Elliott G. Duncan

Contribution of Arsenic Species in Unicellular Algae to the Cycling of Arsenic in Marine Ecosystems

This review investigates the arsenic species produced by and found in marine unicellular algae to determine if unicellular algae contribute to the formation of arsenobetaine (AB) in higher marine organisms. A wide variety of arsenic species have been found in marine unicellular algae including inorganic species (mainly arsenate--As(V)), methylated species (mainly dimethylarsenate (DMA)), arsenoribosides (glycerol, phosphate, and sulfate) and metabolites (dimethylarsenoethanol (DMAE)). Subtle differences in arsenic species distributions exist between chlorophyte and heterokontophyte species with As(V) commonly found in water-soluble cell fractions of chlorophyte species, while DMA is more common in heterokontophyte species. Additionally, different arsenoriboside species are found in each phyla with glycerol and phosphate arsenoribosides produced by chlorophytes, whereas glycerol, phosphate, and sulfate arsenoribosides are produced by heterokontophytes, which is similar to existing data for marine macro-algae. Although arsenoribosides are the major arsenic species in many marine unicellular algal species, AB has not been detected in unicellular algae which supports the hypothesis that AB is formed in marine animals via the ingestion and further metabolism of arsenoribosides. The observation of significant DMAE concentrations in some unicellular algal cultures suggests that unicellular algae-based detritus contains arsenic species that can be further metabolized to form AB in higher marine organisms. Future research establishing how environmental variability influences the production of arsenic species by marine unicellular algae and what effect this has on arsenic cycling within marine food webs is essential to clarify the role of these organisms in marine arsenic cycling.

Uptake and metabolism of arsenate, methylarsonate and arsenobetaine by axenic cultures of the phytoplankton Dunaliella tertiolecta

Axenic cultures of the phytoplankton Dunaliella tertiolecta were dosed with either arsenate wAs(V)x, methylarsonate (MA) or arsenobetaine (AB) at environmentally realistic concentrations (2 m gl -1 ) to investigate incorporation and transformation of arsenic species. Total arsenic concentrations in cultures dosed with As(V) were significantly higher than those dosed with MA and AB (6–10 m gg -1 compared to 1–3 m gg -1 ). Arsenic concentrations in As(V)-dosed cultures increased over time, whereas arsenic concentrations in MAand AB-dosed cultures remained constant, indicating that As(V) is rapidly and continuously incorporated into the cell in comparison to MA and AB. Arsenic was found predominantly in the residue fractions (29–55%) of all cultures, irrespective of culture age. This was attributed to the ability of inorganic arsenic as arsenite wAs(III)x to bind to proteins and/ or phytochelatins. 2-Dimethylarsinoylethanol (DMAE) was the main arsenic species found in lipid cell fractions. Within the water-soluble and residue cell fractions, arsenic was isolated primarily as As(V), although it is likely the extraction procedure may have converted some As(III) to As(V). Small amounts of As(III), MA, dimethylarsinate (DMA) and arsenoribosides were also found in the lipid and water soluble As fractions. All cultures showed an increase in DMA and arsenoriboside species over time. The significance of these results is that while AB, the major arsenical in marine animals, is not produced or accumulated by this phytoplankton species, the possible precursors to AB formation are produced.

Toxicity of arsenic species to three freshwater organisms and biotransformation of inorganic arsenic by freshwater phytoplankton (Chlorella sp. CE-35)

In the environment, arsenic (As) exists in a number of chemical species, and arsenite (As(III)) and arsenate (As(V)) dominate in freshwater systems. Toxicity of As species to aquatic organisms is complicated by their interaction with chemicals in water such as phosphate that can influence the bioavailability and uptake of As(V). In the present study, the toxicities of As(III), As(V) and dimethylarsinic acid (DMA) to three freshwater organisms representing three phylogenetic groups: a phytoplankton (Chlorella sp. strain CE-35), a floating macrophyte (Lemna disperma) and a cladoceran grazer (Ceriodaphnia cf. dubia), were determined using acute and growth inhibition bioassays (EC₅₀) at a range of total phosphate (TP) concentrations in OECD medium. The EC₅₀ values of As(III), As(V) and DMA were 27 ± 10, 1.15 ± 0.04 and 19 ± 3 mg L(-1) for Chlorella sp. CE-35; 0.57 ± 0.16, 2.3 ± 0.2 and 56 ± 15 mg L(-1) for L. disperma, and 1.58 ± 0.05, 1.72 ± 0.01 and 5.9 ± 0.1 mg L(-1) for C. cf. dubia, respectively. The results showed that As(III) was more toxic than As(V) to L. disperma; however, As(V) was more toxic than As(III) to Chlorella sp. CE-35. The toxicities of As(III) and As(V) to C. cf. dubia were statistically similar (p>0.05). DMA was less toxic than iAs species to L. disperma and C. cf. dubia, but more toxic than As(III) to Chlorella sp. CE-35. The toxicity of As(V) to Chlorella sp. CE-35 and L. disperma decreased with increasing TP concentrations in the growth medium. Phosphate concentrations did not influence the toxicity of As(III) to either organism. Chlorella sp. CE-35 showed the ability to reduce As(V) to As(III), indicating a substantial influence of phytoplankton on As biogeochemistry in freshwater aquatic systems.

Influence of culture regime on arsenic cycling by the marine phytoplankton Dunaliella tertiolecta and Thalassiosira pseudonana

Arsenic cycling by the marine phytoplankton Dunaliella tertiolecta and the marine diatom Thalassiosira pseudonana was influenced by culture regime. Arsenic was associated with the residue cell fractions of batch cultured phytoplankton (D. tertiolecta and T. pseudonana), due to the accumulation of dead cells within batch cultures. Greater arsenic concentrations were associated with water-soluble and lipid-soluble cell fractions of continuously cultured phytoplankton. Arsenoribosides (as glycerol (Gly-), phosphate (PO4-) and sulfate (OSO3-)) were ubiquitous in D. tertiolecta (Gly- and PO4- only) and T. pseudonana (all three species). Additionally, arsenobetaine (AB) was not detectedinanyphytoplanktontissues,illustratingthatmarinephytoplanktonthemselvesarenotanalternatesourceofAB. Arsenic species formation was influenced by culture regime, with PO4-riboside produced under nutrient rich conditions, whereas Dimethylarsenoacetate (DMAA) was found in old (.42 days old) batch cultures, with this arsenic species possiblyproducedbythedegradationofarsenoribosides-arsenolipidsfromdecomposingcellsratherthanbybiosynthesis. Nutrient availability, hence culture regime was thus influential in directly and indirectly influencing arsenic cycling and the arsenic species produced by D. tertiolecta and T. pseudonana. Future research should thus utilise continuous culture regimes to study arsenic cycling as these are far more analogous to environmental processes. Additional keywords: arsenic species, arsenoribosides, batch culture, continuous culture, lipid-soluble arsenic.

The nitrification inhibitor 3,4,-dimethylpyrazole phosphate strongly inhibits nitrification in coarse-grained soils containing a low abundance of nitrifying microbiota

The effectiveness of the nitrification inhibitor 3,4,-dimethylpyrazole phosphate (DMPP) on sandy soils containing low nitrifying microbial abundance has not been established. Two coarse-grained soils, representative of Western Australia’s agricultural zones, were incubated with 100mgNkg–1 soil, added as either urea, urea+DMPP or urea+nitrapyrin as an alternative nitrification inhibitor for comparative purposes. Ammonium (NH4+) and nitrate (NO3–) concentrations, potential nitrification rates (PNR) and the abundance of ammonia-oxidising bacteria (AOB) and archaea (AOA) were measured over time. Interactions between soil type and inhibitor type altered the extent of nitrification observed in these soils. When N was supplied as urea alone, NH4+-N concentrations decreased from 100mgNkg–1 soil to approximately 20mgNkg–1 soil in the high nutrient soil (Williams) and approximately 60mgNkg–1 soil in the low nutrient soil (Vasse). These differences were reflected in AOB abundance, which was higher (~105genecopiesg–1 soil) in Williams soil than in Vasse soil (<104genecopiesg–1 soil). This difference could have been attributable to differences in soil pH between Williams and Vasse (5.4 vs 4.0 respectively) and/or copper (Cu) availability (~1.5 vs ~0.5mgCukg–1 soil respectively), both of which have been demonstrated to reduce AOB abundance or limit nitrification. On the Williams soil, DMPP limited nitrification, resulting in approximately 80mgNkg–1 soil being retained as NH4+-N. Nitrapyrin was similarly effective for the first 56 days of incubation, but declined considerably in effectiveness between Days 56 and 100. Changes in soil nitrification rates were accompanied by changes in AOB abundance, which was below 103genecopiesg–1 soil when nitrification was impaired. Both DMPP and nitrapyrin inhibit nitrification via chelating Cu and, because these soils contained low Cu concentrations, it may be possible that interactions between DMPP, naturally low abundance of AOB and low Cu availability facilitated the long-term inhibition of nitrification in these soils.

The formation and fate of organoarsenic species in marine ecosystems: do existing experimental approaches appropriately simulate ecosystem complexity?

Environmental context In marine environments, inorganic arsenic present in seawater is transformed to organoarsenic species, mainly arsenoribosides in algae and arsenobetaine in animals. These transformations decrease the toxicity of arsenic, yet the fate of arsenoribosides and arsenobetaine when marine organisms decompose is unknown. We review the current literature on the degradation of these organoarsenic species in marine systems detailing the drivers behind their degradation, and also discuss the environmental relevance of laboratory-based experiments. Abstract Despite arsenoribosides and arsenobetaine (AB) being the major arsenic species in marine macro-algae and animals they have never been detected in seawater. In all studies reviewed arsenoribosides from marine macro-algae were degraded to thio-arsenoribosides, dimethylarsinoylethanol (DMAE), dimethylarsenate (DMA), methylarsenate (MA) with arsenate (AsV) the final product of degradation. The use of different macro-algae species and different experimental microcosms did not influence the arsenoriboside degradation pathway. The use of different experimental approaches, however, did influence the rate and extent at which arsenoriboside degradation occurred. This was almost certainly a function of the complexity of the microbial community within the microcosm, with greater complexity resulting in rapid and more complete arsenoriboside degradation. AB from decomposing animal tissues is degraded to trimethylarsine oxide (TMAO) or dimethylarsenoacetate (DMAA), DMA and finally AsV. The degradation of AB unlike arsenoribosides occurs via a dual pathway with environmental or microbial community variability influencing the pathway taken. The environmental validity of different experimental approaches used to examine the fate of organoarsenic species was also reviewed. It was evident that although liquid culture incubation studies are cheap and reproducible they lack the ability to culture representative microbial communities. Microcosm studies that include sand and sediment are more environmentally representative as they are a better simulation of marine ecosystems and are also likely to facilitate complex microbial communities. An added benefit of microcosm studies is that they are able to be run in parallel with field-based research to provide a holistic assessment of the degradation of organoarsenic species in marine environments.

Arsenoriboside degradation in marine systems: The use of bacteria culture incubation experiments as model systems

Arsenoribosides (as glycerol; phosphate; sulfate and sulfonate) persisted in all bacteria-inoculated cultures irrespective of the source of bacteria (seawater, macro-algae surface) or the culture media used (DIFCO Marine Broth 2216 or novel blended Hormosira banksii tissue-based). This is unlike observations from traditional macro-algae tissue decomposition studies or in nature. In addition known arsenoriboside degradation products such as dimethylarsenoethanol (DMAE), dimethylarsenate (DMA), methylarsenate (MA) and arsenate - As(V) were not detected in any cultures. Consequently, the use of bacterial culture incubation experiments to explain the fate of arsenoribosides in marine systems appears limited as the processes governing arsenoriboside degradation in these experiments appear to be different to those in macro-algae tissue decomposition studies or in nature.

The degradation of arsenoribosides from Ecklonia radiata tissues decomposed in natural and microbially manipulated microcosms

We investigated the influence of microbial communities on the degradation of arsenoribosides from E. radiata tissues decomposing in sand and seawater-based microcosms. During the first 30 days, arsenic was released from decomposing E. radiata tissues into seawater and sand porewaters in all microcosms. In microcosms containing autoclaved seawater and autoclaved sand, arsenic was shown to persist in soluble forms at concentrations (9-18mg per microcosm) far higher than those present initially (,3mg per microcosm). Arsenoribosides were lost from decomposing E. radiatatissues in all microcosms with previously established arsenoriboside degradation products, such as thio-arsenic species, dimethylarsinoylethanol (DMAE), dimethylarsenate (DMA) and arsenate (As V ) observed in all microcosms. DMAE and DMA persisted in the seawater and sand porewaters of microcosms containing autoclaved seawater and autoclavedsand.Thissuggeststhatthedegradationstepfromarsenoribosides-DMAEoccursonalgalsurfaces,whereas the step from DMAE-As V occurs predominantly in the water-column or sand-sediments. This study also demonstrates that disruptions to microbial connectivity (defined as the ability of microbes to recolonise vacant habitats) result in alterations to arsenic cycling. Thus, the re-cycling of arsenoribosides released from marine macro-algae is driven by microbial complexity plus microbial connectivity rather than species diversity as such, as previously assumed. Additional keywords: algal decomposition, arsenic cycling, macro-algae, microbial ecology.

Ecological factors affecting the accumulation and speciation of arsenic in twelve Australian coastal bivalve molluscs

Environmental context Knowledge of the pathways by which arsenic is accumulated and transferred in marine ecosystems is scarce. Molluscs are important keystone organisms providing a link between primary producers (micro and macroalgae) and higher trophic levels such as fish. The present study examines the accumulation and species of arsenic in common bivalve molluscs from south-east Australia to understand the cycling of arsenic in marine food webs. Abstract The present paper reports the whole-tissue total arsenic concentrations and water-soluble arsenic species in 12 common coastal Australian bivalve mollusc species. Mean arsenic concentrations ranged from 18 to 57 µg g−1 dry mass. Planktivores had significantly less arsenic (20–40 µg g−1; 22 ± 3 µg g−1) than did suspension and deposit feeders (36–57 µg g−1; 43 ± 7 µg g−1), with those associated with fine clay–silt sediments (49 ± 7 µg g−1) having significantly more arsenic than those associated with sand substrates (31 ± 11 µg g−1 ). Most planktivores and suspension feeders had similar arsenic species, with high proportions of arsenobetaine (AB) (64–92 %) and relatively low proportions of other arsenic species (0.55–15.8 %). Lower proportions of AB (13–57 %) and larger proportions of inorganic arsenic (6–7 %) were found in deposit feeders, reflecting increased exposure to inorganic arsenic in sediments. The study indicated that at lower trophic levels, organisms feed on algae and suspended matter containing a range of arsenic species including arsenosugars and AB. The implications for arsenic cycling are that as all bivalve molluscs accumulate AB and are a source of AB in benthic food webs. Because all bivalve molluscs also contained appreciable concentrations of arsenoriboses, precursors are present for the de novo synthesis of AB. As well, deposit feeders have higher proportions of inorganic arsenic that can be metabolised to different end products when ingested by higher trophic organisms

Crop and microbial responses to the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) in Mediterranean wheat-cropping systems

Nitrification inhibitors (NIs) such as 3,4,-dimethylpyrazole phosphate (DMPP), are used to suppress the abundance of ammonia-oxidising micro-organisms responsible for nitrification. In agriculture, NIs are used to retain soil mineral nitrogen (N) as ammonium to minimise the risk of losses of N from agricultural soils. It is currently unclear whether DMPP-induced nitrification inhibition can prevent losses of N from the light soils prevalent across the main rain-fed cropping regions of Western Australia, or whether it can improve the productivity or N uptake by broadacre crops such as wheat. Herein, we report on a series of glasshouse and field studies that examined the effect of applications of DMPP in conjunction with urea (as ENTEC urea; Incitec Pivot, Melbourne, Vic., Australia) on: (1) soil nitrification rates; (2) the abundance of ammonia-oxidising bacteria and archaea (AOB and AOA respectively); and (3) wheat performance (grain yield, protein content and N accumulation). A glasshouse study demonstrated that DMPP inhibited nitrification (for up to ~40 days after application) and reduced the abundance of AOB (by 50%), but had no effect on AOA abundance, wheat grain yield or protein content at any fertiliser N rate. Across six field experiments, DMPP also limited nitrification rates and reduced AOB abundance for approximately the first 40 days after application. However, by the end of the growing season, DMPP use had not increased soil mineral N resources or impaired AOB abundance compared with urea-only applications. In addition, DMPP had no effect on AOA abundance in any trial and did not improve crop performance in most trials.