In this work, an integrative passive sampler based on a silicone membrane filled with a suspension of γ-Fe2O3 at pH 3.5 was developed. The novel device was calibrated for the measurement of microcystin concentrations in water. Laboratory calibration studies of the passive sampling devises under controlled conditions of temperature, water turbulence, and analyte concentration were conducted in order to establish how variable environmental conditions affect the novel sampler's performance. The chemical uptake of microcystin (MC)-RR, -LR, and -YR into the passive sampler remained linear and integrative throughout the 28-day exposure. The relative standard deviations of mean concentrations obtained using silicone-based sampler ranged from 1.42 to 3.74% for microcystin-LR, -RR, and -YR. The values for reproducibility from triplicate samplers ranged from 3.5 to 7.1% for microcystin-LR, -RR, and -YR. The detection limits on high performance liquid chromatography (HPLC) with PDA detection for microcystins LR, RR, and YR were 24.7, 17.2, and 23.8 μg L−1 respectively, calculated as three times the signal to noise ratio. The rate of accumulation of most of the MC compounds tested was dependent on temperature and flow velocity. Furthermore, the sample matrix, e.g. humic substances, had no significant effect on the concentration of compounds trapped in the acceptor solution and once these MC compounds were trapped in the acceptor phase they did not diffuse back during the deployment period.
The aim of this work is to develop a passive, in situ, integrative sampler for monitoring of biotoxins in water bodies based on a functionalised silicone membrane. The specific objectives of this project are as follows:
To synthesise and characterise γ-Fe2O3 nanoparticles.
To functionalise the silicone membrane by filling it with γ-Fe2O3 nanoparticles for the purpose of using it to sample cylindrospermopsin and MCs at pH values (3 < pH < 12), as demonstrated by Lee & Walker (2011).
To devise a constant concentration flow through exposure system to allow calibration of the passive sampling devices to be made under controlled conditions of temperature, water turbulence and analyte concentration.
To demonstrate the potential of modified silicone membrane for passive sampling of cylindrospermopsin and MCs-LR, -YR, and -RR in water bodies.
Materials and methods
Chemicals and materials
CyanoBiotech GmbH, MC standards (MC-LR, MC-RR, and MC-YR) were purchased from Germany and supplied by Industrial Analytical (Pty) Ltd, South Africa. Other solvents and reagents used in this work were of high purity (Analytical grade and/or high performance liquid chromatography (HPLC) grade, >99%) and were purchased from Sigma-Aldrich (South Africa) and Merck (South Africa). Solid-phase extraction SPE cartridges (Oasis™ HLB cartridges, Water and ODS C-18 cartridges) and C18 Empore disks, 47 mm diameter, were purchased from Waters, Inc., USA, supplied by Microsep (Pty) Ltd, South Africa. Silicone membranes were obtained from Technical Products, Inc. (Georgia, USA).
With regards to the relatively large amounts of MCs needed for calibration experiments, and high costs of the toxin analytical standards, MCs for the experiments were isolated from the natural cyanobacterial biomass, collected into amber glass bottles from Hartebeespoort dam, by repeated extraction with 50% methanol and partial purification with solid-phase extraction using an ODS cartridge. The final extract (in 75% v/v methanol: water), containing dominant MC variants, was aliquoted and stored at −18 °C.
Synthesis of iron oxide (maghemite) nanoparticles
Flow through exposure system
Fabrication of passive sampling devices
Silicone-membrane/γ-Fe2O3-nanoparticle-sorbent-based passive sampler
Silicone membranes used for the optimization process were bought as long tubes and cut to appropriate lengths (48 cm × 0.1575 cm ID × 0.2413 cm OD giving a volume of 0.9349 cm3 ∼1000 μL). Eighteen silicone membranes, previously soaked in deionised water, were filled with a pH 3.5 acceptor buffer of the maghemite suspension (2.3 g L−1) using a 1000 μL micropipette. The membranes were tightened together and made in the form of a loop about 5 cm in diameter. The outside was rinsed with deionised water thoroughly to remove any buffer spills. After exposure, the samplers were taken out of the sample vessel, the outside flushed with deionised water, and contents transferred into a 1.5 mL vials. Prior to HPLC analysis, the extracts were sonicated in a Branson 5800 ultrasonic bath (Danbury, USA) for 5 minutes and then centrifuged using an Eppendorf 5430 centrifuge (Hamburg, USA) for 10 minutes at 7500 rpm, and the supernatants were filtered through 0.45-μm polyvinylidene fluoride (PVDF) membrane syringe filters. The extracts were either analysed immediately or stored in the refrigerator at −18 °C.
Polar organic chemical integrative sampler
The commercial polar organic chemical integrative sampler (POCIS) samplers (pharmaceutical configuration) for the comparison experiment were obtained from Exposmeter AB (Tavelsjo, Sweden). In-house made POCIS samplers (pharmaceutical configuration) for the comparison experiment were fabricated in the laboratory. These POCIS samplers contained 300 mg of Oasis HLB sorbent enclosed between two polyethersulphone (PES) membranes. The membrane-sorbent-membrane layers were compressed between two holder washers. The total exchange surface area of the membrane (both sides) was ∼41 cm2 and the surface area per mass of sorbent ratio was ∼300 cm2 g−1. Thereafter, exposed samplers were taken out of the sample vessel, the outside flushed with deionised water and the holder disassembled. Membranes with the enclosed sorbent were transferred into a 15 mL centrifuge tube and extracted two times with 5 mL of aqueous methanol (90% v/v acidified with 0.1% trifluoroacetic acid) for 15 minutes in an ultrasonic bath. After centrifugation (10 minutes at 7500 rpm); supernatants were pooled, evaporated to dryness at 40 °C under a stream of nitrogen and reconstituted with 500 μL of HPLC grade methanol. The extracts were either analysed immediately or stored in the refrigerator at −18 °C.
Solid phase extraction technique
Optimisation of extraction parameters
Influence of maghemite suspension on the sampler performance
In order to assess the influence of maghemite suspension on the uptake of the MC compounds by the silicone tube, fifteen tubes filled with a pH 3.5 acceptor buffer without the maghemite suspension (control samplers) were exposed alongside fifteen POCIS. The exposures lasted for 28 days, during which triplicate samplers of both samplers under investigation were removed at set time intervals (0, 7, 14, 21, and 28 days). Every time a POCIS was removed for analysis it was replaced by an empty sampler body. Grab samples of water (100 mL) from the outlet of exposure tank were also taken each time the samplers were removed, and the concentration of biotoxins in the water pre-concentrated by an SPE technique prior to HPLC PDA according to the ISO 20179 method (ISO20179 2005). Figure 4 shows a depiction of the experimental set-up to measure the uptake of target analytes at different combinations of exposure times, temperatures, sample matrix and hydrodynamic conditions.
In order to assess the influence of exposure time on the uptake of the MC compounds, the silicone membrane-based passive samplers filled with a suspension of iron oxide nanoparticles were exposed for 0, 7, 14, 21, and 28 days. Deionised water spiked with 50 ng mL−1 mixtures of MCs was extracted at ambient temperature. In these experiments, up to fifteen silicone membranes, previously soaked in deionised water were used. After exposure, a set of three passive samplers were treated in the same way as described in the extraction procedure.
Effect of hydrodynamics on the uptake kinetics of MCs
The effect of hydrodynamics on the uptake rates of individual MC compounds into the samplers was studied at six different stirring speeds (i.e. 0, 20, 40, 60, 80, and 100 rpm). Deionised water spiked with 50 ng mL−1 mixtures of MCs was extracted for 14 days at ambient temperature. In these influence-of-hydrodynamic experiments, up to 18 silicone membranes, previously soaked in deionised water, were filled with a pH 3.5 acceptor buffer of the maghemite suspension (2.3 g L−1). A set of three passive samplers were exposed in appropriate stirring speeds. After exposure, the passive samplers were treated in the same way as described in the extraction procedure.
Effect of temperature on the uptake kinetics of MCs
The relationship between sampling rates of three MC compounds and temperature was compared at four temperatures (4, 17, 23, and 40 °C). In these influence-of-temperature experiments, up to twelve silicone membranes, previously soaked in deionised water, were filled with a pH 3.5 acceptor buffer of the maghemite suspension (2.3 g L−1). A set of three passive samplers were exposed in appropriate temperature-controlled systems. Deionized water containing ±50 ng mL−1 of a mixture of MCs was used as sample solution. Four sample vessels water were used: (1) vessel placed in a cooler box filled with ice and maintained at 4 °C, (2) vessel maintained at 17 °C, (3) vessel with a heating element set at 23 °C and (4) vessel heated and maintained at 40 °C. Before exposing the silicone-membrane/γ-Fe2O3-passive samplers, the water samples were allowed to equilibrate for at least 2 hours at appropriate temperature. Passive exposure period was for 14 days.
Effect of humic substances on the uptake kinetics of MCs
To study the effects of sample matrix on the performance of the passive sampler, deionised water containing ±50 ng mL−1 of a mixture of MCs and 10 mg L−1 of humic substances was used as a sample solution. This sample solution was extracted with three silicone membranes filled with a pH 3.5 acceptor buffer of the maghemite suspension (2.3 g L−1). Exposure period was 14 days. Afterwards, the sampling rates obtained were compared with sampling rates of samplers exposed to deionised water without any humic substances.
Effects of humic substances on sampler performance
To study whether compounds trapped in the acceptor phase can diffuse back, a series of experiments were performed. A buffer solution of the maghemite suspension (2.3 g L−1) was spiked with 1.0 mg L−1 of MC compounds and filled into the three silicone membranes as before. These were deployed in deionized water spiked with 10 mg L−1 of humic substances only. The exposure period was 14 days. The contents of the acceptor solutions was analysed to check for any loss of MCs from the acceptor solution.
Chromatographic separation of MCs
Extract were analysed by the Surveyor Plus™ modular LC system and the ChromQuest™ data system, products of Thermo Fisher Scientific San Jose, on a 150 mm × 4.6 mm, 5 μm column (waters) at 30 °C with a mobile phase composition of 60% water + 0.1% trifluoroacetic acid and 40% acetonitrile + 0.1% trifluoroacetic acid at a flow rate of 1.0 mL min−1. Chromatograms at 238 nm were recorded with the Surveyor PDA Plus Detector, and MCs were identified by retention time and characteristic UV absorption spectra (200–300 nm). Quantification was based on external calibrations of MC-RR, -LR, and -YR, respectively.
In ensuring that the concentration determined using the sampling devices reflect the true picture in the aquatic media, quality control procedures to address issues such as contamination and loss of the trapped analytes, accuracy and precision of the results were conducted. Inspection for signs of puncture, discoloration, or any malfunctioning upon retrieval to see any possible sources of contamination and/or loss of the trapped analytes (EPA 1986; Paschke et al. 2006) was performed. Procedural blanks, certified reference materials, and control samples were used for identification of the contamination from the process (EPA 1986; Paschke et al. 2006).
RESULTS AND DISCUSSION
Identity of the synthesised iron oxide (maghemite) nanoparticles
The identity and purity of the synthesised iron oxides was verified by XRD, TEM, SEM/EDS, surface zeta potential analyser, and N2-BET.
X-ray diffraction of synthesised iron oxide) nanoparticles
Transmission electron microscopy of synthesised iron oxide nanoparticles
Surface zeta potentials and N2-BET of synthesised iron oxide nanoparticles
Scanning electron microscopy with energy dispersive X-ray spectroscopy
Flow through exposure system
Suitability studies for integrative sampling: silicone membrane vs. POCIS
Optimization of novel silicone membrane passive sampler
When passive samplers are deployed in the environment, they accumulate micro-pollutants. Chemicals diffuse from the bulk water through the boundary layer to the sampler surface and then partition between the sampler and the water. Depending on the sampler design, the mass of pollutant accumulated by a sampler reflects either the equilibrium or the time-averaged concentration. The use of integrative passive samplers enables direct estimation of time weighted average (TWA) concentrations provided the sampling rates are known (Petty et al. 2004; Alvarez et al. 2004; Namiesnik et al. 2005).
The limits of detection for MCs LR, RR, and YR were 24.7 μg L−1, 17.2 μg L−1, and 23.8 μg L−1, respectively, calculated as three times the signal to noise ratio. Comparing these data with published data for POCIS (Kohoutek et al. 2010); the sensitivity of the silicone based sampler is lower than POCIS. This could be due to the fact that hydrophilic MCs have large molecular mass (∼1000 Da) and thus do not easily pass across a silicone membrane into the receiving phase. However, mass transfer processes of these biotoxins across a silicone membrane are dependent on the physical form of the biotoxin molecules and the temperature. Geinoz et al. (2002), reported that exposure of silicone material to solvents may result in some swelling of the membrane, thus allowing the permeation of even larger compounds through the material.
Effect of temperature
At elevated temperature of 40°C, the cyclic heptapeptide MCs are de-natured and this is seen by the decline of the analyte concentration trapped in the sampler. Therefore, from the results obtained it is quite clear that increased temperature of the environmental media can to some extent enhance mass transfer of MC compounds into the silicone membrane based passive sampler. This is in agreement with what Harada et al. (1996) reported about MCs slowly breaking down at high temperature (40 °C), and at either very low (<1) or high (>9) pH. Tsuji et al. (1995) also reported that MCs’ half-life at pH 1 and 40 °C is 3 weeks, but this can stretch to 10 weeks at typical ambient conditions. In full sunlight, especially when water-soluble pigments are present, these toxins also break down slowly. Although MCs and can be broken down by some bacterial proteases, in many circumstances these bacteria are not present in the cooler, dark, natural water bodies (Jones et al. 1995; Rapala et al. 2005). Thus, these toxins may persist for months or even years once released in such areas. MCs can still persist after boiling, indicating that cooking is not sufficient to destroy them (WHO 1999).
Figure 12(b) is a typical chromatogram (HPLC–PDA) obtained after passive extraction of deionised water (50 L) spiked with 50 μg L−1 mixture of MC-RR, -LR, and -YR compounds.
In this study, one sampler consisted of a polyethersulphone limiting membrane while the other consisted of polyethylene. Both of these samplers used the same 47 mm C18 Empore disk as receiving phase. In both cases, an increase in water temperature led to an increase in sampling rate. The effects of temperature on sampling rates have also been observed in semi-permeable membrane devices (SPMDs) (Yusà et al. 2005) and in membrane enclosed sorptive coating (MESCO) sampler (Vrana et al. 2001). For practical purposes, it is therefore necessary to determine the effects of temperature in the laboratory for each compound of interest and also measure the temperature during field deployment.
Effect of hydrodynamics on sampler's uptake kinetics
Effects of humic substances on sampler's performance
An alternative and more informative, cost-effective approach has been developed and calibrated in the laboratory to obtain a time-weighted average (TWA) concentration of MCs, which forms a fundamental part of an ecological risk assessment process. The potential of the silicone membrane functionalised with γ-Fe2O3 nanoparticles for passive sampling of MC-LR, -YR, and -RR in water bodies has been demonstrated. Variable environmental conditions (e.g. effects of temperature, sample matrix, and hydrodynamics) were found to affect the sampler performance. Furthermore, the sample matrix, e.g. humic substances, do not have an effect in the concentration of compounds trapped in the acceptor solution. More research is necessary to provide an understanding of the effect of bio-fouling on the sampler performance. Other future research will focus on incorporating the performance reference compounds (PRC) concept into the sampler configurations and bioassays in the trapping media as well as field application to test the sampler performance alongside spot sampling and commercially available passive samplers.
The authors would like to thank the financial support from the Nano-Science Centre, Department of Applied Chemistry, University of Johannesburg as well as their valuable inputs, seminars and guidance.