This research project comprises two parts, describing the development of methods for the extraction and measurement of mercury (Hg) species in marine sediments and fish tissues and selenium (Se) species in marine fish tissues. Two optimised methods for the speciation analysis of Hg and Se were developed. The release of Hg and Se from geological sources has been accelerated over the years due to anthropogenic activities. These elements when released into the atmosphere are deposited in the sediments and transferred to aquatic food chains making them bioavailable to fish and other organisms in aquatic ecosystems. The measurement of total Hg and Se is essential to provide elemental concentrations, but it is insufficient information as the biological actions of Hg and Se are dependent on the physical properties of their various chemical species. Mercury is converted into the toxic methyl mercury (MeHg (I)) form by undergoing biogeochemical transformation processes and bioaccumulates and biomagnifies in aquatic food webs to concentrations of toxicological concern. Selenium is taken up by animals and mainly present as selenomethionine (SeMet) and selenocysteine (SeCys) bound within proteins. Although Se is an essential element required in our diet at certain concentrations, high doses can be very toxic. Speciation information is, therefore, required to understand the biogeochemical cycling of the Hg and Se species in the environment and assessing their ecotoxicological risk. Part A: Development of a method to measure mercury species in marine sediments and fish tissues A procedure for the extraction and determination of MeHg (I) and inorganic mercury (Hg (II)) in sediments and fish muscle tissues using High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS) after extraction using 2-mercaptoethanol was developed. Various extraction protocols were applied for the extraction of MeHg (I) and Hg (II) from estuarine sediment certified reference material CRM BCR 580 to achieve the best recoveries of Hg species. Initially the extraction of Hg species from sediment CRM BCR 580 was carried out using thiourea, l-cysteine or 2-mercaptoethanol solutions because of the affinity of Hg towards thiol groups. The best recoveries were achieved using aqueous 2-mercaptoethanol. The effect of sample mass,2-mercaptoethanol concentration, and extraction time and extraction temperature were evaluated using recoveries of MeHg (I) concentrations from sediment reference material CRM BCR 580. The optimum extraction procedure was the use of 0.2 g of the sediment with 5 ml of 0.5% (v/v) mercaptoethanol and an extraction temperature of 120°C and an extraction time of 15 min. The HPLC separation of Hg species (Hg (II) and MeHg (I)) was also evaluated. When a mobile phase containing cysteine is used, Hg (II) elutes before MeHg (I) allowing the measurement of higher MeHg (I) concentrations in samples such as fish tissues which contain low Hg (II) concentrations. For sediment samples, however, elution of MeHg (I) species prior to the Hg (II) species is required for accurate quantitation of this species to avoid peak tailing and memory effects in the ICPMS due to the low MeHg (I) concentrations and very high Hg (II) concentrations (e.g. sediment CRM BCR 580 contains total Hg 132 ± 3 mg kg-1 and MeHg (I) 75.5 ± 3.7 μg kg-1 on a dry mass basis). The very high concentrations of Hg (II) in the sediments samples will then lead to severe peak tailing over the MeHg (I) peak if eluted first on the HPLC column. Various mobile phases and a variety of C8 or C18 HPLC columns were trialled to evaluate the separation and elution of Hg species. When 2-mercaptoethanol was used as an extractant and present in the mobile phase, MeHg (I) elutes before Hg (II). A mobile phase containing 0.5% (v/v) 2-mercaptoethanol and 5% (v/v) MeOH (pH 5.5) with the Perkin-Elmer 3 μm C8 (30 mm × 3 mm) HPLC column at a flow rate 1.5 ml min-1 at 25°C in conjunction with ICPMS gave the best separation of Hg species. The recoveries obtained for the sediment CRM BCR 580 using 0.5% (v/v) 2-mercaptoethanol were 92 ± 3%. Spike recoveries with 5,10,20 and 40 μg g-1 of MeHg (I) gave recoveries of 82 ± 12,92 ± 3,96 ± 1 and 89 ± 2%,respectively,for the sediment CRM. MeHg (I) concentrations of biological certified reference materials, NRCC Dorm-2 Dogfish muscle, Dogfish liver NRCC DOLT-3,Albacore tuna NIST RM, tuna fish IRMM IMEP-20 and a range of fish tissues were also measured after extraction with 2-mercaptoethanol and separation using this mobile phase. The MeHg (I) concentrations of biological certified reference materials were in agreement with the certified values after extraction with 2-mercaptoethanol. The recoveries of the Hg species after 2-mercaptoethanol extraction were compared against an enzymatic hydrolysis using protease type XIV which has been previously published. Similar results for the Hg species concentrations were obtained for both extraction procedures. An alternative method for the separation of Hg species in the enzymatic extracts of fish tissues was also developed using High Performance Liquid Chromatography - Hydride Generation Atomic Absorption Spectrometry (HPLC-HGAAS). The effect of HCl and NaBH4 concentrations and gas flow for the generation of hydrides were trialled to achieve maximum absorbance signal for Hg species on the HGAAS. The best absorbance signals were obtained when 2 % (v/v) HCl,0.75 % (w/v) NaBH4 in 0.05% NaOH (w/v) were used with a gas flow of 0.7 ml min-1. A mobile phase containing 0.06 M ammonium acetate in 5% (v/v) methanol and 0.1% (w/v) l-cysteine at 25 ◦C with a Phenomenox 5μm Luna C18 (250 mm × 4.6 mm) column resulted in good separation of Hg species with MeHg (I) peak eluting after the Hg (II) peak. This elution order of the peaks allowed accurate quantification of higher MeHg (I) concentrations in the fish tissues. The enzymatic extracts of fish tissues was when analysed by both HPLC-ICPMS and HPLC-HGAAS approaches gave equivalent results. Part B: Development of method to measure selenium species in marine fish tissues Initially, marine fish tissue CRMs Lobster hepatopancreas NRCC TORT, Dogfish liver NRCC DOLT and Oyster tissue NIST 1566b were extracted and total Se concentrations were measured with the aim of evaluating the efficiency of different extraction procedures. Aqueous leaching, enzymatic hydrolysis, acidic leaching with ultrasound or microwave assisted heating and conventional heating were applied to extract Se from certified reference marine tissue materials. Better recoveries were obtained with the use of methanesulfonic acid as an extractant (89 - 92 %). Due to the low pH values, however, the extraction solution is not compatible with most HPLC columns. Also, degradation of Se species (SeMet and SeCys) occurs in solution with low pH values. Enzymatic hydrolysis gave recoveries of 63 - 67 %,however, only SeMet was stable in the extract, while, SeCys was degraded. Concentrations of SeMet were measured using anion exchange PRP X100 (4.1 × 250 mm,10 μm) column with a mobile phase containing 20 mM ammonium phosphate, pH 4.6 at a flow rate of 1.5 ml min-1 at 40°C as a separation technique after enzymatic hydrolysis. Separation of SeCys and SeMet in the enzymatic extract was also carried out using a cation exchange Supelco SCX (4.1 × 250 mm,10 μm) column with a mobile phase containing 20 mM pyridine formate, pH 2.5 at a flow rate of 1.5 ml min-1 at 40°C. The concentrations of Se species (SeMet and SeCys) measured were similar by using both types of chromatography.
|Date of Award
|Bill Maher (Supervisor), Simon Foster (Supervisor) & Michael J. Ellwood (Supervisor)