Simple on-site extraction and GC-MS analysis of rotenone and degradation products for monitoring invasive fish eradication treatments in fresh and brackish waters
- Photo: Journal of Chromatography A 2024, 1730, 465063: Fig. 1. Molecular structures of rotenoids commonly present in the piscicide formulation CFT Legumine (ROT, DEG), their most abundant degradation products (ROH, TEP), and the surrogate standard used in this work for their analysis (MIL).
In the research article published recently in the Journal of Chromatography A the researchers from the Institute of Environmental Assessment and Water Research and Centre for Advanced Studies of Blanes, Spanish National Research Council, and Sorelló - Estudis al Medi Aquàtic, Parc Científic de la Universitat de Girona, Spain, presented a simple on-site SPE extraction, followed by GC-MS for analyzing rotenone-containing piscicides, like CFT Legumine, in aquatic environments.
This study presents a method for analyzing rotenoids in aquatic environments, with a focus on rotenone-based piscicides, such as CFT Legumine, commonly used for eradicating invasive fish species. The method addresses challenges such as the rapid degradation of rotenone and the varying salinity of water by using a simplified on-site solid-phase extraction. Gas chromatography coupled with mass spectrometry allows selective analysis of rotenoids and their byproducts. The technique demonstrated high accuracy and precision, with quantification limits below ecological thresholds, making it effective for monitoring piscicide treatments. Results revealed fast rotenoid degradation and accumulation in particulates, which could impact eradication efforts.
The original article
Simple on-site extraction and GC-MS analysis of rotenone and degradation products for monitoring invasive fish eradication treatments in fresh and brackish waters
Raimon M. Prats, Marc Ventura, Quim Pou-Rovira, Teresa Buchaca, Pilar Fernández, Joan O. Grimalt, Barend L. van Drooge
Journal of Chromatography A 2024, 1730, 465063
https://doi.org/10.1016/j.chroma.2024.465063.
licensed under CC-BY 4.0
Selected sections from the article follow.
Highlights
- A simple on-site SPE extraction is proposed for analyzing rotenone in lakes.
- Immediate extraction limits the degradation of rotenoids in samples before analysis.
- GC-MS analysis enabled monitoring rotenoid degradation after piscicide treatments.
- LOQs under 2 µg/L ensure adequate analysis below the no-effect levels of rotenone.
Abstract
The introduction of invasive fish species to aquatic ecosystems has been demonstrated to cause disastrous ecological effects. Current conservation strategies regard rotenone-containing piscicide formulations, such as commercial product CFT Legumine, as a potentially viable alternative to the cumbersome traditional approaches to fish eradication. This consideration relies on the fast degradation of rotenone and its relatively rapid dissipation from the environment. Piscicide treatments in fragile aquatic ecosystems should thus monitor not only rotenone concentrations following application, but also other byproducts and degradation products. We present a methodology for the analysis of rotenoids in fresh and brackish waters that addresses two main challenges: the accurate determination of applied concentrations in different salinity concentrations by performing a simplified on-site solid-phase extraction, overcoming the fast degradation of rotenone in sample storage conditions, and the selective analysis of rotenoid byproducts and degradation products by gas chromatography coupled to mass spectrometry. Limits of quantification were below the ecological no-effect concentration of rotenone (2 µg/L) and average recoveries exceeded 80%. Accuracy (compared to expected values) and precision (deviation of replicates) ranged from 78 to 103% and 3 to 14%, respectively, across various rotenoid concentrations. These metrics are more than satisfactory for the intended application of this simplified procedure. The method was applied to piscicide-treated samples, revealing significant and fast degradation of parent rotenoids in storage conditions, as well as a non-negligible accumulation of rotenone in the particulate fraction of water that could impact the effectivity of eradication efforts.
1. Introduction
Rotenone (Fig. 1) is a naturally occurring compound primarily extracted from the roots of select plants within the Leguminosae family, notably from the genera Derris, Lonchocarpus, and Tephrosia. Rotenone inhibits mitochondrial cellular respiration and has thus found widespread use as an insecticide and piscicide, applied in freshwater lakes, rivers, and fisheries to control fish populations and to eradicate invasive species [[1], [2], [3], [4]]. Piscicidal formulations of rotenone, such as CFT Legumine, often contain other rotenoids like deguelin, compounds of the same class that are byproducts of the extraction process which may also exhibit toxic activity [5].
Journal of Chromatography A 2024, 1730, 465063: Fig. 1. Molecular structures of rotenoids commonly present in the piscicide formulation CFT Legumine (ROT, DEG), their most abundant degradation products (ROH, TEP), and the surrogate standard used in this work for their analysis (MIL).
The introduction of invasive fish species into new environments can have severe ecological consequences, particularly in delicate, naturally-fishless ecosystems like alpine lakes [[6], [7], [8]]. Attempts to eradicate such fish in high-mountain environments have demonstrated positive effects on the ecosystem [9,10]. Similarly, brackish water environments usually host endangered fish species threatened by the introduction of invasive species such as mosquito fish [11]. However, complete eradication through traditional fishing techniques can take several years and become a costly endeavor, especially in alpine areas of difficult access or in brackish ecosystems with high vegetation cover and saline waters [3]. Rotenone emerges as a quick alternative with significant potential for application in these sites [[12], [13], [14]]. While it may exhibit nontarget impacts on aquatic communities other than fish [15], rotenone and other rotenoids are highly degradable in water [[16], [17], [18]] and concentrations tend to rapidly decrease post-treatment [1,2,19]. Nonetheless, under certain environmental conditions, some degradation products may persist in the lake waters for several months [20].
A periodic and accurate analytical control during rotenone treatments is thus essential in aquatic ecosystems. This not only ensures the application of effective concentrations but also allows monitoring the dissipation and degradation of rotenone and other rotenoid byproducts over time. Traditional laboratory methods for rotenoid extraction and analysis in freshwaters have primarily involved nonpolar solid phase extraction (SPE) followed by liquid chromatography (LC) coupled with UV-vis or mass spectrometry (MS) detection [2,[21], [22], [23]]. SPE followed by micellar electrokinetic chromatography (MEKC) has also been explored [24]. Gas chromatography (GC) separation has been less popular, but significant benefits over some LC approaches for analyzing rotenoids at the time of publication, even without derivatization, were reported [25].
The analysis of rotenoids in samples taken from sites of difficult access, as is the case for many lakes in protected natural areas, presents a unique analytical challenge: the rapid degradation of rotenone can significantly impact the accurate analysis of concentrations as samples degrade between water sampling and analysis in the laboratory. A recently proposed on-site SPE extraction and LC-UV detection method during fish control treatments prevents this degradation [26]. However, this method requires access to a mobile laboratory with power generation capabilities, making it impractical for deployment in less accessible sites. Hence, we present an alternative, simplified methodology that achieves three crucial objectives: 1) immediate on-site SPE extraction of water samples to avoid rotenoid degradation; 2) the ability to perform all sampling and extraction operations in sites that are difficult to reach and without access to an energy source; and 3) GC-MS analysis for monitoring rotenone fate and the appearance of rotenoid degradation products after fish control treatments.
2. Material and methods
2.1. Chemicals
Rotenone >95% (ROT) was purchased in solid form from Sigma-Aldrich (St. Louis, MI, US). Rotenolone 99% (ROH), deguelin >95% (DEG), and millettone >95% (MIL, surrogate standard) were purchased in solid form from Biosynth (Staad, Switzerland). 1-Phenyldodecane 99.8% (1PD) was purchased in solid form from Sigma-Aldrich. All standard solutions were prepared in acetonitrile except for 1PD in toluene. All solvents (acetonitrile >99.9%, methanol >99.9%, isooctane >99%, toluene >99.9%, water) were HPLC-grade and purchased from Merck (Darmstadt, Germany). The piscicide formulation CFT Legumine (3.3% rotenone) was purchased from VESO (Veterinærmedisinsk Oppdragssenter AS, Oslo, Norway).
2.2. Water sampling
All analyses were performed using water collected from three lakes in Catalonia, Spain, in November of 2022. The Pyrenean, high-mountain, freshwater lakes Lake Dellui (42.5475 °N, 0.9477 °E, 2349 m above sea level) and Lake Collada (42.5437 °N, 0.9417 °E, 2453 m), and the brackish coastal lagoon of Santes Creus (40.8621 °N, 0.7812 °E, 0 m). Lake Dellui and Lake Collada are oligotrophic alpine lakes that present the following characteristics, respectively: conductivity 0.04 and 0.02 mS/cm, dissolved organic carbon (DOC) 2.9 and 2.2 mg/L, and chlorophyll a concentration 0.6 and 0.7 µg/L. The lagoon of Santes Creus is a mesotrophic to eutrophic coastal lake with the following characteristics: conductivity 38.9 mS/cm, DOC 8.6 mg/L, chlorophyll a concentration 3.2 µg/L.
Some samples were grab-sampled and filtered (47 mm ⌀ GF/F glass microfibre filters from Whatman, Maidstone, England) for independent analysis of the dissolved and particulate fractions. Otherwise, the method we present was optimized for analyzing small volumes of unfiltered water samples from the lakes. Thus, the sampling procedure was simplified compared to more traditional water sampling techniques, aiming for a sampler device with low weight, high transportability, and with the capacity for obtaining samples at different depths up to three meters below the water surface. Other more conventional but perhaps bulkier equipment like a Ruttner Water Sampler can be used for sampling at lower depths. Low enough detection and quantification limits of the analytical method (shown in following sections) allowed the collection of 60 mL water samples using conveniently sized glass vials. A custom tool was built for this purpose which we named GADGET (Graduated Aquatic Depth Grabber for Environmental Testing) (Fig. 2A). It uses a telescopic 3 m clip stick (Beta Climbing Designs, Sheffield, England) and a 3D-printed vial housing and silicone rubber-lined lid held by three tensioned springs that keep the vial shut and water-tight. Once the vial is lowered underwater to the desired depth, the lid is opened by pulling on a rope, water enters the vial, and releasing tension on the rope closes the lid again. This simple mechanism ensures that only water from the desired depth is collected. This sampler design proved to be easy to operate, very effective, and highly transportable, weighing less than 1 kg and measuring 70 cm long when collapsed.
Journal of Chromatography A 2024, 1730, 465063: Fig. 2. Simplified methodologies for sampling and extracting water in alpine sites. A: custom-built GADGET sampler for collecting water at different depths; B: solid-phase extraction equipment configuration for immediate on-site water extraction.
2.3. Extraction
C18 solid-phase extraction (SPE) cartridges were used for extracting the rotenoids from the water samples. Three cartridges were tested: HyperSep (40–60 µm, 500 mg, 6 mL from Thermo Fisher Scientific, Waltham, MA, US), Bond Elut (40 µm, 500 mg, 6 mL from Agilent Technologies, Santa Clara, CA, US), and Empore (50 µm, 35 mg of C18-SD, 6 mL from 3M, Saint Paul, MN, US). Note that the Empore cartridges differ in sorbent amount and presentation, but they were the only C18 SPE cartridge option offered by the supplier. HyperSep C18 SPE cartridges were chosen as the optimal option, as explained in the following sections. The extraction procedure was optimized for allowing on-site extraction, favoring simplicity of operations, portability of materials, and immediacy of extraction after sample collection to avoid degradation. The SPE cartridges were pre-conditioned on-site by successive elution of 2 mL each of acetonitrile, methanol, and HPLC-grade water with the help of a syringe plunger. All discarded solvents were collected for proper disposal. The 60 mL water samples were passed through the SPE cartridges by manually suctioning with a syringe, as shown in Fig. 2B. The extraction of each sample took around 2 min, so the extraction flow was approximately 30 mL/min on average. As shown later, minimal breakthrough was observed, which indicates that the manual extraction did not impact the performance of the SPE cartridges. After extraction, the cartridges were dried to the greatest achievable extent with the syringe plunger, refrigerated during transport, and stored at -20°C. In the laboratory, they were once again dried before extraction and after reaching room temperature by letting air pass through them for 3 to 5 min in a vacuum manifold. The elution was performed with 3 × 2 mL of acetonitrile, which were collected in glass tubes, evaporated under a gentle nitrogen gas stream, transferred into injection vials, further evaporated to dryness, and reconstituted in 50 µL of isooctane and 25 µL of injection standard 1PD in toluene (1.2 ng/µL).
In order to determine the distribution of rotenoids between the dissolved and the particulate (mostly phytoplankton) phase, some CFT-treated samples from the coastal lagoon were filtered beforehand on 47 mm ⌀ GF/F glass microfibre filters. For the analysis of particle-bound rotenoids, the filters were directly spiked with recovery standard, refrigerated during transport, and stored at -20°C until extracted in the laboratory by sonication with 2 × 10 mL of acetonitrile. The extracts were filtered (25 mm ⌀ MGF glass microfibre discs from Sartorius, Göttingen, Germany), concentrated down to 2 mL by rotary evaporation, transferred into injection vials, and then treated like the other extracts.
2.4. Instrumental analysis
The analysis of the extracts was performed with an Agilent 7890A gas chromatograph coupled to a 5975C single quadrupole mass spectrometer (GC–MS) (Agilent Technologies, Santa Clara, CA, US) operating in electron impact mode (70 eV). An HP-5MS fused capillary column was used (60 m × 0.25 mm i.d. × 25 μm, Agilent Technologies). The injector, ion source, quadrupole, and transfer line temperatures were 280, 230, 150, and 280°C, respectively. Helium was used as carrier gas (1 mL/min). The oven program was as follows: 90°C held for 1 min, increased to 150°C at 10°C/min and then to 320°C at 6°C/min, held for 15 min. In full scan mode, all compounds were identified by their retention times and m/z ratios (Table 1) and their quantification was performed from their respective extracted ion chromatograms.
Journal of Chromatography A 2024, 1730, 465063: Table 1. Compounds and GC-MS identification parameters (retention time, tR, and quantification and confirmation mass-to-charge ratios, m/z).
2.5. Evaluation of method performance
The performance of the method and its suitability for the objectives of this work were evaluated based on extraction parameters (breakthrough, recovery), instrumental parameters (fitness of regression), and whole-method analytical parameters (blanks, limits of detection and quantification, accuracy, precision).
The quantification of the amounts of compounds was performed using internal standard power calibration curves (y=axb where, for simplicity in the calculation, y is the ratio between amounts of rotenoid and internal standard and x is the ratio between their respective areas). Six calibration points were used, containing concentrations of ROT, DEG, and ROH between 67 and 10,000 pg/µL, and constant MIL and 1PD concentrations of 836 and 401 pg/µL, respectively.
An assessment of SPE cartridge breakthrough was performed for each cartridge type by extracting lake water samples treated with CFT Legumine (3.3% rotenone) at an approximate concentration of 300 µg/L of rotenone using two SPE cartridges placed in series. The average (n=2) breakthrough was calculated as the amount of each rotenoid in the second cartridge over the total amount in both cartridges, expressed as a percentage. The extraction recovery (percentage of recovered analyte) and precision of the analysis (repeatability expressed as relative standard deviation percent, RSD) of each SPE cartridge type were assessed for those rotenoids with available analytical standards from the analysis of the first SPE cartridges in the series, with additional sample replicates (n=4).
Once the optimal SPE cartridge had been selected and all procedures optimized, the performance of the whole analytical method was further assessed from replicates of filtered alpine lake water samples spiked with ROT, DEG, and ROH analytical standards at three concentration levels: L1 (<2 µg/L, n=6), L2 (10 µg/L, n=3), and L3 (100 µg/L, n=3). The concentration at L1 was chosen so it was below typical piscicidal treatment ranges (5 to 200 µg/L) and below 2 µg/L, the lowest 96-hour median lethal concentration (LC50) among those reported for many tested fish species (i.e., the LC50 reported for the most susceptible tested species. For reference, reported LC50 values frequently exceed 50 µg/L and range up to >1000 µg/L) [27]. Twenty-five microliters of each standard solution, diluted in acetonitrile to the required concentrations, were directly added to the water in the 60 mL vials. Two blanks were performed by omitting the addition of target rotenoid analytical standards. The limits of detection (LOD) and quantification (LOQ) were calculated as the concentrations of analyte corresponding to a signal-to-noise ratio (S/N) of 3 and 10, respectively, determined at the lowest concentration level (L1). The final recovery, accuracy (recovery-corrected quantified amount of analyte expressed as a percentage of the expected spiked amount), and precision (repeatability expressed as RSD percent) of the whole method were calculated at each concentration level.
2.6. Additional performance assessments: effects of degradation and salinity
Although the elution with solvent of rotenoids from the SPE cartridge can be performed on-site immediately after extraction following steps similar to cartridge pre-conditioning, we opted for performing it in the laboratory. To assess whether significant degradation could occur in storage conditions between extraction and elution, the stability of the rotenoids retained in the HyperSep C18 SPE cartridges was tested. Four 60 mL water samples from Lake Collada were spiked with rotenoid standards and simultaneously extracted with SPE cartridges following the procedure described above. One cartridge was eluted every day for four consecutive days beginning on the same day of the extraction. The cartridges awaiting elution were stored at -20°C. This experimental design allowed the direct comparison of relative rotenoid amounts after 0, 1, 2, and 3 days of storage in the SPE columns, with three being the maximum number of days that an extracted sample is expected to remain stored before elution. The resulting extracts were then treated like all other extracts.
The effect of water salinity on the SPE extraction efficiency was also assessed. Different amounts of sea salt mixture (Instant Ocean, Blacksburg, VA, USA) were added to HPLC-grade water, resulting in four salinity levels with three 60 mL replicates per level. The target salinities were 0, 10, 30, and 50 mS/cm, which were approximately achieved after the addition of 0, 1.27, 3.79, and 7.00 g of the salt mixture, respectively. CFT Legumine (3.3% rotenone) was then added to each test sample to achieve a concentration of ROT in water of around 230 µg/L. These samples were extracted, eluted, and analyzed as described previously.
3. Results and discussion
3.1. Identification of rotenoids
Purchasing analytical standards at the time of analysis was economically reasonable for only three out of the five main rotenoids observed in test injections of the CFT Legumine formulation used in this study: ROT, ROH, and DEG. This allowed for the unequivocal identification of these three compounds by comparing retention times and mass-to-charge ratios (m/z) of their most characteristic fragments (Table 1). Two other peaks were observed in the chromatograms at 38.506 and 40.784 min (Fig. 3). The first one was confidently identified as tephrosin (TEP) based on the m/z of its most relevant fragments (208 and 410), its retention time relative to other rotenoids, and its relative abundance in the extracts. Moreover, the degradation behavior observed in some samples also supported this identification: TEP is a hydroxylated degradation product of DEG, similar to how ROH is a hydroxylated degradation product of ROT (Fig. 1), and we observed a comparable increase in concentration of TEP and ROH over time when CFT-treated samples were allowed to degrade.
Journal of Chromatography A 2024, 1730, 465063: Fig. 3. GC-MS extracted ion chromatograms for m/z 192, 208 and 176 (offset by 105 counts from each other for clarity) from a full scan mode injection in a 60 m fused silica capillary column. See Table 1 for compound abbreviations.
3.2. Method optimization
A primary objective of this work was to simplify the extraction of rotenoids from water, enabling on-site processing directly after sampling. This approach aimed to address two key challenges: 1) the potential degradation of rotenone between sampling and analysis, and 2) the logistical difficulty of transporting heavy and bulky sampling and extraction equipment to difficult-to-access sites like high mountain lakes.
Numerous studies in the literature employ nonpolar SPE techniques for the extraction of rotenoids from water, commonly utilizing C18 disks or cartridges [2,[21], [22], [23], [24],26]. We initially tested C18 disks, but they revealed two distinct drawbacks for the present application: the required water volumes at the working rotenone concentrations were too small for effective operation, and the extraction equipment needed precludes direct on-site operation. Consequently, we opted for the more practical C18 SPE cartridges, which were manually operated as depicted in Fig. 2B. Three options, detailed in Table 2, were tested for precision of the analysis, recovery, and breakthrough using CFT-treated water.
Journal of Chromatography A 2024, 1730, 465063:Table 2. Extraction optimization results for three different solid-phase extraction cartridge alternatives. See Table 1 for compound abbreviations.
All cartridge types demonstrated satisfactory performance in extraction tests performed with lake water treated with piscicide (around 300 µg/L). Precision RSDs were consistently below 10%, there was minimal breakthrough of all rotenoids (generally below 1%), and recoveries of ROT, ROH and DEG generally exceeded 70% and reached up to 100%. Based on recovery values alone, the Empore C18-SD cartridges exhibited the highest extraction efficiency. However, the solid phase of this cartridge differed in density and presentation from the other two options, requiring a significantly stronger vacuum during extraction. This complication for the manual on-site extraction proposed in this study led to discarding the Empore cartridges. Considering the relatively similar performance of the remaining two options, HyperSep cartridges were chosen for their ease of extraction and availability at the time of analysis.
3.3. Effect of degradation and salinity on the extraction
Two main factors were identified as likely critical for the extraction and elution of rotenoids in water samples, especially for the intended application to a broad range of water ecosystems with potentially difficult accessibility: degradation of rotenoids in the SPE cartridge and the effect of salinity on the efficiency of the extraction.
The possibility of degradation (mostly biodegradation) of rotenoids retained in the C18 SPE cartridges between on-site extraction and elution in the laboratory could have an impact on their apparent concentrations in water. The analysis of simultaneously extracted, spiked samples that were eluted over four consecutive days showed no evidence of significant degradation of the parent rotenoids (i.e., ROT and DEG). Their relative amounts only varied by -8 to +17% and -9 to +10%, respectively, compared to their levels at day zero. Relative amounts of the degradation product ROH similarly varied by -1 to +18%, and the appearance of the other main degradation product TEP was not observed above limits of detection after three days. None of these variations showed clear increasing or decreasing trends, suggesting that they could be attributed to the variability in the analysis. The ROH/ROT ratio (degradation product/parent compound) showed a similar behavior compared to the DEG/ROT ratio (byproducts present in the piscicide formulation), thus also indicating analytical uncertainty as the source of variation. Three days is the maximum number of days that samples would be stored between on-site extraction and elution in the laboratory (with one single day being the usual time), so these results justify delaying the elution without impacting the results. However, care must be taken for different water bodies with different microbiological characteristics and diversities that could influence this assessment differently.
Since the methodology that we present is intended for application in a wide variety of ecosystems ranging from alpine lakes to brackish coastal lagoons, the effect that the salinity of water could have on the retention of rotenoids in the C18 material was assessed. Previous studies have reported negative impacts of water salinity on the extraction efficiency of ROT [26]. However, we did not observe any obvious and substantial hindrance in the extraction of rotenoids. Fig. 4A shows the percentage change in relative amounts of rotenoids in test water samples spiked with piscicide at four different salinities (conductivities between 0 and 50 mS/cm). There may be changes in relative amounts between samples and conflicting trends seem to exist (e.g., possible decrease in ROT and DEG levels while ROH and TEP levels increase). However, compared to the uncertainty in these analyses, these variations are not enough to claim the existence of a significant effect of salinity on the extraction efficiency of the C18 SPE. A two-way ANOVA with replication (n=3) shows that the salinity factor did not cause the means to be different from each other with enough statistical significance (p=0.135). While we did not notice significant effects on the analysis or results with salinity, or at least not as clear and severe as those previously reported [26], the uncertainty in the analyses is relatively large and may have been affected by it. Thus, no specific conclusions could be drawn that indicate the absence of an impact of salinity either. Still, we argue that the present method can be applied to multiple water bodies besides seawater with enough confidence in its efficiency, always considering that the simplification of the procedure may yield moderate uncertainties in the results.
Journal of Chromatography A 2024, 1730, 465063: Fig. 4. A: Effect of water salinity on SPE extraction efficiency: increase in amount of compound relative to measurement at base salinity level 0 mS/cm (n=3 at each salinity level, error bars show standard deviations). B: effect of degradation on relative rotenoid levels in extracted vs. non-extracted CFT-treated samples under storage conditions (n=3, error bars show standard deviations). See Table 1 for compound abbreviations.
3.4. Method performance
The analytical performance of the whole method (extraction to analysis) was determined from the analysis of lake water samples spiked with ROT, DEG, and ROH standards. The figures of merit of this methodology are reported in Table 3.
Journal of Chromatography A 2024, 1730, 465063: Table 3. Method performance parameters for rotenoids with commercially available standards. LOD and LOQ refer to water volumes before extraction. See Table 1 for compound abbreviations.
Power calibration curves were chosen over linear ones due to their superior performance in the lower concentration range, where samples around the no-effect levels of rotenone (2 µg/L) are expected. That is, linear regressions tend to yield large residuals relative to the low concentrations at the lowest calibration levels. Fig. S2 in the Supplementary materials shows the calibration curves and their residuals both for linear and power regression models. Percent residuals are within 15% of the two lowest calibration levels for the power curves, but go up to 990% for the linear ones. Although residuals at the higher end of the power calibration range are higher in percentage (within 13% compared to 6% for the linear regressions), this method benefits more from being able to quantify lower concentrations more accurately than higher ones. The power regression determination coefficients for all rotenoids were above 0.99.
Both the LOD and LOQ for all three rotenoids were below 2 µg/L of water, except for the LOQ for DEG at 2.5 µg/L. This ensures the effective detection and quantification of rotenone, most CFT Legumine byproducts, and their degradation products even at low levels. This is crucial for monitoring dilution and degradation after treatment application in fish control efforts. Despite employing low volumes of water with no sample pre-treatment, these LOQs were not much higher or within the range of those reported for other procedures in the literature (1–10 µg/L) [2,19,20,24].
The average extraction recoveries were above 80% for ROT and ROH, and slightly lower for DEG at 74%. The recovery for ROH seems notably higher, albeit within reasonable margins of error, than the one obtained in previous tests (Table 2). Although uncertainty probably plays a larger role in this difference, a number of slight adaptations that were made to the extraction and elution for achieving a more streamlined procedure (e.g., improvement to order of operations, increased number of samples processed in parallel, increased pre-conditioning volumes and reduced time between pre-conditioning and extraction) may have contributed. Still, no definite effects could be attributed to specific changes. Compound-specific recoveries were applied for these rotenoids with available analytical standards. For all other rotenoids, in the absence of specific surrogate standards, we suggest that recovery corrections can be adequately estimated using the surrogate standard MIL, which in our extractions showed an average recovery of 74.8±8.2% (n=6). The accuracy of the analysis at all spiking levels normally showed a difference below 10% between expected and measured concentrations, except for ROT (within 15%) and DEG at the lowest concentration level, L1 (just above 20%). The latter case is likely attributed to uncertainties related to the method LOQ for DEG being slightly higher than the concentration at L1. It must also be noted that some replicates at concentration level L1 also fell below the LOQ for ROT and ROH, but only by a maximum amount of 0.2 µg/L. While this may affect the confidence in the reported accuracy and precision values at L1, the results are essentially the same as those obtained for L2 and L3. We believe these figures to be useful and argue that the method can confidently operate at concentrations around 2 µg/L. Precision RSDs at all spiking levels were predominantly below 10%. We consider an accuracy below a 15% difference from expected values and a precision RSD lower than 10% more than adequate for the purpose and application of the simplified methodology presented in this study: the straightforward, immediate, and rapid on-site extraction of rotenoids from areas of challenging access such as alpine lakes.
3.5. Application to samples
The method we present is designed for the periodic measurement of rotenone concentrations in lake waters and its degradation over time following fish eradication treatments, particularly in sites where the immediacy and simplicity of extraction are a priority. Before the full-scale application of this methodology in future treatments, we analyzed the concentration of ROT, ROH, DEG, and TEP in CFT-treated waters from a brackish coastal lagoon. This allowed for an assessment of two critical operational parameters regarding the application of rotenone treatments and the quantification of rotenone levels: the degradation of rotenone in non-extracted, CFT-treated samples during storage and the distribution of rotenoids between the dissolved and particulate phases.
For the assessment of rotenoid degradation, CFT-treated water was extracted and analyzed in triplicate at two different times: immediately after sampling at the coastal sampling site and four days later, after storage of the water sample in an amber glass bottle refrigerated at 4°C in a dark, cold chamber. These samples were not filtered, so the results of their analysis likely represent bulk concentrations (dissolved + particle-bound). It is worth noting that, at the small sample volumes for which the method is designed, the SPE extraction could adequately handle the particulate phase without clogging even in this extreme application scenario that is a eutrophic lake. Fig. 4B illustrates the percentage difference in the relative amount ratios of three pairs of rotenoids between both analyses. The DEG/ROT ratio remained unchanged after four days in storage (+1%), as both products are present in the CFT Legumine piscicide formulation. In contrast, the ratios of the two pairs of degradation product/parent compound increased significantly (+67% ROH/ROT, +89% TEP/DEG), suggesting that ROT was likely being degraded into ROH and DEG into TEP. Moreover, the calculated ROT concentration between both samples decreased by approximately 51%, from 70±16 µg/L to 36±4 µg/L. This indicates that degradation (most likely microbiological degradation) of the parent rotenoids is prominent even in samples that are kept refrigerated and in the dark for just a few days. This finding supports the argument for performing a quick on-site extraction of water samples to accurately track in-lake degradation and dissipation profiles of rotenone in fish control treatments since, as we have shown, rotenoids remain mostly stable in SPE cartridges after extraction.
The analysis of these piscicide-treated waters also allowed the assessment of rotenoid distribution between the dissolved and particulate phases, so we applied our method to filtered samples from the coastal lagoon. The distribution of rotenoids between phases merits consideration, as the success of a fish eradication treatment with rotenone greatly depends on achieving effective concentrations in the dissolved phase of water. The freely-dissolved fractions were above 85% for all rotenoids, which is reasonable since they are relatively polar compounds and are expected to remain mostly dissolved. In agreement with that, ROH and TEP showed the highest relative abundances in the dissolved phase (96% and 92%, respectively) due to their higher water solubility. Still, a fraction of 5 to 15% of compounds trapped in the particulate phase could reduce the effectiveness of fish eradication treatments, especially in highly eutrophic lakes, and must be considered during application of the piscicide.
4. Conclusions
We present a simple procedure for analyzing rotenone and other rotenoid byproducts and degradation products in fresh and brackish waters, specifically designed for monitoring fish control and eradication treatments in areas of challenging access and no energy supply like high-altitude alpine lakes. A collapsible and lightweight custom sampling device was constructed to collect 60 mL samples at various depths below the water surface. The water extraction methodology was streamlined through the use of C18 SPE cartridges, enabling on-site operations to minimize the degradation of rotenone between sampling and analysis. An assessment of rotenoid stability once retained in the SPE phase showed that no significant degradation seems to occur for the first few days between sample extraction and extract elution. While the salinity of water (in a range between 0 and 50 mS/cm) did not seem to have an obvious effect on the efficiency of the extraction, it may have had one on its precision. Thus, no conclusive evidence could be found to support the opposite.
The analysis was performed by GC-MS, which provided enough resolution for monitoring rotenone degradation profiles and the emergence of degradation products following piscicide applications. The resulting method is both simple and effective, enabling the detection and quantification of rotenoids with LOD and LOQ of approximately 0.4 and 1.5 µg/L, respectively. Average recoveries exceeded 80%, and the method demonstrated average accuracy and precision RSD ranging from 78 to 103% and 3 to 14%, respectively, across various rotenoid concentrations (2 to 100 µg/L). These performance metrics are deemed more than satisfactory for the intended application of this procedure.
The advantages of this method were evident during its application to the analysis of piscicide-treated water samples, which revealed significant degradation of parent rotenoids within a few days under dark and refrigerated storage conditions. Instead, the simplified on-site solid phase extraction proposed here allowed a much more accurate determination of the concentrations of not only rotenone, but also other rotenoid byproducts and degradation products. Additionally, the degree of distribution of rotenoids towards the particulate phase of water was found to be potentially relevant for the correct application of piscicide treatments, especially in eutrophic lakes. Regardless, the proposed method is versatile and capable of analyzing bulk rotenone concentrations (dissolved + particle-bound) in unfiltered water without saturation during the extraction.
- Simple on-site extraction and GC-MS analysis of rotenone and degradation products for monitoring invasive fish eradication treatments in fresh and brackish waters. Raimon M. Prats, Marc Ventura, Quim Pou-Rovira, Teresa Buchaca, Pilar Fernández, Joan O. Grimalt, Barend L. van Drooge. Journal of Chromatography A 2024, 1730, 465063. https://doi.org/10.1016/j.chroma.2024.465063.
