The dynamics of pyrethroid residues and Cyp P450 gene expression in insects depends on the circadian clock.

A circadian clock may underlie pesticide resistance mechanisms in organisms that are very important for humans, for example, in the honey bee (Apis mellifera). Using the gas chromatography, we evaluated the daily variability in the λ-cyhalothrin degradation rate in bodies of guards and forager bees, Apis mellifera. Additionally, using the RT-qPCR method, we studied expression levels of selected cytochrome P450 genes after exposure to λ-cyhalothrin. During 48-h-tests, we exposed bees to λ-cyhalothrin at four crucial times of the day: at 04:30 a.m., 11:30 a.m., 06:30 p.m., and 11:30 p.m. The results obtained indicate that in bees the intensity of the λ-cyhalothrin degradation is the highest during first 6 h after intoxication, when it disappeared at the rate of 14.29% h-1, 11.43% h-1, 13.15% h-1, and 12.50% h-1 in bees treated at noon, sunset, midnight, and sunrise, respectively. In the later period (6-48 h of the experiment), the degradation stopped and its rate did not exceed 1.0% h-1. In the control group of bees we demonstrated that the increase in the Cyp9Q1 and Cyp9Q3 expression was the highest during the experiments started at 04:30 a.m., while the highest elevation in the Cyp9Q2 expression was observed in the group for which the experiments started at 11:30 p.m.In intoxicated honey bees, the highest increase in the Cyp9Q1 expression occurred in the group treated with the pesticide at 11:30 a.m. In the case of genes encoding Cyp9Q2 and Cyp9Q3, the highest rise in the expression took place at 06:30 p.m.The obtained results indicate that honey bees activate detoxifying mechanisms partly protecting them against the effects of hazardous substances absorbed from the environment more efficiently during foraging than at other times of the day.

Assessment of risk to honey bees and honey consumers resulting from the insect exposure to captan, thiacloprid, penthiopyrad, and λ-cyhalothrin used in a commercial apple orchard.

Samples of leaves, flowers, soil, pollen, bee workers, bee brood, honey, and beeswax were collected to assess the possibility of a transfer of captan, thiacloprid, penthiopyrad, and λ-cyhalothrin from apple trees of Idared variety to honey bee (Apis mellifera) hives. Chemical analyses were performed using the Agilent 7890 Gas Chromatograph equipped with the Micro-cell Electron Capture Detector. It was found that significant amounts of penthiopyrad, the active ingredient of Fontelis 200 SC, were present in leaves, flowers, pollen, bee workers, and beeswax. Simultaneously, captan was present in the brood, worker bees, and honey samples. Significant levels of the captan residues were also detected on the soil surface. In honey samples, captan residue levels exceeded the acceptable standard, reaching 160% of its maximum residue level. However, in no case the amounts of captan, thiacloprid, penthiopyrad, and λ-cyhalothrin ingested with honey by an adult consumer exceeded the level of 0.02% of the acceptable daily intake. Despite the trace amounts of pesticide residues in honey samples collected during the field trial, bee honey consumption can be considered safe. An adult consumer can safely consume about 16 kg of honey.

Treating honey bees with an extremely low frequency electromagnetic field and pesticides: Impact on the rate of disappearance of azoxystrobin and λ-cyhalothrin and the structure of some functional groups of the probabilistic molecules.

The purpose of these laboratory tests was to assess the impact of 50 Hz EMF (electromagnetic field) on the disappearance of azoxystrobin (active ingredient (AI) of Amistar 250 SC) and λ-cyhalothrin (AI of Karate Zeon 050 CS) in the body of honey bees (Apis mellifera) and the structure of some functional groups of the probabilistic molecules in their organisms. Amistar 250 SC (an azoxystrobin-based fungicide; ABF) and Karate Zeon 050 CS (a λ-cyhalothrin-based insecticide; CBI) are plant protection products (PPPs) applied to bee-pollinated-crops. Chromatographic methods were used to assess the rate of AI disappearance. EMF affected the rate of disappearance of azoxystrobin and λ-cyhalothrin in bees within 6 h of intoxication. When these substances were used separately their disappearance in the presence of EMF slowed from 12.6% to 10.5% h-1 and from 9.2% to 4.8% h-1, respectively, and accelerated when used in a mixture, from 14.1% to 14.7% h-1 and from 9.3% to 11.5% h-1 respectively. Fourier Transform Infrared (FTIR) spectroscopy was used to analyze changes in the functional groups of the probabilistic molecules of the tested bees. To obtain the information about the spectra variations we used the Principal Component Analysis. It has been shown, that EMF statistically significantly interferes with amide I and II, symmetric PO32- group from DNA, RNA and phospholipids vibrations. It also increased the number of changes of functional groups of the probabilistic molecules caused by ABF, but at the same time limited the changes in the functional groups studied in bees treated with CBI and a mixture containing both of them. In addition, exposure to EMF in bees treated with fungicide or insecticide, separately, and with both preparations caused differences (p < 0.05) in the secondary structure of proteins compared to controls. The obtained results indicate that EMF may affect the rate of metabolism and the detoxification process of pesticides in bees, depending on the AI of PPPs, applied individually or together. However, further detailed research is required to explain the mechanism of EMF as a detoxification modulator.

Influence of a Commercial Biological Fungicide containing Trichoderma harzianum Rifai T-22 on Dissipation Kinetics and Degradation of Five Herbicides in Two Types of Soil.

Biological crop protection is recommended to be applied alternately or together with chemical one, to protect human health from the excessive use of toxic pesticides. Presence of microorganisms can influence the concentration of chemical pollutants in soil. The aim of this study is to estimate the influence of a commercial biological fungicide containing Trichoderma harzianum Rifai T-22 on dissipation kinetics and degradation of five herbicides belonging to different chemical classes: clomazone, fluazifop-P-butyl, metribuzin, pendimethalin, and propyzamide, in two types of soil. Results of the study revealed that T. harzianum T-22 influences pesticide degradation and dissipation kinetics of the non-persistent herbicides: clomazone, fluazifop-P-butyl, and metribuzin. In soil with a higher content of nitrogen, phosphorus, and organic matter, degradation increased by up to 24.2%, 24.8%, and 23.5% for clomazone, fluazifop-P-butyl, and metribuzin, respectively. In soil with lower organic content, degradation was on a low level, of 16.1%, 17.7%, and 16.3% for clomazone, fluazifop-P-butyl, and metribuzin, respectively. In our study, the addition of the biological preparation shortened herbicide dissipation half-lives, from 0.3 days (2.9%) for fluazifop-P-butyl, to 18.4 days (25.1%) for clomazone. During the degradation study, no significant differences were noticed for pendimethalin, belonging to persistent substances. Biological protection of crops can modify pesticide concentrations and dissipation rates. On one hand, this may result in the reduced effectiveness of herbicide treatments, while on the other, it can become a tool for achieving cleaner environment.

Influence of Bacillus subtilis and Trichoderma harzianum on Penthiopyrad Degradation under Laboratory and Field Studies.

In plant protection, biological preparations are used alternately with chemical pesticides. The applied microorganism can influence the concentration of chemical substances. Laboratory and field studies were conducted to assess the influence of Bacillus subtilis and Trichoderma harzianum on the penthiopyrad concentration. In laboratory studies, the effectiveness of penthiopyrad degradation by B. subtilis was approximately 5% during 14 days of the experiment. For penthiopyrad treated with T. harzianum strains, the degradation effectiveness ranged from 34.2% on Day 3 to 56.9% on Day 14. In experiments testing the effects of mixed culture of microorganisms, the effectiveness of penthiopyrad degradation ranged from 23.7% on Day 3 to 29.1% on Day 14. After treatment of apple trees of Gala and Golden Delicious varieties with a biological preparation, a maximum degradation of penthiopyrad of 20% was found in both varieties. Samples of apples were prepared by the quick, easy, cheap, effective, rugged and safe (QuEChERS) method, and penthiopyrad was analyzed by gas chromatography with a mass detector. A determined value of the chronic exposure to penthiopirad was 1.02% of the acceptable daily intake, both for children and for adults. The acute exposure amounted to 7.2% and 1.9% of the acute reference dose for children and adults, respectively. These values were considered to be acceptable and not threatening to health.

Transfer of the Active Ingredients of Some Plant Protection Products from Raspberry Plants to Beehives.

Plant protection products (PPPs) have been found increasingly in the environment. They pose a huge threat to bees, contributing to honeybee colony losses and consequently to enormous economic losses. Therefore, this field investigation was designed to determine whether their active ingredients (AIs) were transferred from raspberry plants to beehives located in the immediate neighbourhood of the crop and to what extent they were transferred. Every week for 2 months, samples of soil, raspberry leaves, flowers and fruits, worker bees, honeybee brood, and honey were collected and analysed for the presence of propyzamide, chlorpyrifos, iprodione, pyraclostrobin, boscalid, cypermethrin, difenoconazole, azoxystrobin, and pyrimethanil residues. Five of these substances were found in the worker bee bodies. Chlorpyrifos, applied to only the soil through the irrigation system, also was detected in the brood. A small amount of boscalid was noted in the honey, but its residues did not exceed the maximum residue level. For chlorpyrifos, boscalid, and pyrimethanil, a positive correlation between the occurrence of PPPs in the crops and the beehives was found. Statistical methods confirmed that the application of PPPs on a raspberry plantation, as an example of nectar-secreting plants, was linked to the transfer of their AIs to beehives.

Pyrethroid residue dynamics in insects depends on the circadian clock.

Many factors may affect pesticide effectiveness against pests. One of the factors that should be considered is circadian rhythmicity. In this study, we evaluated daily variations in pyrethroid susceptibility in the house cricket, Acheta domesticus L. Crickets were exposed to a standard dose of ß-cyfluthrin at different times of a day, and pesticide residue levels were evaluated using gas chromatography. Results demonstrate that the time of pyrethroid disappearance is correlated with the circadian clock, with the highest decomposition rate at night. Furthermore, crickets also showed the highest resistance to the insecticide at night, expressed as a high survival rate. Moreover, ß-cyfluthrin induced significant changes in thermal preferences of intoxicated crickets. This is the first report showing that pyrethroid residue levels in the crickets' body depend on its circadian clock.

Assessment of boscalid and pyraclostrobin disappearance and behavior following application of effective microorganisms on apples.

The aim of this study is to assess the disappearance of boscalid (IUPAC name: 2-chloro-N-[2-(4-chlorophenyl)phenyl]pyridine-3-carboxamide) and pyraclostrobin (IUPAC name: methyl N-[2-[[1-(4-chlorophenyl)pyrazol-3-yl]oxymethyl]phenyl]-N-methoxycarbamate) residues in apple fruit, and to verify whether an organic fertilizer enriched with strains of antagonistic microorganisms can reduce pesticide residue levels. Field trials were conducted in a commercial orchard on apples of the Gloster variety, during 21 days after the treatment with Bellis 38 WG and the subsequent application of Zumba Plant formulation containing Bacillus spp., Trichoderma spp. and Glomus spp. In control samples, the decrease rate of boscalid and pyraclostrobin residue levels followed an exponential function, described by formulae Rt = 0.2824e-0.071t and Rt = 0.1176e-0.060t, with the coefficient of determination of r2 = 0.8692 and r2 = 0.9268, respectively. These levels dropped to half (t1/2) of their initial values after 9.8 and 11.5 days, respectively. The treatment with Zumba Plant resulted in a reduction in boscalid and pyraclostrobin residue levels by 52% and 41%, respectively. The results of this study are of importance for horticulture sciences and for producers of apples using plant protection products (PPPs).

The transfer of active ingredients of insecticides and fungicides from an orchard to beehives.

This investigation was undertaken to determine whether active ingredients (AIs) of currently recommended plant protection products (PPPs) could be transferred to beehives from apple and pear trees. A field trial was carried out with apple trees of Ligol and Idared variety, and pear trees of Conference variety. For pest and diseases control of fungal origin, recommended PPPs were applied. Samples of flowers from the above-mentioned varieties of fruit trees, of bees, brood and honey from beehives located in their direct neighborhood were collected regularly and analyzed for the presence of lambda-cyhalothrin (an insecticide) and cyprodinil, captan, fluopyram, kresoxim-methyl, penthiopyrad and trifloxystrobin (fungicides). In samples of flowers of Ligol variety, fluopyram residues (on average 0.621 µg single flower-1) were at the highest levels, whereas in samples of pear flowers of Conference variety, and in flowers of Idared variety, captan residues (on average, respectively, 0.705 and 165.7 µg single flower-1). In samples of bees and honey, residues of five AIs were detected, and in brood six AIs, whereby in each case captan residues prevailed, respectively, up to 585.2, 51.52 and 126.5 µg kg-1 bees and honey. In the honey, significantly larger residues of captan were found out than maximum residue level (MRL) for this AI - 103.04% MRL. In the case of any AI, the daily intake did not exceed 0.002% acceptable daily intake (ADI).

Transfer of plant protection products from raspberry crops of Laszka and Seedling varieties to beehives.

Field studies were conducted to evaluate the transfer of active ingredients (AIs) of plant protection products (PPPs) to beehives. They were applied in two commodity red raspberry plantations of two varieties: Laszka (experiment 1) and Seedling (experiment 2). Samples of flowers, leaves, bees, brood, and honey were examined for the presence of chlorpyrifos, cypermethrin, difenoconazole, cyprodinil, and trifloxystrobin (experiment 1) and chlorpyrifos, boscalid, pyraclostrobin, cypermethrin, difenoconazole, and azoxystrobin (experiment 2). In experiment 1, the highest levels of trifloxystrobin were observed on the surface of flowers, (0.04 µg/flower) and for difenoconazole on the inside (0.023 µg/flower). Leaves contained only trace residues of cypermethrin and cyprodinil (0.001 µg/cm2 of leaves each) and trifloxystrobin (0.01 µg/cm2 of leaves) on the surface; inside the leaves, the highest levels of trifloxystrobin were observed (0.042 µg/cm2 of leaves). In experiment 2, boscalid was found on the surface and inside the flowers and leaves (0.063 and 0.018 µg/flower and 0.057 and 0.033 µg/cm2 of leaves, respectively). In bees, brood, and honey (experiment 1), chlorpyrifos was present in the highest quantity (7.3, 1.6, and 4.7 µg/kg, respectively). Additionally, cypermethrin and trifloxystrobin were found in bees, and trifloxystrobin was present in honey. Bees, brood, and honey from plantation 2 contained all studied AIs, with the highest levels of boscalid (28.6 µg/kg of bees, 37.0 µg/kg of brood, and 33.9 µg/kg of honey, respectively). In no case did the PPP residues in honey exceed acceptable maximum residue levels (MRLs)-from a formal and legal point of view, in terms of the used plant protection products, the analysed honey was fit for human consumption.

Behavior of fluopyram and tebuconazole and some selected pesticides in ripe apples and consumer exposure assessment in the applied crop protection framework.

The supervised field trials were conducted in a commercial apple orchard in 2016. The trials were an attempt to determine a model for dissipation and toxicological evaluation of fluopyram, tebuconazole, captan, tetrahydrophthalimide (THPI), pirimicarb, spirodiclofen, and boscalid residues detected in fruit of Red Jonaprince, Lobo, and Gala varieties immediately before harvest. The analysis also covered amounts of pesticides still present in remnants of calyx in Lobo and Gala varieties immediately before harvest. Laboratory samples of ripe apples were collected within 14 days of the treatment. Levels of pesticide residues detected in the samples changed at a constant exponential rate, and the residue levels found in ripe apples of Red Jonaprince, Gala, and Lobo varieties immediately before harvest were below maximum residue levels (MRL). Overall, captan residues in remnants of calyx were at a level of 22.3% for the Gala variety and 9.3% for the Lobo variety. Likewise, the long-term daily intake of the detected substances by a Polish adult consumer was low, ranging from 0.02% ADI for pirimicarb to 0.72% ADI for captan.

A case study on toxicological aspects of the pest and disease control in the production of the high-quality raspberry (Rubus idaeus L.).

The field studies on the residue levels of the fungicides and insecticides used in commercial raspberry (Rubus idaeus L.) plantation have been performed. Starting on the first day of harvesting (on June 19), 20 laboratory samples of fruit, 10 laboratory samples of leaves and 4 samples of soil were analyzed and the residue levels were compared to the Maximum Residue Limits (MRL) and Acceptable Daily Intakes (ADI). All analyses were carried out using extraction method and gas chromatography technique. Esfenwalerate (Sumi-alpha 050 EC) and beta-cyfluthrin (Bulldock 025 EC), the insecticides belonging to the group of synthetic pyrethroids, were not found in harvested ripe fruits, while cypermethrin residues (Cyperkill 25 EC) applied on May 24, 25 days later was still found on low levels in fruits (0.026 mg kg(-1)) and in leaves (2.58 mg kg(-1)). In turn, residues of chlorpyrifos (Dursban 480 EC), applied to the soil on May 15 against the cockchafers Melolontha melolontha and Otiorhynchus sp., were found at the level 0.004 mg kg(-1). The content of pesticides in ripe fruits depended mainly on the dose and on the time that has elapsed from the date of their application and were as follows: boscalid -0.950, pyrimethanil -0.917, pyraclostrobin -0.253 cypermethrin -0.026 and chlorpyrifos -0.004 mg kg(-1) while in leaves: boscalid -30.64, pyrimethanil -8.13, pyraclostrobin -15.82, cypermethrin -2.58 and chlorpyrifos -0.15 mg kg(-1). The highest average daily intake was in the case of boscalid, and in fruits and leaves reached the levels 0.205 and 6.63, in total 0.33% and 12.18% of ADI, respectively.

Behavior of pyrimethanil, pyraclostrobin, boscalid, cypermethrin and chlorpyrifos residues on raspberry fruit and leaves of Laszka variety.

The purpose of the research conducted was to investigate and evaluate the behavior of pyrimethanil, pyraclostrobin, boscalid, cypermethrin and chlorpyrifos, the active ingredients of selected fungicides and insecticides, on ripe fruit and in fully developed leaves of raspberry of the Laszka variety. The field trial was carried out in the period of one month starting from the first fruit picking. The results obtained indicated that residue levels on the day of the first crop picking did not even approximate the corresponding EU-MRLs (http://ec.europa.eu/sanco_pesticides). Individual substances in raspberry fruits and leaves disappeared at a similar rate. As a result of chlorpyrifos application to the soil, its residue in fruits and leaves occurred for the whole period of fruit bearing, though in fruit they dropped successively. To produce raspberries with residues below or equal to 0.01 µg g(-1), the application of pesticides should be stopped at least 2-3 weeks before the first crop picking, and on condition that an appropriate preparation (active in low doses) is applied to the last treatments.

Insecticide and fungicide effect on thermal and olfactory behavior of bees and their disappearance in bees' tissues.

Plant protection products may affect the behavior of organisms which are not a target of control. The effect of Karate Zeon 050 CS (λ-Cyhalothrin -based insecticide; λ-CBI) and Amistar 250 SC (Azoxystrobin-based fungicide; ABF) was determined on Apis mellifera worker attraction towards their own colony odour, along with temperature preferences. Bees exposed to pesticides prefer the environment with the odour of their nest less often than the control group, and that insecticide-treated bees chose warmer environments than the control insects. The observed differences in the bees, especially with attraction towards their own colony, were dependent on the time of day. Chromatographic analyses indicated that λ-Cyhalothrin elimination was half that of Azoxystrobin in bee organisms, and both agents retarded each other's clearance. Mathematical modeling estimated that despite a relatively high disappearance rate, both compounds might have been bio-accumulated at relatively high level.

The Effect of the Queen's Presence on Thermal Behavior and Locomotor Activity of Small Groups of Worker Honey Bees.

We examined effects of the queen's presence on diurnal rhythms of temperature preference (TP) and locomotor activity (LA) in worker honeybees' groups. TP and LA of six queenless and six queenright (with the queen) groups of bees, consisting of 7-8 worker bees, were recorded in a thermal gradient system for four days, under light to darkness (LD) 12:12 photoperiod. The same experiments were conducted on five virgin queens (of the same age as those in the queenright groups), which were placed individually in the gradient chambers. The single virgin queens showed signs of distress and no rhythms of TP and LA. In contrast, there were diurnal rhythms of TP and LA in both group variants with daytime activity and nighttime rest. However, the queen's presence exerted a strong calming effect, reducing LA of bees both at day- and nighttime. The nighttime minimum LA of queenright groups was five times lower than that in queenless groups. Moreover, there was a reversal of the diurnal pattern of TP in queenright groups. The results are discussed in terms of the bee colony organization as a superorganism.

A study on residue levels of fungicides and insecticides applied according to the program of raspberry protection.

This paper presents surveys on residue levels of fungicides and insecticides applied according to the raspberry protection program. The field trials were conducted in 2013-2014 on a plantation of raspberry of the Laszka variety dessert raspberry very popular in Poland. Laboratory samples were collected starting from a day of the first fruit picking to the end of harvest. The highest mean residue levels were found for boscalid and pyraclostrobin (2.395 mg/kg and 0.732 mg/kg, respectively), in both cases they were at a level of about 24% of their maximum residue levels (MRLs); and for cypermethrin (0.235 mg/kg; i.e. close to 50% of its MRL). The long-term dietary intakes of those substances by Polish adult consumers were also at low levels of 0.52, 0.22, and 0.04% of acceptable daily intake (ADI), respectively. Therefore, the results obtained indicated that even on day zero of picking ripe raspberries, the pesticide residues not only were well below their corresponding MRLs, but also their daily intakes did not even approach 1% of the ADI. In 2013, pesticide residues in ripe fruit evolved according to a pattern different than in a subsequent year; while in 2014 they changed at a constant exponential rate.

Comparative study on disappearance trends of captan and trifloxystrobin residues on fruit and apple tree leaves using internal normalisation method.

Supervised field trials were carried out in a commercial orchard in 2011. The purpose of the study was to assess the usefulness of the comparative method to examine the mechanism of disappearance of pesticide residues. Captan and trifloxystrobin residues were determined with the use of gas chromatograph equipped with a micro-electron capture detector. Disappearance trends of captan and trifloxystrobin residues in fruit and leaves were estimated using the method of internal normalisation, and based on that, the courses of concentration changes of these substances on fruit and leaves and the amount of these substances in one apple were established. The initial deposits of trifloxystrobin on leaves and fruits dropped by 50% within 8 and 4 days after treatment, respectively, in both varieties, whereas captan residues dropped by 50% within 29 days in leaves and 7 days in apples of the Olive Yellow varieties.