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

Transfer of the Active Ingredients of Some Plant Protection Products from Raspberry Plants to... Plant protection products (PPPs) have been found increasingly in the environment. They pose a huge threat to bees, con- tributing 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. The honeybee (Apis mellifera F.) is an insect species of sig- million. In general, the profit earned through pollination by nificant importance to the biosphere and the economy (Free bees is approximately 25–30% of the total yield of the crop 1993; Delaplane and Mayer 2000). This pollinator influences (Sanjerehei 2014; Giannini et al. 2015). According to San- the yields of approximately 70% of cultivated plants, which jerehei (2014), this value is 54 times higher than the value represents approximately 35% of the total global food pro- of honey produced by bees. A. mellifera is the main pollina- duction (Klein et al. 2007), which, in turn, yields $150 bil- tor that generates 86.8% of the gains generated by all the lion per year. In Brazil, the value of the work performed by pollinators. The use of plant protection products (PPPs) on all pollinators is estimated at nearly $ 12 billion (Giannini nectar-secreting plants goes hand in hand with the problem et al. 2015). In Great Britain, for Gala apples, the value of of exposing pollinators to such substances (Piechowicz et al. bees as pollinators is estimated at £5.7 million a year (Gar- 2018a, b). ratt et al. 2014). Majewski (2014) showed that a decrease PPPs may enter hives due to foraging by worker bees in the number of the honeybee colonies in Poland caused (Balayiannis and Balayiannis 2008; Mao et al. 2013; McMe- a decline in total crops valued at approximately €728.5 namin and Genersch 2015), because their active ingredients (AIs), especially those that have a contact activity, are pre- sent in crops, and consequently, they can be collected from * Przemysław Grodzicki flowers and leaves and then transferred to the hive. In turn, grodzick@umk.pl AIs that have deep-seated and systemic activity can be col- Department of Analytical Chemistry, Institute lected by bees together with pollen and nectar. of Biotechnology, University of Rzeszów, Werynia, Poland The AIs of PPPs, transferred by the worker bees to Laboratory of Pesticide Residues, Institute of Plant the hives, may result in miscellaneous, distinct effects. Protection, National Research Institute, Rzeszów, Poland Łozowicka (2013) investigated cases of honeybee colony Faculty of Mathematics and Natural Sciences, University intoxication. A presence of cypermethrin (pyrethroid insec- of Rzeszów, Rzeszów, Poland ticide, detected in 51% samples), chlorpyrifos (organophos- Department of Animal Physiology, Faculty of Biology phorus insecticide, detected in 27% samples), and bifenthrin and Environmental Protection, Nicolaus Copernicus (pyrethroid insecticide, detected in 21% samples) was found University, Toruń, Poland Vol.:(0123456789) 1 3 46 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 in 33 worker bee samples. Likewise, in intoxicated worker Materials and Methods bee bodies, Walorczyk and Gnusowski (2009) found an occurrence of tebuconazole (triazole fungicide, detected in Field Trial 48% samples), omethoate (oxygenated form of dimethoate, organophosphorus insecticide, detected in 44% samples), The field trial was performed from May 20 to July 15, and fipronil (phenylpyrazole insecticide, detected in 40% 2014, on a raspberry (Rubus idaeus), Laszka variety, plan- samples). In turn, among 19 of the detected compounds, tation in the village Grabówka Kolonia, in the province of Barganska et al. (2014) most frequently found heptenophos Lublin, which is protected from pests using conventional (organophosphorus insecticide, detected in 68% samples), methods, in accordance with current programmes. All bifenthrin (pyrethroid insecticide, detected in 53% samples), preparations were applied according to the labels posted. and pyrazophos and diazinon (organophosphorus insecti- A sprayer, model RA 10/80 (Lochmann, Vilpiano, Italy) cide, detected in 32% samples). However, cases of acute with nozzles ALBUZ ATR 80, was used. Within 2 km of honeybee poisoning by the PPPs may be only marginal. In the studied raspberry plantation, there were no plantations most of the cases, the presence of pesticides in the hive is at of any other blossoming plants secreting nectar that could such a low level that it does not affect the honeybee colony have interfered with the test results. The honeybee colo- well-being. Pohorecka et al. (2017) suggest that the phenom- nies were transported from an area where the bees had no enon of winter colony collapse could be caused by honey- contact with pesticides. On May 17, 2014, the colonies bee parasites. Studies of Piechowicz et al. (2018a, b) on the were placed approximately 3 m from the raspberry planta- transfer of plant protection products from oilseed rape crops tion on an area of 4 ha. and orchards to beehives showed a presence of pesticides On the raspberry plantation, four rows of plants were cho- both in bee bodies (5/7 detected compounds at rape planta- sen for the study, each approximately 150 m long. On each tion 1; 3/5 at rape plantation 2; and 5/6 AIs in orchards) and sampling date, from each of the four selected rows, a sample in honeybee brood (4 and 2 AIs in hives located near rape of 16 leaves from randomly selected plants was taken (only crops and 6 AIs in bees in the orchard), and in honey (3 and fully developed leaves were collected from the outside of 3 AIs in rape honey and 4 AIs in apple-pear honey). In the the bush), and then analytical portions, which consisted of studied cases, when the worker bees were directly exposed 16 disks 1 cm in diameter, were cut. On the same sampling to pesticides originating from the crops, no deterioration in day, samples of flowers and fruits, consisting of 8 and 16 honeybee colony well-being was observed. It does not mean pieces, respectively, were collected from the same randomly that PPP AIs, especially in the case of their simultaneous selected plants. presence in the hive, could not have affected the bees. Some During the e fi ld trial, from each of the four hives, one lab - investigators indicate that for bees endowed with only 46 oratory sample of worker bees (retrieved from the frames), genes responsible for the detoxification system functioning the brood (from non-sealed cells, 4–6 days before hatch- (Claudianos et al. 2006), which additionally have few genes ing), and honey (from non-sealed cells) also were collected. controlling detoxification of the plant protection products Each sample weighed at least 5 g. Additionally, every week, (The honeybee genome sequencing consortium 2006), a syn- soil samples were collected using an Egner stick, with one ergistic action of small, sublethal residues of two or more sample from each of the four rows. Each sample consisted AIs (Thompson 1996; Thompson and Wilkins 2003; Mullin of eight portions taken from randomly selected places in the et al. 2010; Glavan and Božič 2013; Johnson et al. 2013) row at a distance no further than 30 cm from the raspberry can be dangerous for them. Even if these compounds are plants. not toxic to bees, this does not mean that they are not harm- ful to the brood (Zhu et al. 2014). This effect is especially relevant to intensively protected crops in which the flowering Chemicals and Pesticides and fruiting periods occur at the same time, so both plants and fruits need protection. Raspberries are one such crop. During the period from January 7 to June 9, 2014, 12 protec- tive treatments were performed on the plantation. The terms, Our study was an initial analysis of whether some AIs of PPPs may be carried by bees from raspberry plants and preparations and applied doses are shown in Table 1. transferred to beehives located in the immediate vicinity of the crop and to what extent these AIs were transferred. Extraction of Pesticide Residues from the Honey, Worker Bees, and Brood for Analysis The samples of the worker bees and the brood were lyophi- lized using a Labconco Freezone 2.5 freeze dryer (Labconco, 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 47 1 3 Table 1 Pesticide spraying program performed on the raspberry plantation Date of treatment PPP, trade name AI, common name Chemical clas- AI structure AI content Mode of applica- Mechanism of Dose of MRL, (IUPAC name) sification tion action PPP (L, kg/ honey (mg/ ha) kg) January 7 Kerb 50 WP (H)* Propyzamide Benzonitrile 500 g/kg (50%) Between rows Systemic 2.00 0.05 (3,5-dichloro-N-(1,1- dimethylprop-2-ynyl) benzamide) March 23 Treol 770 EC (I) Paraffin oil – – 770 g/L Foliar By contact— 20.00 0.01 plants; By contact— insects May 7 Amistar 250 SC Azoxystrobin Strobilurin 250 g/L (22.81%) Foliar Systemic 0.50 0.05 (F) (methyl (E)-2-{2-[6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl}-3-methoxy- acrylate) May 7 Score 250 EC (F) Difenoconazole Triazole 250 g/L (23.58%) Foliar Systemic 0.50 0.05 (1-[2-[2-chloro- 4-(4-chloro- phenoxy)-phenyl]-4 methyl[1,3]dioxolan- 2-ylmethyl]-1H-1,2,4- triazole) May 14 Mythos 300 SC (F) Pyrimethanil Anilinopyrimidine 300 g/L (28.3%) Foliar By contact and 2.50 0.05 (4,6-dimethyl-N-phe- deep-seated nylpyrimidin-2-amine) May 14 Dursban 480 Chlorpyrifos Organophosphate 460 g/L (44.86%) To the soil through By contact and 2.00 0.05 EC*(I) (O,O-diethyl O-3,5,6- the irrigation deep-seated— trichloropyridin-2-yl system plants; phosphorothioate) By ingestion— insects May 16 Mospilan 20 SP (I) Acetamiprid Neonicotinoids 200 g/kg (20%) Foliar Systemic—plants; 0.20 0.05 (N-[(6-chloro-3-pyridyl) by ingestion— methyl]-N’-cyano- insects N-methyl-acetamidine) May 19/May 29 Cyperkil Super Cypermethrin Pyrethroids 250 g/L (25.92%) Foliar By contact— 0.15 0.05 250 EC (I) ([cyano-(3-phenoxy- plants; phenyl)methyl]3-(2,2- By contact, 0.15 0.05 dichloroethenyl)- by ingestion— 2,2-dimethylcyclopro- insects pane-1-carboxylate) 48 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 1 3 Table 1 (continued) Date of treatment PPP, trade name AI, common name Chemical clas- AI structure AI content Mode of applica- Mechanism of Dose of MRL, (IUPAC name) sification tion action PPP (L, kg/ honey (mg/ ha) kg) June 4 Rovral Aquaflo Iprodione Dicarboximide 500 g/L (42.91%) Foliar By contact 2.00 0.05 500 SC (F) (3-(3,5-dichlorophenyl)- N-isopropyl-2,4-diox- oimidazolidine-1-car- boxamide) June 5 Bellis 38 WG (F) Boscalid Anilide 252 g/kg (25.2%) Foliar Systemic 1.50 0.50 (2-chlor-N-(4′- chlorbiphenyl-2-yl) nicotinamid) Pyraclostrobin Strobilurin 128 g/kg (12.8%) 0.05 (methyl-N-(2-[1- (4-chlorphenyl)- 1H-pyrazol-3-yl] oxymethylphenyl)-(N- methoxy)carbamat) June 9 Signum 33 WG (F) Boscalid Anilide 267 g/kg (26.7%) Foliar Systemic 1.80 0.50 (2-chlor-N-(4′- chlorbiphenyl-2-yl) nicotinamid) Pyraclostrobin Strobilurin 67 g/kg (6.7%) 0.05 (methyl-N-(2-[1- (4-chlorphenyl)- 1H-pyrazol-3-yl] oxymethylphenyl)-(N- methoxy)carbamat) *H herbicide, I insecticide, F fungicide Archives of Environmental Contamination and Toxicology (2018) 75:45–58 49 USA) (pressure: 0.024  mbar; temperature: 50  °C, time: Poland) and stirred carefully. Analytical portions of 20 g 168 h). were taken from the samples and shaken for 1  h with a Analytical portions of 5 g of the lyophilized animals or 50-mL mixture of dichloromethane:acetone (9:1; v/v) on honey were shaken with 10 mL of acetonitrile (Chempur, a GFL 3006 shaker (GFL, Germany). The extracts were Poland). Then, a mixture of salts containing 4 g of anhy- allowed to stand for 10 min and then decanted through a drous magnesium sulfate (VI) (Chempur, Poland), 1 g of layer of anhydrous sodium sulfate (VI) that had been placed sodium chloride (Chempur, Poland), 1 g of trisodium citrate in the funnel. The soil samples were washed twice with (Chempur, Poland), and 0.5 g of sesquihydrate disodium 20 mL of dichloromethane, and the combined extracts were hydrogen citrate (Chempur, Poland) was added. The con- evaporated to dryness on a Heidolph Laborota 4000 Effi- tents were shaken for 2 min and centrifuged for 5 min at cient rotary evaporator. The residues were then dissolved in 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase 10 mL of petroleum ether. The resulting extracts were puri- was transferred to a polypropylene test tube that contained fied using a Florisil mini-column (Sadło et al. 2014, 2015), 150 mg of PSA (primary secondary amine) (Agilent, USA) and the residues were eluted with 70 mL of 3:7 (v/v) diethyl and 900 mg of anhydrous sodium sulfate(VI) (Chempur, ether:petroleum ether, followed by elution with 70 mL of 3:7 Poland). The extract was vigorously shaken for 2 min and (v/v) acetone:petroleum ether. The combined eluates were centrifuged for 5 min as described above. Four millilitres evaporated to dryness, and the residues were quantitatively of the obtained extract was taken and transferred to a glass transferred using petroleum ether into a 10-mL volumetric tube, evaporated to dryness on a Heidolph Efficient Labware measuring flask. 4000 rotary evaporator (Heidolph, Germany), and then dis- solved in 4 mL of petroleum ether (Chempur, Poland). Chromatographic Determination of Pesticide Residues Extraction of Pesticide Residues from the Leaves, Flowers and Fruits for Analysis The extracts were analysed using an Agilent 7890 (Agi- lent, USA) gas chromatograph equipped with a micro-cell The analytical portions of the leaves (16 disks, 1 cm in diam- electron capture detector (µECD) and a nitrogen–phospho- eter each) and flowers (8 pieces), both with the addition of rus detector (NPD). The chromatograph was controlled by 100 mL of water, and fruits (16 pieces) were homogenized ChemStation software (Agilent, USA). It also was equipped in a Waring Commercial 8010 EG blender (Waring, USA) with an autosampler and an HP-5MS, 30-m  ×  0.32- with 150  mL of acetone (Chempur, Poland) and filtered mm × 0.25-µm column. The instrumental analysis conditions through a Büchner funnel under vacuum. The blender jar were as follows: an NPD detector temperature of 300 °C, a was flushed with 50 mL of acetone, and the washings were μECD detector temperature of 290 °C, and an injector tem- used to wash the filter cake. One-fifth of the filtrate (the perature of 250 °C. The oven temperature was programmed equivalent of approximately 15.4 g of fruit and approxi- as follows: 100  °C at 0  min →  10  °C per min →  4  min mately 0.1  g of leaves) was used for further analysis. It at 180 °C → 3 °C per min → 15 min at 220 °C → 10 °C was placed in a separatory funnel together with 100 mL per min → 11 min at 260 °C. The total analysis time was of 2.5% sodium sulfate (VI) (Chempur, Poland) solution. 55.3 min, and the injection volume was 2 µL. The pesticide residues were extracted three times with 20, 10, and 10 mL of dichloromethane (Chempur, Poland). The Statistical Analysis of the Results combined extracts were evaporated to dryness, dissolved in 10 mL of petroleum ether and purified using a Florisil Recovery studies were performed by spiking each matrix (Chempur, Poland) mini-column (Sadło et al. 2014, 2015). with the substances used in field trials at a single concentra- The pesticide residues were eluted with a 70-mL mixture of tion (Table 2). The pesticide residues (R ) in the samples 3:7 (v/v) ethyl ether:petroleum ether (Chempur, Poland) as were recalculated (R ) using the results of the recovery rec well as with a 70-mL mixture of 3:7 (v/v) acetone:petroleum study (Rec in %; Table 3) according to Eq. 1. ether. The solvents were evaporated to dryness, and the resi- R = 100 × R ∕Rec due was transferred quantitatively using petroleum ether into (1) rec i a 10-mL volumetric measuring flask. The transfer factor (TF) from the soil to the plants was calculated according to Eq. 2. Extraction of Pesticide Residues in the Soil TF = 100 × R ∕R i (plant) i (soil) (2) for Analysis To determine the similarity in the residue concentrations The soil laboratory samples were air dried and pulver- of the individual substances between different sample types, ized with a Testchem LMG grinder (Testchem Sp. z.o.o., a cluster analysis was performed. The Euclidean metric was 1 3 50 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 2 Recoveries (expressed in %) of AIs of PPPs applied on the raspberry plantation Sample Propyzamide Chlorpyrifos Iprodione Pyraclos- Boscalid Cypermethrin Difenocona- Azoxystrobin Pyrimethanil trobin zole Worker bees 96.3 95.7 92.5 95.0 90.1 108.3 105.2 86.7 101.8 Brood 95.6 89.9 111.2 90.5 95.0 110.4 120.0 111.7 107.3 Soil 90.0 88.9 86.8 108.5 100.8 114.3 110.4 86.7 87.4 Honey 100.1 88.4 76.9 76.0 63.4 87.0 75.6 61.4 93.2 Flowers 94.2 92.4 98.5 117.4 89.0 85.7 93.2 82.2 93.2 Leaves 98.7 92.1 101.3 112.9 91.6 86.1 85.7 82.5 94.3 Fruits 92.0 91.9 88.2 118.1 85.1 85.7 85.3 81.6 93.7 used to describe the similarities. The Ward method was used ha. The residues of those substances were found in only the as the agglomeration algorithm. The Friedman test was used samples collected on May 27 (13 and 20 days since applica- to determine whether the residue concentrations in the sam- tion) at concentrations of 0.09 and 0.14 mg/kg of the flowers, ple types varied significantly. The Spearman’s rank correla- respectively. The TF from the soil to flowers for chlorpyrifos tion coefficient was used to assess the strength, direction was 150%, which indicated that a distinct part of that sub- and statistical significance of dependencies between the stance penetrated the flowers from the soil with the transpi- residue concentrations in the various sample types, assum- ration current. On only two sampling days, in the samples ing α < 0.05 as statistically significant. collected on May 27 and June 4, difenoconazole residues (triazole fungicide with a systemic mode of action) were found at a relatively high level (1.03 and 0.84 mg/kg). This Results substance was applied on May 7 in the form of Score 250 EC (0.5 L/ha). Pyrimethanil, which was applied in the form In general, pesticide residue recoveries should be in the of Mythos 300 SC at an application rate of 2.5 L/ha on May range of 70–120% of the substance introduced into the 14, occurred at the highest level. The initial residue of this sample, and the repeatability should be ≤ 20% (Document compound, with slight fluctuations in the levels, decreased SANTE 2015). In our study, satisfactory values of both of in concentration until the last sampling day. Boscalid (ani- these parameters were obtained for nine AIs of PPPs in lide fungicide) and pyraclostrobin (strobilurin fungicide) seven sample types. However, for boscalid and azoxystrobin, residues, after the application of Bellis 38 WG (1.5 kg/ha) the recovery from honey did not exceed 70%; the recoveries on June 5 and Signum 33 WG (1.8 kg/ha) on June 9 (both were 63.4 and 61.4%, respectively (Table 2). The limit of preparations with a systemic mode of action in plants), were quantification (LOQ) of propyzamide, chlorpyrifos, iprodi- found on four sampling days, and their values gradually one, pyraclostrobin, boscalid, cypermethrin, difenoconazole, decreased, excluding the samples collected on June 2, when azoxystrobin, and pyrimethanil in all studied matrices was none of the two AIs was found, from 7.2 to 0.36 mg/kg in the 0.01 mg/kg. flower samples collected on June 10 to 1.08 and 0.08 mg/kg in the flower samples from July 8. Cypermethrin (pyrethroid Pesticide Residues in Raspberry Plantation insecticide with a contact mode of action) was applied on May 19 and May 29 in the form of Cyperkill Super 250 EC, Table 3 shows the average concentrations of pesticide resi- in both cases at an application rate of 0.15 L/ha. Its residues dues in the seven sample types. were observed in the flower samples collected on all sam- pling days, while after the first application (samples from Flowers May 20), its residues were 0.33 mg/kg, and after the second application, they were 0.51 and 0.57 mg/kg, respectively. Seven of the nine studied AIs were found in the flowers. The From that time until June 25, the concentrations decreased to residues of chlorpyrifos (an organophosphorus insecticide 0.18 mg/kg and then increased to the 0.63 mg/kg on July 2. with a deep-seated mode of action in plants) were applied to the soil by the irrigation system on May 14 in the form of Leaves Dursban 480 EC at an application rate of 2 L/ha, and azox- ystrobin (a strobilurin fungicide with a systemic mode of In the laboratory leaf samples, chlorpyrifos, pyraclostrobin, action in plants) was applied by foliar spraying on May 7 in boscalid, cypermethrin, difenoconazole, azoxystrobin, the form of Amistar 250 SC at an application rate of 0.5 L/ and pyrimethanil were found. Chlorpyrifos residues were 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 51 Table 3 The average residues ± standard deviations (in mg/kg) of AIs applied on the raspberry plantation Sampling Propyza- Chlorpy- Iprodione Pyraclos- Boscalid Cyperme- Difenocona- Azox- Pyrimethanil date mide rifos trobin thrin zole ystrobin Flowers May 20 < LOQ < LOQ < LOQ < LOQ < LOQ 0.33 ± 0.21 < LOQ < LOQ 24.50 ± 6.60 May 27 < LOQ 0.09 ± 0.04 < LOQ < LOQ < LOQ 0.24 ± 0.15 1.03 ± 0.15 0.14 ± 0.05 3.41 ± 1.02 June 4 < LOQ < LOQ < LOQ < LOQ < LOQ 0.51 ± 0.51 0.84 ± 0.54 < LOQ 1.72 ± 0.68 June 10 < LOQ < LOQ < LOQ 0.36 ± 0.27 7.20 ± 3.84 0.57 ± 0.09 < LOQ < LOQ 1.75 ± 0.76 June 17 < LOQ < LOQ < LOQ 0.30 ± 0.11 6.34 ± 2.40 0.41 ± 0.03 < LOQ < LOQ 0.03 ± 0.03 June 25 < LOQ < LOQ < LOQ 0.04 ± 0.04 1.94 ± 0.77 0.18 ± 0.11 < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ 0.63 ± 0.11 < LOQ < LOQ 0.10 ± 0.03 July 8 < LOQ < LOQ < LOQ 0.08 ± 0.16 1.08 ± 2.16 0.51 ± 0.44 < LOQ < LOQ 0.06 ± 0.07 Leaves May 20 < LOQ 0.13 ± 0.15 < LOQ < LOQ < LOQ 0.90 ± 1.27 < LOQ < LOQ 206.28 ± 61.02 May 27 < LOQ 0.11 ± 0.22 < LOQ < LOQ < LOQ 0.11 ± 0.13 31.98 ± 8.09 5.61 ± 0.91 30.29 ± 17.85 June 4 < LOQ < LOQ < LOQ < LOQ < LOQ 2.80 ± 1.11 9.71 ± 2.40 1.39 ± 0.90 34.05 ± 25.18 June 10 < LOQ < LOQ < LOQ 12.30 ± 4.49 7.40 ± 1.60 1.54 ± 0.32 4.92 ± 0.48 < LOQ 2.50 ± 0.42 June 17 < LOQ < LOQ < LOQ 19.59 ± 7.52 7.61 ± 3.62 0.96 ± 0.44 7.20 ± 3.72 0.67 ± 0.67 3.25 ± 1.91 June 25 < LOQ < LOQ < LOQ 11.70 ± 4.34 5.51 ± 1.96 1.33 ± 0.33 4.17 ± 1.57 0.44 ± 0.44 5.44 ± 3.58 July 2 < LOQ 0.06 ± 0.06 < LOQ 6.49 ± 2.39 4.41 ± 1.40 1.20 ± 0.40 3.78 ± 0.53 < LOQ 0.27 ± 0.27 July 8 < LOQ < LOQ < LOQ 2.84 ± 1.26 2.23 ± 0.82 0.47 ± 0.10 1.54 ± 0.92 < LOQ < LOQ July 15 < LOQ < LOQ < LOQ 0.60 ± 0.35 1.11 ± 0.42 0.34 ± 0.09 2.09 ± 1.25 < LOQ < LOQ Fruits June 17 < LOQ < LOQ < LOQ 0.04 ± 0.01 1.34 ± 0.29 0.27 ± 0.07 0.02 ± 0.00 0.01 ± 0.00 0.16 ± 0.05 June 25 < LOQ < LOQ < LOQ 0.03 ± 0.00 0.84 ± 0.18 0.14 ± 0.05 0.01 ± 0.00 < LOQ 0.04 ± 0.00 July 2 < LOQ < LOQ < LOQ < LOQ 0.41 ± 0.08 0.07 ± 0.02 0.01 ± 0.00 < LOQ 0.02 ± 0.00 July 8 < LOQ < LOQ < LOQ < LOQ 0.23 ± 0.06 0.02 ± 0.00 0.01 ± 0.00 < LOQ 0.01 ± 0.00 July 15 < LOQ < LOQ < LOQ < LOQ 0.05 ± 0.00 0.02 ± 0.00 0.01 ± .0.00. < LOQ 0.01 ± 0.00 Soil May 20 0.16 ± 0.08 0.08 ± 0.08 < LOQ < LOQ < LOQ. < LOQ < LOQ < LOQ < LOQ May 27 0.10 ± 0.05 0.06 ± 0.05 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 0.07 ± 0.03 0.04 ± 0.03 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 0.04 ± 0.02 0.03 ± 0.02 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 17 0.03 ± 0.01 0.02 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 0.02 ± 0.01 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 0.01 ± 0.01 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 0.01 ± 0.00 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 0.01 ± 0.00 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Honey May 20* < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ May 27 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 < LOQ < LOQ < LOQ < LOQ 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ June 17 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Worker honeybees May 20 < LOQ 0.03 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 0.09 ± 0.03 May 27 < LOQ 0.03 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 0.03 ± 0.01 June 4 < LOQ 0.05 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 1 3 52 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 3 (continued) Sampling Propyza- Chlorpy- Iprodione Pyraclos- Boscalid Cyperme- Difenocona- Azox- Pyrimethanil date mide rifos trobin thrin zole ystrobin June 10 < LOQ 0.04 ± 0.01 0.09 ± 0.09 < LOQ 0.08 ± 0.05 < LOQ < LOQ < LOQ < LOQ June 17 < LOQ 0.01 ± 0.04 0.11 ± 0.13 < LOQ 0.06 ± 0.10 < LOQ 0.02 ± 0.07 < LOQ < LOQ June 25 < LOQ 0.05 ± 0.00 0.63 ± 0.16 < LOQ 0.11 ± 0.05 < LOQ < LOQ < LOQ < LOQ July 2 < LOQ 0.02 ± 0.01 0.01 ± 0.01 < LOQ 0.01 ± 0.01 < LOQ 0.12 ± 0.03 < LOQ < LOQ July 8 < LOQ 0.02 ± 0.01 0.02 ± 0.02 < LOQ 0.01 ± 0.01 < LOQ 0.09 ± 0.03 < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Worker brood May 20 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ May 27 < LOQ 0.02 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 17 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ *In worker honeybees and the brood as well in honey samples collected on May 16 (a day before transporting hives to the crop), none of the determined PPP AIs were shown detected on May 20 and May 27 (0.13 and 0.11  mg/kg, decreased to 0.27 mg/kg in the samples from July 2 and to an respectively) and on July 2 (0.06 mg/kg). In this case, the amount < LOQ during the subsequent leaf sampling days. TF was 162.5, 183.3, and 600%, respectively. Pyraclostrobin and boscalid residues were found in samples collected on six Fruits sampling days, and their highest values occurred on June 17 (19.59 and 7.61 mg/kg, respectively). After that, since the The harvest period for fruits began on June 17, and pyr- last sampling day, a systematic decrease in their level was aclostrobin, boscalid, cypermethrin, difenoconazole, observed. It is worth mentioning that the residues of pyra- pyrimethanil, and azoxystrobin were detected at that time. clostrobin were significantly higher than those of boscalid, The pyraclostrobin residues were found only at a level 3–4 which was in contradiction to proportions of those AIs in times higher than the limit of quantification (LOQ) on June the applied preparations (1:1.97 in the case of Bellis 38 WG 17 and June 25 (0.04 and 0.03 mg/kg, respectively), while and 1:3.99 in the case of Signum 33 WG). The cypermethrin boscalid, applied together with pyraclostrobin, in all sam- residues were detected in all the samples, while after the r fi st ples, ranged from 1.34 mg/kg on June 17 to 0.05 mg/kg on application of Cyperkill Super 250 EC, they were 0.9 mg/kg July 15. The cypermethrin residues were found in all sam- (samples collected on May 20), and after the second appli- ples of fruit, and its residue decreased from 0.27 mg/kg on cation, they were 2.8 mg/kg (samples collected on June 4). June 17 to 0.02 mg/kg on the last sampling day. Similarly, From that time until the end of the investigation, a decrease difenoconazole and pyrimethanil were found in fruit sam- in the cypermethrin residue level was observed. Likewise, in ples on each sampling date, while in the case of difenocona- all samples, excluding those collected on May 20, the pres- zole, its residues occurred at the level closest to the LOQ ence of the applied difenoconazole was found; its residues (0.02 mg/kg on June 17 and 0.01 mg/kg on other dates), appeared to be high (31.98 mg/kg) on May 27; and after that and the pyrimethanil residue level decreased with time from day, they decreased until the last sampling day (2.09 mg/ 0.16 mg/kg on June 17 to 0.01 mg/kg on June 15. Trace kg). Azoxystrobin was detected in samples collected on azoxystrobin residues were found only in samples collected four application days. Its highest residue level was found in on June 17 (0.01 mg/kg). leaves collected on May 27 (5.61 mg/kg), and in subsequent application dates, this value decreased to 0.44 mg/kg in the Soil samples collected on June 25, while the residues of this AI were not found in the samples from June 10. After foliar Only two AIs of PPPs applied were detected in the 10-cm application on May 14, pyrimethanil left significant resi - soil layer. In all studied samples, the residue of chlorpyrifos dues on leaves (206 mg/kg in the samples from May 20) that decreased systematically from 0.08 mg/kg of soil on May 20 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 53 to 0.01 mg/kg in the samples collected from June 25 to July associated distance matrix analysis, differences in the resi- 15, and propyzamide, with an AI of Kerb 50 WP, belonging due concentrations of the individual substances could be to benzonitrile group. The herbicide was applied between seen in the various sample types. However, when using the rows on January 7 at an application rate of 2 L/ha, and its Friedman test, statistically significant differences between residues decreased systematically from 0.16 mg/kg on the the residue concentrations of specific substances were not first sampling day to 0.01 mg/kg in the samples collected demonstrated. during the period July 2 to July 15. Spearman’s rank correlation coefficient was used to deter - mine whether there was a mutual relationship between the Pesticide Residues in Samples of Honey, Worker residue concentrations in the samples analysed, regardless Bees and Brood of the collection date (Table 4). Spearman’s rank correlation coefficient was positive in all Honey cases, which indicated a quantitative relationship between the application rate and the concentration of the residues Only a trace residue of boscalid at the level of the LOQ of AIs of the PPP, located at sites frequented by the worker (0.01 mg/kg on June 10) was detected in honey on one sam- bees and in the hive interior. pling date. Spearman’s rank correlation coefficient was also used to determine whether a mutual relationship existed between Worker Bees and Brood the residue concentrations of the AIs of PPPs in different combinations of samples and for different sampling condi- Chlorpyrifos, iprodione, boscalid, difenoconazole, and tions, and the results of the analysis of these relationships pyrimethanil were found in worker bee samples. Chlorpyri- are presented in Table 5. fos residues were detected most frequently and were found In most cases, a positive correlation was noted between in samples taken for analysis on eight of nine sampling dates the residue concentration of AIs of PPPs in the crop and in (May 20 and 27; June 4, 10, 17, and 25; and July 2 and the hive. Only certain worker bees collected on May 27, June 8), although in all cases, its residues were at low levels (to 10, and July 17 showed negative correlation coefficients. 0.05 mg/kg in the samples collected on June 4). However, No positive correlations were noted between the AI of PPP animal bodies contained iprodione residues (dicarboximide residues in the environment and in the worker bees. group; the AI of Rovral Aquaflo 500 SC, used on June 4 at an application rate of 2 L/ha), a fungicide with a contact mode of action on plants, which was not detected in other Discussion matrices. The iprodione residues increased during the period from June 10 to June 25 from 0.09 to 0.63 mg/kg and then Raspberries, including the Laszka variety, are nectar- started to decrease below LOQ on the last sampling day. secreting plants. Their yields depend largely on the effi- The boscalid residues were detected at low levels (no more ciency of their pollination by insects (Chauzat et al. 2009). than 0.11 mg/kg on June 25), but pyraclostrobin, which was Worker bees, when foraging on entomophilous plants, may applied in the form of Bellis 38 WG and Signum 33 WG together with boscalid, was not detected at all. Similarly, in Table 4 The relationship between residue concentrations found in dif- the worker bee samples, small amounts of systemic difecon- ferent matrix combinations azole were detected (in the samples from 3 sampling days; to 0.12 mg/kg in samples from July 2) as well as pyrimethanil AI, common name Matrix combination Correlation level with a contact and deep-seated action (0.09 and 0.03 mg/ Chlorpyrifos Soil-worker brood 0.75 kg in the samples from May 20 and May 27, respectively). Iprodione All Lack of relationship In brood samples, the pesticide residues at a level higher Pyraclostrobin All Lack of relationship than the LOQ were collected only on three sampling days. Boscalid Flowers-honey 0.80 Only a trace amount of chlorpyrifos was found (no more Flowers-worker honeybees 0.88 than 0.02 mg/kg of the brood) on May 27. Leaves-honey 0.87 Leaves-worker honeybees 0.89 Statistical Analysis Honey-worker honeybees 0.88 Cypermethrin All Lack of relationship The similarity degree for the residue concentrations of the Difenoconazole All Lack of relationship individual substances found in the various sample types, Azoxystrobin All Lack of relationship regardless of the sampling date, was assessed using a clus- Pyrimethanil Flowers-worker honeybees 0.76 ter analysis. Based on the cluster analysis results and the 1 3 54 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 5 The relationship AI, common name Sampling date Relation Correlation coefficient between the residue levels of AIs of PPPs in various matrices, Propyzamide All All Lack of relationship depending on terms of sampling Chlorpyrifos May 27 Leaves-worker honeybees − 0.88 Iprodione June 17 Soil-worker honeybees − 0.89 Pyraclostrobin All All Lack of relationship Boscalid June 10 Leaves-honey 0.81 June 10 Leaves-worker honeybees − 0.90 June 25 Leaves-worker honeybees 0.82 July 2 Flowers-honey 0.90 July 2 Flowers-worker honeybees 0.87 July 2 Fruits-worker honeybees 0.99 Cypermethrin All All Lack of relationship Difenoconazole All All Lack of relationship Azoxystrobin All All Lack of relationship Pyrimethanil May 27 Flowers-worker honeybees − 0.88 simultaneously collect various contaminants and transfer aboveground parts of plants at approximately 5 days after them to the hive (Anderson and Wojtas 1986; Chauzat et al. their introduction to the soil, which may partially explain 2009; Cresswell and Thompson 2012; Oruc et  al. 2012; the obtained results. The cypermethrin behaviour, which Piechowicz et al. 2018a, b). Some pesticides used to protect was applied in the form of Cyperkill Super 250 EC on May raspberry plantations from pests and diseases show a pos- 19 and May 29, indicates the impacts of intensive rainfall sibility of accumulation in the bee bodies. They also pollute on May 19 and May 20. On May 20, the first day after the bee products (Rissato et al. 2006). Therefore, some reports first application, the cypermethrin residue on leaves was indicate that some AIs of PPPs may be transferred into the 0.9 mg/kg. In the samples obtained on June 4, i.e., 6 days beehive (Anderson and Wojtas 1986; Southwick and South- after the second treatment (in both cases, 0.15 L/ha), this wick 1992; Pettis et al. 2004; Panseri et al. 2014). value increased to 2.8 mg/kg; therefore, without taking into The AIs of PPPs differ in their chemical structure (they account natural disappearance, it was threefold higher than belong to different chemical groups) and mode of actions, after the first treatment. which determine how they are distributed in the environ- Pyrimethanil, which was applied to the crop in the form ment, how they spread in plants and penetrate plants, and of Mythos 300 SC at the same time as chlorpyrifos (Dursban how they penetrate animal bodies. Furthermore, the PPPs are 480 EC), i.e., on May 14, and cypermethrin, used in the form characterized by long half-lives and consequently by their of Cyperkill Super 250 EC on May 19 and May 29, which persistence in the environment (Gerolt 1983; Różański 1992; are substances with contact action and more adherent to the Leroux 1996; Mileson et al. 1998; Seńczuk 2012; Szpyrka plant surface, were found on leaves and flowers since the and Walorczyk 2017). The above-mentioned factors may first sampling day (24.5 mg/kg on flowers and 206 mg/kg on result in the occurrence of chlorpyrifos (deep-seated and leaves in the case of pyrimethanil and 0.33 mg/kg on flow - probably, which is indicated by the results in Table 3, semi- ers and 0.90 mg/kg on leaves in the case of cypermethrin). systemic), azoxystrobin or difenoconazole residues (both However, the residue of iprodione (AI of Rovral Aquaflo 500 with a systemic action in plants), which were not detected SC), also with a contact mode of action, was not detected in the samples collected on May 20 (all 3 compounds; 6, 13, on any part of plant, although probably in this second case, and 13 days after application, respectively), nor the leaves the preparation, which was still damp, was washed away (azoxystrobin and difenoconazole). They were present only from the plant, which is partly indicative of the necessity of in samples collected on May 27, which indicates that their the subsequent fungicide treatment (Bellis 38 WG) that was residues are linked to the secondary absorption of those applied on June 5, i.e., the day after the application of Rovral compounds from the deeper layers of the soil, where they Aquaflo 500 SC. Most likely, not the lack of application but were probably diluted due to the intensive rainfalls rather washing away the preparation from the plant resulted in the than their primary presence at the plant surface. In addi- presence of marked amounts of iprodione on bee bodies, tion, because only chlorpyrifos, at an application rate of because the fungicide treatments were performed during the 0.08 mg/kg on May 20 and of 0.06 mg/kg on May 27, was daily period of worker bee foraging. observed in the surface layer. As Kubik et al. (2000) notes, Small amounts of difenoconazole and pyrimethanil at plant protection products with systemic action appear in the a level close to LOQ were observed on all fruit samples. 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 55 Additionally, the presence of azoxystrobin was detected on be found. However, its long persistence in the soil (Table 3) June 17, which indicates that preparation of systemic and and its continuous transport and concurrent transpiration to extensive action can occur in a plant for a long period (69, aboveground plant parts resulted in its occurrence in the 62, and 41 days after application, respectively). flowers at a higher concentration than the LOQ, which con - In the preparations, which were applied on June 5 and sequently constituted a threat to pollinators. The presence of June 9 and contained pyraclostrobin and boscalid (Bel- some pesticides in brood indicates that the worker bees have lis 38 WG and Signum 33 WG), the proportions between been exposed to PPPs since the earliest stages of their ontog- these substances were 1:1.97 and 1:3.99, respectively. The eny, at a time of intensive nervous system development. amount of pyraclostrobin was reduced relative to the amount Chlorpyrifos, applied to the soil on May 14, was present of boscalid that occurred in flowers (proportions from 1:13.5 in the soil samples until the last sampling date (i.e., July 8) on July 8 to 1:48.5 on June 25, respectively) and in fruits and consequently also occurred in the flowers and leaves. (1:33.5 on June 17 and 1:28.0 on June 25); however, in the The highest amounts of chlorpyrifos residues, compared case of leaves, we observed a complete reversal of the pro- with its residue in the soil, were detected on July 2 in fully portion, excluding samples from July 15 (the proportion developed leaves (TF = 600%), whose surfaces and weights pyraclostrobin to boscalid 1:1.85), in plants for a long period did not change, which could suggest that this compound was (69, 62, and 41 days after application, respectively). In the still actively transported from the ground by the plant root remaining sampling days, a larger residue of pyraclostrobin system. In flowers, these residues were observed on May 27 than boscalid (to 1:0.37 July 2 and July 8) was noted. The (TF = 150%). reversal of those proportions in the case of leaves may result Iprodione, the fungicide with a contact mode of action, from the increased transpiration of boscalid by the leaves was found in worker bees (up to 0.63 mg/kg found in the compared with that of pyraclostrobin. Such a phenomenon sample on June 25), but the residue concentrations in the was observed in the raspberry crop, for the pesticide residues leaves, flowers, and fruits did not exceed the LOQ. An expla- from the surface of leaves, flowers, and fruits (material in nation of this effect may be the temporal bioaccumulation preparation) when analysed exclusively. of this substance in animal tissues or foraging by bees on Our surveys confirmed the possibility of transferring other crops. However, in a 2-km area around the crop, no measurable (i.e., above the LOQ) amounts of some PPP other nectar-secreting blossoming plants were present, and AIs from the sprayed dessert raspberry bushes to the bee- such a large pesticide residue concentration would point to hives. Most often, residues were found in worker bees; for the transfer of large amounts of pesticides from other crops, example, chlorpyrifos was found in samples from eight of where the preparation was applied between June 4 and June total nine sampling dates (from May 20 to July 8), iprodi- 10, when it was first observed in bee samples. It seems to be one from five of six sampling dates (from June 10 to July more likely, as it was mentioned above, that the preparation 8), boscalid from five of six dates (from June 10 to July 8), was used during daily period of the bee foraging, shortly difenoconazole from three of nine dates (on June 17, July 2 before intense rainfall, which could remove the preparation and 8), and pyrimethanil from two of nine sampling dates from plants and top layer of the soil. However, it is worth (on May 20 and 27). Residues were found less frequently in mentioning that close to the studied plantation, there were the honey and brood and were found in only samples on one small home cultivations of plants that did not secrete nectar, of eight sampling dates. Chlorpyrifos was found in broods so they were unlikely to be attractive to bees (Lycopersicon from four of nine sampling dates (Tables 1, 3). The larger Mill. or Cucumis L.); however, they were protected using content of the pesticides in the worker bee bodies is a result preparations containing iprodione and constituted a place of direct contact of the bee worker foragers with the sprayed where the bees could stop, for example, to get water. plants, as well as their direct contact with PPPs, because the Despite the fact that various concentrations of PPP AI treatments were performed during the daily period of bee residues were found in the samples, it was not easy to active foraging. estimate the significance of the differences between the Preparations with deep-seated and systemic action on concentrations. When the sampling conditions were not plants more frequently reached the hives than those with considered, a positive correlation between the presence of contact action. Chlorpyrifos, an organophosphorus insec- boscalid, pyrimethanil and chlorpyrifos in the crop and in ticide and the AI of Dursban 480 EC, which was included the hive (Table 4) was found, which meant that an increase in our study for the control of May bug larvae (Melolon- in the residues of AIs of PPPs in the beehive was linked to tha melolontha), is an example of the abovementioned their higher concentrations in the crop. A similar relation- rule. Unlike the other PPPs for which a foliar application ship was observed when the sampling dates were consid- was used, Dursban 480 EC was applied to the soil only via ered (Table 5), although for chlorpyrifos (sampled on May the irrigation system, and it had no direct contact with the 27), iprodione (sampled on June 1), boscalid (sampled on aboveground parts of plants, on which foraging bees might June 10), and pyrimethanil (sampled on May 27), negative 1 3 56 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 correlations also were found, indicating that the increases if, as suggested Kessler et al. (2015), honeybees prefer a diet in the AI residues in the crops were related to lower con- containing neonicotinoids. centrations in the worker bee tissues. A probable reason Bees in the crop also were exposed to six AIs of fungi- for this phenomenon could have been the relatively intense cides (i.e., azoxystrobin, difenoconazole, pyrimethanil, ipro- and long rainfall that limited foraging intensity (personal dione, boscalid, and pyraclostrobin), from which iprodione, communication from the crop owner). boscalid, difenoconazole, and pyrimethanil were detected in Our studies were limited to 2 months. During this time worker bee bodies and boscalid was detected in honey. Addi- period, no clear-cut declines in the strength of the tested tionally, pesticide adjuvants, which increased the probabil- honeybee colonies were observed. However, as previously ity of adverse interactions (Mullin et al. 2015) were found. mentioned, some AIs might be toxic to honeybees even Furthermore, because of the possibility of AI accumulation at sublethal doses (Weick and Thorn 2002; Williamson in the wax (Serra-Bonvehí and Orantes-Bermejo 2010), the and Wright 2013). As Leonardi et al. (1996) discusses, adverse effects of those substances might extend far beyond the AIs of pesticides even at a level lower than the LOQ the raspberry flowering period. can affect insects. They can act synergistically with each Even though chlorpyrifos, iprodione, boscalid, difenocon- other (Thompson 1996; Thompson and Wilkins 2003, Gla- azole, and pyrimethanil were found in the bees and chlor- van and Božič 2013), e.g., via competition for metabolic pyrifos was found in the brood, a small amount of boscalid enzymes (Johnson et  al. 2009) or cellular efflux (Haw - (0.01 mg/kg of honey) was detected in the honey on one thorne and Dively 2011) and with other environmental sampling date, i.e., in samples collected on June 10. The stressors (Renzi et al. 2016; Doublet et al. 2015). Thomp- amount of boscalid was so small that it did not exceed the son (1996) suggested that even PPPs considered safe for MRL of 0.05 mg/kg (EU Pesticides Database 2017). The bees could intensify their activity against those insects by results indicate that honey from the beehive adjacent to the two orders of magnitude when used in combination with raspberry plantation, protected against pests and diseases, other PPPs. was a completely safe product in terms of the presence of The honeybees in the present study were exposed to the nine studied plant protection products. four insecticides: paraffin oil, acetamiprid, chlorpyrifos, Our surveys confirmed the possibility of transferring and cypermethrin. Paraffin oil physically disturbs the gas measurable amounts of some PPP AIs from the sprayed exchange process in pests (Card of Characteristics the prep- dessert raspberry bushes to the beehives. Five of the nine aration Treol 770 EC). Acetamiprid, being the agonist of applied were detected in worker bee bodies. The honeybee nicotine acetylcholine receptors in the synapse, influences brood was polluted by small amounts of chlorpyrifos applied survival, including impairment of learning and memory, to only the soil through the irrigation system. Only trace disruption of the navigation, and reduction of the honeybee amounts of boscalid residues were detected in honey, which foraging activity (Belzunces et al. 2012; Blacquiere et al. indicated that it was completely safe for consumption. The 2012; Henry et al. 2012). Acetamiprid significantly impairs obtained results confirm occurrence of the phenomenon of olfactory learning in laboratory-based studies (Decourtye active transferring the active ingredients of plant protection et  al. 2004; Han et al. 2010). The next AI, chlorpyrifos, products (PPP AIs) by the honeybees from the crops to bee blocks the active sites of acetylcholinesterase in the synapse hives. space by phosphorylation, and as a consequence, it inten- Acknowledgements The authors are thankful to Prof. Stanisław Sadło, sifies the action of acetylcholine, which is distributed by Mr. Kazimierz Czepiela, Mr. Waldemar Mitrut, Mrs. Liliana Pastuła, chlorpyrifos, and influences honeybee learning and memory Mrs. Katarzyna Stats, and Mrs. Natalia Kopeć for their assistance and abilities in sublethal doses (Guez et al. 2010). Finally, cyper- help during the performance of these studies. methrin causes elongation of sodium channel opening states Open Access This article is distributed under the terms of the Creative in insect nervous cells (Wang and Wang 2003). It also is Commons Attribution 4.0 International License (http://creativecom- an agonist of the T-type calcium channel in insect muscles mons.org/licenses/by/4.0/), which permits unrestricted use, distribu- and is involved in the excretion of acetylcholine and dopa- tion, and reproduction in any medium, provided you give appropriate mine in the synaptic space (Aldridge 1990). Moreover, it credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. inhibits the mitochondrial complex I (Gassner et al. 1997), disrupts the protein phosphorylation process, and modifies the function of the gap junctional protein. Among the above- mentioned insecticides, the presence of chlorpyriphos was References detected in the worker bee bodies and brood. Unfortunately, Aldridge WN (1990) An assessment of the toxicological properties of the presence of acetamiprid (AI of Mospilan 20 SP, neoni- pyrethroids and their neurotoxicity. 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Transfer of the Active Ingredients of Some Plant Protection Products from Raspberry Plants to Beehives

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Environment; Ecotoxicology; Pollution, general; Environmental Health; Environmental Chemistry; Soil Science & Conservation; Monitoring/Environmental Analysis
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Abstract

Plant protection products (PPPs) have been found increasingly in the environment. They pose a huge threat to bees, con- tributing 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. The honeybee (Apis mellifera F.) is an insect species of sig- million. In general, the profit earned through pollination by nificant importance to the biosphere and the economy (Free bees is approximately 25–30% of the total yield of the crop 1993; Delaplane and Mayer 2000). This pollinator influences (Sanjerehei 2014; Giannini et al. 2015). According to San- the yields of approximately 70% of cultivated plants, which jerehei (2014), this value is 54 times higher than the value represents approximately 35% of the total global food pro- of honey produced by bees. A. mellifera is the main pollina- duction (Klein et al. 2007), which, in turn, yields $150 bil- tor that generates 86.8% of the gains generated by all the lion per year. In Brazil, the value of the work performed by pollinators. The use of plant protection products (PPPs) on all pollinators is estimated at nearly $ 12 billion (Giannini nectar-secreting plants goes hand in hand with the problem et al. 2015). In Great Britain, for Gala apples, the value of of exposing pollinators to such substances (Piechowicz et al. bees as pollinators is estimated at £5.7 million a year (Gar- 2018a, b). ratt et al. 2014). Majewski (2014) showed that a decrease PPPs may enter hives due to foraging by worker bees in the number of the honeybee colonies in Poland caused (Balayiannis and Balayiannis 2008; Mao et al. 2013; McMe- a decline in total crops valued at approximately €728.5 namin and Genersch 2015), because their active ingredients (AIs), especially those that have a contact activity, are pre- sent in crops, and consequently, they can be collected from * Przemysław Grodzicki flowers and leaves and then transferred to the hive. In turn, grodzick@umk.pl AIs that have deep-seated and systemic activity can be col- Department of Analytical Chemistry, Institute lected by bees together with pollen and nectar. of Biotechnology, University of Rzeszów, Werynia, Poland The AIs of PPPs, transferred by the worker bees to Laboratory of Pesticide Residues, Institute of Plant the hives, may result in miscellaneous, distinct effects. Protection, National Research Institute, Rzeszów, Poland Łozowicka (2013) investigated cases of honeybee colony Faculty of Mathematics and Natural Sciences, University intoxication. A presence of cypermethrin (pyrethroid insec- of Rzeszów, Rzeszów, Poland ticide, detected in 51% samples), chlorpyrifos (organophos- Department of Animal Physiology, Faculty of Biology phorus insecticide, detected in 27% samples), and bifenthrin and Environmental Protection, Nicolaus Copernicus (pyrethroid insecticide, detected in 21% samples) was found University, Toruń, Poland Vol.:(0123456789) 1 3 46 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 in 33 worker bee samples. Likewise, in intoxicated worker Materials and Methods bee bodies, Walorczyk and Gnusowski (2009) found an occurrence of tebuconazole (triazole fungicide, detected in Field Trial 48% samples), omethoate (oxygenated form of dimethoate, organophosphorus insecticide, detected in 44% samples), The field trial was performed from May 20 to July 15, and fipronil (phenylpyrazole insecticide, detected in 40% 2014, on a raspberry (Rubus idaeus), Laszka variety, plan- samples). In turn, among 19 of the detected compounds, tation in the village Grabówka Kolonia, in the province of Barganska et al. (2014) most frequently found heptenophos Lublin, which is protected from pests using conventional (organophosphorus insecticide, detected in 68% samples), methods, in accordance with current programmes. All bifenthrin (pyrethroid insecticide, detected in 53% samples), preparations were applied according to the labels posted. and pyrazophos and diazinon (organophosphorus insecti- A sprayer, model RA 10/80 (Lochmann, Vilpiano, Italy) cide, detected in 32% samples). However, cases of acute with nozzles ALBUZ ATR 80, was used. Within 2 km of honeybee poisoning by the PPPs may be only marginal. In the studied raspberry plantation, there were no plantations most of the cases, the presence of pesticides in the hive is at of any other blossoming plants secreting nectar that could such a low level that it does not affect the honeybee colony have interfered with the test results. The honeybee colo- well-being. Pohorecka et al. (2017) suggest that the phenom- nies were transported from an area where the bees had no enon of winter colony collapse could be caused by honey- contact with pesticides. On May 17, 2014, the colonies bee parasites. Studies of Piechowicz et al. (2018a, b) on the were placed approximately 3 m from the raspberry planta- transfer of plant protection products from oilseed rape crops tion on an area of 4 ha. and orchards to beehives showed a presence of pesticides On the raspberry plantation, four rows of plants were cho- both in bee bodies (5/7 detected compounds at rape planta- sen for the study, each approximately 150 m long. On each tion 1; 3/5 at rape plantation 2; and 5/6 AIs in orchards) and sampling date, from each of the four selected rows, a sample in honeybee brood (4 and 2 AIs in hives located near rape of 16 leaves from randomly selected plants was taken (only crops and 6 AIs in bees in the orchard), and in honey (3 and fully developed leaves were collected from the outside of 3 AIs in rape honey and 4 AIs in apple-pear honey). In the the bush), and then analytical portions, which consisted of studied cases, when the worker bees were directly exposed 16 disks 1 cm in diameter, were cut. On the same sampling to pesticides originating from the crops, no deterioration in day, samples of flowers and fruits, consisting of 8 and 16 honeybee colony well-being was observed. It does not mean pieces, respectively, were collected from the same randomly that PPP AIs, especially in the case of their simultaneous selected plants. presence in the hive, could not have affected the bees. Some During the e fi ld trial, from each of the four hives, one lab - investigators indicate that for bees endowed with only 46 oratory sample of worker bees (retrieved from the frames), genes responsible for the detoxification system functioning the brood (from non-sealed cells, 4–6 days before hatch- (Claudianos et al. 2006), which additionally have few genes ing), and honey (from non-sealed cells) also were collected. controlling detoxification of the plant protection products Each sample weighed at least 5 g. Additionally, every week, (The honeybee genome sequencing consortium 2006), a syn- soil samples were collected using an Egner stick, with one ergistic action of small, sublethal residues of two or more sample from each of the four rows. Each sample consisted AIs (Thompson 1996; Thompson and Wilkins 2003; Mullin of eight portions taken from randomly selected places in the et al. 2010; Glavan and Božič 2013; Johnson et al. 2013) row at a distance no further than 30 cm from the raspberry can be dangerous for them. Even if these compounds are plants. not toxic to bees, this does not mean that they are not harm- ful to the brood (Zhu et al. 2014). This effect is especially relevant to intensively protected crops in which the flowering Chemicals and Pesticides and fruiting periods occur at the same time, so both plants and fruits need protection. Raspberries are one such crop. During the period from January 7 to June 9, 2014, 12 protec- tive treatments were performed on the plantation. The terms, Our study was an initial analysis of whether some AIs of PPPs may be carried by bees from raspberry plants and preparations and applied doses are shown in Table 1. transferred to beehives located in the immediate vicinity of the crop and to what extent these AIs were transferred. Extraction of Pesticide Residues from the Honey, Worker Bees, and Brood for Analysis The samples of the worker bees and the brood were lyophi- lized using a Labconco Freezone 2.5 freeze dryer (Labconco, 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 47 1 3 Table 1 Pesticide spraying program performed on the raspberry plantation Date of treatment PPP, trade name AI, common name Chemical clas- AI structure AI content Mode of applica- Mechanism of Dose of MRL, (IUPAC name) sification tion action PPP (L, kg/ honey (mg/ ha) kg) January 7 Kerb 50 WP (H)* Propyzamide Benzonitrile 500 g/kg (50%) Between rows Systemic 2.00 0.05 (3,5-dichloro-N-(1,1- dimethylprop-2-ynyl) benzamide) March 23 Treol 770 EC (I) Paraffin oil – – 770 g/L Foliar By contact— 20.00 0.01 plants; By contact— insects May 7 Amistar 250 SC Azoxystrobin Strobilurin 250 g/L (22.81%) Foliar Systemic 0.50 0.05 (F) (methyl (E)-2-{2-[6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl}-3-methoxy- acrylate) May 7 Score 250 EC (F) Difenoconazole Triazole 250 g/L (23.58%) Foliar Systemic 0.50 0.05 (1-[2-[2-chloro- 4-(4-chloro- phenoxy)-phenyl]-4 methyl[1,3]dioxolan- 2-ylmethyl]-1H-1,2,4- triazole) May 14 Mythos 300 SC (F) Pyrimethanil Anilinopyrimidine 300 g/L (28.3%) Foliar By contact and 2.50 0.05 (4,6-dimethyl-N-phe- deep-seated nylpyrimidin-2-amine) May 14 Dursban 480 Chlorpyrifos Organophosphate 460 g/L (44.86%) To the soil through By contact and 2.00 0.05 EC*(I) (O,O-diethyl O-3,5,6- the irrigation deep-seated— trichloropyridin-2-yl system plants; phosphorothioate) By ingestion— insects May 16 Mospilan 20 SP (I) Acetamiprid Neonicotinoids 200 g/kg (20%) Foliar Systemic—plants; 0.20 0.05 (N-[(6-chloro-3-pyridyl) by ingestion— methyl]-N’-cyano- insects N-methyl-acetamidine) May 19/May 29 Cyperkil Super Cypermethrin Pyrethroids 250 g/L (25.92%) Foliar By contact— 0.15 0.05 250 EC (I) ([cyano-(3-phenoxy- plants; phenyl)methyl]3-(2,2- By contact, 0.15 0.05 dichloroethenyl)- by ingestion— 2,2-dimethylcyclopro- insects pane-1-carboxylate) 48 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 1 3 Table 1 (continued) Date of treatment PPP, trade name AI, common name Chemical clas- AI structure AI content Mode of applica- Mechanism of Dose of MRL, (IUPAC name) sification tion action PPP (L, kg/ honey (mg/ ha) kg) June 4 Rovral Aquaflo Iprodione Dicarboximide 500 g/L (42.91%) Foliar By contact 2.00 0.05 500 SC (F) (3-(3,5-dichlorophenyl)- N-isopropyl-2,4-diox- oimidazolidine-1-car- boxamide) June 5 Bellis 38 WG (F) Boscalid Anilide 252 g/kg (25.2%) Foliar Systemic 1.50 0.50 (2-chlor-N-(4′- chlorbiphenyl-2-yl) nicotinamid) Pyraclostrobin Strobilurin 128 g/kg (12.8%) 0.05 (methyl-N-(2-[1- (4-chlorphenyl)- 1H-pyrazol-3-yl] oxymethylphenyl)-(N- methoxy)carbamat) June 9 Signum 33 WG (F) Boscalid Anilide 267 g/kg (26.7%) Foliar Systemic 1.80 0.50 (2-chlor-N-(4′- chlorbiphenyl-2-yl) nicotinamid) Pyraclostrobin Strobilurin 67 g/kg (6.7%) 0.05 (methyl-N-(2-[1- (4-chlorphenyl)- 1H-pyrazol-3-yl] oxymethylphenyl)-(N- methoxy)carbamat) *H herbicide, I insecticide, F fungicide Archives of Environmental Contamination and Toxicology (2018) 75:45–58 49 USA) (pressure: 0.024  mbar; temperature: 50  °C, time: Poland) and stirred carefully. Analytical portions of 20 g 168 h). were taken from the samples and shaken for 1  h with a Analytical portions of 5 g of the lyophilized animals or 50-mL mixture of dichloromethane:acetone (9:1; v/v) on honey were shaken with 10 mL of acetonitrile (Chempur, a GFL 3006 shaker (GFL, Germany). The extracts were Poland). Then, a mixture of salts containing 4 g of anhy- allowed to stand for 10 min and then decanted through a drous magnesium sulfate (VI) (Chempur, Poland), 1 g of layer of anhydrous sodium sulfate (VI) that had been placed sodium chloride (Chempur, Poland), 1 g of trisodium citrate in the funnel. The soil samples were washed twice with (Chempur, Poland), and 0.5 g of sesquihydrate disodium 20 mL of dichloromethane, and the combined extracts were hydrogen citrate (Chempur, Poland) was added. The con- evaporated to dryness on a Heidolph Laborota 4000 Effi- tents were shaken for 2 min and centrifuged for 5 min at cient rotary evaporator. The residues were then dissolved in 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase 10 mL of petroleum ether. The resulting extracts were puri- was transferred to a polypropylene test tube that contained fied using a Florisil mini-column (Sadło et al. 2014, 2015), 150 mg of PSA (primary secondary amine) (Agilent, USA) and the residues were eluted with 70 mL of 3:7 (v/v) diethyl and 900 mg of anhydrous sodium sulfate(VI) (Chempur, ether:petroleum ether, followed by elution with 70 mL of 3:7 Poland). The extract was vigorously shaken for 2 min and (v/v) acetone:petroleum ether. The combined eluates were centrifuged for 5 min as described above. Four millilitres evaporated to dryness, and the residues were quantitatively of the obtained extract was taken and transferred to a glass transferred using petroleum ether into a 10-mL volumetric tube, evaporated to dryness on a Heidolph Efficient Labware measuring flask. 4000 rotary evaporator (Heidolph, Germany), and then dis- solved in 4 mL of petroleum ether (Chempur, Poland). Chromatographic Determination of Pesticide Residues Extraction of Pesticide Residues from the Leaves, Flowers and Fruits for Analysis The extracts were analysed using an Agilent 7890 (Agi- lent, USA) gas chromatograph equipped with a micro-cell The analytical portions of the leaves (16 disks, 1 cm in diam- electron capture detector (µECD) and a nitrogen–phospho- eter each) and flowers (8 pieces), both with the addition of rus detector (NPD). The chromatograph was controlled by 100 mL of water, and fruits (16 pieces) were homogenized ChemStation software (Agilent, USA). It also was equipped in a Waring Commercial 8010 EG blender (Waring, USA) with an autosampler and an HP-5MS, 30-m  ×  0.32- with 150  mL of acetone (Chempur, Poland) and filtered mm × 0.25-µm column. The instrumental analysis conditions through a Büchner funnel under vacuum. The blender jar were as follows: an NPD detector temperature of 300 °C, a was flushed with 50 mL of acetone, and the washings were μECD detector temperature of 290 °C, and an injector tem- used to wash the filter cake. One-fifth of the filtrate (the perature of 250 °C. The oven temperature was programmed equivalent of approximately 15.4 g of fruit and approxi- as follows: 100  °C at 0  min →  10  °C per min →  4  min mately 0.1  g of leaves) was used for further analysis. It at 180 °C → 3 °C per min → 15 min at 220 °C → 10 °C was placed in a separatory funnel together with 100 mL per min → 11 min at 260 °C. The total analysis time was of 2.5% sodium sulfate (VI) (Chempur, Poland) solution. 55.3 min, and the injection volume was 2 µL. The pesticide residues were extracted three times with 20, 10, and 10 mL of dichloromethane (Chempur, Poland). The Statistical Analysis of the Results combined extracts were evaporated to dryness, dissolved in 10 mL of petroleum ether and purified using a Florisil Recovery studies were performed by spiking each matrix (Chempur, Poland) mini-column (Sadło et al. 2014, 2015). with the substances used in field trials at a single concentra- The pesticide residues were eluted with a 70-mL mixture of tion (Table 2). The pesticide residues (R ) in the samples 3:7 (v/v) ethyl ether:petroleum ether (Chempur, Poland) as were recalculated (R ) using the results of the recovery rec well as with a 70-mL mixture of 3:7 (v/v) acetone:petroleum study (Rec in %; Table 3) according to Eq. 1. ether. The solvents were evaporated to dryness, and the resi- R = 100 × R ∕Rec due was transferred quantitatively using petroleum ether into (1) rec i a 10-mL volumetric measuring flask. The transfer factor (TF) from the soil to the plants was calculated according to Eq. 2. Extraction of Pesticide Residues in the Soil TF = 100 × R ∕R i (plant) i (soil) (2) for Analysis To determine the similarity in the residue concentrations The soil laboratory samples were air dried and pulver- of the individual substances between different sample types, ized with a Testchem LMG grinder (Testchem Sp. z.o.o., a cluster analysis was performed. The Euclidean metric was 1 3 50 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 2 Recoveries (expressed in %) of AIs of PPPs applied on the raspberry plantation Sample Propyzamide Chlorpyrifos Iprodione Pyraclos- Boscalid Cypermethrin Difenocona- Azoxystrobin Pyrimethanil trobin zole Worker bees 96.3 95.7 92.5 95.0 90.1 108.3 105.2 86.7 101.8 Brood 95.6 89.9 111.2 90.5 95.0 110.4 120.0 111.7 107.3 Soil 90.0 88.9 86.8 108.5 100.8 114.3 110.4 86.7 87.4 Honey 100.1 88.4 76.9 76.0 63.4 87.0 75.6 61.4 93.2 Flowers 94.2 92.4 98.5 117.4 89.0 85.7 93.2 82.2 93.2 Leaves 98.7 92.1 101.3 112.9 91.6 86.1 85.7 82.5 94.3 Fruits 92.0 91.9 88.2 118.1 85.1 85.7 85.3 81.6 93.7 used to describe the similarities. The Ward method was used ha. The residues of those substances were found in only the as the agglomeration algorithm. The Friedman test was used samples collected on May 27 (13 and 20 days since applica- to determine whether the residue concentrations in the sam- tion) at concentrations of 0.09 and 0.14 mg/kg of the flowers, ple types varied significantly. The Spearman’s rank correla- respectively. The TF from the soil to flowers for chlorpyrifos tion coefficient was used to assess the strength, direction was 150%, which indicated that a distinct part of that sub- and statistical significance of dependencies between the stance penetrated the flowers from the soil with the transpi- residue concentrations in the various sample types, assum- ration current. On only two sampling days, in the samples ing α < 0.05 as statistically significant. collected on May 27 and June 4, difenoconazole residues (triazole fungicide with a systemic mode of action) were found at a relatively high level (1.03 and 0.84 mg/kg). This Results substance was applied on May 7 in the form of Score 250 EC (0.5 L/ha). Pyrimethanil, which was applied in the form In general, pesticide residue recoveries should be in the of Mythos 300 SC at an application rate of 2.5 L/ha on May range of 70–120% of the substance introduced into the 14, occurred at the highest level. The initial residue of this sample, and the repeatability should be ≤ 20% (Document compound, with slight fluctuations in the levels, decreased SANTE 2015). In our study, satisfactory values of both of in concentration until the last sampling day. Boscalid (ani- these parameters were obtained for nine AIs of PPPs in lide fungicide) and pyraclostrobin (strobilurin fungicide) seven sample types. However, for boscalid and azoxystrobin, residues, after the application of Bellis 38 WG (1.5 kg/ha) the recovery from honey did not exceed 70%; the recoveries on June 5 and Signum 33 WG (1.8 kg/ha) on June 9 (both were 63.4 and 61.4%, respectively (Table 2). The limit of preparations with a systemic mode of action in plants), were quantification (LOQ) of propyzamide, chlorpyrifos, iprodi- found on four sampling days, and their values gradually one, pyraclostrobin, boscalid, cypermethrin, difenoconazole, decreased, excluding the samples collected on June 2, when azoxystrobin, and pyrimethanil in all studied matrices was none of the two AIs was found, from 7.2 to 0.36 mg/kg in the 0.01 mg/kg. flower samples collected on June 10 to 1.08 and 0.08 mg/kg in the flower samples from July 8. Cypermethrin (pyrethroid Pesticide Residues in Raspberry Plantation insecticide with a contact mode of action) was applied on May 19 and May 29 in the form of Cyperkill Super 250 EC, Table 3 shows the average concentrations of pesticide resi- in both cases at an application rate of 0.15 L/ha. Its residues dues in the seven sample types. were observed in the flower samples collected on all sam- pling days, while after the first application (samples from Flowers May 20), its residues were 0.33 mg/kg, and after the second application, they were 0.51 and 0.57 mg/kg, respectively. Seven of the nine studied AIs were found in the flowers. The From that time until June 25, the concentrations decreased to residues of chlorpyrifos (an organophosphorus insecticide 0.18 mg/kg and then increased to the 0.63 mg/kg on July 2. with a deep-seated mode of action in plants) were applied to the soil by the irrigation system on May 14 in the form of Leaves Dursban 480 EC at an application rate of 2 L/ha, and azox- ystrobin (a strobilurin fungicide with a systemic mode of In the laboratory leaf samples, chlorpyrifos, pyraclostrobin, action in plants) was applied by foliar spraying on May 7 in boscalid, cypermethrin, difenoconazole, azoxystrobin, the form of Amistar 250 SC at an application rate of 0.5 L/ and pyrimethanil were found. Chlorpyrifos residues were 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 51 Table 3 The average residues ± standard deviations (in mg/kg) of AIs applied on the raspberry plantation Sampling Propyza- Chlorpy- Iprodione Pyraclos- Boscalid Cyperme- Difenocona- Azox- Pyrimethanil date mide rifos trobin thrin zole ystrobin Flowers May 20 < LOQ < LOQ < LOQ < LOQ < LOQ 0.33 ± 0.21 < LOQ < LOQ 24.50 ± 6.60 May 27 < LOQ 0.09 ± 0.04 < LOQ < LOQ < LOQ 0.24 ± 0.15 1.03 ± 0.15 0.14 ± 0.05 3.41 ± 1.02 June 4 < LOQ < LOQ < LOQ < LOQ < LOQ 0.51 ± 0.51 0.84 ± 0.54 < LOQ 1.72 ± 0.68 June 10 < LOQ < LOQ < LOQ 0.36 ± 0.27 7.20 ± 3.84 0.57 ± 0.09 < LOQ < LOQ 1.75 ± 0.76 June 17 < LOQ < LOQ < LOQ 0.30 ± 0.11 6.34 ± 2.40 0.41 ± 0.03 < LOQ < LOQ 0.03 ± 0.03 June 25 < LOQ < LOQ < LOQ 0.04 ± 0.04 1.94 ± 0.77 0.18 ± 0.11 < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ 0.63 ± 0.11 < LOQ < LOQ 0.10 ± 0.03 July 8 < LOQ < LOQ < LOQ 0.08 ± 0.16 1.08 ± 2.16 0.51 ± 0.44 < LOQ < LOQ 0.06 ± 0.07 Leaves May 20 < LOQ 0.13 ± 0.15 < LOQ < LOQ < LOQ 0.90 ± 1.27 < LOQ < LOQ 206.28 ± 61.02 May 27 < LOQ 0.11 ± 0.22 < LOQ < LOQ < LOQ 0.11 ± 0.13 31.98 ± 8.09 5.61 ± 0.91 30.29 ± 17.85 June 4 < LOQ < LOQ < LOQ < LOQ < LOQ 2.80 ± 1.11 9.71 ± 2.40 1.39 ± 0.90 34.05 ± 25.18 June 10 < LOQ < LOQ < LOQ 12.30 ± 4.49 7.40 ± 1.60 1.54 ± 0.32 4.92 ± 0.48 < LOQ 2.50 ± 0.42 June 17 < LOQ < LOQ < LOQ 19.59 ± 7.52 7.61 ± 3.62 0.96 ± 0.44 7.20 ± 3.72 0.67 ± 0.67 3.25 ± 1.91 June 25 < LOQ < LOQ < LOQ 11.70 ± 4.34 5.51 ± 1.96 1.33 ± 0.33 4.17 ± 1.57 0.44 ± 0.44 5.44 ± 3.58 July 2 < LOQ 0.06 ± 0.06 < LOQ 6.49 ± 2.39 4.41 ± 1.40 1.20 ± 0.40 3.78 ± 0.53 < LOQ 0.27 ± 0.27 July 8 < LOQ < LOQ < LOQ 2.84 ± 1.26 2.23 ± 0.82 0.47 ± 0.10 1.54 ± 0.92 < LOQ < LOQ July 15 < LOQ < LOQ < LOQ 0.60 ± 0.35 1.11 ± 0.42 0.34 ± 0.09 2.09 ± 1.25 < LOQ < LOQ Fruits June 17 < LOQ < LOQ < LOQ 0.04 ± 0.01 1.34 ± 0.29 0.27 ± 0.07 0.02 ± 0.00 0.01 ± 0.00 0.16 ± 0.05 June 25 < LOQ < LOQ < LOQ 0.03 ± 0.00 0.84 ± 0.18 0.14 ± 0.05 0.01 ± 0.00 < LOQ 0.04 ± 0.00 July 2 < LOQ < LOQ < LOQ < LOQ 0.41 ± 0.08 0.07 ± 0.02 0.01 ± 0.00 < LOQ 0.02 ± 0.00 July 8 < LOQ < LOQ < LOQ < LOQ 0.23 ± 0.06 0.02 ± 0.00 0.01 ± 0.00 < LOQ 0.01 ± 0.00 July 15 < LOQ < LOQ < LOQ < LOQ 0.05 ± 0.00 0.02 ± 0.00 0.01 ± .0.00. < LOQ 0.01 ± 0.00 Soil May 20 0.16 ± 0.08 0.08 ± 0.08 < LOQ < LOQ < LOQ. < LOQ < LOQ < LOQ < LOQ May 27 0.10 ± 0.05 0.06 ± 0.05 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 0.07 ± 0.03 0.04 ± 0.03 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 0.04 ± 0.02 0.03 ± 0.02 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 17 0.03 ± 0.01 0.02 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 0.02 ± 0.01 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 0.01 ± 0.01 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 0.01 ± 0.00 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 0.01 ± 0.00 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Honey May 20* < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ May 27 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 < LOQ < LOQ < LOQ < LOQ 0.01 ± 0.00 < LOQ < LOQ < LOQ < LOQ June 17 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Worker honeybees May 20 < LOQ 0.03 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 0.09 ± 0.03 May 27 < LOQ 0.03 ± 0.00 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 0.03 ± 0.01 June 4 < LOQ 0.05 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 1 3 52 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 3 (continued) Sampling Propyza- Chlorpy- Iprodione Pyraclos- Boscalid Cyperme- Difenocona- Azox- Pyrimethanil date mide rifos trobin thrin zole ystrobin June 10 < LOQ 0.04 ± 0.01 0.09 ± 0.09 < LOQ 0.08 ± 0.05 < LOQ < LOQ < LOQ < LOQ June 17 < LOQ 0.01 ± 0.04 0.11 ± 0.13 < LOQ 0.06 ± 0.10 < LOQ 0.02 ± 0.07 < LOQ < LOQ June 25 < LOQ 0.05 ± 0.00 0.63 ± 0.16 < LOQ 0.11 ± 0.05 < LOQ < LOQ < LOQ < LOQ July 2 < LOQ 0.02 ± 0.01 0.01 ± 0.01 < LOQ 0.01 ± 0.01 < LOQ 0.12 ± 0.03 < LOQ < LOQ July 8 < LOQ 0.02 ± 0.01 0.02 ± 0.02 < LOQ 0.01 ± 0.01 < LOQ 0.09 ± 0.03 < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Worker brood May 20 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ May 27 < LOQ 0.02 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 4 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 10 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 17 < LOQ 0.01 ± 0.01 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ June 25 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 2 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 8 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ July 15 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ *In worker honeybees and the brood as well in honey samples collected on May 16 (a day before transporting hives to the crop), none of the determined PPP AIs were shown detected on May 20 and May 27 (0.13 and 0.11  mg/kg, decreased to 0.27 mg/kg in the samples from July 2 and to an respectively) and on July 2 (0.06 mg/kg). In this case, the amount < LOQ during the subsequent leaf sampling days. TF was 162.5, 183.3, and 600%, respectively. Pyraclostrobin and boscalid residues were found in samples collected on six Fruits sampling days, and their highest values occurred on June 17 (19.59 and 7.61 mg/kg, respectively). After that, since the The harvest period for fruits began on June 17, and pyr- last sampling day, a systematic decrease in their level was aclostrobin, boscalid, cypermethrin, difenoconazole, observed. It is worth mentioning that the residues of pyra- pyrimethanil, and azoxystrobin were detected at that time. clostrobin were significantly higher than those of boscalid, The pyraclostrobin residues were found only at a level 3–4 which was in contradiction to proportions of those AIs in times higher than the limit of quantification (LOQ) on June the applied preparations (1:1.97 in the case of Bellis 38 WG 17 and June 25 (0.04 and 0.03 mg/kg, respectively), while and 1:3.99 in the case of Signum 33 WG). The cypermethrin boscalid, applied together with pyraclostrobin, in all sam- residues were detected in all the samples, while after the r fi st ples, ranged from 1.34 mg/kg on June 17 to 0.05 mg/kg on application of Cyperkill Super 250 EC, they were 0.9 mg/kg July 15. The cypermethrin residues were found in all sam- (samples collected on May 20), and after the second appli- ples of fruit, and its residue decreased from 0.27 mg/kg on cation, they were 2.8 mg/kg (samples collected on June 4). June 17 to 0.02 mg/kg on the last sampling day. Similarly, From that time until the end of the investigation, a decrease difenoconazole and pyrimethanil were found in fruit sam- in the cypermethrin residue level was observed. Likewise, in ples on each sampling date, while in the case of difenocona- all samples, excluding those collected on May 20, the pres- zole, its residues occurred at the level closest to the LOQ ence of the applied difenoconazole was found; its residues (0.02 mg/kg on June 17 and 0.01 mg/kg on other dates), appeared to be high (31.98 mg/kg) on May 27; and after that and the pyrimethanil residue level decreased with time from day, they decreased until the last sampling day (2.09 mg/ 0.16 mg/kg on June 17 to 0.01 mg/kg on June 15. Trace kg). Azoxystrobin was detected in samples collected on azoxystrobin residues were found only in samples collected four application days. Its highest residue level was found in on June 17 (0.01 mg/kg). leaves collected on May 27 (5.61 mg/kg), and in subsequent application dates, this value decreased to 0.44 mg/kg in the Soil samples collected on June 25, while the residues of this AI were not found in the samples from June 10. After foliar Only two AIs of PPPs applied were detected in the 10-cm application on May 14, pyrimethanil left significant resi - soil layer. In all studied samples, the residue of chlorpyrifos dues on leaves (206 mg/kg in the samples from May 20) that decreased systematically from 0.08 mg/kg of soil on May 20 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 53 to 0.01 mg/kg in the samples collected from June 25 to July associated distance matrix analysis, differences in the resi- 15, and propyzamide, with an AI of Kerb 50 WP, belonging due concentrations of the individual substances could be to benzonitrile group. The herbicide was applied between seen in the various sample types. However, when using the rows on January 7 at an application rate of 2 L/ha, and its Friedman test, statistically significant differences between residues decreased systematically from 0.16 mg/kg on the the residue concentrations of specific substances were not first sampling day to 0.01 mg/kg in the samples collected demonstrated. during the period July 2 to July 15. Spearman’s rank correlation coefficient was used to deter - mine whether there was a mutual relationship between the Pesticide Residues in Samples of Honey, Worker residue concentrations in the samples analysed, regardless Bees and Brood of the collection date (Table 4). Spearman’s rank correlation coefficient was positive in all Honey cases, which indicated a quantitative relationship between the application rate and the concentration of the residues Only a trace residue of boscalid at the level of the LOQ of AIs of the PPP, located at sites frequented by the worker (0.01 mg/kg on June 10) was detected in honey on one sam- bees and in the hive interior. pling date. Spearman’s rank correlation coefficient was also used to determine whether a mutual relationship existed between Worker Bees and Brood the residue concentrations of the AIs of PPPs in different combinations of samples and for different sampling condi- Chlorpyrifos, iprodione, boscalid, difenoconazole, and tions, and the results of the analysis of these relationships pyrimethanil were found in worker bee samples. Chlorpyri- are presented in Table 5. fos residues were detected most frequently and were found In most cases, a positive correlation was noted between in samples taken for analysis on eight of nine sampling dates the residue concentration of AIs of PPPs in the crop and in (May 20 and 27; June 4, 10, 17, and 25; and July 2 and the hive. Only certain worker bees collected on May 27, June 8), although in all cases, its residues were at low levels (to 10, and July 17 showed negative correlation coefficients. 0.05 mg/kg in the samples collected on June 4). However, No positive correlations were noted between the AI of PPP animal bodies contained iprodione residues (dicarboximide residues in the environment and in the worker bees. group; the AI of Rovral Aquaflo 500 SC, used on June 4 at an application rate of 2 L/ha), a fungicide with a contact mode of action on plants, which was not detected in other Discussion matrices. The iprodione residues increased during the period from June 10 to June 25 from 0.09 to 0.63 mg/kg and then Raspberries, including the Laszka variety, are nectar- started to decrease below LOQ on the last sampling day. secreting plants. Their yields depend largely on the effi- The boscalid residues were detected at low levels (no more ciency of their pollination by insects (Chauzat et al. 2009). than 0.11 mg/kg on June 25), but pyraclostrobin, which was Worker bees, when foraging on entomophilous plants, may applied in the form of Bellis 38 WG and Signum 33 WG together with boscalid, was not detected at all. Similarly, in Table 4 The relationship between residue concentrations found in dif- the worker bee samples, small amounts of systemic difecon- ferent matrix combinations azole were detected (in the samples from 3 sampling days; to 0.12 mg/kg in samples from July 2) as well as pyrimethanil AI, common name Matrix combination Correlation level with a contact and deep-seated action (0.09 and 0.03 mg/ Chlorpyrifos Soil-worker brood 0.75 kg in the samples from May 20 and May 27, respectively). Iprodione All Lack of relationship In brood samples, the pesticide residues at a level higher Pyraclostrobin All Lack of relationship than the LOQ were collected only on three sampling days. Boscalid Flowers-honey 0.80 Only a trace amount of chlorpyrifos was found (no more Flowers-worker honeybees 0.88 than 0.02 mg/kg of the brood) on May 27. Leaves-honey 0.87 Leaves-worker honeybees 0.89 Statistical Analysis Honey-worker honeybees 0.88 Cypermethrin All Lack of relationship The similarity degree for the residue concentrations of the Difenoconazole All Lack of relationship individual substances found in the various sample types, Azoxystrobin All Lack of relationship regardless of the sampling date, was assessed using a clus- Pyrimethanil Flowers-worker honeybees 0.76 ter analysis. Based on the cluster analysis results and the 1 3 54 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 Table 5 The relationship AI, common name Sampling date Relation Correlation coefficient between the residue levels of AIs of PPPs in various matrices, Propyzamide All All Lack of relationship depending on terms of sampling Chlorpyrifos May 27 Leaves-worker honeybees − 0.88 Iprodione June 17 Soil-worker honeybees − 0.89 Pyraclostrobin All All Lack of relationship Boscalid June 10 Leaves-honey 0.81 June 10 Leaves-worker honeybees − 0.90 June 25 Leaves-worker honeybees 0.82 July 2 Flowers-honey 0.90 July 2 Flowers-worker honeybees 0.87 July 2 Fruits-worker honeybees 0.99 Cypermethrin All All Lack of relationship Difenoconazole All All Lack of relationship Azoxystrobin All All Lack of relationship Pyrimethanil May 27 Flowers-worker honeybees − 0.88 simultaneously collect various contaminants and transfer aboveground parts of plants at approximately 5 days after them to the hive (Anderson and Wojtas 1986; Chauzat et al. their introduction to the soil, which may partially explain 2009; Cresswell and Thompson 2012; Oruc et  al. 2012; the obtained results. The cypermethrin behaviour, which Piechowicz et al. 2018a, b). Some pesticides used to protect was applied in the form of Cyperkill Super 250 EC on May raspberry plantations from pests and diseases show a pos- 19 and May 29, indicates the impacts of intensive rainfall sibility of accumulation in the bee bodies. They also pollute on May 19 and May 20. On May 20, the first day after the bee products (Rissato et al. 2006). Therefore, some reports first application, the cypermethrin residue on leaves was indicate that some AIs of PPPs may be transferred into the 0.9 mg/kg. In the samples obtained on June 4, i.e., 6 days beehive (Anderson and Wojtas 1986; Southwick and South- after the second treatment (in both cases, 0.15 L/ha), this wick 1992; Pettis et al. 2004; Panseri et al. 2014). value increased to 2.8 mg/kg; therefore, without taking into The AIs of PPPs differ in their chemical structure (they account natural disappearance, it was threefold higher than belong to different chemical groups) and mode of actions, after the first treatment. which determine how they are distributed in the environ- Pyrimethanil, which was applied to the crop in the form ment, how they spread in plants and penetrate plants, and of Mythos 300 SC at the same time as chlorpyrifos (Dursban how they penetrate animal bodies. Furthermore, the PPPs are 480 EC), i.e., on May 14, and cypermethrin, used in the form characterized by long half-lives and consequently by their of Cyperkill Super 250 EC on May 19 and May 29, which persistence in the environment (Gerolt 1983; Różański 1992; are substances with contact action and more adherent to the Leroux 1996; Mileson et al. 1998; Seńczuk 2012; Szpyrka plant surface, were found on leaves and flowers since the and Walorczyk 2017). The above-mentioned factors may first sampling day (24.5 mg/kg on flowers and 206 mg/kg on result in the occurrence of chlorpyrifos (deep-seated and leaves in the case of pyrimethanil and 0.33 mg/kg on flow - probably, which is indicated by the results in Table 3, semi- ers and 0.90 mg/kg on leaves in the case of cypermethrin). systemic), azoxystrobin or difenoconazole residues (both However, the residue of iprodione (AI of Rovral Aquaflo 500 with a systemic action in plants), which were not detected SC), also with a contact mode of action, was not detected in the samples collected on May 20 (all 3 compounds; 6, 13, on any part of plant, although probably in this second case, and 13 days after application, respectively), nor the leaves the preparation, which was still damp, was washed away (azoxystrobin and difenoconazole). They were present only from the plant, which is partly indicative of the necessity of in samples collected on May 27, which indicates that their the subsequent fungicide treatment (Bellis 38 WG) that was residues are linked to the secondary absorption of those applied on June 5, i.e., the day after the application of Rovral compounds from the deeper layers of the soil, where they Aquaflo 500 SC. Most likely, not the lack of application but were probably diluted due to the intensive rainfalls rather washing away the preparation from the plant resulted in the than their primary presence at the plant surface. In addi- presence of marked amounts of iprodione on bee bodies, tion, because only chlorpyrifos, at an application rate of because the fungicide treatments were performed during the 0.08 mg/kg on May 20 and of 0.06 mg/kg on May 27, was daily period of worker bee foraging. observed in the surface layer. As Kubik et al. (2000) notes, Small amounts of difenoconazole and pyrimethanil at plant protection products with systemic action appear in the a level close to LOQ were observed on all fruit samples. 1 3 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 55 Additionally, the presence of azoxystrobin was detected on be found. However, its long persistence in the soil (Table 3) June 17, which indicates that preparation of systemic and and its continuous transport and concurrent transpiration to extensive action can occur in a plant for a long period (69, aboveground plant parts resulted in its occurrence in the 62, and 41 days after application, respectively). flowers at a higher concentration than the LOQ, which con - In the preparations, which were applied on June 5 and sequently constituted a threat to pollinators. The presence of June 9 and contained pyraclostrobin and boscalid (Bel- some pesticides in brood indicates that the worker bees have lis 38 WG and Signum 33 WG), the proportions between been exposed to PPPs since the earliest stages of their ontog- these substances were 1:1.97 and 1:3.99, respectively. The eny, at a time of intensive nervous system development. amount of pyraclostrobin was reduced relative to the amount Chlorpyrifos, applied to the soil on May 14, was present of boscalid that occurred in flowers (proportions from 1:13.5 in the soil samples until the last sampling date (i.e., July 8) on July 8 to 1:48.5 on June 25, respectively) and in fruits and consequently also occurred in the flowers and leaves. (1:33.5 on June 17 and 1:28.0 on June 25); however, in the The highest amounts of chlorpyrifos residues, compared case of leaves, we observed a complete reversal of the pro- with its residue in the soil, were detected on July 2 in fully portion, excluding samples from July 15 (the proportion developed leaves (TF = 600%), whose surfaces and weights pyraclostrobin to boscalid 1:1.85), in plants for a long period did not change, which could suggest that this compound was (69, 62, and 41 days after application, respectively). In the still actively transported from the ground by the plant root remaining sampling days, a larger residue of pyraclostrobin system. In flowers, these residues were observed on May 27 than boscalid (to 1:0.37 July 2 and July 8) was noted. The (TF = 150%). reversal of those proportions in the case of leaves may result Iprodione, the fungicide with a contact mode of action, from the increased transpiration of boscalid by the leaves was found in worker bees (up to 0.63 mg/kg found in the compared with that of pyraclostrobin. Such a phenomenon sample on June 25), but the residue concentrations in the was observed in the raspberry crop, for the pesticide residues leaves, flowers, and fruits did not exceed the LOQ. An expla- from the surface of leaves, flowers, and fruits (material in nation of this effect may be the temporal bioaccumulation preparation) when analysed exclusively. of this substance in animal tissues or foraging by bees on Our surveys confirmed the possibility of transferring other crops. However, in a 2-km area around the crop, no measurable (i.e., above the LOQ) amounts of some PPP other nectar-secreting blossoming plants were present, and AIs from the sprayed dessert raspberry bushes to the bee- such a large pesticide residue concentration would point to hives. Most often, residues were found in worker bees; for the transfer of large amounts of pesticides from other crops, example, chlorpyrifos was found in samples from eight of where the preparation was applied between June 4 and June total nine sampling dates (from May 20 to July 8), iprodi- 10, when it was first observed in bee samples. It seems to be one from five of six sampling dates (from June 10 to July more likely, as it was mentioned above, that the preparation 8), boscalid from five of six dates (from June 10 to July 8), was used during daily period of the bee foraging, shortly difenoconazole from three of nine dates (on June 17, July 2 before intense rainfall, which could remove the preparation and 8), and pyrimethanil from two of nine sampling dates from plants and top layer of the soil. However, it is worth (on May 20 and 27). Residues were found less frequently in mentioning that close to the studied plantation, there were the honey and brood and were found in only samples on one small home cultivations of plants that did not secrete nectar, of eight sampling dates. Chlorpyrifos was found in broods so they were unlikely to be attractive to bees (Lycopersicon from four of nine sampling dates (Tables 1, 3). The larger Mill. or Cucumis L.); however, they were protected using content of the pesticides in the worker bee bodies is a result preparations containing iprodione and constituted a place of direct contact of the bee worker foragers with the sprayed where the bees could stop, for example, to get water. plants, as well as their direct contact with PPPs, because the Despite the fact that various concentrations of PPP AI treatments were performed during the daily period of bee residues were found in the samples, it was not easy to active foraging. estimate the significance of the differences between the Preparations with deep-seated and systemic action on concentrations. When the sampling conditions were not plants more frequently reached the hives than those with considered, a positive correlation between the presence of contact action. Chlorpyrifos, an organophosphorus insec- boscalid, pyrimethanil and chlorpyrifos in the crop and in ticide and the AI of Dursban 480 EC, which was included the hive (Table 4) was found, which meant that an increase in our study for the control of May bug larvae (Melolon- in the residues of AIs of PPPs in the beehive was linked to tha melolontha), is an example of the abovementioned their higher concentrations in the crop. A similar relation- rule. Unlike the other PPPs for which a foliar application ship was observed when the sampling dates were consid- was used, Dursban 480 EC was applied to the soil only via ered (Table 5), although for chlorpyrifos (sampled on May the irrigation system, and it had no direct contact with the 27), iprodione (sampled on June 1), boscalid (sampled on aboveground parts of plants, on which foraging bees might June 10), and pyrimethanil (sampled on May 27), negative 1 3 56 Archives of Environmental Contamination and Toxicology (2018) 75:45–58 correlations also were found, indicating that the increases if, as suggested Kessler et al. (2015), honeybees prefer a diet in the AI residues in the crops were related to lower con- containing neonicotinoids. centrations in the worker bee tissues. A probable reason Bees in the crop also were exposed to six AIs of fungi- for this phenomenon could have been the relatively intense cides (i.e., azoxystrobin, difenoconazole, pyrimethanil, ipro- and long rainfall that limited foraging intensity (personal dione, boscalid, and pyraclostrobin), from which iprodione, communication from the crop owner). boscalid, difenoconazole, and pyrimethanil were detected in Our studies were limited to 2 months. During this time worker bee bodies and boscalid was detected in honey. Addi- period, no clear-cut declines in the strength of the tested tionally, pesticide adjuvants, which increased the probabil- honeybee colonies were observed. However, as previously ity of adverse interactions (Mullin et al. 2015) were found. mentioned, some AIs might be toxic to honeybees even Furthermore, because of the possibility of AI accumulation at sublethal doses (Weick and Thorn 2002; Williamson in the wax (Serra-Bonvehí and Orantes-Bermejo 2010), the and Wright 2013). As Leonardi et al. (1996) discusses, adverse effects of those substances might extend far beyond the AIs of pesticides even at a level lower than the LOQ the raspberry flowering period. can affect insects. They can act synergistically with each Even though chlorpyrifos, iprodione, boscalid, difenocon- other (Thompson 1996; Thompson and Wilkins 2003, Gla- azole, and pyrimethanil were found in the bees and chlor- van and Božič 2013), e.g., via competition for metabolic pyrifos was found in the brood, a small amount of boscalid enzymes (Johnson et  al. 2009) or cellular efflux (Haw - (0.01 mg/kg of honey) was detected in the honey on one thorne and Dively 2011) and with other environmental sampling date, i.e., in samples collected on June 10. The stressors (Renzi et al. 2016; Doublet et al. 2015). Thomp- amount of boscalid was so small that it did not exceed the son (1996) suggested that even PPPs considered safe for MRL of 0.05 mg/kg (EU Pesticides Database 2017). The bees could intensify their activity against those insects by results indicate that honey from the beehive adjacent to the two orders of magnitude when used in combination with raspberry plantation, protected against pests and diseases, other PPPs. was a completely safe product in terms of the presence of The honeybees in the present study were exposed to the nine studied plant protection products. four insecticides: paraffin oil, acetamiprid, chlorpyrifos, Our surveys confirmed the possibility of transferring and cypermethrin. Paraffin oil physically disturbs the gas measurable amounts of some PPP AIs from the sprayed exchange process in pests (Card of Characteristics the prep- dessert raspberry bushes to the beehives. Five of the nine aration Treol 770 EC). Acetamiprid, being the agonist of applied were detected in worker bee bodies. The honeybee nicotine acetylcholine receptors in the synapse, influences brood was polluted by small amounts of chlorpyrifos applied survival, including impairment of learning and memory, to only the soil through the irrigation system. Only trace disruption of the navigation, and reduction of the honeybee amounts of boscalid residues were detected in honey, which foraging activity (Belzunces et al. 2012; Blacquiere et al. indicated that it was completely safe for consumption. The 2012; Henry et al. 2012). Acetamiprid significantly impairs obtained results confirm occurrence of the phenomenon of olfactory learning in laboratory-based studies (Decourtye active transferring the active ingredients of plant protection et  al. 2004; Han et al. 2010). The next AI, chlorpyrifos, products (PPP AIs) by the honeybees from the crops to bee blocks the active sites of acetylcholinesterase in the synapse hives. space by phosphorylation, and as a consequence, it inten- Acknowledgements The authors are thankful to Prof. Stanisław Sadło, sifies the action of acetylcholine, which is distributed by Mr. Kazimierz Czepiela, Mr. Waldemar Mitrut, Mrs. Liliana Pastuła, chlorpyrifos, and influences honeybee learning and memory Mrs. Katarzyna Stats, and Mrs. Natalia Kopeć for their assistance and abilities in sublethal doses (Guez et al. 2010). Finally, cyper- help during the performance of these studies. methrin causes elongation of sodium channel opening states Open Access This article is distributed under the terms of the Creative in insect nervous cells (Wang and Wang 2003). 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Archives of Environmental Contamination and ToxicologySpringer Journals

Published: Dec 15, 2017

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