Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites.

Since their discovery in the late 1980s, neonicotinoid pesticides have become the most widely used class of insecticides worldwide, with large-scale applications ranging from plant protection (crops, vegetables, fruits), veterinary products, and biocides to invertebrate pest control in fish farming. In this review, we address the phenyl-pyrazole fipronil together with neonicotinoids because of similarities in their toxicity, physicochemical profiles, and presence in the environment. Neonicotinoids and fipronil currently account for approximately one third of the world insecticide market; the annual world production of the archetype neonicotinoid, imidacloprid, was estimated to be ca. 20,000 tonnes active substance in 2010. There were several reasons for the initial success of neonicotinoids and fipronil: (1) there was no known pesticide resistance in target pests, mainly because of their recent development, (2) their physicochemical properties included many advantages over previous generations of insecticides (i.e., organophosphates, carbamates, pyrethroids, etc.), and (3) they shared an assumed reduced operator and consumer risk. Due to their systemic nature, they are taken up by the roots or leaves and translocated to all parts of the plant, which, in turn, makes them effectively toxic to herbivorous insects. The toxicity persists for a variable period of time-depending on the plant, its growth stage, and the amount of pesticide applied. A wide variety of applications are available, including the most common prophylactic non-Good Agricultural Practices (GAP) application by seed coating. As a result of their extensive use and physicochemical properties, these substances can be found in all environmental compartments including soil, water, and air. Neonicotinoids and fipronil operate by disrupting neural transmission in the central nervous system of invertebrates. Neonicotinoids mimic the action of neurotransmitters, while fipronil inhibits neuronal receptors. In doing so, they continuously stimulate neurons leading ultimately to death of target invertebrates. Like virtually all insecticides, they can also have lethal and sublethal impacts on non-target organisms, including insect predators and vertebrates. Furthermore, a range of synergistic effects with other stressors have been documented. Here, we review extensively their metabolic pathways, showing how they form both compound-specific and common metabolites which can themselves be toxic. These may result in prolonged toxicity. Considering their wide commercial expansion, mode of action, the systemic properties in plants, persistence and environmental fate, coupled with limited information about the toxicity profiles of these compounds and their metabolites, neonicotinoids and fipronil may entail significant risks to the environment. A global evaluation of the potential collateral effects of their use is therefore timely. The present paper and subsequent chapters in this review of the global literature explore these risks and show a growing body of evidence that persistent, low concentrations of these insecticides pose serious risks of undesirable environmental impacts.

Effects of neonicotinoids and fipronil on non-target invertebrates.

We assessed the state of knowledge regarding the effects of large-scale pollution with neonicotinoid insecticides and fipronil on non-target invertebrate species of terrestrial, freshwater and marine environments. A large section of the assessment is dedicated to the state of knowledge on sublethal effects on honeybees (Apis mellifera) because this important pollinator is the most studied non-target invertebrate species. Lepidoptera (butterflies and moths), Lumbricidae (earthworms), Apoidae sensu lato (bumblebees, solitary bees) and the section "other invertebrates" review available studies on the other terrestrial species. The sections on freshwater and marine species are rather short as little is known so far about the impact of neonicotinoid insecticides and fipronil on the diverse invertebrate fauna of these widely exposed habitats. For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large-scale and wide ranging negative biological and ecological impacts on a wide range of non-target invertebrates in terrestrial, aquatic, marine and benthic habitats.

Environmental fate and exposure; neonicotinoids and fipronil.

Systemic insecticides are applied to plants using a wide variety of methods, ranging from foliar sprays to seed treatments and soil drenches. Neonicotinoids and fipronil are among the most widely used pesticides in the world. Their popularity is largely due to their high toxicity to invertebrates, the ease and flexibility with which they can be applied, their long persistence, and their systemic nature, which ensures that they spread to all parts of the target crop. However, these properties also increase the probability of environmental contamination and exposure of nontarget organisms. Environmental contamination occurs via a number of routes including dust generated during drilling of dressed seeds, contamination and accumulation in arable soils and soil water, runoff into waterways, and uptake of pesticides by nontarget plants via their roots or dust deposition on leaves. Persistence in soils, waterways, and nontarget plants is variable but can be prolonged; for example, the half-lives of neonicotinoids in soils can exceed 1,000 days, so they can accumulate when used repeatedly. Similarly, they can persist in woody plants for periods exceeding 1 year. Breakdown results in toxic metabolites, though concentrations of these in the environment are rarely measured. Overall, there is strong evidence that soils, waterways, and plants in agricultural environments and neighboring areas are contaminated with variable levels of neonicotinoids or fipronil mixtures and their metabolites (soil, parts per billion (ppb)-parts per million (ppm) range; water, parts per trillion (ppt)-ppb range; and plants, ppb-ppm range). This provides multiple routes for chronic (and acute in some cases) exposure of nontarget animals. For example, pollinators are exposed through direct contact with dust during drilling; consumption of pollen, nectar, or guttation drops from seed-treated crops, water, and consumption of contaminated pollen and nectar from wild flowers and trees growing near-treated crops. Studies of food stores in honeybee colonies from across the globe demonstrate that colonies are routinely and chronically exposed to neonicotinoids, fipronil, and their metabolites (generally in the 1-100 ppb range), mixed with other pesticides some of which are known to act synergistically with neonicotinoids. Other nontarget organisms, particularly those inhabiting soils, aquatic habitats, or herbivorous insects feeding on noncrop plants in farmland, will also inevitably receive exposure, although data are generally lacking for these groups. We summarize the current state of knowledge regarding the environmental fate of these compounds by outlining what is known about the chemical properties of these compounds, and placing these properties in the context of modern agricultural practices.

Toxicity of the systemic insecticide, imidacloprid, to forest stream insects and microbial communities.

Imidacloprid was added to laboratory aquatic microcosms at concentrations of 12, 24, 48 and 96 microg/L to determine effects on leaf-shredding aquatic insect survival and feeding rates, and on aquatic microbial decomposition of leaf material. Survival of the stonefly, Pteronarcys dorsata, was significantly reduced at 48 and 96 microg/L. There was no significant mortality of the cranefly, Tipula sp., but most surviving tipulids were very sluggish and non-responsive to prodding at 48 and 96 microg/L. Leaf decomposition by these leaf-shredding insects was significantly reduced at all test concentrations. There were no significant adverse effects on microbial decomposition of leaf material.

Fate and effects of azadirachtin in aquatic mesocosms--1: fate in water and bottom sediments.

The fate and effects of azadirachtin were examined using in situ enclosures deployed in a typical forest pond of northern Ontario. A commercial azadirachtin-based insecticide formulation, Neemix 4.5, was applied as the test substance. Fate studies were conducted to determine kinetics and persistence of azadirachtin isomers A and B in the aqueous phase and whether either isomer partitioned significantly to bottom sediments or pore water. Aqueous azadirachtin residues dissipated following slow linear kinetics with time to 50% dissipation of 25, 45, and 30 days for azadirachtin A, azadirachtin B, and total residues, respectively. Sediment pore water concentrations increased slowly, reaching low-level equilibrium with the overlying water column toward the end of the summer season. No significant sorption to bottom sediments was observed. Results demonstrated that fate and dissipation of azadirachtin residues are consistent from year to year and that biota may be chronically exposed to diminishing levels of azadirachtins A and B in aqueous phase under conditions of a typical forest pond environment.

Applications of computer-intensive statistical methods to environmental research.

Conventional statistical approaches rely heavily on the properties of the central limit theorem to bridge the gap between the characteristics of a sample and some theoretical sampling distribution. Problems associated with nonrandom sampling, unknown population distributions, heterogeneous variances, small sample sizes, and missing data jeopardize the assumptions of such approaches and cast skepticism on conclusions. Conventional nonparametric alternatives offer freedom from distribution assumptions, but design limitations and loss of power can be serious drawbacks. With the data-processing capacity of today's computers, a new dimension of distribution-free statistical methods has evolved that addresses many of the limitations of conventional parametric and nonparametric methods. Computer-intensive statistical methods involve reshuffling, resampling, or simulating a data set thousands of times to empirically define a sampling distribution for a chosen test statistic. The only assumption necessary for valid results is the random assignment of experimental units to the test groups or treatments. Application to a real data set illustrates the advantages of these methods, including freedom from distribution assumptions without loss of power, complete choice over test statistics, easy adaptation to design complexities and missing data, and considerable intuitive appeal. The illustrations also reveal that computer-intensive methods can be more time consuming than conventional methods and the amount of computer code required to orchestrate reshuffling, resampling, or simulation procedures can be appreciable.

Infectivity and effects of gypsy moth and spruce budworm nuclear polyhedrosis viruses ingested by rainbow trout.

Rainbow trout fingerlings were fed dried krill injected with gypsy moth or spruce budworm nuclear polyhedrosis virus (LdNPV and CfNPV, respectively) at a total dose of 1.4 x 10(7) occlusion bodies (OBs) per fish. By the end of the 21-day experimental period there were no adverse effects on fish survival or behavior and no significant differences in feeding rates or growth between treated and control fish. The internal organs of all fish were examined at the end of the experiment and there were no signs of lesions, discoloration, swelling, hemorrhaging, or other aberrations. Visceral tissues were analyzed with a horseradish peroxidase-labeled whole genomic DNA probe (enhanced chemiluminescence procedure) to detect infection by the NPVs. There were no indications of NPV infection (no positive signals) in stomach and intestinal tract tissues of treated fish. High background signals were obtained from liver samples, but further analyses indicated that these were not due to the presence of LdNPV or CfNPV. The protocols outlined here should be applicable to determining infectivity and effects of genetically modified insect viruses on fish.

Nontarget effects of neem-based insecticides on aquatic invertebrates.

Neem-based insecticides are being investigated as alternatives to synthetic insecticides for the control of forest insect pests in Canada. Because the proposed use pattern against forest defoliators often necessitates spraying near shoreline areas, an assessment of adverse effects of neem on nontarget aquatic invertebrates was warranted. Acute lethal effects of two neem-based formulations on eight species of macroinvertebrates were determined in flow-through screening tests at 10 times the expected environmental concentration (0.35 mg/liter). Significant mortality occurred only in the mayfly Isonychia bicolor/rufa exposed to one formulation, and further tests were conducted to determine a concentration-dependent response. The LC50 for I. bicolor/rufa was estimated at 1.12 mg/liter; the LC10 was 0.63 mg/liter. Three detritivorous species were tested at expected environmental concentrations (0.035 mg/liter) at longer exposures in aquatic microcosms to determine effects of the two formulations on feeding rates and survival. Neither formulation caused significant mortality or antifeedant effects after a 28-day exposure. The implications of these results for forest pest management operations are discussed.

Functional effects of the bacterial insecticide Bacillus thuringiensis var. kurstaki on aquatic microbial communities.

Epilithic microbial communities were colonized on leaf disks and exposed to commercial preparations of Bacillus thuringiensis var. kurstaki (Btk) in aquatic microcosms. Responses in terms of microbial respiration, bacterial cell density, protozoan density, and microbial decomposition activity were measured. Test concentrations for treatments with Dipel 64AF and Dipel 8AF in microcosms were the expected environmental concentration (EEC) of 20 IU/ml, 100x the EEC, and 1000x the EEC. Bacterial cell density in the biofilm of leaf disks was significantly increased at concentrations as low as the EEC. There were no concomitant alterations in protozoan density. Microbial respiration was significantly increased, and decomposition activity was significantly decreased, but only at the artificially high concentration of 1000x the EEC. This effect was attributed to the spore-crystal component rather than formulation ingredients. Microbial decomposition of leaf material was also determined in outdoor stream channels treated at concentrations ranging from the EEC to 100x the EEC. Although there tended to be reduced decomposition activity in treated channels, there were no significant differences in mass loss of leaf material between treated and control channels. Various regression, classification, and ordination procedures were applied to the experimental data, and none indicated significant treatment effects. These results from laboratory and controlled field experiments indicate that contamination of watercourses with Btk is unlikely to result in significant adverse effects on microbial community function in terms of detrital decomposition.

Effects of a new molt-inducing insecticide, tebufenozide, on zooplankton communities in lake enclosures.

: A potent ecdysone agonist, tebufenozide, has recently been developed as a molt-inducing insecticide to control defoliating lepidopterans. As part of continuing research efforts to assess the effectiveness and environmental safety of this material for insect pest management in Canadian forests, tebufenozide (RH-5992-2F) was applied to large lake enclosures and the effects on zooplankton communities were evaluated. There were significant treatment effects at all test concentrations (0.07-0.66 mg L(-1) tebufenozide). Concentration-dependent reductions in the abundance of cladocerans indicated that there were direct toxic effects of tebufenozide on this group of macrozooplankton. There were no indications of direct toxic effects on copepods. Significant increases in abundance of rotifers in treated enclosures at the three higher test concentrations were coincident with reductions in cladocerans and indicated secondary effects of the insecticide on the abundance of microzooplankton. There were no significant differences among treated and control enclosures in chlorophyll a concentrations, indicating that tebufenozide did not have direct effects on phytoplankton biomass, nor did the alterations in the zooplankton communities of treated enclosures have measurable secondary effects on phytoplankton biomass. Daytime dissolved oxygen concentrations were significantly higher in treated enclosures than in controls, indicating that the perturbation to biotic communities of some treated enclosures was sufficient to induce measurable changes in system-level functional attributes. Recovery of zooplankton communities in the enclosures occurred within 1-2 months at 0.07 and 0.13 mg l(-1) and by the following summer (12-13 months) at 0.33 and 0.66 mg l(-1).

Toxicity of a new molt-inducing insecticide (RH-5992) to aquatic macroinvertebrates.

The molt-inducing insecticide RH-5992, a potent ecdysone agonist, is being evaluated for potential use in forestry to control defoliating lepidopterans. The possible adverse effects of RH-5992 on nontarget aquatic organisms were studied in two test systems. Acute lethal effects were determined for one aquatic amphipod and 11 species of aquatic insects in laboratory flowthrough toxicity tests. Lethal and behavioral effects (drift response) on the amphipod and 8 species of stream insects were also evaluated under natural environmental conditions and more realistic exposure regimens in outdoor stream channels. There were no significant effects on drift or survival of the test species exposed to RH-5992 at the maximum test concentration of 3.5 mg/liter (100x the worst-case expected environmental concentration) in laboratory toxicity tests and stream channel treatments. Mortality of the amphipod Gammarus sp. in one toxicity test was considered an artifact, because there was no significant mortality in subsequent tests at concentrations up to 7.0 mg/liter, or in stream channels treated at 3.5 mg/liter. Yellow birch leaves were sprayed with RH-5992 at a rate of 50 g/ha and tested for residual toxic effects on two species of shredding invertebrates in the outdoor stream channels. There was no feeding inhibition or lethal effect on either test species resulting from consumption of the contaminated foliage. The candidate insecticide RH-5992 does not appear to pose undue risk of direct adverse effects to aquatic macroinvertebrates, particularly in water bodies where residues are likely to be short lived following aerial applications (e.g., lotic systems).

Effects of the herbicides hexazinone and triclopyr ester on aquatic insects.

Experiments were conducted to measure acute lethal response of aquatic insects to hexazinone (Velpar L) and triclopyr ester (Garlon 4) in flow-through laboratory bioassays, and to determine lethal and behavioral effects of these herbicides on insects in outdoor stream channels. No significant mortality (chi 2 P greater than 0.05) occurred in 13 test species exposed to hexazinone in laboratory flow-through bioassays (1-hr exposure, 48-hr observation) at the maximum test concentration of 80 mg/liter. The survival of insects exposed to 80 mg/liter hexazinone in outdoor stream channels was likewise unaffected. Significant drift (chi 2 P less than 0.001) of Isonychia sp. occurred during a hexazinone treatment of the stream channels, but only at the maximum concentration of 80 mg/liter, and survival of the displaced Isonychia sp. was not affected. In flow-through bioassays with triclopyr ester, 10 of 12 test species showed no significant mortality at concentrations greater than 80 mg/liter. Survival of Isogenoides sp. and Dolophilodes distinctus was significantly affected at less than 80 mg/liter. Lethal concentrations were estimated by probit analysis of concentration-response data (1-hr exposure, 48-hr observation) for Simulium sp. (LC50 = 303 mg/liter), Isogenoides sp. (LC50 = 61.7 mg/liter), and D. distinctus (LC50 = 0.6 mg/liter). Triclopyr ester applications to the stream channels resulted in significant drift and mortality of D. distinctus at 3.2 mg/liter (no effects at 0.32 mg/liter), Isogenoides sp. at 32 mg/liter, and Hydropsyche sp. and Epeorus vitrea at 320 mg/liter. The risk to aquatic insects of these herbicides used in forest vegetation management is discussed.

Impact of a pulse application of permethrin on the macroinvertebrate community of a headwater stream.

This study evaluated the impact of concentrated pulse (16 microg litre(-1)) of the insecticide permethrin (emulsifiable concentrate) on the macroinvertebrate community of a northern Ontario headwater stream. Post-treatment drift increased by a factor of 2400 within minutes of the arrival of the insecticide. There was a significant (P<0.05) reduction in the abundance of invertebrates in most families as far as 260 m below the point of injection in both kick and artificial substrate samples. Greatest impact was observed in the mayflies, Baetis flavistriga. Heptagenia flavescens, and Epeorus sp., the stonefly, Leuctra tenuis, and the caddisfly, Dolophilodes distinctus. Diptera were not significantly reduced. The number of species occurring 100 m from the point of injection was reduced by 47%, but only by 17% at 260 m. There was no change in the per cent composition of functional feeding groups at any point after treatment. Recovery of most invertebrates was complete within 6 weeks of treatment.