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The apiary which reported the sudden bee death is located 300 m from the paper mill sludge processing facility in Hwasan County, Youngcheon city, GB, Korea (Fig. 1). The beekeeper keeps honey bees in 3 different sites nearby, but only one apiary site was severely damaged, leaving most of the vacant 150 honey bee hives. Based on the beekeeper, few days with damping humid hot days in Early August 2019, all adult bees were disappeared with only developing larvae and pupae remaining (Personal communication with animal quarantine inspector, 2019). One and half month after the event, two different sludge samples ((recycled solid paper mill sludge (SPMS) and leachate paper mill sludge (LPMS)) were collected from the site on September 16, 2019.

Fig. 1. 
Map of the sampling location (marked by A) in Hwasan County, Youngcheon city, GB, Korea for the paper mill sludges used in this study.

Recycled solid paper mill sludge (SPMS) was the final recycled by-product, and used as a fertilizer. In the present study, SPMS was collected from the field storage one month before distributing to the farmers. In the recycling process, SPMS produced at the plant was being composted by blending the paper mill sludge with pig manure then, after dewatering, dried and took the form of granules (SPMS). Leachate paper mill sludge (LPMS) was a thick liquid derived from SPMS at the paper mill waste water treatment plant (WWTP) pond.

Individual paper mill sludge (5 g each) was suspended in distilled water (100 mL each at 15, 25, 40℃) and were poured into the separatory funnel. Hexane (50 mL) was added to it. The mixture was then shaken vigorously for 5 min and then allowed to stand for 30 min. Following this, the organic and aqueous layers were separated. The aqueous layer was then further extracted again by shaking with hexane (50 mL). Both hexane extracts were combined, and the residual water was removed from the extract by treating with an anhydrous sodium sulphate (5 g). The extract was filtered and transferred into a round-bottom flask, followed by evaporation. The extract was stored in a refrigerator for further examination.

Hewlett-Packard HP 5977A Mass Spectrometer coupled with an HP-7890B GC was used to identify and quantify the VOC of SPMS and LPMS. Separation was performed on a non-polar column (HP-5MS-fused silica column; 5% phenyl methylpolysiloxane; 30 m×0.25 mm id; 0.25 μm film thickness, Agilent Technologies). VOC of SPMS and LPMS were diluted in acetone (1:100) prior to GC-MS analysis. Sample (1 μL) was injected in split mode with a 1:10 split ratio. Helium was used as a carrier gas at a flow rate of 1.0 mL/min. The oven was initially set at 40℃ for 3 min, then the temperature was programmed to increase by 6ºC/min until it reached to 150℃, the temperature was further programmed to increase by 10ºC/min until it reached a final temperature of 320ºC, which was kept for 3 min. The MS readings were scanned in the range of 40~500 amu in full-scan mode with electron impact ionization energy of 70 eV. The temperatures of the ion source and injector base were maintained at 230 and 270℃, respectively. Compounds were identified by comparing the mass spectrum of individual VOC with those listed in the MS Library Database.

We have collected young adult workers from healthy honey bee (Apis mellifera) colonies in the experimental apiary of Andong National University. Honey bee colonies are all from the same breeding lines and requeened in the previous autumn season. The collected bees were released into the ventilated test cage made of stainless steel. In each cage 10 bees were apportioned. Then the bees were starved for 2 hr in the experimental room at 25±2℃ and 50~70% relative humidity.

A Y-tube olfactometer system previously described by Li et al. (2014) was used to test the orientation responses of honey bees towards to odors emanating from SPMS and LPMS (50 mg/mL in 50% sugar solution, each). The Y-tube olfactometer (Fig. 2) comprised of a central tube and two lateral arms (each 8 cm long and 18 mm internal diameter). Each arm was connected to an odor chamber (cryogenic vial; 4.3 cm in height, 3.0 cm inner diameter; 20 mL) holding the test sample. Each odor chamber had inlets for the incoming air and outlets for odors to exit the Y-tube. A charcoal-filtered and humidified air stream was passed into each arm at a flow rate of 250 mL/min (DK-800 Air Pump) to allow experimental odors moved towards the decision-making area. A mesh screen was placed at each of the endpoints of the olfactometer to prevent honey bees escaped from the test area and from a direct contact with the test samples. Honey bees were starved for over 2 hr before being tested. The test samples were separately applied to pieces of cotton. A 10 μL aliquot of each sample in sugar solution was dripped onto a piece of cotton that had been placed inside one arm, and another piece of cotton permeated with 10 μL of sugar solution was placed in the other arm of the Y-section as a control. To prevent any positional bias in the behavior of the honey bees, the relative position of the tested stimulus and its corresponding control were alternated between replicates. A clean Y-tube was used for each test in order to avoid carryover of odors. The Y-tube was illuminated with red light by means of an incandescent light bulb to preclude their use of visual cues during the experiments. One honey bee at a time was introduced into the Y-tube after the airflow had been initiated. It was assumed to have made a choice when the honey bee walked more than 2/3 length of the treated source or control arm and stayed there for approximately 1 min or when it frequently visited the arm. A ‘‘no choice’’ decision was recorded if the honey bee had not moved after 5 min. Each treated or control cotton was used only once and was then replaced with a fresh cotton for the next individual, and each individual honey bee was used only once in the experiment. 50 honey bees were used for individual test samples. Response rate (%) and repulsion rate (%) of SPMS and LPMS on honey bees in the Y-tube olfactometer was computed using a formula reported by Li et al. (2014).

Fig. 2. 
Bioassay set-ups: (A): Y-tube set-up; (B): Oral toxicity set-up; (C): Fumigant toxicity set-up.

Test samples (100 mg/mL) of SPMS and LPMS were prepared in 50% sucrose solution. A plastic Kovax-Syringe (5 mL) with the open end narrowed to about 2 mm diameter was used as feeder unit (Fig. 2). An amount of 5 mL of prepared doses was taken into the feeder unit without any air bubbles. The feeder unit containing 50% sucrose solution alone was considered as control. The experiment was replicated thrice. Then the feeder volumes were taken and placed into the respective test cages. The honey bees were allowed to consume the dose for maximum of 6 hr and there after the feeder units were removed from the test cage and weighed to measure the dose consumed. The feeder unit containing sucrose solution alone was replaced into the test cage as soon as the dosage was removed. Then the bees are observed for the mortality on 3, 12, 24, 48, 72, 96 and 120 hours.

SPMS and LPMS (2.9 g/L Air, for each) in a cap were placed at the bottom of a rectangular fumigation box (6.2 cm in length, 6.2 cm in width, 17 cm in height; 695 mL), with mesh-holes fitted approximately 7.5 cm above the bottom of the fumigation box (Fig. 2). Batches of 10 honey bees were introduced into each box by placing them in separate compartment containing food (50% sucrose solution), thereby preventing their direct contact with the test samples. Both ends of the box were sealed with using parafilm to prevent from leaking. The controls had no test sample. Honey bees were maintained at 23±1℃ and 30% relative humidity. Triplicates were set up for each treatment. Mortality of adults was observed at 3, 12, 24, 48, 72, 96 and 120 hours. Honey bees were considered dead if they did not move.

Olfactory responses of honey bees to test samples were compared using the Chi-square test (SPSS Statistics, v. 16) with p<0.05 as an indicator of statistical significance. All toxicity assays were carried out in triplicate, and the results were presented as the mean mortality (%). Two-way ANOVA analysis, followed by Tukey multiple comparison tests, was used to compare test the effect of test samples on honey bees at each time point.