4.1. Direct effects on organisms
Mortality of non-target organisms exposed to insecticides is mostly due to acute toxicity, particularly in the case of carbamates. However, with systemic compounds there are many observations of long-term suppression of populations that suggest a chronic lethal impact over time. The latter impacts are likely due to persistence of residual activity in the soil, foliage or water in the case of reversible toxicants (i.e. carbamates), or to irreversible and persistent binding in other cases. (note: all application rates and concentrations here refer to the active ingredient).
4.1.1. Acetylcholinesterase inhibitors
These compounds can have serious impacts on soil organisms of various taxa. Aldicarb and phorate applied to a cotton crop soil at 0.5 and 1 kg/ha, respectively, eliminated or reduced significantly non-target mesofauna, including mites and springtails. Populations of the latter taxa were reduced for more than 60 days (phorate) and 114 days (aldicarb) [17, 225], with the highest effects peaking after 18 days . Granular applications of phorate (250 mg/kg dry soil) killed almost all earthworms, Collembola, Acarina, free-living saprophytic and parasitic nematodes and Protozoa, with populations of Collembola recovering only when residues went below 2 mg/kg . After a single aldicarb application to soil at 2.5 g/m2, Gamasina predatory mites went to extinction within a year . Bendiocarb impacts on predaceous arthropods and oribatid mites were less severe and temporary compared to the impacts of non-systemic OPs, but increased trap catches of ants two weeks after application , possibly as a result of a longer-term effect. Many soil arthropods, in particular mites and springtails, were the most affected by dimethoate –and its metabolite omethoate– residues in soil after sprays of 1-2 ml/L in the farms of the Zendan valley, Yemen . Similar observations were made when dimethoate was sprayed on vegetation of arable fields  or in soil microcosms ; the springtail populations recovered but attained lower densities a year later, while their dominance structure had changed. However, dimethoate or phosphamidon applied in mustard fields produced only a temporary decline, compared to the long-lasting effect of monocrotophos . Collembola populations do not seem to be affected by pirimicarb applications on cereal crops .
Earthworm populations were affected initially after application of phorate and carbofuran to turfgrass, but not thiofanox, and their numbers recovered subsequently . Reduction of earthworm populations by bendiocarb was the highest (99% in one week) among 17 insecticides applied at label rates on turfgrass, with significant effects lasting up to 20 weeks . Juveniles and species living in the surface layers or coming to the soil surface to feed (e.g., Lumbricus terrestris) are most affected, since a high degree of exposure is usually found in the first 2.5 cm of soil . However, systemic carbamates can be selective to plant-parasitic nematodes without affecting fungal or microbial communities . Thus, cholinesterase inhibitors do not have significant impacts on bacteria, fungi and protozoa in soil , and consequently do not alter the soil biochemical processes . Nevertheless, a combined dimethoate-carbofuran application reduced active hyphal lengths and the number of active bacteria in a treated forest soil .
Populations of beneficial predators can be decimated initially as much as the target pests, but they usually recover quickly. For example, thiodicarb or its degradation product, methomyl, applied at 0.5 kg/ha on soybean crops, significantly reduced populations of the predatory bugs Tropiconabis capsiformis and Nabis roseipennis within two days after treatment only . Demeton-S-methyl reduced populations of predatory insects on strawberry patches, whereas pirimicarb and heptenophos had no significant effect on spiders, staphylinids and anthocorids, or on hymenopteran parasitoids . While populations of web spiders and carabid beetles are severely reduced by dimethoate applied to cabbage fields and cereal crops , pirimicarb does not seem to have much impact on these taxa [97, 195], affecting mainly aphids . Pirimicarb on wheat crops does not impact on ladybirds, but larvae of Episyrphus balteatus are affected . By contrast, longer impacts have been observed with acephate applied at 0.5 kg/ha on rice paddies, which reduced populations of predatory bugs (Cyrtorrhinus lividipennis and Paederus fuscipes) for at least 10 days . Similar rates of acephate on rice and soybean crops reduced spiders populations for three weeks, but they recovered afterwards . In addition, acephate is deadly to three species of whitefly parasitoid species .
Direct mortality of bumble bees (Bombus terrestris) in short exposures to dimethoate is much higher than for heptenophos or ethiofencarb . However, what matters most is the chronic toxicity to the entire bee colony not just the workers. For example, methamidophos contaminated syrup (2 mg/L) produced significant losses of eggs and larvae of honey bees without any appreciable loss of workers after one week of exposure; the colonies would recover completely within 13 weeks if the insecticide was applied only once , indicating a long-term impact on the colony. Similarly, the mortality of non-target adult chrysomelid beetles (Gastrophysa polygoni) after foliar treatment with dimethoate on the host plants was low (1.9-7.6%), but because this insecticide was most toxic to the egg stage, the overall beetle population decreased over time due to hatching failure .
Primary poisoning of birds and mammals by ingestion of OP and carbamate granules or coated seeds is still a problem despite the many attempts to reduce these impacts [189, 190]. For example, mortality of birds that ingested granules of carbofuran in a corn field was extensive, affecting waterfowl, small songbirds and mice within 24 hours. Residues up to 17 mg/kg body weight (b.w.) were found in the dead animals . The granular formulation of this carbamate was banned in the mid-1990s by the US EPA after numerous cases of direct poisoning by animals; however, the liquid formulation applied to alfalfa and corn is just as deadly to bees, because this systemic insecticide is present in the pollen of those plants . Phosphamidon sprayed at 1 kg/ha to larch forests in Switzerland caused many bird deaths ; large bird mortality was also observed in Canadian spruce forests sprayed with phosphamidon (0.55 kg/ha), particularly among insectivorous warblers. There was good evidence that birds picked up the insecticide from sprayed foliage within a few hours of application . Carbofuran and phosphamidon were the most common pesticides implicated in deaths of wild birds in Korea between 1998-2002 , and ducklings died in large numbers when phorate was applied to South Dakota wetlands . Usually birds die when their brain AChE depression is over 75% [92, 114]. Thus, 11 out of 15 blue jays (Cyanocitta cristata) which had depression levels ranging 32-72% after disulfoton was sprayed to pecan groves would die , but their carcasses would probably not be found. In orchards sprayed with methomyl, oxamyl or dimethoate, the daily survival rates for nests of Pennsylvania mourning dove (Zenaida macroura) and American robin (Turdus migratorius) were significantly lower than in non-treated orchards, and the species diversity was also lower. Repeated applications of these and other insecticides reduced the reproductive success of doves and robins and may have lowered avian species diversity .
Secondary poisoning with bendiocarb was attributed to 22 birds that had depressed AChE activity after eating contaminated mole crickets and other soil organisms on the applied turfgrass . Several species of raptors were killed or debilitated after consuming waterfowl contaminated with phorate – the fowl had ingested granules of the insecticide that were applied to potato fields a few months earlier . Equally, ladybugs (Hippodamia undecimnotata) fed upon Aphis fabae, which were reared on bean plants treated with carbofuran, experienced a 67% population reduction due to secondary poisoning . Pirimicarb caused 30-40% mortality of Tasmanian brown lacewing (Micromus tasmaniae) larvae when feeding on contaminated 1st instar lettuce aphid (Nasonovia ribisnigri) for three days .
Impacts on aquatic organisms usually do not last more than a month. For example, thiodicarb applied at 0.25-1.0 kg/ha had severe impacts on copepods, mayflies and chironomids in experimental ponds for three weeks, but not so much on aquatic beetle’s larvae; eventually there was recovery of all populations . Pirimicarb can be lethal to common frog (Rana temporaria) tadpoles, but does not appear to have chronic effects . However, vamidothion and acephate are most lethal to non-target organisms in rice crops, and are not recommended in IPM programs . Carbofuran and phorate are very toxic to aquatic invertebrates , particularly amphipods and chironomids but not so much to snails, leeches or ostracods [72, 249]. Small negative effects in zooplankton communities (cladocerans copepods and rotifers) were observed in rice paddies treated with carbofuran at recommended application rates, but fish were not affected . Carbofuran should not be used in rice paddies, whether in foliar or granular formulations: not only induces resurgence of the brown planthoppers (Nilaparvata lugens) , but it is also more toxic to the freshwater flagellate Euglena gracilis than the non-systemic malation . It reduces populations of coccinellid beetles, carabid beetles, dragonfly and damselfly nymphs, but does not impact much on spiders . However, it appears that carbofuran at 0.2% per ha can double the densities of Stenocypris major ostracods in rice paddies, whereas other insecticides had negative effects on this species . Repeated applications of carbofuran can also have a significant stimulation of the rhizosphere associated nitrogenase activity, with populations of nitrogen-fixing Azospirillum sp., Azotobacter sp. and anaerobic nitrogen-fixing bacteria increasing progressively up to the third application of this insecticide .
4.1.2. Insecticides acting on nAChR
Direct toxicity of cartap to fish species is not as high as that of other neurotoxic insecticides, with 3-h LC50s between 0.02 and 6.8 mg/L [161, 308]. However, cartap affects negatively several species of Hymenoptera and aphid parasitoids used to control a number of crop pests [14, 77, 147, 270]. This insecticide also inhibits hatching of eggs of the nematode Agamermis unka, a parasite of the rice pest Nilaparvata lugens , and reduces significantly the populations of ladybugs and other predatory insects in cotton crops when applied at the recommended rates, i.e. 20 g/ha [109, 169]. In rice paddies, cartap hydrochloride reduced populations of coccinellid beetles, carabid beetles, dragonflies and damselflies by 20-50% . Pollinators such as honey bees and bumble bees can also be seriously reduced in numbers when feeding on crops treated with cartap hydrochloride, which is included among the most toxic insecticides to bees after neonicotinoids and pyrethroids [179, 278]. For all its negative impacts on parasitoids and predatory insects it is hard to understand why cartap was the third most common insecticide (19% of all applications) used in IPM programs in Vietnam a decade ago , and is still among the most widely used in rice farms in China .
Cumulative toxicity of neonicotinoids over time of exposure results in long-term pest control compared to the impact of cholinesterase inhibitor insecticides. For example, soil treated with clothianidin at 0.05-0.15% caused increasing mortality in several species of wireworms (Coleoptera: Elateridae), reaching 30-65% after 70 days, whereas chlorpyrifos at 0.15% produced 35% mortality within 30 days but no more afterwards . Soil application of imidacloprid did not eliminate rapidly Asian citrus psyllid (Diaphorina citri) and leafminer (Phyllocnistis citrella) populations, but resulted in chronic residues in leaf tissue and long-term suppression of both pests . Also, soil applications of neonicotinoids are very effective in controlling soil grubs and berry moths (Paralobesia viteana) in vineyards provided there is no irrigation or rain that washes off the insecticide . For the same reason, however, the impact of neonicotinoids on non-target organisms is long-lasting. For example, repeated corn-seed treatement with imidacloprid caused a significant reduction in species richness of rove beetles in three years, even though the abundance of the main species was not affected . In addition to long-term toxicity, acute toxicity of acetamiprid, imidacloprid and thiomethoxam to planthopper and aphid species is similar to that of synthetic pyrethroids, and higher than that of endosulfan or acetylcholinesterease inhibitors [219, 246]. Thus, combinations of pyrethroid-neonicotinoid have been hailed as the panacea for most pest problems as it suppresses all insect resistance . Mixtures of imidacloprid and thiacloprid had additive effects on the toxicity to the nematode Caenorhabditis elegans but not on the earthworm Eisenia fetida .
Acute toxicity of imidacloprid, thiamethoxam, clothianidin, dinotefuran and nitenpyram to honey bees is higher than that of pyrethroids, while toxicity of acetamiprid and thiacloprid is increased by synergism with ergosterol-inhibiting fungicides [134, 242] and antibiotics . Thus, neonicotinoids can pose a high risk to honey bees, bumble bees [176, 263] and wasps . Bees can be killed immediately by direct contact with neonicotinoid droplets ejected from seed drilling machines. Thus, numerous worker bees were killed when seed was coated with clothianidin during drilling of corn in the Upper Rhine Valley (Germany) in spring 2008 . The same problem happened in Italy with thiamethoxam, imidacloprid and clothianidin [105, 285], leading to the banning of this application method on sunflower, canola and corn during 2008-09 . However, most of the time bee colonies are intoxicated by feeding on contaminated pollen and nectar [9, 228]. It has been observed that bee foraging was notably reduced when Indian mustard was treated with 178 mg/ha imidacloprid . Imidacloprid residues in sunflowers are below the no-adverse-effect concentration to honey bees of 20 μg/kg at 48-h , with surveys in France showing residue levels in pollen from treated crops in the range 0.1-10 μg/kg and average in nectar of 1.9 μg/kg . However, bees feeding on such contaminated pollen or nectar will reach first sublethal and later lethal levels, with 50% mortality occurring within 1-2 weeks [228, 266]. Such data was disputed [89, 240] as it was in conflict with some long-term field observations of honey bees feeding on sunflowers grown from imidacloprid-treated seeds at 0.24 mg/seed . However, recent evidence suggest that chronic lethality by imidacloprid is implicated in the colony collapse disorder (CCD) that affects honey bees . Based on the fast degradation of imidacloprid in bees (4-5 hours), it is assumed that honey bees which consume higher amounts of imidacloprid die already outside of the hive, before the colony’s demise and before samples are taken, though residues of imidacloprid in bees at 5-8 μg/kg have been found in some cases . Clothianidin residues of 6 μg/kg in pollen from canola fields reduced the number of bumble bee (Bombus impatients) workers slightly (~20%) , but exposure to clothianidin-treated canola for three weeks appeared not to have affected honey bee colonies in Canada . Thiamethoxam applied to tomatoes (~150 g/ha) through irrigation water does not have impacts on bumble bees (Bombus terrestris) , whereas pollen contaminated with this insecticide causes high mortality and homing failure .
Negative impacts of neonicotinoids on non-target soil arthropods are well documented. A single imidacloprid application to soil reduced the abundance of soil mesofauna as well as predation on eggs of Japanese beetle (Popillia japonica) by 28-76%, with impacts lasting four weeks. The same level of impact was observed with single applications of clothianidin, dinotefuran and thiamethoxam, so the intended pest control at the time of beetle oviposition runs into conflict with unintended effects – disruption of egg predation by non-target predators . Among several insecticides applied to home lawns, only imidacloprid suppressed the abundance of Collembola, Thysanoptera and Coleoptera adults, non-oribatid mites, Hymenoptera, Hemiptera, Coleoptera larvae or Diptera taxonomic groups by 54-62% . Imidacloprid applied to the root of eggplants (10 mg/plant) greatly reduced most arthropod communities and the species diversity during the first month. Small amounts of soil residues that moved into the surrounding pasture affected also some species; however, non-target ground arthropods both inside and outside the crop showed significant impacts only in the two weeks after planting , probably due to compensatory immigration from nearby grounds.
Foliar applications of thiamethoxam and imidacloprid on soybean crops are preferred to seed treatments, as neonicotinoids appear to have lesser impacts on non-target communities than pyrethroids . However, a foliar application of thiacloprid (0.2 kg/ha) to apple trees reduced the population of earwigs (Forficula auricularia), an important predator of psyllids and woolly apple aphid, by 60% in two weeks, while remaining below 50% after six weeks . Branchlets of hemlock (Tsuga canadensis) treated with systemic imidacloprid (1-100 mg/kg) reduced the populations of two non-target predators of the hemlock woolly adelgid (Adelges tsugae) and had both lethal and sublethal effects on them . Clothianidin, thiamethoxam and acetamiprid were as damaging to cotton crop predators as other broad-spectrum insecticides and cartap . All neonicotinoids are lethal to the predatory mirid Pilophorus typicus, a biological control agent against the whitefly Bemisia tabaci, since their residual activity can last for 35 days on the treated plants . The ladybug Serangium japonicum, also a predator of the whitefly, is killed in large numbers when exposed to residues of imidacloprid on cotton leaves applied at the recommended rate (40 ppm) or lower; apparently, the predator was not affected when imidacloprid was applied as systemic insecticide . Clothianidin is 35 times more toxic to the predatory green miridbug (Cyrtorhinus lividipennis 48-h LC50 = 6 μg/L) than to the main pest of rice (Nilaparvata lugens 48-h LC50 = 211 μg/L), thus questioning seriously its application in such crops . Not surprisingly, populations of predatory miridbugs and spiders suffered an initial set back when rice paddies were treated with a mixture of ethiprole+imidacloprid (125 g/ha), and their recovery was slow and never attained the densities of the control plots . Mixtures of ethiprole+imidacloprid and thiamethoxam+l λ-cyhalothrin on rice paddies are also highly toxic to mirid and veliid natural enemies of rice pests, with 100% mortalities recorded in 24 h .
Secondary poisoning with neonicotinoids reduces or eliminates eventually all predatory ladybirds in the treated areas, compromising biological control in IPM programs. Indeed, exposure of larval stages of Adalia bipunctata to imidacloprid, thiamethoxam, and acetamiprid, and adult stages to imidacloprid and thiamethoxam, significantly reduced all the demographic parameters in comparison with a control –except for the mean generation time–, thus resulting in a reduced coccinellid population; adult exposures produced a significant population delay . Eighty percent of 3rd and 4th instar larvae of the ladybug Harmonia axyridis died after feeding for 6 hours on corn seedlings grown from seeds treated with clothianidin, compared to 53% mortality caused by a similar treatment with thiamethoxam; recovery occurred only in 7% of cases . Survival of the ladybird Coleomegilla maculata among flower plants treated with imidacloprid at the label rate was reduced by 62% , and Hippodamia undecimnotata fed upon aphids reared on bean plants treated with imidacloprid, experienced a 52% population reduction . Equally, 96% of Tasmanian brown lacewing (Micromus tasmaniae) larvae died after feeding on 1st instar lettuce aphid (Nasonovia ribisnigri) for three days. Low doses did not increase mortality but from days 3 to 8, lacewing larvae showed significant evidence of delayed developmental rate into pupae . Grafton-Cardwell and Wu  demonstrated that IGRs, neonicotinoid insecticides, and pyrethroid insecticides have a significant, negative impact on vedalia beetles (Rodolia cardinalis), which are essential to control scale pests in citrus; neonicotinoids were toxic to vedalia larvae feeding on cottony cushion scale that had ingested these insecticides, and survival of adult beetles was also affected but to a lesser extent than other insecticides.
Recent evidence of the negative impacts of neonicotinoids on parasitoids reinforces that these insecticides are not suitable for IPM . All neonicotinoids are deadly to three whitefly parasitoid species (Eretmocerus spp. and Encarsia formosa), with mortality of adults usually greater than the pupae . Thiamethoxam appears to be less toxic to whitefly parasitoids compared to imidacloprid . Imidacloprid, thiamethoxam and nitenpyram appeared to be the most toxic to the egg parasitoids Trichogramma spp. [231, 299]. For example, the acute toxicity of thiomethoxam and imidacloprid to Trichogramma chilonis, an egg parasitoid of leaf folders widely used in cotton IPM, is about 2000 times higher than that of other insecticides used in rice crops in India, such as acephate or endosulfan . Acute toxicity of imidacloprid is more pronounced on Braconidae parasitoids than on T. chilonis, whereas thiacloprid only reduced the parasitization on Microplitis mediator . Thiacloprid is as toxic to the cabbage aphid Brevicoryne brassicae as to its parasitoid (Diaeretiella rapae), whereas pirimicarb and cypermethrin are more toxic to the aphid and are, therefore, preferred in IPM .
Neonicotinoids pose also risks to aquatic taxa. The synergistic toxicity of imidacloprid+thiacloprid on Daphnia magna  implies the combined effect of neonicotinoids on aquatic arthropods would be higher than expected, even if Daphnia is very tolerant of neonicotinoids . Other contaminants, such as the nonylphenol polyethoxylate (R-11) act also synergistically with imidacloprid . Thiacloprid causes delayed lethal and sublethal effects in aquatic arthropods, which can be observed after 4 to 12 d following exposure to single 24-h pulses . Thus, its 5% hazardous concentration (0.72 μg/L) is one order of magnitude lower than predicted environmental concentrations in water . Also, thiacloprid LC50 for survival of midges (Chironomus riparius) is only 1.6 μg/L, and EC50 for emergence 0.54 μg/L , so both acute and chronic toxicity reduce the survival and growth of C. tentans and Hyalella azteca . Acute toxicity of neonicotinoids to red swamp crayfish (Procambarus clarkii) is 2-3 orders of magnitude lower than that of pyrethroids ; comparative data such as this gives the neonicotinoids an apparent better environmental profile. However, experimental rice mesocosms treated with imidacloprid at label rates (15 kg/ha) eliminated all zooplankton communities for two months, and their recovery did not reach the control population levels four months later. Equally, mayflies, coleoptera larvae and dragonfly nymphs were significantly reduced while residues of imidacloprid in water were above 1 μg/L [117, 237]. Similarly, streams contaminated with a pulse of thiacloprid (0.1-100 μg/L) resulted in long-term (7 months) alteration of the overall invertebrate community structure . However, while aquatic arthropods with low sensitivity to thiacloprid showed only transient effects at 100 μg/L, the most sensitive univoltine species were affected at 0.1 μg/L and did not recover during one year .
Fipronil is very efficient in controlling locust outbreaks, but causes more hazards than chlorpyrifos and deltamethrin to non-target insects in the sprayed areas, although it is more selective to specific taxa [214, 252]. Thus, abundance, diversity and activity of termites and ants were all reduced in northern Australia after spraying several areas with fipronil for locust control , and 45% of the termite colonies died within 10 months of a spraying operation with fipronil for controlling locusts in Madagascar . Reducing the recommended application rates by seven times (0.6-2 g/ha) still achieves 91% elimination of locusts while having lesser impacts on non-target organisms, comparable to those inflicted by carbamate and OP insecticides .
Despite its selectivity, fipronil in maize crops reduced the abundance of arthropod populations of the soil mesofauna more significantly than other systemic insecticides, i.e. carbofuran , although springtails are little affected as they avoid feeding on litter contaminated with fipronil and are more tolerant of this insecticide . When applied to citrus orchards, fipronil was among the most detrimental insecticides affecting two Euseius spp. of predatory mites . In rice crops, the effectiveness of fipronil in controlling pests was overshadowed by its negative impact on the predatory miridbugs Cyrtorhinus lividipennis and Tytthus parviceps .
Of greater concern is the impact of this systemic chemical on honey bees and wild bee pollinators. With an acute contact LD50 of 3.5 ng/bee  and acute oral LD50 of 3.7-6.0 ng/bee , fipronil is among the most toxic insecticides to bees ever developed. Even more worrying is the finding that the adjuvant Sylgard, used to reduce the toxicity of most insecticidal products on bees, increases the toxic effects of fipronil . The systemic nature of this chemical implies that chronic feeding of the bees on nectar contaminated with fipronil caused 100% honey bee mortality after 7 days, even if the residue concentration was about 50 times lower than the acute lethal dose . Residues of fipronil in pollen have been measured as 0.3-0.4 ng/g, which are 30-40 times higher than the concentration inducing significant mortality of bees by chronic intoxication . Unlike neonicotinoids, no residues of fipronil have been found in guttation drops .
The acute toxicity of fipronil to cladocerans is similar to the toxicity to estuarine copepods, with 48-h LC50 in the range 3.5-15.6 μg/L [47, 259], but the chronic toxicity with time of exposure is what determines the fate of the populations exposed. For example, populations of Daphnia pulex went to extinction after exposure to 80 μg/L for 10 days, equivalent to LC75 , and 40% of a population of grass shrimps (Palaemonetes pugio) died in 28 days after being exposed to fipronil concentrations of 0.35 μg/L in marsh mesocosms, and none of the shrimps survived when exposed to 5 mg/L during the same period . Such impacts on zooplankton are likely to occur in estuaries, where waters have been found to contain 0.2-16 μg/L of fipronil residues [45, 163], even if no apparent effect on amphipods, mussels nor fish has been observed [37, 303]. Fipronil sprays on water surfaces to control mosquito larvae have negative impacts not only on cladocerans but also on chironomid larvae exposed to chronic feeding on contaminated residues [183, 264]. Studies on rice mesocosms have shown that significant population reductions due to fipronil application at the recommended rates (50 g per seedling box) are not restricted to zooplankton and benthic species, but affect most species of aquatic insects. Moreover, fipronil impacts on aquatic arthropods were more pronounced after a second application in the following year , indicating persistence of this insecticide in rice paddies. Chronic toxicity over time explains the long-term toxicity of this systemic compound, so it is not surprising that concentrations of 1.3 μg/L in paddy water were sufficient to kill 100% of dragonfly (Sympetrum infuscatum) nymphs in nine days .
4.1.4. Insect growth regulators
There is little information about the effect of systemic chitin inhibitors on non-target organisms. Obviously these compounds are harmless to fish at levels above 1 mg/L for a week-long exposures , and to all vertebrates in general. IGRs affect mainly the larval stages of Lepidoptera, Coleoptera and Hymenoptera, and their activity last longer than that of other pest control products . The effectiveness of these compounds in controlling target pests is demonstrated by comparing the dietary LC50 of hexaflumuron (0.31 mg/L) to the target cotton worm (Helicoverpa sp.), which is 35 times lower than that of the systemic carbamate thiodicarb and less damaging to non-target predators . Aquatic communities of non-target arthropods in rice fields (e.g. Cladocera, Copepoda, Odonata, Notonectidae, Coleoptera and Chironomidae taxa) were not affected by teflubenzuron applied at rates to control mosquitoes (5.6 mg/ha), even though this IGR remained active for several weeks during autumn and winter periods .
After application of IGRs to a crop, affected insect pests are prey to many species of spiders, some of which are also susceptible to the toxicity of these products, in particular the ground hunter spiders . Larvae and eggs of pests contaminated with systemic IGR are consumed by a number of predators, including earwigs, which undergo secondary poisoning and stop growing beyond the nymph stage . Chitin inhibitors only show effects on the larvae of predatory insects that had consumed treated-prey, not on the adult insects. As a consequence, predatory populations collapse, as it happened with the ladybeetle Chilocorus nigritus that fed on citrus red scales (Aonidiella aurantii) in African orchards that had been treated with teflubenzuron . Teflubenzuron sprayed at 16.4 g/ha for locust control in Mali did not affect the non-target arthropods in the herb layer, whereas ground-living Collembola, Thysanura, Coleoptera and Lepidoptera larvae were reduced by about 50% . Moreover, teflubenzuron has multigenerational impacts: experiments with springtails exposed to artificial soil contaminated with this IGR showed that the F2 generation suffered significantly from its effects even when only the F0 generation had been exposed for 10 days . Secondary poisoning with chitin inhibitors can be detrimental also to parasitoids such as Diadegma semiclausum, which may fail to produce enough cocoons in the treated hosts, but do not seem to affect the parasitism of other Hymenoptera . For instance, novaluron did not affect the parasitisation of Trichogramma pretiosum on mill moth’s caterpillars, a pest of tomato crops . On the other hand, teflubenzuron appears to be harmless to predatory mites . IPM programs must always consider the implications of using systemic chitin inhibitors to control specific pests without destroying their natural predators in the first place.
Halofenozide does not appear to cause any acute, adverse effects through topical, residual, or dietary exposure of the ground beetle Harpalus pennsylvanicus. In contrast to the negative effects of other systemic insecticides (i.e. imidacloprid), the viability of eggs laid by females fed halofenozide-treated food once, or continuously for 30 days, was not reduced .
4.2. Sublethal effects
Very often, sublethal effects of systemic insecticides are a first step towards mortality, as they are caused by the same neurotoxic mechanisms. Apart from these, there may be other effects on reproduction, growth, longevity, etc. when organisms are exposed to low, sublethal doses or concentrations. These effects are only observable in individuals that survive the initial exposure, or in species that are tolerant to insecticides. For a review see .
4.2.1. Acetylcholinesterase inhibitors
Longevity of the parasitoid Microplitis croceipes that fed on nectar from cotton treated with aldicarb was affected for at least 10 days after application, and its foraging ability of the parasitoid’s host was severely impaired for 18 days . Carbofuran caused a significant reduction of adult weight and longevity of the predator ladybug Hippodamia undecimnotata, as well as a 55% reduction in fecundity when fed on aphids contaminated with this insecticide . Longevity and survival of Aphidius ervi, an important parasitoid of the pea aphid (Acyrthosiphon pisum), were significantly reduced after treating with LC25 concentrations of dimethoate or pirimicarb . A significant reduction in body size of females of the predator carabid Pterostichus melas italicus and altered sexual dimorphism were observed after long-term exposure in olives groves treated with dimethoate at a rate that caused 10% mortality after three days . Unlike other insecticides, no behavioural effects of dimethoate or triazamate on honey bees were recorded .
Earthworms (Lumbricus terrestris) experienced significant reduction in growth rate and total protein content after soil applications of aldicarb at LC10 or LC25, but only small amounts of residues were detected in the worms . Aldicarb and phorate can also increase infections by Rhizoctonia stem canker in potato fields .
A typical pattern of sublethal intoxication was revealed when red-winged blackbirds (Agelaius phoeniceus) were exposed to increasing doses of dimethoate: 2 mg/kg b.w. doses produced ataraxia, defecation and diarrhoea; neuromuscular dysfunctions and breathing complications appeared at 3 mg/kg, and by 5 mg/kg muscle paralysis and death occurred. The estimated LC50 was 9.9 mg/kg, and all birds died at doses above 28 mg/kg . Although sublethal AChE depression by acephate (25% brain) did not affect the attack behaviour in American kestrels (Falco sparverius) , nor did alter breeding behaviour in American robins (Turdus migratorius) , exposure to 256 mg/kg b.w. acephate impaired the migratory orientation of the white-throated sparrow (Zonotrichia albicollis) . Similarly, low doses of demeton–S-methyl did not affect starlings (Sturnus vulgaris) behaviour , but doses of 2.5 mg/kg b.w. of dicrotophos administered to female starlings significantly reduced their parental care and feeding of nestlings . Carbofuran orally administered to pigeons (Columba livia) had profound effects on flight time, with pigeons falling off the pace of the flock when doses were between 0.5 and 1.0 mg/kg b.w. .
AChE activities in adductor muscle were depressed in freshwater mussels (Elliptio complanata) exposed for 96 h at concentrations as low as 0.1 mg/L and 1.3 mg/L of aldicarb and acephate respectively, while increasing the water temperature from 21 to 30 oC resulted in mortality . High AChE inhibition (70%) by acephate was not associated with immobility of Daphnia magna, but increasing the concentration of acephate further had a strong detrimental effect on mobility, suggesting that binding sites other than AChE may be involved in acephate toxicity .
Exposure of bluegill fish (Lepomis macrochirus) to 30 μg/L carbofuran decreased significantly adenylate parameters in gill, liver, muscle and stomach tissues after 10 days, and then returned to normal . Also, concentrations of carbofuran at half the LC50 dose for fathead minnow (Pimephales promelas) larvae caused reductions in swimming capacity, increased sensitivity to electric shocks, and a reduction in upper lethal temperature . Enzymes of protein and carbohydrate metabolism were altered (some increased, others decreased) in liver and muscle tissues of the freshwater fish, Clarias batrachus when exposed to 7.7 mg/L of carbofuran for six days, recovering later to normal levels . Exposure of guppies (Brachydanio rerio) to half the recommended dose for dimethoate (0.025 μl/L) caused morphological changes in hepatocytes within three days, as well as necrosis and other abnormalities . When exposed to a range of monocrotophos concentrations (0.01-1.0 mg/L), male goldfish (Carassius auratus) showed higher levels of 17-β-estradiol and vitollogenin and lower levels of testosterone than normal, interfering with gonadotropin synthesis at the pituitary gland . Eggs of the toad Bufo melanostictus exposed to acephate hatched normally, but the tadpoles exhibited deformities such as tail distortions and crooked trunk; decreased pigmentation, peeling of the skin, inactivity, delay in emergence of limbs and completion of metamorphosis were also apparent .
Insecticide mixtures can enhance not only the acute but also the sublethal effects. For example, disulfoton together with endosulfan caused cytological and biochemical changes in liver of rainbow trout (Oncorrhynchus mykiss), independently of their respective modes of action . Mixtures of aldicarb and other insecticides enhanced significantly the establishment of parasitic lungworm nematodes (Rhandias ranae) in leopard frogs (Rana pipiens) some 21 days after infection , as the frog’s immune response was suppressed or altered . Similarly, laboratory rats exposed to sublethal mixtures of aldicarb, methomyl and a herbicide (metribuzin) showed learning impairment, immune response and endocrine changes .
4.2.2. Insecticides acting on nAChR
Laboratory experiments have shown a number of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects in zebrafish (Danio rerio) embryos exposed to a range of cartap concentrations. The most sensitive organ was the notochord, which displayed defects at concentrations as low as 25 μg/L . It is obvious that essential enzymatic processes are disturbed during embryo development, among which the inhibition of lysyl oxidase is responsible for the notochord undulations observed.
Imidacloprid does not cause high mortality among eggs or adults of the preparasite nematode Agamermis unka, but impairs the ability of the nematode to infect nymphs of the host brown planthopper (Nilaparvata lugens) . Contrary to this, a synergistic effect of imidacloprid on reproduction of entomopathogenic nematodes against scarab grubs may increase the likelihood of infection by subsequent generations of nematodes, thereby improving their field persistence and biological potential to control grubs. Acetamiprid and thiamethoxam, however, do not show synergist interactions with nematodes . Imidacloprid at 0.1-0.5 mg/kg dry soil disturbs the burrowing ability of Allolobophora spp. earthworms , and the highest concentration can also induce sperm deformities in the earthworm Eisenia fetida . Reduction in body mass (7-39%) and cast production (42-97%) in Allolobophora spp. and Lumbricus terrestris have also been observed after 7 days exposure to relevant environmental concentrations of imidacloprid . Residues of imidacloprid in maple leaves from treated forests (3–11 mg/kg) did not affect survival of aquatic leaf-shredding insects or litter-dwelling earthworms. However, feeding rates by aquatic insects and earthworms were reduced, leaf decomposition (mass loss) was decreased, measurable weight losses occurred among earthworms, and aquatic and terrestrial microbial decomposition activity was significantly inhibited, thus reducing the natural decomposition processes in aquatic and terrestrial environments .
The dispersal ability of the seven-spotted ladybirds (Coccinella septempunctata) sprayed with imidacloprid was compromised, and this may have critical consequences for biological control in IPM schemes . A significant reduction of adult weight and longevity of the ladybug Hippodamia undecimnotata, as well as 33% reduction in fecundity were observed when this predatory bug fed on aphids contaminated with imidacloprid . Imidacloprid and fipronil had adverse effects on the immune response of the wolf-spider Pardosa pseudoannulata, reducing significantly its phenoloxidase activity, the total number of hemocytes and encapsulation rate ; the implications of such effects on this natural enemy of rice pests are unknown. When applied in the egg-larval or pupal stages, acetamiprid or imidacloprid reduced the parasitisation capacity of F1 and F2 generation females of Trichogramma pretiosum on mill moth’s caterpillars (Anagasta kuehniella), a pest of tomato crops . Longevity of females of the parasitoid Microplitis croceipes that fed on nectar from imidacloprid-treated cotton was affected for at least 10 days after application, while the parasitoid's host foraging ability was severely affected from day 2 onwards . Exposure of western subterranean termites (Reticulitermes hesperus) to acetamiprid (1 mg/kg sand) or imidacloprid also impaired locomotion of termites within 1 hour .
Bumble bees (Bombus terrestris) interrupt their activity for several hours when exposed to imidacloprid sprayed on plants , and soil treatment at the highest recommended doses extended the handling times of B. impatiens on the complex flowers . Such an impairment affects the bees foraging behaviour and can result in a decreased pollination, lower reproduction and finally in colony mortality due to a lack of food . Although Franklin et al.  found that clothianidin residues of 6 μg/kg in canola pollen reduced the production of queens and increased the number of males in B. impatients, their study did not find significant differences with controls due to a high variability in the results. Larval development in wild bees (Osmia lignaria and Megachile rotundata) was delayed significantly when fed pollen contaminated with either imidacloprid or clothianidin at 30 or 300 μg/kg . Honey bees are more sensitive to neonicotinoids than bumble bees: at 6 μg/kg, imidacloprid clearly induced a decrease in the proportion of active bees , and 50-500 μg/L affect significantly their activity, with bees spending more time near the food source . Other authors found that lower activity of honey bees during the hours following oral exposure to 100-500 μg/L imidacloprid in syrup is transitory . In any case, that may explain the delayed homing behaviour of honey bees exposed to 100 μg/L imidacloprid in syrup and their disappearance at higher doses [34, 304]. Honey bees fed on syrup contaminated with acetamiprid increased their sensitivity to antennal stimulation by sucrose solutions at doses of 1 μg/bee and had impaired long-term retention of olfactory learning at 0.1 μg/bee. Contact exposure at 0.1 and 0.5 μg/bee increased locomotor activity and water-induced proboscis extension reflex but had no effect on behaviour . Similar response was obtained with honey bees exposed to thiomethoxam by contact, having impaired long-term retention of olfactory learning at 1 ng/bee . Winter bees surviving chronic treatment with imidacloprid and its metabolite (5-OH-imidacloprid) had reduced learning performances than in summer: the lowest-effect concentration of imidacloprid was lower in summer bees (12 μg/kg) than in winter bees (48 μg/kg), indicating a greater sensitivity of honey bees behaviour in summer bees compared to winter bees .
Honey bees infected with the microsporidian Nosema ceranae experienced 7 or 5 times higher mortality than normal when fed syrup contaminated with sublethal doses of thiacloprid (5 mg/L) or fipronil (1 μg/L), respectively [293). N. ceranae is a key factor in the CCD in honey bees , and the synergistic effect of these systemic insecticides on Nosema is probably its underlying cause . Suppression of the immune system is not restricted to bees, as a massive infection of medaka fish by a protozoan ectoparasite (Trichodina spp.) when exposed to imidacloprid in rice mesocosms has been documented .
Imidacloprid residues in water as low as 0.1 μg/L are sufficient to reduce head and torax length in mayfly nymphs of Baetis and Epeorus, whether applied as pulses or in continuous exposures for 20 days . At 1 μg/L the insecticides caused feeding inhibition. However, 12-h pulses induced emergence because of stress, whereas constant exposure reduced survivorship progressively. Also, the aquatic worm Lumbriculus variegatus experienced immobility during 4 days when exposed to 0.1-10 μg/L imidacloprid .
Apart from the extreme acute toxicity of this insecticide to bees, honey bees fed on sucrose syrup contaminated with fipronil (2 μg/kg) reduced significantly their attendance to the feeder . It has also been demonstrated that sublethal concentrations of this insecticide as low as 0.5 ng/bee, whether orally or topically applied, reduce the learning performance of honey bees and impair their olfactory memory but not their locomotor activity [67, 82]. Furthermore, chronic feeding exposure at 1 μg/kg or 0.01 ng/bee reduced learning and orientation, whilst oral treatment of 0.3 ng/bee reduced the number of foraging trips among the exposed workers . In addition to their activity, honey bees fed with sucrose syrup containing 1 μg/L fipronil increased significantly the mortality of bees infected with the endoparasite Nosema ceranae, suggesting a synergistic effect between the insecticide and the pathogen . All these sublethal effects reduce the performance of the hive and help explain the decline in honey bee and wild bee pollinators in many countries , although fipronil is not alone in causing this demise – neonicotinoids are equally implicated.
Female zebra finches (Taeniopygia guttata) fed with single sublethal doses of fipronil (1, 5, and 10 mg/kg b.w.) failed to hatch 6 out of 7 eggs laid. The only chick born was underdeveloped and had fiprole residues in the brain, liver and adipose tissues. By contrast, 12-day-old chicken eggs injected with fipronil (5.5 to 37.5 mg/kg egg weight) hatched normally although the chicks from the highest dose group showed behavioural and developmental abnormalities .
Low residues of fipronil in estuary waters (0.63 μg/L) inhibited reproduction of the copepod Amphiascus tenuiremis by 73-89%, and this effect seems to be more prevalent on males than on females . Even lower residue levels (0.22 μg/L) halted egg extrusion by 71%, whereas exposure to 0.42 μg/L nearly eliminated reproduction (94% failure) on this species. Based on these results from chronic and sublethal toxicity, a three-generation Leslie matrix model predicted a 62% decline in population size of A. tenuiremis at only 0.16 μg/L . Unlike other insecticides, the stress on Ceriodaphnia dubia caused by predatory cues of bluegill fish (Lepomis macrochirus) was significantly exacerbated when the cladocerans were exposed to 80-160 μg/L of fipronil ; however, these concentrations are much higher than the residue levels usually found in waters [99, 163].
While fipronil applied at the recommended rates in rice fields induces biochemical alterations in carp (Cyprinus carpio), such metabolic disturbances do not appear to have any effect on growth nor mortality of this fish after 90 days exposure at <0.65 μg/L . However, similar residue levels (<1 μg/L) reduced significantly the growth of adult medaka fish (Oryzias latipes) after two weeks of exposure, as well as growth of their offspring in the first 35 days, even if residues of fipronil by that time were below the analytical detection limit (0.01 μg/L) .
4.2.4. Insect growth regulators
Longevity of predatory bug Podisus maculiventris was reduced after preying on Colorado potato beetles that fed on foliage treated with novaluron at 85 g/ha. Females produced fewer eggs and their hatching was significantly suppressed, while 5th instars that also preyed on the beetles failed to moult into adults . Novaluron and hexaflumuron significantly decrease (<30%) the total protists population in the guts of termites (Reticultermes flavipes), thus upsetting their digestive homeostasis .
4.3. Indirect effects on populations and communities
Indirect effects result from the dynamics of ecosystems. Thus, applications of granular phorate to soil eliminate most soil invertebrates (see 4.1) except for Enchytraeidae worms, which increase in large numbers and take over the leaf-litter decomposition function carried out by the eliminated springtails .
Resurgence or induction of pests by altering the prey-predator relationships in favour of the herbivore species is most common. When carbofuran was applied to corn plantations in Nicaragua, the population levels of the noctuid pest Spodoptera frugiperda increased because of lesser foraging activity by predatory ants . Methomyl eliminated the phytoseiid predatory mite Metaseiulus occidentalis for 10 days, thus causing an increase in Pacific spider mites (Tetranychus pacificus) and leafhopper (Eotetranychus willamettei) populations in the treated vineyards . Unexpected outbreaks of a formerly innocuous herbivore mite (Tetranychus schoenei) were observed after imidacloprid applications to elms in Central Park, New York. A three-year investigation on the outbreaks showed that elimination of its predators and the enhanced fecundity of T. schoenei by this insecticide were responsible for that outcome .
The widespread use of insecticides usually tips the ecological balance in favour of herbivore species. For example, dimethoate sprayed on clover fields indirectly reduced the populations of house mice (Mus musculus) in the treated areas as the insect food source was depleted. However, herbivore species such as prairie voles (Microtus ochrogaster) and prairie deer mouse (Peromyscus maniculatus) increased in density levels , since they had more clover available due to either higher clover yields or through less competition with the house mice or both.
A reduction in arthropod populations often implies starvation of insectivorous animals. For example, densities of two species of lizards and hedgehogs in Madagascar were reduced 45-53% after spraying with fipronil to control a locust outbreak, because their favourite termite prey was almost eliminated (80-91%) by this chemical . However, this type of indirect impact is difficult to observe and measure in birds, since they can move to other areas or change their resource diet. For example, hemlock forests treated with imidacloprid to control hemlock woolly adelgid (Adelges tsugae) reduced significantly Hemiptera and larval Lepidoptera, but not other insect taxa. Although larval Lepidoptera are the primary prey for insectivorous foliage-gleaning birds, many birds were able to find other food resources in the mixed hemlock-deciduous stands that were not treated . Similarly, post-treatment with fipronil for grasshopper control in Wyoming did not affect bird densities, perhaps due to the large initial insect populations; fipronil plots generally had higher avian population densities (nongregarious, insectivores and total birds) than other areas treated with carbaryl . Although some early studies found that fipronil did not have much impact on aquatic communities of Sahelian ponds , nor in predatory invertebrates in the Camargue marshes, herons in the latter region avoid rice fields treated with fipronil because of the scarcity of invertebrate food in there .
Food aversion to pesticide-treated seeds or plants is a mechanism that may indirectly ameliorate the toxic effects of systemic insecticides such as carbofuran in mice and other small rodents . Some Collembola species (i.e. Folsomia fimetaria) avoid dimethoate sprayed areas , and female parasitoids (Cotesia vestalis) are discouraged from getting to their host –the diamond-back month (Plutella xylostella) – in turnip plants treated with methomyl, whereas clothianidin does not produce aversion . Equally, dimethoate and oxydemeton-methyl sprayed on peach trees discourage honey bees from visiting in the first two days after application, while treatments with imidacloprid, acetamiprid and thiamethoxam allow honey bees visits . This helps explain the high long-term impact of neonicotinoids on bees compared to the effect of OP insecticides, even if imidacloprid at high experimental concentrations in syrup (>0.5 mg/L) may also have repellent effect on honey bees .