2.1. Site description
The monitoring study was carried out in Humleoreskov, a temperate forest located 60 km west of Copenhagen, Denmark (N 55° 28' 29.7" E 11° 54' 26.1", Figure 1a). The study area is located inside a forest glade dominated by bracken and surrounded by deciduous tree species such as European beech (Fagus sylvatica L.) and Common oak (Quercus robur L.) (Fig. 1a,b). In the area, bracken is present in form of dense stands in glades composed almost exclusively of bracken, as well as forest floor vegetation at a lower density. Bracken completely cover all surface at high density, with a homogeneous canopy surpassing two meters height when fully developed. The criteria for selecting the area was to have a minimal disturbance from the tree canopy on precipitation, a flat topography, and homogeneous bracken biomass in the plot (Figure 1b).
Humleore soil is categorized as sandy loam by the United States Department of Agriculture (USDA) classification and identified as an Oxyaquic Hapludalf in the USDA soil taxonomy [38,39]. The soil is composed of morainic till with a content of clay and silt in deeper horizons up to 18 and 26%, respectively. The soil presents pH values ranging from 4.3 at the surface to 6.9 at 36 cm depth. For a more detailed description of the soil profile, horizons and physicochemical characteristics, the reader is refered to Section 2 in the Supplementary Information (SI).
2.2. Field monitoring and lab experiments
The monitoring began May 2018 and finished March 2020 (Fig. 1c). For the entire period, the frequency of sampling ranged from one to three weeks, depending on the time of the year. High frequency sampling of plant material and soil pore water was centered in the period from beginning of the growing season until the full transition of bracken into litter, i.e., from May to December.
Two different plots of each 25 m2 were set in the forest glade, with 5 meter side length, located in the center of the forest glade. Plots were separated by approximately 70 meters from each other (Fig. 1a,d). Moreover, each plot was divided into 25 subplots of 1 m2 each for sampling purposes. Bracken biomass and PTA content was measured in both plots, while PTA in pore water, soil water content and climate variables (temperature, relative humidity, precipitation) were only monitored in the B-plot (Fig. 1b,d). Rain collectors were set in the borders of the B plot to monitor precipitation, temperature and relative humidity. Moreover, nine suction cells were installed in the center of the plot in March of 2018 to sample soil solution under bracken canopy for the entire calendar year (Fig. 1d).
2.2.1. Environmental variables: from March 2019 until March 2020, precipitation, relative humidity and temperature were constantly monitored on-site (Figure 1d). Precipitation was measured using three rain collectors comprising a PronamicRain-O-Matic small rain gauge (Ringkoebing, Denmark) coupled with an ONSET HOBO® Pendant® Event Data Logger (Bourne, USA). The rain collectors were installed on poles placed in the middle of three different sides of the plot, at 250 cm height and above the bracken canopy (Fig. 1b). Temperature and relative humidity were monitored with two ONSET® HOBO® External Temperature/RH Sensor Data Loggers (Bourne, USA). For measuring the water content in the soil profile, three Delta-T PR2 soil profile probes were installed at 100 cm depth in the B-plot. Measurements were carried out with a Delta-T HH2 Moisture Meter (Cambridge, UK).
2.2.2. Bracken biomass: during the entire monitoring period, bracken biomass was monitored in plots A and B (Fig. 1a) comprising a total of 22 sampling days. In each sampling day, six different subplots were sampled for bracken biomass, each for both plots A and B. All subplots to be sampled were randomly selected at the beginning of each season.
For each subplot and measuring day, the number of fronds, frond height, number of pinnae and pinna length were determined on-site. One frond from the most representative class was collected from each subplot. This was selected visually considering height and apparent biomass of all fronds for the specific subplot. The length of the second and third pinnae of the selected frond were also measured on site. The collected fronds were placed in plastic bags and kept on ice for up to two hours until arrival to the lab. Once in the lab, the diameter of the rachis, as well as the weight of the frond, pinna and rachis were determined separately. The total aboveground biomass per square meter was calculated as the sum of pinna and rachis biomass, multiplied by the number of fronds in each plot. For description of the morphology of bracken, the reader is referred to Fig. 1 in SI.
Moreover, below-ground biomass was determined once on 1st July of 2020, at the time of frond maturity. For that, three plots of 0.5x2.0 m were established at 20-60 m west-southwest of the B-plot (Fig. 1a). Each plot was then excavated until no further rhizomes were found for 20 cm. The soil was removed from the rhizomes, washed and divided into frond-bearing and storage rhizomes based on the occurrence of short-shots (frond-bearing only). The length of all rhizome-fragments was measured and the number of terminal buds counted. The dry-matter content was determined after drying for 48 h at 110 oC in a fan-ventilated oven.
2.2.3. PTA in the canopy: Initially, the pinna and rachis were separated with common scissors. Then, bracken pinnae were cut into 5-10 cm pieces, mixed by hand and a subsample of approximately 15 gr stored in a plastic bag. Rachis used for PTA determination was cut longitudinally in pieces with an approximate length of 10 cm and placed in plastic bags. Thereafter, all plant samples were stored at -80 oC until sample preparation and measurement.
2.2.4. Soil pore water: from March 2019, pore water was monitored for one full calendar year (Figure 1c). Nine Prenart Super Steel suction cells (Frederiksberg, Denmark), with a dimension of 110 mm length, 19.5 mm outer diameter, porous area of 40 cm2 and an average pore size of 50 nm were used. The suction cells were installed at approximately 50 cm depth and at an angle of 45 degrees to minimize disturbance of the soil column above the cell. Suction cells were connected to 100 mL glass collector bottles on the soil surface via 2 mm PTFE tubing. Between sampling days, vacuum was applied with a hand vacuum pump until a pressure of -20 kPa was reached. At the time of sampling, the content in the bottle was emptied, followed by the application of a vacuum of -60 kPa for the extraction of fresh soil pore water. A subsample, if possible, of 10 mL was transferred to 15 mL polypropylene conical centrifuge tubes. Samples were immediately stored on ice, for a two 2-hour period, until arrival at the laboratory. Afterwards, samples were preserved by adding 0.5 mL of 0.3 M ammonium acetate buffer per 20 mL of sample, following the protocol of Clauson-Kaas et al. [8]. Thereafter, the samples were immediately stored at -18 oC until sample purification.
2.3. PTA release: for quantification of the PTA being released by precipitation, we sampled the throughfall water under bracken, i.e., intercepted rainwater spilling off the canopy, during four precipitation events. The precipitation events that were monitored took place in 25th and 30th of August 2018, and 5th and 30th July of 2019. For collecting the throughfall water, glass jars were placed under the bracken canopy prior to the precipitation event. The jars were cylindrical with an opening diameter of 8 cm. Moreover, a plastic mesh with grid size of 1.1 mm was placed on the openings of the jars to avoid plant materials entering the container. 2 mL of 0.3 M ammonium acetate buffer was added to the containers before each rain event for minimizing PTA degradation until collection of the water [40]. A total of 9 (2018) or 12 (2019) jars were placed in the middle of the B plot, with the same distribution pattern than suction cells in the soil (Fig. 1d). However, during the precipitation event taking place on 30th July 2019, a total of 9 jars were placed in both plots A and B, to determine the variation in wash off amount between plots. The mass of PTA washed off the canopy during a precipitation event [mg PTA m-2] was calculated as the concentration of PTA measured in throughfall water [mg L-1], multiplied by the volume of throughfall water collected in the glass containers [L] and divided by the area of the jar [m2].
2.4. Degradation of PTA in soil: We conducted 6 rounds of batch experiments estimating PTA degradation kinetics for the three uppermost horizons of the soil, i.e. A1, A2 and AE For this, a soil profile was excavated under bracken canopy near plot B. Moreover, we tested the influence of soil moisture content on PTA degradation kinetics, by carrying out incubations in both unsaturated (matric potential of -100 cm or pF 2) and near saturated (matric potential of -10 cm or pF 1) conditions. An aliquot of PTA solution was added to the soil samples to reach an initial concentration of 10 µg g-1 dry weight (DW). The stock solution of PTA used for addition was concentrated bracken extract purified by preparative HPLC with a Perkin Elmer Series 10 liquid chromatograph (Connecticut, USA) equipped with a Shimadzu SPD-10A UV-VIS Detector (Kyoto, Japan). The purification followed the method by Rasmussen et al. [29]. For more detailed information of the standards use and the procedure followed see Section 5.1 in SI.
After the soil water mixture was homogenized, an aliquot equivalent to 2 grams DW was added into 15 mL centrifuge vials. Three different replicates were prepared for each sampling time, making a total of 180 samples incubated during the experiment. All samples were stored on ice during sample preparation to avoid degradation. Thereafter, samples were placed in a climate chamber at 10 oC, 70% humidity and darkness. To avoid anaerobic conditions, the tubes were incubated covered with aluminum foil. The samples were left undisturbed throughout the experiment until the moment of extraction and analysis.
The PTA degradation kinetic data were fitted as a pseudo first-order reaction with respect to PTA. TableCurve 2D software was used (v 5.01, Jandel Scientific, AISN Software, San Rafael, CA, USA) for non-linear regression. The nominal initial concentration at time 0 was not included in the regression analysis but was estimated by regression. The values are expressed as degradation, as sorption of PTA is negligible compared with microbial degradation [29,30,41].
2.5. PTA determination methods
2.5.1. Solvents and chemicals: HPLC-grade methanol for bracken extraction and determination of PTA in bracken was obtained from Sigma-Aldrich (Denmark). HPLC-grade hexane was obtained from VWR (HiPerSolv Chromanorm, Denmark). LC-MS grade methanol was obtained from Honeywell (LC-MS Chromasolv, Germany), while LC-MS grade acetonitrile was obtained from Merck Millipore (LiChrosolv hypergrade for LC-MS, Germany). All acids and bases (sodium hydroxide, formic and trifluoroacetic acid) were analytical grade obtained from Sigma-Aldrich (Denmark). Polyamide was obtained from Sigma-Aldrich (Polyamide for column chromatography 6, Denmark). Loganin used as internal standard was purchased from Sigma-Aldrich (Denmark).
2.5.2. PTA determination in bracken tissue samples: frozen plant samples for PTA determination were freeze dried in a Labogene Scanvac Cool Safe freezer dryer, at 1 hPa and -96oC for 48 hours. The freeze-dried samples, both pinna and rachis, were milled into a fine powder in a Kenwood KVC3100 W kitchen machine adapted with a Kenwood Multi Mill For extraction, an aliquot of 0.5 g of powder was placed into polyethylene centrifuge tubes of 50 mL, followed by the addition of 20 mL of 80% v/v methanol into the centrifuge tubes and shaking for 20 mins. For extraction, both methanol and ethanol were tested, and methanol was selected because it showed a better extraction efficiency. Thereafter, the tubes were centrifuged at 20,000 g and 4 oC for 15 minutes. The supernatant was transferred to another centrifuge tube and kept on ice. Then, an aliquot of 4 mL of the extract was filtered through a 0.45 µm regenerated cellulose (RC) syringe filter (Sigma-Aldrich, Denmark) into a 15 mL centrifuge tube. For removal of pigments, 4 mL of hexane was added. The tube was shaken by hand for 10 seconds and the two phases then allowed to separate. The methanol phase was transferred to a 15 mL centrifuge tube using a Pasteur pipette. The cleaning with hexane was repeated twice, to remove as much interfering substances as possible. Thereafter, 2 mL of the methanolic extract was diluted with DI to reach a 40% methanol solution, filtered with a 0.22 µm PTFE syringe filter into a 1.5 LC amber vial and stored on ice until analysis. All samples were done in duplicates. Extraction and analysis of all samples were carried out in the same day.
The method for quantification of PTA and PTB was adapted from the method of Ayala-Luis et al. and Rasmussen and Pedersen [31,42]. Quantification of PTA and PTB in bracken extracts took place on an Agilent 1200 series diode array detector (DAD) HPLC system equipped with a Phenomenex Hyperclone C8-DBS (150 mm x 4.6 mm, 3 µm) column thermostated at 35 oC; a Phenomenex Gemini C6-Phenyl guard column was used. The analytes were separated with a mobile phase composed of 10% acetonitrile (eluent A) and 100% acetonitrile (eluent B). The elution gradient was: 0-6 min 11% B, 7 min 47% B, 7-10 min 47% B, 11 min 11% B, and 11-13 min 11% B. The column was flushed with ?? at end of each run, with a total time of analysis of 10 minutes. The sample injection volume was 50 µl. UV detection was performed at 214 nm for PTA and 220 nm for PTB. All samples were prepared in duplicates.
The LOD and LOQ of the method for PTA was 82 and 276 µg g-1, respectively, while for PTB it was 16 and 55 ug g-1. The LOD and LOQ for PTA was calculated as 3 and 10 times the standard deviation of the injection of the lowest standard for all runs, divided by the average slope of the calibration curves. Since PTB is present in low amounts in the canopy and assumed to not be produced in the plant but formed as degradation of PTA, we decide to proceed with molar sum as a calculation of the total PTA produced (PTATOT). The final concentration of PTATOT in each sample was obtained adding the measured concentration of PTA and concentration of PTB, applying 1:1 molar conversion ratio from PTA to PTB. For more information regarding calculations of toxin in the biomass, the reader is referred to Section 7 in SI.
2.5.3. PTA determination in soil solution: the soil water samples were purified and concentrated by solid phase extraction (SPE) using Waters Oasis MAX (1 cc Flangeless Vac Cartridge, 10 mg, USA) using a SPE method adapted from Skrbic et al. (2020) [9]. The SPE cartridges were conditioned consecutively with 0.33 mL of 100% methanol and 0.33 mL of deionized water, with the cartridges running dry for 20 seconds between additions. A total of 3 mL of sample was loaded to each SPE, by consecutive 1 mL additions. The cartridges were washed with 0.33 mL of deionized water, eluted with 1 mL of 100% methanol and the eluate collected into 1.5 mL LC amber vials. The methanol in the samples was then evaporated in a heat block (Mikrolab Aarhus, Denmark), thermostated at 30 oC, with a gentle air flow. After evaporation, the eluate was dissolved in 40% methanol + 0.1 M ammonium acetate solution, buffered at pH 5 [22]. For ensuring the recovery of all compounds in the vial, these were shaken on a vortex shaker for approximately 10 seconds. The solution was then transferred to a LC amber vial of 200 µl and stored at -20 oC until analysis.
The method used for quantification is adapted from Kisielius et al. [22]. The chromatographic separation and quantification of analytes were performed using Agilent 1260 Infinity HPLC system equipped with an Agilent 6130 Single Quadrupole mass spectrometer. The LC system was thermostated at 35 oC and a flow of 1 mL min-1. The analytes were separated with a mobile phase comprised of LCMS-grade water (eluent A) and acetonitrile (eluent B), both containing 0.1% formic acid. The gradient of elution was: 0-1 min 20% B, 4.5 min 52% B, 5 min 95% B, 5-5.5 min 95% B, 5.6 min 20% B, and 5.6-6 min 20% B. Injection volume was 100 µL including 2 µL internal standard. Loganin was used as an internal standard in all determinations, by the addition of 2 ul of 500 ug L-1 into each sample by the sampling robot connected to the LC instrument [40]. The mass spectrometer operated in single ion mode, targeting 219.1 m/z fragments for both PTA and PTB [22]. Total ion scans were included for the m/z window between 200 and 460.
The LOD and LOQ of the analytical method for PTA with external standards, including preconcentration, was 14.7 and 45.3 ng L-1, respectively. On the other hand, the LOD and LOQ for PTB was 2 and 6 ng L-1, respectively. The recovery of the full method, including preconcentration, was 74.3 ± 0.02 % and 99.6 ± 0.03 % for PTA and PTB, respectively. For handling of the results, in cases where a signal at the specific retention time of PTA or PTB was recorded, but with areas below the LOQ, the concentrations were set at half of the LOQ. In cases where no signal was detected, the concentration was set at half of the LOD.
For quality control, a positive and a negative sample was extracted with SPE as any other field sample every 15 field samples. Positive sample was composed of a solution of PTA and PTB with known concentration, while the negative was a field blank from a soil in Humleore without bracken presence (SI, Section 8). All determinations were performed using purified external standards with known concentrations provided by Vaidotas Kisielius [22]. No matrix effect nor instrumental drift were identified.
2.5.4. PTA determination in soil: the extraction of PTA from soils was carried out following the method by Jensen et al. [27]. After extraction, PTA was converted to PTB using the method by Rasmussen and Pedersen (2017) [42]. First, 6 mL of deionized water was added to each sample tube, followed by shaking on a flatbed shaker for 10 minutes at 18 Hz. After shaking, samples were centrifuged at 20,000 g at 1 oC for 10 minutes. The supernatant was then filtered through a RC syringe filter of 0.2 µm. However, the top horizons had to be filtered with 0.45 µm instead, due to clogging of the filter by the organic-rich topsoil materials. The cleaning up of impurities and PTB in the extract was done by passage through solid phase extraction columns dry packed with polyamide. The PTA in the filtered extract was then converted to PTB by stepwise addition of 75 µL of sodium hydroxide (1 M) and 75 µL of trifluoroacetic acid (2.5 M) [42]. Samples were stored at -18 oC until HPLC analysis. PTB concentrations were analyzed by HPLC using the same analytical instrument used for PTA and PTB in bracken samples (section 2.5.1) but with optimized settings for the chromatographic separation of PTB. The analytes were separated with a mobile phase composed of 43:57 water: acetonitrile. Flow of 1 mL min-1 and 50 µl of injection volume. The LOD and LOQ of the analytical method for PTB was 8.3 and 28 µg L-1, respectively. For more details, the reader is referred to SI (Section 4.1).
2.5.5. PTA concentration in throughfall: the quantification of PTA and PTB in the throughfall water from the wash off field experiments was carried out using the same method as described above. The samples from the events taking place in the 2018 growing season were analyzed by transformation to PTB and quantification by HPLC (section 2.5.1). Samples from the 2019 release experiments were quantified using a Waters Acquity UPLC I-class module equipped with a Waters Xevo TQD triple quadrupole mass spectrometer, with adjusted parameters based on the method by Kisielius et al. [22].