UPLC-MS/MS determination of ptaquiloside and pterosin B in preserved natural water.

The naturally occurring carcinogen ptaquiloside and its degradation product pterosin B are found in water leaching from bracken stands. The objective of this work is to present a new sample preservation method and a fast UPLC-MS/MS method for quantification of ptaquiloside and pterosin B in environmental water samples, employing a novel internal standard. A faster, reliable, and efficient method was developed for isolation of high purity ptaquiloside and pterosin B from plant material for use as analytical standards, with purity verified by 1H-NMR. The chemical analysis was performed by cleanup and preconcentration of samples with solid phase extraction, before analyte quantification with UPLC-MS/MS. By including gradient elution and optimizing the liquid chromatography mobile phase buffer system, a total run cycle of 5 min was achieved, with method detection limits, including preconcentration, of 8 and 4 ng/L for ptaquiloside and pterosin B, respectively. The use of loganin as internal standard improved repeatability of the determination of both analytes, though it could not be employed for sample preparation. Buffering raw water samples in situ with ammonium acetate to pH ∼5.5 decisively increased sample integrity at realistic transportation and storing conditions prior to extraction. Groundwater samples collected in November 2015 at the shallow water table below a Danish bracken stand were preserved and analyzed using the above methods, and PTA concentrations of 3.8 ± 0.24 µg/L (±sd, n = 3) were found, much higher than previously reported. Graphical abstract Workflow overview of ptaquiloside determination.

Ptaquiloside in Irish Bracken Ferns and Receiving Waters, with Implications for Land Managers.

Ptaquiloside, along with other natural phytotoxins, is receiving increased attention from scientists and land use managers. There is an urgent need to increase empirical evidence to understand the scale of phytotoxin mobilisation and potential to enter into the environment. In this study the risk of ptaquiloside to drinking water was assessed by quantifying ptaquiloside in the receiving waters at three drinking water abstraction sites across Ireland and in bracken fronds surrounding the abstraction sites. We also investigated the impact of different management regimes (spraying, cutting and rolling) on ptaquiloside concentrations at plot-scale in six locations in Northern Ireland, UK. Ptaquiloside concentrations were determined using recent advances in the use of LC-MS for the detection and quantification of ptaquiloside. The results indicate that ptaquiloside is present in bracken stands surrounding drinking water abstractions in Ireland, and ptaquiloside concentrations were also observed in the receiving waters. Furthermore, spraying was found to be the most effective bracken management regime observed in terms of reducing ptaquiloside load. Increased awareness is vital on the implications of managing land with extensive bracken stands.

Ptaquiloside from bracken in stream water at base flow and during storm events.

The bracken fern (Pteridium spp.) densely populates both open and woodland vegetation types around the globe. Bracken is toxic to livestock when consumed, and a group of potent illudane-type carcinogens have been identified, of which the compound ptaquiloside (PTA) is the most abundant. The highly water soluble PTA has been shown to be leachable from bracken fronds, and present in the soil and water below bracken stands. This has raised concerns over whether the compound might pose a risk to drinking water sources. We investigated PTA concentrations in a small stream draining a bracken-infested catchment at base flow and in response to storm events during a growth season, and included sampling of the bracken canopy throughfall. Streams in other bracken-dominated areas were also sampled at base flow for comparison, and a controlled pulse experiment was conducted in the field to study the in-stream dynamics of PTA. Ptaquiloside concentrations in the stream never exceeded 61 ng L-1 in the base flow samples, but peaked at 2.2 µg L-1 during the studied storm events. The mass of PTA in the stream, per storm event, was 7.5-93 mg from this catchment. A clear temporal connection was observed between rainfall and PTA concentration in the stream, with a reproducible time lag of approx. 1 h from onset of rain to elevated concentrations, and returning rather quickly (about 2 h) to base flow concentration levels. The concentration of PTA behaved similar to an inert tracer (Cl-) in the pulse experiment over a relative short time scale (minutes-hours) reflecting no PTA sorption, and dispersion and dilution considerably lowered the observed PTA concentrations downstream. Bracken throughfall revealed a potent and lasting source of PTA during rainfall, with concentrations up to 169 µg L-1, that did not decrease over the course of the event. In the stream, the throughfall contribution to PTA cannot be separated from a possible below-ground input from litter, rhizomes and soil. Catchment-specific factors such as the soil pH, topography, hydrology, and bracken coverage will evidently affect the level of PTA observed in the receiving stream, as well as the distance from bracken, but time since precipitation seems most important. Studying PTA loads and transport in surface streams fed by bracken-infested catchments, simply taking occasional grab samples will not capture the precipitation-linked pulses. The place and time of sampling governs the findings, and including event-based sampling is essential to provide a more complete picture of PTA loads to surface water.

Dissipation of pterosin B in acid soils - Tracking the fate of the bracken fern carcinogen ptaquiloside.

Bracken ferns (Pteridium spp.) are well-known for their carcinogenic properties, which are ascribed to the content of ptaquiloside and ptaquiloside-like substances. Ptaquiloside leach from the ferns and may cause contamination of drinking water. Pterosin B is formed by hydrolysis of ptaquiloside. In soil, Pterosin B is adsorbed more strongly and it is expected to have a slower turnover than ptaquiloside. We thus hypothesized that pterosin B may serve as an indicator for any past presence of ptaquiloside. Pterosin B degradation was studied in acid forest soils from bracken-covered and bracken-free areas. Soil samples were incubated with pterosin B at 3 and 8 µg g-1 for 10 days, whereas sterile (autoclaved) samples were incubated for 23 days. Pterosin B showed unexpected fast degradation in soils with full degradation in topsoils in 2-5 days. Pterosin B dissipation followed the sum of two-first order reactions. The initial fast reaction with half-lives of 0.7-3.5 h contributed 11-59% of the total pterosin B degradation, while the slow reaction was 20-100 times slower than the fast reaction. Total dissipation half-lives were shorter for loamy sand (4 h) than for sandy loam soils (28 h). No degradation of pterosin B took place under sterile conditions assuming observed dissipation during the first 3 h could be attributed to irreversible sorption. Our results demonstrate that pterosin B is microbially degraded and that pterosin B is as unstable as ptaquiloside and hence cannot be used as an indicator for former presence of ptaquiloside in soil.

The naturally occurring carcinogen ptaquiloside is present in groundwater below bracken vegetation.

The present study demonstrates unequivocally the presence of the natural carcinogen ptaquiloside and its transformation product pterosin B in groundwater and surface water. Groundwater concentrations up to 0.23 nmol/L (92 ng/L) ptaquiloside and up to 2.2 nmol/L (0.47 µg/L) pterosin B were found. Of 21 groundwater samples, 5 contained ptaquiloside, exceeding the estimated threshold for drinking water (1.3-40 pmol/L). The results are critical for water abstraction in bracken-infested areas.