified outcome, similar to multiple regression), we examined the direct and indirect effects of a glyphosate-based herbicide and nutrient enrichment on wetland communities. The latter did not impact any measured endpoints. The strongest drivers of macrophyte, benthic invertebrate, and amphibian assemblages were the ephemerality and the size of wetlands, factors which were not altered by herbicide applications. The herbicide had a direct negative effect on macrophyte cover, amphibian larval abundance, and the proportion of predatory benthic invertebrates. However, both amphibians and invertebrates were positively affected by the reduction in the macrophyte cover caused by the herbicide applications. The opposing directions of the direct and indirect effects lead to no net change in either group. The compensatory dynamics observed herein highlight the need for a better understanding of indirect effects pathways to determine whether common anthropogenic disturbances alter the ecological communities in small wetland ecosystems.

Aquatic macroinvertebrate communities are often used to assess the ecological integrity of streams. However, conventional methods involving morphometric identification of macroinvertebrates are usually costly and time-consuming. Here we compare stream macroinvertebrate community metrics based on conventional morphometrics vs. non-destructive DNA metabarcoding from storage ethanol to assess forest management impacts on headwater streams across a gradient of intensively managed forest catchments in eastern Canada. The two approaches demonstrated substantial congruence in the detection of taxa (81% and 69% at the family and genus level, respectively) and in the characterization of community composition and richness. However, DNA metabarcoding from preservative ethanol identified significantly fewer genera (3.3 on average, 15.9%) and families (2.0, 11.5%) than conventional morphometrics. Taxa missed by metabarcoding of storage ethanol were typically those low in proportional mass or poorly represented in the CO1 reference database. This led to some differences in the explanatory variables identified as being related to macroinvertebrate metrics, which could have implications on conclusions and management actions that might result therefrom. For example, the negative relationships between richness and reach-scale variables associated with forest management intensity were weaker when richness was based on metabarcoding as compared to conventional morphometrics. Discriminatory power was greater when data at the genus level were used. The congruence between functional feeding group results derived from morphometric (based on relative abundance) vs. metabarcoding (based on relative frequency and read abundance) identifications was group specific (r = 0.16-0.63), but low overall. We conclude that DNA metabarcoding of storage ethanol provides a promising approach for characterizing stream macroinvertebrate communities, but that its full deployment in biomonitoring projects requires developing more complete reference libraries and enhancing the sensitivity for detecting taxa with low sample biomass.

Cost-effective, ecologically relevant, sensitive, and standardized indicators are requisites of biomonitoring. DNA metabarcoding of macroinvertebrate communities is a potentially transformative biomonitoring technique that can reduce cost and time constraints while providing information-rich, high resolution taxonomic data for the assessment of watershed condition. Here, we assess the utility of DNA metabarcoding to provide aquatic indicator data for evaluation of forested watershed condition across Canadian eastern boreal watersheds, subject to natural variation and low-intensity harvest management. We do this by comparing the similarity of DNA metabarcoding and morphologically derived macroinvertebrate metrics (i.e. richness, % Ephemeroptera, Plecoptera and Trichoptera, % chironomid), and the ability of DNA metabarcoding and morphological metrics to detect key gradients in stream condition linked to forested watershed features. Our results show consistency between methods, where common DNA metabarcoding and morphological macroinvertebrate metrics are positively correlated and indicate the same key gradients in stream condition (i.e. dissolved oxygen, and dissolved organic carbon, total nitrogen and conductivity) linked to watershed size and shifts in forest composition across watersheds. Our study demonstrates the potential usefulness of macroinvertebrate DNA metabarcoding to future application in broad-scale biomonitoring of watershed condition across environmental gradients.

We report morphometric and demographic characteristics of 2 distinct populations of Glyptemys insculpta (Wood Turtle), at the northwestern periphery of the range in Canada, where they are designated as a species at risk. Our surveys of the 2 study watersheds (2012-2015) were assisted by a trained canine unit which was demonstrably more efficient than human crews in detecting Wood Turtles. We observed that both populations were large-214 and 114 uniquely marked individuals documented over time. We found no significant differences (P >= 0.05) in age structure, sex ratios, sexual size-dimorphism, body condition, number of observed mating attempts, or frequency and type of injuries between populations. We observed female-biased sex ratios in both populations (1:1.47 and 1:1.84, respectively) that were not attributable to sampling bias. Our data generally support the postulate of an inverse relationship between Wood Turtle body size and number of frost-free days or latitude. The general health of the 2 study populations was evidenced by the numerous large and reproductively mature individuals of both sexes, relatively high percentage of juveniles observed (average = 26%), and size-class frequency distributions that indicated sustained juvenile recruitment over several years in both watersheds. Our data suggest that high-quality forested watershed habitats, even at the northwestern extreme of the species range in Canada, can and do support large, healthy populations of Wood Turtles.

BACKGROUNDThe Asian long-horned beetle [ALB; Anoplophora glabripennis (Motschulsky)] is an invasive, wood-boring insect posing significant economic and ecological threats to the deciduous forests of North America. An efficacious and environmentally acceptable chemical control technique is a requirement of a comprehensive, integrated response strategy. RESULTSResults of this study demonstrate statistically significant, concentration-dependent effects of azadirachtins, a family of natural compounds derived from the neem tree, on both ALB larval and adult life stages. Growth inhibitory effects on ALB larvae were greatest on early life stages. Significant effects on adults included inhibition of female feeding, oviposition effort and fecundity for adults exposed to azadirachtins via maturation feeding on systemically loaded twigs. CONCLUSIONThese quarantine laboratory experiments verify multi-mechanistic, deleterious effects on both larval and adult life stages of ALB, an exotic, invasive insect pest of critical importance in North America. Field efficacy studies are required to further understand dose acquisition by larval and adult ALB life stages following systemic injections to host trees under semi-operational use scenarios. Such studies could also be used to test postulates regarding optimal deployment strategies to meet objectives such as slowing the spread of this pest and protection of high-value deciduous forest resources. (c) 2017 Her Majesty the Queen in Right of Canada Pest Management Science (c) 2017 Society of Chemical Industry

Automated recording units are increasingly being used to sample wildlife populations. These devices can produce large amounts of data that are difficult to process manually. However, the information in the recordings can be summarized with semiautomated sound recognition software. Our objective was to assess the utility of the semiautomated bird song recognizers to produce data useful for conservation and sustainable forest management applications. We compared detection data generated from expert-interpreted recordings of bird songs collected with automated recording units and data derived from a semiautomated recognition process. We recorded bird songs at 109 sites in boreal forest in 2013 and 2014 using automated recording units. We developed bird-song recognizers for 10 species using Song Scope software (Wildlife Acoustics) and each recognizer was used to scan a set of recordings that was also interpreted manually by an expert in birdsong identification. We used occupancy models to estimate the detection probability associated with each method. Based on these detection probability estimates we produced cumulative detection probability curves. In a second analysis we estimated detection probability of bird song recognizers using multiple 10-minute recordings for a single station and visit (35-63, 10-minute recordings in each of four one-week periods). Results show that the detection probability of most species from single 10-min recordings is substantially higher using expert-interpreted bird song recordings than using the song recognizer software. However, our results also indicate that detection probabilities for song recognizers can be significantly improved by using more than a single 10-minute recording, which can be easily done with little additional cost with the automate procedure. Based on these results we suggest that automated recording units and song recognizer software can be valuable tools to estimate detection probability and occupancy of boreal forest birds, when sampling for sufficiently long periods.

The sensitivity of 15 aquatic species, including primary producers, benthic invertebrates, cladocerans, mollusks, and fish, to MON 0818, a commercial surfactant mixture of polyoxyethylene tallow amines, was evaluated in standard acute (48–96‐h) laboratory tests. In addition, the potential for chronic toxicity (8 d) was evaluated with Ceriodaphnia dubia. Exposure concentrations were confirmed. No significant effects on any endpoint were observed in the chronic test. A tier‐1 hazard assessment was conducted by comparing species sensitivity distributions based on the generated data, as well as literature data, with 4 exposure scenarios. This assessment showed moderate levels of hazard (43.1% of the species exposed at or above median effective concentration levels), for a chosen worst‐case scenario—unintentional direct over‐spray of a 15‐cm‐deep body of water with the maximum label application rate for the studied formulations (Roundup Original, Vision Forestry Herbicide; 12 L formulation ha−1, equivalent to 4.27 kg acid equivalent [a.e.] ha−1). The hazard decreased to impairment of 20.9% of species under the maximum application rate for more typical uses (6 L formulation ha−1, 2.14 kg a.e. ha−1), and down to 6.9% for a more frequently employed application rate (2.5 L formulation ha−1, 0.89 kg a.e. ha−1). Finally, the percentage (3.8%) was less than the hazardous concentration for 5% of the species based on concentrations of MON 0818 calculated from maximum measured concentrations of glyphosate in the environment. Environ Toxicol Chem 2017;36:501–511. © 2016 SETAC

Glyphosate-based herbicides are the dominant products used internationally for control of vegetation in planted forests. Few international, scientific syntheses on glyphosate, specific to its use in planted forests, are publically available. We provide an international overview of the current use of glyphosate-based herbicides in planted forests and the associated risks. Glyphosate is used infrequently in planted forests and at rates not exceeding 4 kg ha(-1). It is used within legal label recommendations and applied by trained applicators. While the highest risk of human exposure to glyphosate is during manual operational application, when applied according to label recommendations the risk of exposure to levels that exceed accepted toxicity standards is low. A review of the literature on the direct and indirect risks of operationally applied glyphosate-based herbicides indicated no significant adverse effects to terrestrial and aquatic fauna. While additional research in some areas is required, such as the use of glyphosate-based products in forests outside of North America, and the potential indirect effects of glyphosate stored in sediments, most of the priority questions have been addressed by scientific investigations. Based on the extensive available scientific evidence we conclude that glyphosate-based herbicides, as typically employed in planted forest management, do not pose a significant risk to humans and the terrestrial and aquatic environments.

Glyphosate-based herbicides are the dominant products used internationally for control of vegetation in planted forests. Few international, scientific syntheses on glyphosate, specific to its use in planted forests, are publically available. We provide an international overview of the current use of glyphosate-based herbicides in planted forests and the associated risks. Glyphosate is used infrequently in planted forests and at rates not exceeding 4 kg ha−1. It is used within legal label recommendations and applied by trained applicators. While the highest risk of human exposure to glyphosate is during manual operational application, when applied according to label recommendations the risk of exposure to levels that exceed accepted toxicity standards is low. A review of the literature on the direct and indirect risks of operationally applied glyphosate-based herbicides indicated no significant adverse effects to terrestrial and aquatic fauna. While additional research in some areas is required, such as the use of glyphosate-based products in forests outside of North America, and the potential indirect effects of glyphosate stored in sediments, most of the priority questions have been addressed by scientific investigations. Based on the extensive available scientific evidence we conclude that glyphosate-based herbicides, as typically employed in planted forest management, do not pose a significant risk to humans and the terrestrial and aquatic environments.

Concentrations of glyphosate observed in the environment are generally lower than those found to exert toxicity on aquatic organisms in the laboratory. Toxicity is often tested in the absence of other expected co-occurring contaminants. By examining changes in the phytoplankton and zooplankton communities of shallow, partitioned wetlands over a 5 month period, we assessed the potential for direct and indirect effects of the glyphosate-based herbicide, Roundup WeatherMax(©) applied at the maximum label rate, both in isolation and in a mixture with nutrients (from fertilizers). The co-application of herbicide and nutrients resulted in an immediate but transient decline in dietary quality of phytoplankton (8.3 % decline in edible carbon content/L) and zooplankton community similarity (27 % decline in similarity and loss of three taxa), whereas these effects were not evident in wetlands treated only with the herbicide. Thus, even at a worst-case exposure, this herbicide in isolation, did not produce the acutely toxic effects on plankton communities suggested by laboratory or mesocosm studies. Indirect effects of the herbicide-nutrient mixture were evident in mid-summer, when glyphosate residues were no longer detectable in surface water. Zooplankton abundance tripled, and zooplankton taxa richness increased by an average of four taxa in the herbicide and nutrient treated wetlands. The lack of significant toxicity of Roundup WeatherMax alone, as well as the observation of delayed interactive or indirect effects of the mixture of herbicide and nutrients attest to the value of manipulative field experiments as part of a comprehensive, tiered approach to risk assessments in ecotoxicology.

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 (2.88 mg acid equivalents/L and 0.21 mg 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. Environ Toxicol Chem 2014; 33:2076–2085. © 2014 SETAC

Variation in toxicity among formulations and species makes it difficult to extrapolate results to all species and all formulations of herbicides. The authors exposed larval wood frogs (Lithobates sylvaticus) from 4 populations to 2 glyphosate-based herbicides, Roundup Weed and Grass Control (R) and Roundup WeatherMax (R). The 96-h median lethal concentration values for both formulations varied among the populations (Roundup Weed and Grass Control, 0.14mg acid equivalents (a.e.)/L to 1.10mg a.e./L; Roundup WeatherMax, 4.94mg a.e./L to 8.26mg a.e./L), demonstrating that toxicity varies among the formulations and that susceptibility may differ among populations. Environ Toxicol Chem 2014;33:2628-2632. (c) 2014 SETAC

Azadirachtins are natural triterpenoid compounds derived from Neem tree extracts with potential for use as systemic insecticides against invasive wood-boring insect pests.

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.