Frank Krikowa

A microwave-assisted sequential extraction of water and dilute acid soluble arsenic species from marine plant and animal tissues

This paper describes the use of dilute nitric acid for the extraction and quantification of arsenic species. A number of extractants (e.g. water, 1.5M orthophosphoric acid, methanol-water and dilute nitric acid) were tested for the extraction of arsenic from marine biological samples, such as plants that have proved difficult to quantitatively extract. Dilute 2% (v/v) nitric acid was found to give the highest recoveries of arsenic overall and was chosen for further optimisation. The optimal extraction conditions for arsenic were 2% (v/v) HNO(3), 6 min(-1), 90 degrees C. Arsenic species were found to be stable under the optimised conditions with the exception of the arsenoriboses which degraded to a product eluting at the same retention time as glycerol arsenoribose. Good agreement was found between the 2% (v/v) HNO(3) extraction and the methanol-water extraction for the certified reference material DORM-2 (AB 17.1 and 16.2microg g(-1), respectively, and TETRA 0.27 and 0.25microg g(-1), respectively), which were in close agreement with the certified concentrations of AB 16.4+/-1.1microg g(-1) and TETRA 0.248+/-0.054microg g(-1). To preserve the integrity of arsenic species, a sequential extraction technique was developed where the previously methanol-water extracted pellet was further extracted with 2% (v/v) HNO(3) under the optimised conditions. Increases in arsenic recoveries between 13% and 36% were found and speciation of this faction revealed that only inorganic and simple methylated species were extracted.

Arsenic Species Determination in Biological Tissues by HPLC–ICP–MS and HPLC–HG–ICP–MS

The use of high-pressure liquid chromatography coupled directly or by a hydride generation system to an inductively coupled plasma mass spectrometer for the unambiguous measurement of 13 arsenic species in marine biological extracts is described. The use of two chromatography systems; a Supelcosil LC-SCX cation-exchange column eluted with a 20 mM pyridine mobile phase adjusted to pH 2.2 and 2.6 with formic acid, with a flow rate of 1.5 mL min−1 at 40°C, and a Hamilton PRP-X100 anion-exchange column eluted with 20 mM NH4H2PO4 buffer at pH 5.6, with a flow rate of 1.5 mL min−1 at 40°C, was required to separate and quantify cation and anion arsenic species. Under these conditions, arsenous acid could not be separated from other arsenic species and required the use of an additional hydride generation step. Arsenic species concentrations in a locally available Tasmanian kelp (Durvillea potatorum), a certified reference material (DORM-2), and a range of commercially available macroalgae supplements and sushi seaweeds have been measured and are provided for use as in-house quality control samples to assess the effectiveness of sample preparation, extraction, and measurement techniques.

Measurement of Trace Elements in Marine Environmental Samples using Solution ICPMS. Current and Future Applications

The strengths and weaknesses of using inductively coupled plasma mass spectrometer (ICPMS) measurements of samples in solution for marine environmental analyses using real world examples is discussed. ICPMS can detect nanogram per litre concentrations of trace elements but suffers from polyatomic interferences generated from the sample matrix. Most of the routine trace elements measured in marine biological tissue and sediment digests, with the notable exceptions of iron, chromium, vanadium, and selenium, are not subject to severe interferences. Low recoveries of trace elements from sediments are due to the inability of extraction acids to remove trace elements such as chromium and nickel from sediment matrices. The use of ICPMS offers the advantage that elements such as phosphorus, which previously required elaborate digestion procedures and a colorimetric determination, can be rapidly determined using nitric acid digestion alone. The use of flow injection coupled with ICPMS allows on-line preconcentration of trace metals and metalloids using chelation by ion-exchange resins or hydride generation and trapping as well as separation from matrix elements. Thus, the routine determination of trace elements and inorganic and methylated arsenic, antimony, mercury, and germanium species in open-ocean waters is possible. The coupling of HPLC and GC to ICPMS allows the measurement of metal and metalloid species in biological and sediment extracts. However, extraction of unaltered species from matrices presents a challenge. Many of the species found in the environmental samples are not known and analytical standards are not available. The concurrent use of HPLC-MS is needed to confirm these species.

Arsenic Species in a Rocky Intertidal Marine Food Chain in NSW, Australia, revisited

Environmental Context. The pathways by which arsenic is accumulated, biotransformed and transferred in aquatic ecosystems are relatively unknown. Examination of whole marine ecosystems rather than individual organisms provides greater insights into the biogeochemical cycling of arsenic. Rocky intertidal zones, which have a high abundance of organisms but low ecological diversity, are an important marine habitat. This study examines the cycling of arsenic within intertidal ecosystems to further understand its distribution and transfer. Abstract. The present study reports total arsenic and arsenic species in a short rocky intertidal marine food chain in NSW, Australia. Total mean arsenic concentrations increased up the food chain in the following order: 4 ± 2 µg g–1 in attached rock microalgae, 31 ± 14 µg g–1 in Bembicium nanum Lamarck, 45 ± 14 µg g–1 in Cellana tramoserica Sowerby, 58 ± 14 µg g–1 in Nerita atramentosa Reeve, 75 ± 15 µg g–1 in Austrocochlea constrica Lamarck (a herbivore) and 476 ± 285 µg g–1 in the carnivore Morula marginalba Blainville. Significant differences in arsenic concentrations of B. nanum, N. atramentosa and M. marginalba were found among locations and may be related to food availability, spawning or differences in age and/or size classes of individuals. Significant differences in arsenic concentrations were also found within locations among species, and increased in the order: rock microalgae < B. nanum < C. tramoserica < N. atramentosa < A. constricta < M. marginalba. Although small differences in total arsenic concentrations were found among locations for some gastropod species, arsenic species proportions were very consistent within gastropod species across locations. The majority of arsenic in Homosira banksii (macroalgae) was oxo-arsenoribosides, with thio-arsenoribosides making up ~10% of the total methanol–water extractable arsenic. The rock microalgae contained arsenobetaine (AB) (59 ± 5%) and arsenoribosides (36 ± 15%). The AB content of the herbivores B. nanum, N. atramentosa and A. constricta ranged from 71 to 95%, and that of the carnivore M. marginalba was 98%. Most gastropods contained thio-arsenosugars (up to 13 ± 3% of total extractable arsenic), with C. tramoserica containing higher proportions of thio-phosphate arsenoriboside (7 ± 2%) and lower proportions of AB (69 ± 4%). Glycerol trimethylarsonioribosides (1.4 ± 0.1%) were also found in most of the herbivorous gastropods. Oxo-dimethylarsinoylethanol (oxo-DMAE) was found in N. atramentosa (<1%).

Changes in proportions of arsenic species within an Ecklonia radiata food chain

Environmental context. The present study examines arsenic species in kelp and associated grazing animals of an Ecklonia radiata food chain. The study focusses on the changes in proportions of arsenoribosides obtained from E. radiata and mechanisms are proposed to explain the transformations of arsenoribosides observed in the organisms that graze on it. Abstract. Total arsenic and arsenic species in the tissues of three growth stages of the macroalgae Ecklonia radiata and within organisms that feed on it are reported. Arsenic concentrations in E. radiata tissues varied from 40 to 153 μg g–1. Growth stage did not influence arsenic concentrations or arsenic species. E. radiata contained glycerol arsenoriboside (1–8.5%), phosphate arsenoriboside (10–22%) and sulfonate arsenoriboside (73–91%). Arsenic concentrations varied significantly among animal species and between tissues (5–123 μg g–1). Animals contained variable quantities of arsenobetaine (14–83%). Haliotis rubra tissues contained high concentrations of glycerol trimethylarsonioriboside (0.7–22%) and the fish Odax cyanomelas contained large quantities of phosphate arsenoriboside (25–64%) with little arsenobetaine (1.5–15%). Arsenoribosides consumed from macroalgae are substantially converted or differentially accumulated as glycerol and phosphate arsenoribosides in animal tissues. In all animals, phosphate arsenoriboside would appear to be conserved or synthesised de novo. In gastropods, glycerol trimethylarsonioriboside and thio arsenic species are formed in the digestive system. Thus, the intermediate arsenic species that form a plausible pathway for the formation of arsenobetaine from dimethylarsenoribosides are present.

Measurement of methyl mercury (I) and mercury (II) in fish tissues and sediments by HPLC-ICPMS and HPLC-HGAAS.

A procedure for the extraction and determination of methyl mercury and mercury (II) in fish muscle tissues and sediment samples is presented. The procedure involves extraction with 5% (v/v) 2-mercaptoethanol, separation and determination of mercury species by HPLC-ICPMS using a Perkin-Elmer 3 μm C8 (33 mm×3 mm) column and a mobile phase 3 containing 0.5% (v/v) 2-mercaptoethanol and 5% (v/v) CH(3)OH (pH 5.5) at a flow rate 1.5 ml min(-1) and a temperature of 25°C. Calibration curves for methyl mercury (I) and mercury (II) standards were linear in the range of 0-100 μgl(-1) (r(2)=0.9990 and r(2)=0.9995 respectively). The lowest measurable mercury was 0.4 μgl(-1) which corresponds to 0.01 μgg(-1) in fish tissues and sediments. Methyl mercury concentrations measured in biological certified reference materials, NRCC DORM - 2 Dogfish muscle (4.4±0.8 μgg(-1)), NRCC Dolt - 3 Dogfish liver (1.55±0.09 μgg(-1)), NIST RM 50 Albacore Tuna (0.89±0.08 μgg(-1)) and IRMM IMEP-20 Tuna fish (3.6±0.6 μgg(-1)) were in agreement with the certified value (4.47±0.32μgg(-1), 1.59±0.12 μgg(-1), 0.87±0.03 μgg(-1), 4.24±0.27 μgg(-1) respectively). For the sediment reference material ERM CC 580, a methyl mercury concentration of 0.070±0.002 μgg(-1) was measured which corresponds to an extraction efficiency of 92±3% of certified values (0.076±0.04 μgg(-1)) but within the range of published values (0.040-0.084 μgg(-1); mean±s.d.: 0.073±0.05 μgg(-1), n=40) for this material. The extraction procedure for the fish tissues was also compared against an enzymatic extraction using Protease type XIV that has been previously published and similar results were obtained. The use of HPLC-HGAAS with a Phenomenox 5 μm Luna C18 (250 mm×4.6 mm) column and a mobile phase containing 0.06 moll(-1) ammonium acetate (Merck Pty Limited, Australia) in 5% (v/v) methanol and 0.1% (w/v) l-cysteine at 25°C was evaluated as a complementary alternative to HPLC-ICPMS for the measurement of mercury species in fish tissues. The lowest measurable mercury concentration was 2 μgl(-1) and this corresponds to 0.1 μgg(-1) in fish tissues. Analysis of enzymatic extracts analysed by HPLC-HGAAS and HPLC-ICPMS gave equivalent results.

Arsenic distribution and species in two Zostera capricorni seagrass ecosystems, New South Wales, Australia

Arsenic concentrations and species were compared in biota from two Zostera capricorni ecosystems. Mean arsenic concentrations were not significantly different for non-vegetative sediment, rhizosphere sediment, Z. capricorni blades, roots, rhizomes, epiphytes, amphipods, polychaetes, molluscs, crustaceans and fish, but were significantly different in detritus. Sediments and plant tissues contained mostly inorganic arsenic and PO4-arsenoriboside. Detritus contained mostly PO4-arsenoriboside. Fish tissues contained predominately arsenobetaine. Other animals had lower proportions of arsenobetaine and variable quantities of minor arsenic species. Bioconcentration but not biomagnification ofarsenicisoccurringwithnoevidenceofarsenichyperaccumulation.Theproportionofarsenobetaineincreasesthrough the food web and is attributed to a shift from a mixed diet at lower trophic levels to animals containing mostly arsenobetaineathighertrophiclevelsandthemoreefficientretentionofarsenobetaine,comparedtootherarsenic species.

Distribution and Speciation of Arsenic in Temperate Marine Saltmarsh Ecosystems

This paper reports the distribution of total arsenic and arsenic species in saltmarsh ecosystems located in south-east Australia. We also investigated the relationship between arsenic, iron, and phosphorus concentrations in saltmarsh halophytes and associated sediment. Total mean arsenic concentrations in saltmarsh plants, S. quinqueflora and S. australis, for leaves ranged from 0.03 ± 0.05 to 0.67 ± 0.48 µ gg −1 and 0.03 ± 0.02 to 0.08 ± 0.06 µ gg −1 , respectively, and for roots ranged from 2 ± 2t o 6± 12 µ gg −1 and 0.39 ± 0.20 to 0.57 ± 1.06 µ gg −1 respectively. Removal of iron plaque from the roots reduced the arsenic concentration variability to 0.40-0.79 µ gg −1 and 0.95-1.05 µ gg −1 for S. quinqueflora and S. australis roots respectively. Significant differences were found between locations for total arsenic con- centrations in plant tissues and these differences could be partially attributed to differences in sediment arsenic concentrations between locations. For S. quinqueflora but not S. australis there was a strong correlation between arsenic and iron concentrations in the leaf and root tissues. A significant negative relationship between arsenic and phosphorus concentrations was found for S. quinqueflora leaves but not roots. Total mean arsenic concentrations in salt marsh animal tissues (7 ± 2-21 ± 13 µ gg −1 ) were consistent with those found for other marine animals. The concentration of total arsenic in gastropods and amphipods could be partially explained by the concentration of total arsenic in the dominant saltmarsh plant S. quinqueflora. Of the extractable arsenic, saltmarsh plants were dominated by arsenic(iii), arsenic(v) (66-99%), and glycerol arsenoribose (17-35%). Arsenobetaine was the dominant extractable arsenic species in the gastropods Salinator soilda (84%) and Ophicardelus ornatus (89%) and the crab Neosarmatium meinerti (89%). Amphipods contained mainly arsenobetaine (44%) with some phosphate arsenoribose (23%). Glycerol trimethyl arsonioribose was found in both gastropods (0.7-0.8%) and the visceral mass of N. meinerti (0.1%). These results show that arsenic uptake into plants from uncontaminated saltmarsh environments maybe dependent on plant iron uptake and inhibited by high phosphorus concentrations. Arsenic in saltmarsh plants is mainly present as inorganic arsenic, but arsenic in animals that eat plant detritus is present as organo arsenic species, primarily arsenobetaine and arsenosugars. The presence of glycerol trimethyl arsonioribose poses the question of whether trimethylated arsonioriboses are transitory intermediates in the formation of arsenobetaine.

Measurement of arsenic species in environmental, biological fluids and food samples by HPLC-ICPMS and HPLC-HG-AFS

The importance of measuring arsenic (As) species has been appreciated for a long time mainly because of the wide spread knowledge of arsenics toxicity and its use as a poison. Increasingly health, environmental and food regulations have been written around As species rather than total concentrations. Knowledge of As speciation is important as the chemical form of As controls its bioavailability, toxicity, mobility and therapeutic benefits. Arsenic is present as inorganic (arsenate, arsenite, thioarsenates), complexed (arsenic glutathionines and phytochelatins), low molecular weight (monomethylarsonate, dimethylarsenate, arsenobetaine, arsenocholine etc.) and high molecular weight (arsenic hydrocarbons and arsenic phospholipids) species. In this review we cover the intergrity of As species during collection, storage, sample preparation and measurement by HPLC-ICPMS and HPLC-HG-AFS. It is essential to ensure that As species, especially in waters and sediments, are not artefacts of the preservation or extraction procedure. Most samples can be stored frozen (−20 °C), but the stability of water and sediment samples is matrix dependent and depends on preservation technique applied. Arsenic cannot be extracted from samples using a single set of conditions but must be optimised for each sample type. Methanol–water mixtures with microwave heating are commonly used to extract polar As species from tissues while As-lipids required a non-polar solvent. Dilute acid can be used to increase the efficiencies of extraction of hard to extract tissue As species. Freeze drying is suitable for the drying of biotic material while sediments should not be dried before analysis. Extraction efficiencies are critically dependent on particle size. Polar As species have a wide variety of ionic characteristics thus complimentary chromatographic approaches utilising ion-exchange or reverse phase columns with modifiers are needed to separate all the As species. Arsenic-lipids require the use of a reverse phase column and gradient elution with high concentrations of organic solvents and require compensation for carbon enhancement effects in the ICPMS. Care must be taken that chromatographic peaks are not misidentified and matrix interferences accounted for that may influence quantification. Finally, to ensure accurate results, mass balances and extraction and column recoveries need to be determined at all steps. Methods need to be evaluated using As spikes and certified reference materials to provide a means of assessing the quality of results.

Observations on the measurement of total antimony and antimony species in algae, plant and animal tissues

This paper describes our experiences with undertaking measurements of total antimony and antimony speciation in algae, plant and animal tissues. Digestion with nitric acid alone is suitable to release antimony from animal tissues. When organisms have high silica contents, e.g. some plants and algae, the addition of tetrafluorboric acid is required to dissolve silica as some antimony is retained by silica in extracts. Antimony in digested extracts is present as Sb5+ and hydride generation procedures can be used to determine total antimony concentrations, as total antimony in extracts will not be under estimated. Relatively non-aggressive solvents such as water, dilute nitric acid, sodium hydroxide and enzymes remove highly variable amounts of antimony (2-84%) from algae, plant and animal tissues. Addition of Sb3+ and Sb5+ to NIST CRM 1572 Citrus Leaves, pre- and post-extraction with water showed that Sb3+ is oxidised to Sb5+ while Sb5+ is redistributed amongst binding sites giving rise to artefacts. DOLT-2 and algae extracts indicated the presence of only inorganic antimony. A moss sample had inorganic antimony and a number of unknown antimony species in extracts. Future studies should explore the nature of the binding of antimony in tissues as solvents commonly used to extract metals and metalloids from algae, plant and animal tissues are not appropriate.