Laboratory bio-assays indicate that reduced risk pesticides can, in some cases, still be toxic to natural enemies and thus an assessment of the toxicity of field aged residues of these products is key to understanding how well laboratory bio-assays translate to a field setting. To encourage a reduction of organophosphate use, the Environmental Protection Agency Office of Pesticide Programs’ called for incentives to promote the development and use of alternative reduced-risk pesticides. Subsequently, the EPA announced the Reduced-Risk Pesticide Initiative, which created incentives to promote registration of pesticides under the following reduced-risk criteria: reducing human health risks and reducing risks to non-target organisms such as birds, beneficial insects, and aquatic organisms . One such insecticide developed under the new criteria is chlorantraniliprole, which has frequently been shown to have little to no effect on several natural enemy traits, including survivor ship, predation rates, parasitoid emergence, or parasitism rates . For example, it was found to be compatible with beneficial insects such as Copidosoma bakeri Howard via direct contact, Harpalus pennsylvanicus DeGeer via treated food, and Tiphia vernalis Rohwer and Bombus impatiens Cresson via residual exposure . However, for two species of green lacewing, Chrysoperla carneaand C. johnsoni Henry, Wells and Pupedis , chlorantraniliprole caused 100% mortality . Lambda-cyhalothrin is another reduced-risk insecticide, but, unlike chlorantraniliprole, it has demonstrated lethal effects on several natural enemies. Lambda-cyhalothrin is lethal to larvae of C. carnea and C. johnsoni . Additionally, it is highly toxic to the parasitoid, Diadegma insulare , and resulted in a change in host preference . At the full field rate and, it caused high mortality in adults of the predatory mite, Galendromus occidentalisand for larvae that hatched from the eggs of treated females . Coccinellid populations topically exposed to lambda-cyhalothrin had significant decreases in suvivorship,25 liter pot including populations of Coleomegilla maculata , Cycloneda sanguinea , HarmoniaRecently, both lambda-cyhalothrin and chlorantraniliprole were formulated into a new combination foliar insecticide .

This combination was developed to provide broad spectrum control of both sucking and chewing insects, including lepidopteran, coleopteran, and hemipteran pests. Additionally, because the compound coats and penetrates the leaf tissue, it affects insects via multiple routes of exposure . Hence, the combination may target feeding and mobile life stages as wells as eggs. A range of other combination insecticides have been developed for use in agricultural crops, however, there is no evidence that they preclude resistance . The toxicity of insecticides to H. convergens is important for biological control efforts throughout the western United States, where it is a well-known generalist aphid predator . It is a particularly important natural enemy in highvalue tree crops, where both chlorantraniliprole and lambda cyhalothrin are used to control codling moth . In this study, we evaluated the acute toxicity to H. convergens of aged residues of a combination insecticide, chlorantraniliprole plus lambda-cyhalothrin. The objectives of this study were to 1) assess acute toxicity of the combination insecticide residues on two life stages of H. convergens , 2) determine how acute toxicity of residues is influenced by age, and 3) evaluate the differences in acute toxicity between residues aged in glass arenas in the laboratory bio-assay versus residues aged on foliage in the field.Aged residues of the combination insecticide, chlorantraniliprole plus lambda-cyhalothrin , were tested in comparison to a distilled water control. The combination insecticide was formulated as 0.835 lb of chlorantraniliprole and 0.417 lb of lambda-cyhalothrin per gallon . The solutions used for both laboratory and field aged residues were the equivalent of 0.73 l per ha . For the laboratory aged residues, the insecticide solution was prepared as 16.89 mg of formulated product in 50 ml of distilled water, and, for the field aged residues, the solution was prepared as 35.8 g of formulated product per 106 l of water. Four variables were manipulated 1) treatment , 2) life stage of H. convergens , 3) residue age, and 4) setting . A minimum of 28 replicates was used in all bio-assays. For all bio-assays, test insects were placed individually in 15 x 45 mm glass vial arenas, with cotton wool placed over vial openings to allow for ventilation.

Insects were kept at 22 ⁰C, 60-70% RH, and 16:8 h photoperiod. Bio-assays lasted for a period of 48 h, after which acute toxicity was recorded. Acute toxicity was estimated from the number of live and dead or moribund insects , where moribund insects were those that were unable to right themselves. For adult bio-assays, equal numbers of each gender were used and acute toxicity was recorded separately for each gender. The effects of laboratory and field aging were evaluated for residues 0, 1, 2, 4, 8, 16, and 38 d old. To test the toxicity of laboratory-aged residues, individual glass vials were treated by adding 4 ml of insecticide solution and turning 360 degrees in order to coat the entire inside surface before the solution was poured out. For residues aged 0 d, vials were inverted on a rack and allowed to dry for 4 h until they were used in the bio-assays. For all other residue ages, vials were inverted, suspended on a rack, allowed to dry in a fume hood for a period of 24 h, and then placed in an incubator maintained at 22 ⁰C, 60-70% RH, and 16:8 h photo period under fluorescent lights until used for the bio-assays. For field-aged residues, individual walnut trees were sprayed in a commercial walnut orchard in Tracy, CA. Eight walnut trees were treated with the insecticide and 7 trees were treated with water on June 19, 2013. Foliar sprays were applied with an orchard sprayer operating at 250 psi with a finished spray volume of approximately 2338.5 l per ha. For each sample date after spray application four leaves were collected from the lower canopy of each tree in each cardinal direction. In the laboratory, 4 cm x 5 cm sections were cut from each of the leaves and arranged as liners in individual glass vials such that the inside surface of each vial was covered by the lower surface of the leaf sections. Cotton wool plugs were placed at the bottom of each vial to secure the leaf sections in place. For both the laboratory and field aged residues, approximately 10 untreated adult A. pisum were then added to each glass vial as a food source, and a single untreated H. convergenswas introduced and allowed to crawl on the treated surface for a period of 48 h. The statistical program R was used for the analysis. Generalized linear models with binomial errors, or quasibinomial errors in cases where there was over dispersion, were used to analyze the effects of treatment , life stage , residue age , and setting on the acute mortality of H. convergens. For all GLMs, model reduction and log likelihood ratio tests were used to assess the statistical significance of each factor and their interactions on acute mortality at α = 0.05.

The uncorrected acute mortality data were analyzed, starting with a full model that included all four factors and interactions. The data set was finally subdivided into four separate models with no significant interaction between factors to compare the relative acute toxicity of treatment and age: 1) adults exposed to field-aged residues, 2) larvae exposed to field-aged residues, 3) adults exposed to laboratory-aged residues, and 4) larvae exposed to laboratory-aged residues. Additionally, we tested the effects of gender on adult mortality. In a separate analysis, the data was corrected to account for variability among control groups , and then used to examine comparative effects of life stage and setting. Due to significant interactions, the corrected data set was subdivided when necessary. Additionally, the decay in insecticide impact over time was fitted to a first-order degradation curve Mt = M0e – at where Mt is the corrected acute mortality of H. convergens exposed to residues of age t , M0 is the corrected acute mortality of H. convergens exposed to residues of age 0, and a is a rate constant . A linearized version of the degradation curve, a plot of ln on residue age,raspberry cultivation pot was used to fit a linear regression model and to estimate the rate constant a from the slope. From this we estimated the residue age that represents a 50% loss of the acute mortality response of H. convergens to the combination pesticide from its initial effect on day 0 from t1/2 = /a.There were substantial differences in the acute mortality responses of H. convergens to aging of field residues of the combination insecticide , but not of laboratory residues. Based on the decay rate of the responses over time, we were able to estimate the effects of differently aged residues on H. convergens mortality, and to categorize the residue ages according to the criteria used for toxicity classification by the International Organization for Biological Control . Field-aged residues were moderately harmful to adults for up to 1 day, but residues were harmless after 18 days from the initial application. Similarly, field-aged residues were estimated to be moderately harmful to larvae for 9 days, and were still slightly harmful after 51 days. In contrast, laboratory-aged residues were estimated to be moderately harmful to adults for a period of 29 days, and harmful to larvae regardless of residue age. Laboratory-aged residues produced higher mortality rate responses than field-aged residues, and the responses of larvae were greater than those of adults. High mortality rates from residual exposure to the combination insecticide were most likely due to the lambda-cyhalothrin component, which has been shown to be toxic to several natural enemies . For example, Neochrysocharis formosaand Ganaspidium nigrimanusexperienced high mortality rates when exposed to lambda-cyhalothrin residues . C. carnea and C. johnsoni adults and larvae experienced 100% mortality when treated with the maximum label rate .

Likewise, lambda-cyhalothrin exposure resulted in 100% mortality for D. brevis adults and nymphs . When exposed to a treated leaf surface, the parasitoid D. insulare and the predator C. maculata each had mortality rates > 80% . Exposure of H. convergens to fresh lambdacyhalothrin residues at the maximum field rate resulted in mortality rates > 90% for adults and larvae , and topically exposed pupae and eggs had mortality rates > 80% . Although the concentration of lambda-cyhalothrin in the maximum field rate dosage used in Chapter 2 was much greater than its concentration in the combination insecticide , the mortality rate response of H. convergens to the combination insecticide remained high for 0-1 d of the field-aged residues and for all ages of the laboratory residues. In contrast to the lambda-cyhalothrin component, it is unlikely that the chlorantraniliprole component of the combination insecticide accounted for the high mortality rates of H. convergens. Chlorantraniliprole was harmless to H. convergens adults and larvae via residual, oral, and topical exposure . Moreover, its primary mode of action is via ingestion, and it is unlikely that adults or larvae of H. convergens received oral exposure in our bio-assays with either field-aged or laboratory-aged residues. Chlorantraniliprole has also been demonstrated to be compatible with several natural enemies . For example, residual exposure was harmless to Orius armatusnymphs and adults . It was also harmless to multiple parasitoid species, including Aphelinus mali Haldeman , Diadegma semiclausum Hellen , and Dolichogenidea tasmanica Cameron . Even in a maximum exposure scenario, where the predator Macrolophus pygmaeuswas treated orally, residually, and topically, chlorantraniliprole had no effect on mortality . In a similar maximum exposure treatment, it had no effect on the mortality of G. occidentalis adults, and an LD50 could not be established due to its low toxicity . Given the low acute toxicity of chlorantraniliprole, the half-life response of H. convergens to the combination insecticide was probably unaffected by its rate of degradation under field conditions or by the difference in substrates used for laboratory and field-aged residues. Chlorantraniliprole was harmless to H. convergens adults and larvae exposed to residues on glass , and to Trichogramma pretiosum Riley as residues on filter paper or cotton leaves .