The risk of harmful algal blooms (HABs) in the oyster-growing estuaries of New South Wales, Australia.

The spatial and temporal variability of potentially harmful phytoplankton was examined in the oyster-growing estuaries of New South Wales. Forty-five taxa from 31 estuaries were identified from 2005 to 2009. Harmful species richness was latitudinally graded for rivers, with increasing number of taxa southward. There were significant differences (within an estuary) in harmful species abundance and richness for 11 of 21 estuaries tested. Where differences were observed, these were predominately due to species belonging to the Pseudo-nitzschia delicatissima group, Dinophysis acuminata, Dictyocha octonaria and Prorocentrum cordatum with a consistent upstream versus downstream pattern emerging. Temporal (seasonal or interannual) patterns in harmful phytoplankton within and among estuaries were highly variable. Examination of harmful phytoplankton in relation to recognised estuary disturbance measures revealed species abundance correlated to estuary modification levels and flushing time, with modified, slow flushing estuaries having higher abundance. Harmful species richness correlated with bioregion, estuary modification levels and estuary class, with southern, unmodified lakes demonstrating greater species density. Predicting how these risk taxa and risk zones may change with further estuary disturbance and projected climate warming will require more focused, smaller scale studies aimed at a deeper understanding of species-specific ecology and bloom mechanisms. Coupled with this consideration, there is an imperative for further taxonomic, ecological and toxicological investigations into poorly understood taxa (e.g. Pseudo-nitzschia).

A new genus for four myobatrachid frogs from the South Western Australian Ecoregion.

The southern Australian endemic genus Geocrinia Blake 1973 (Anura: Myobatrachidae) currently contains seven species, with five restricted to Western Australia and two in the south-eastern states covering parts of New South Wales, Victoria, Tasmania and South Australia. All species have a modified life history with at least some or all of the larval stage being completed terrestrially. Four of the Western Australian species have terrestrial, non-feeding tadpoles nourished by yolk until metamorphosis. The remaining species have a biphasic development with embryos developing on land followed by an aquatic tadpole stage. The presence of species groups within the Geocrinia has been recognised since the 1970s, with all relevant subsequent studies supporting a model of two groups within the genus, recovered as reciprocally monophyletic in phylogenetic analyses. We examined character traits of the seven recognised Geocrinia species, concluding that distinction of the two monophyletic groups is supported by differences in life history strategy, larval morphology, adult morphology, call structure, breeding season and geographic distribution. The differences between the two groups correspond to phylogenetic structuring for all traits except distribution. Given reciprocal monophyly, and greater variation in traits than present within other myobatrachid genera, we conclude that the two groups should be given generic distinction. We therefore describe a new genus, Anstisia gen. nov., for four Western Australian Geocrinia species, retaining three species in Geocrinia. This increases the number of recognised myobatrachid genera to 14: five are endemic to south-western Australia.

Quantitative biomonitoring of polycyclic aromatic compounds (PACs) using the Sydney rock oyster (Saccostrea glomerata).

Increasing our understanding of the bioavailable fractions of polycyclic aromatic compounds (PACs) in an aquatic environment is important for the assessment of the environmental and human health risks posed by PACs. More importantly, the behaviour of polar polycyclic aromatic hydrocarbons (polar PAHs), which are metabolites of legacy PAHs, are yet to be understood. We, therefore, carried out a study involving Sydney rock oysters (Saccostrea glomerata) sourced from two locations, that had been exposed to PAH contamination, within an Australian south-east estuary. Biomonitoring of these oysters, following relocation from the estuary to a relatively isolated waterway, was done at 24 and 72 h after deployment and subsequently at 7, 14, 28, 52 and 86 days. Control samples from Camden Haven River were sampled for PAC analyses just before deployment, after 28 days and at the end of the study (day 86). Lipid-normalised concentrations in oyster tissues across the 86-day sampling duration, elimination rate constants (k2), biological half-lives (t1/2) and time required to reach 95% of steady-state (t95) were reported for parent PAHs and the less-monitored polar PAHs including nitrated/oxygenated/heterocyclic PAHs (NPAHs, oxyPAHs and HPAHs) for the three differently sourced oyster types. Most of the depurating PAHs and NPAHs, as well as 9-FLO (oxyPAH), had k2 values significantly different from zero (p < 0.05). All other oxyPAHs and HPAHs showed no clear depuration, with their concentrations remaining similar. The non-depuration of polar PAHs from oyster tissues could imply greater human health risk compared to their parent analogues.

Discriminating the intraerythrocytic lifecycle stages of the malaria parasite using synchrotron FT-IR microspectroscopy and an artificial neural network.

Synchrotron Fourier transform infrared (FT-IR) spectra of fixed single erythrocytes infected with Plasmodium falciparum at different stages of the intraerythrocytic cycle are presented for the first time. Bands assigned to the hemozoin moiety at 1712, 1664, and 1209 cm(-1) are observed in FT-IR difference spectra between uninfected erythrocytes and infected trophozoites. These bands are also found to be important contributors in separating the trophozoite spectra from the uninfected cell spectra in principal components analysis. All stages of the intraerythrocytic lifecycle of the malarial parasite, including the ring and schizont stage, can be differentiated by visual inspection of the C-H stretching region (3100-2800 cm(-1)) and by using principal components analysis. Bands at 2922, 2852, and 1738 cm(-1) assigned to the nu(asym)(CH(2) acyl chain lipids), nu(sym)(CH(2) acyl chain lipids), and the ester carbonyl band, respectively, increase as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage. Training of an artificial neural network showed that excellent automated spectroscopic discrimination between P. falciparum-infected cells and the control cells is possible. FT-IR difference spectra indicate a change in the production of unsaturated fatty acids as the parasite matures. The ring stage spectrum shows bands associated with cis unsaturated fatty acids. The schizont stage spectrum displays no evidence of cis bands and suggests an increase in saturated fatty acids. These results demonstrate that different phases of the P. falciparum intraerthyrocytic life cycle are characterized by different lipid compositions giving rise to distinct spectral profiles in the C-H stretching region. This insight paves the way for an automated infrared-based technology capable of diagnosing malaria at all intraerythrocytic stages of the parasite's life cycle.

Aggregated enhanced Raman scattering in Fe(III)PPIX solutions: the effects of concentration and chloroquine on excitonic interactions.

Resonance Raman spectra of hematin and hemin solutions are reported for 413 and 514 nm excitation wavelengths. Enhancement of A1g modes (1569 and 1370 cm(-1)) and B1g modes (1124 and 755 cm(-1)) as a function of increased concentration are observed when irradiating with 514 nm laser excitation but not 413 nm. This can be rationalized by considering an excitonic coupling mechanism. As the concentration of hematin increases there is an increased probability of supramolecular interactions between iron(III) protoporphyrin IX (Fe(III)PPIX) units occurring. The Fe(III)PPIX concentration reaches a saturation point in solution and excitonic coupling reaches a maximum causing the enhancement profile to plateau when applying 514 nm excitation. In contrast, when using 413 nm excitation there were no changes in band intensity with increased concentration showing that excitonic coupling through supramolecular interactions for aggregated solutions is wavelength dependent. Electronic absorption spectra show that as the concentration of Fe(III)PPIX increases in solution the Soret band is slightly blue shifted and the Q-band significantly broadens supporting the excitonic hypothesis. Understanding the mechanism that accounts for the Raman photophysical behavior of hemes at high concentrations provided an indirect method to monitor antimalarial drug interactions. A second aim was to investigate chloroquine binding to Fe(III)PPIX-OH/H2O monomers, pi-pi dimers and micro-oxo dimers formed in highly concentrated solutions approaching those of the digestive vacuole of the P. falciparum malaria parasite using excitonic Raman enhancement. It was hypothesized that the Raman excitonic enhancement mechanism could be impeded in heme aggregated solutions by the addition of chloroquine. This would result in a reduction in heme bands associated with the A1g modes including nu4. Resonance Raman spectra recorded using 514 nm excitation show that chloroquine (CQ) acts as a molecular spacer and binds noncovalently through dispersion interactions giving rise to pi-pi interactions, between micro-oxo dimer units of Fe(III)PPIX as evinced by the decrease in intensity of nu4 in the Raman spectrum as a function of increasing CQ mole ratio. In comparison, electronic spectra show that CQ can bind to the unligated face of Fe(III)PPIX-OH/H2O monomers, potentially reducing the formation of pi-pi dimers. This study has important implications in determining the effectiveness of potential antimalarial compounds that are thought to exert their effectiveness by binding through supramolecular interactions to the unligated faces of Fe(III)PPIX-OH/H2O monomers and micro-oxo dimers.

Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics.

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

The impact of lead co-contamination on ecotoxicity and the bacterial community during the bioremediation of total petroleum hydrocarbon-contaminated soils.

The continued increase in the global demand for oil, which reached 4,488 Mtoe in 2018, leads to large quantities of petroleum products entering the environment posing serious risks to natural ecosystems if left untreated. In this study, we evaluated the impact of co-contamination with lead on the efficacy of two bioremediation processes, natural attenuation and biostimulation of Total Petroleum Hydrocarbons (TPH) as well as the associated toxicity and the changes in the microbial community in contaminated soils. The biostimulated treatment resulted in 96% and 84% reduction in TPH concentration in a single and a co-contamination scenario, respectively, over 28 weeks of a mesocosm study. This reduction was significantly more in comparison to natural attenuation in a single and a co-contamination scenario, which was 56% and 59% respectively. In contrast, a significantly greater reduction in the associated toxicity of in soils undergoing natural attenuation was evident compared with soils undergoing biostimulation despite the lower TPH degradation when bioassays were applied. The earthworm toxicity test showed a decrease of 72% in the naturally attenuated toxicity versus only 62% in the biostimulated treatment of a single contamination scenario. In a co-contamination scenario, toxicity decreased only 30% and 8% after natural attenuation and biostimulation treatments, respectively. 16s rDNA sequence analysis was used to assess the impact of both the co-contamination and the bioremediation treatment. NGS data revealed major bacterial domination by Nocardioides spp., which reached 40% in week 20 of the natural attenuation treatment. In the biostimulated soil samples, more than 50% of the bacterial community was dominated by Alcanivorax spp. in week 12. The presence of Pb in the natural attenuation treatment resulted in an increased abundance of a few Pb-resistant genera such as Sphingopyxis spp. and Thermomonas spp in addition to Nocardioides spp. In contrast, Pb co-contamination completely shifted the bacterial pattern in the stimulated treatment with Pseudomonas spp. comprising approximately 45% of the bacterial profile in week 12. This study confirms the effectiveness of biostimulation over natural attenuation in remediating TPH and TPH-Pb contaminated soils. In addition, the presence of co-contaminants (e.g. Pb) results in serious impacts on the efficacy of bioremediation of TPH in contaminated soils, which must be considered prior to designing any bioremediation strategy.

Resonance Raman spectroscopy can detect structural changes in haemozoin (malaria pigment) following incubation with chloroquine in infected erythrocytes.

Resonance Raman spectroscopy was applied to monitor the effects of chloroquine (CQ) treatment on cultures of Plasmodium falciparum trophozoites. A number of bands assigned to A(1g) and B(1g) modes characteristic of the haemozoin aggregate are reduced in intensity in the CQ-treated cells, however, no bands from the CQ are observed. The intensity changes are attributed to intermolecular drug binding of the CQ in a sandwich type complex between ferriprotoporphyrin IX (FePPIX) dimer units. It is postulated that the CQ binds via pi-pi interactions between adjacent and orientated porphyrins thereby disrupting the haemozoin aggregate and reducing excitonic interactions between adjacent haems. The results show the potential of Raman microscopy as a screening tool for FePPIX:drug interactions in live cells.

Diarrhetic Shellfish Toxin Monitoring in Commercial Wild Harvest Bivalve Shellfish in New South Wales, Australia.

An end-product market survey on biotoxins in commercial wild harvest shellfish (Plebidonax deltoides, Katelysia spp., Anadara granosa, Notocallista kingii) during three harvest seasons (2015⁻2017) from the coast of New South Wales, Australia found 99.38% of samples were within regulatory limits. Diarrhetic shellfish toxins (DSTs) were present in 34.27% of 321 samples but only in pipis (P. deltoides), with two samples above the regulatory limit. Comparison of these market survey data to samples (phytoplankton in water and biotoxins in shellfish tissue) collected during the same period at wild harvest beaches demonstrated that, while elevated concentrations of Dinophysis were detected, a lag in detecting bloom events on two occasions meant that wild harvest shellfish with DSTs above the regulatory limit entered the marketplace. Concurrently, data (phytoplankton and biotoxin) from Sydney rock oyster (Saccostrea glomerata) harvest areas in estuaries adjacent to wild harvest beaches impacted by DSTs frequently showed elevated Dinophysis concentrations, but DSTs were not detected in oyster samples. These results highlighted a need for distinct management strategies for different shellfish species, particularly during Dinophysis bloom events. DSTs above the regulatory limit in pipis sampled from the marketplace suggested there is merit in looking at options to strengthen the current wild harvest biotoxin management strategies.

Rapid prediction of total petroleum hydrocarbons in soil using a hand-held mid-infrared field instrument.

This manuscript reports on the performance of a hand-held diffuse reflectance (mid)-infrared Fourier transform (DRIFT) spectrometer for the prediction of total petroleum hydrocarbons (TPH) in three different diesel-contaminated soils. These soils include: a carbonate dominated clay, a kaolinite dominated clay and a loam from Padova Italy, north Western Australia and southern Nigeria, respectively. Soils were analysed for TPH concentration using a standard laboratory methods and scanned in DRIFT mode with the hand-held spectrometer to determine TPH calibration models. Successful partial least square regression (PLSR) predictions, with coefficient of determination (R(2)) ~0.99 and root mean square error (RMSE) <200mg/kg, were obtained for the low range TPH concentrations of 0 to ~3,000mg/kg. These predictions were carried out using a set of independent samples for each soil type. Prediction models were also tested for the full concentration range (0-60,000mg/kg) for each soil type model with R(2) and RMSE values of ~0.99 and <1,255mg/kg, respectively. Furthermore, a number of intermediate concentration range models were also generated for each soil type with similar R(2) values of ~0.99 and RMSE values <800mg/kg. This study shows the capability of using a portable mid-infrared (MIR) DRIFT spectrometer for predicting TPH in a variety of soil types and the potential for being a rapid in-field screening method for TPH concentration levels at common regulatory thresholds. A novel hand-held mid-infrared instrument can accurately detect TPH across different soil types and concentrations, which paves the way for a variety of applications in the field.

Detecting the early onset of shear-induced fibril formation of insulin in situ.

A new approach is presented for detecting the early onset of amyloid fibril formation of insulin in a fluidic environment. The fibrillogenesis of insulin in a well-characterized Taylor-Couette flow cell was analyzed in situ using Raman spectroscopy in combination with principal components analysis (PCA). Raman spectra recorded using a 532.5 nm excitation laser revealed a more rapid fibrillogenesis process during the first 90 min of shearing than previously reported for samples exposed to flow. Bands corresponding to intermolecular H-bonded ß-sheet structure of insulin at 1678, 1630, and 1625 cm(-1) observed in the Raman difference spectra between unsheared insulin and sheared insulin show an increase in intensity as a function of shear exposure time, which is characteristic of fibril formation, with the first changes detected after 10 min. Additional analysis of samples removed from the flow cell after specific time periods provided conformation of the flow-enhanced fibrillogenesis process, including the detection of early fibril formation after only 1 min of shearing. FT-IR spectra of the insulin solutions showed evolution of bands at 1673 and 1633 cm(-1) from an increase in H-bonded ß-turn and ß-sheet structures, respectively, while fluorescence emission spectra detected the presence of a new emission band at 482 nm. TEM images confirmed the early onset of fibril formation at 1 min shear exposure, before a maturation and concentration increase of fibrils with further shearing. This study highlights the ability of fluid flows to accelerate insulin fibril formation, which has important implications for biotechnology applications such as the purification process of insulin therapeutic drugs in the pharmaceutical industry, as well as the use of optical-based methods for detecting fibrillogenesis.

Supramolecular interactions playing an integral role in the near-infrared Raman "excitonic" enhancement observed in ß-hematin (malaria pigment) and other related heme derivatives.

To gain more understanding into the mechanism that enables the dramatic resonant Raman enhancement of totally symmetric modes observed in hemozoin (malaria pigment) and other related heme supramolecular arrays when applying near-infrared excitation wavelengths, the iron(III) porphyrins Fe(TPP)Cl, [Fe(TPP)](2)O, Fe(OEP)Cl, and [Fe(OEP)](2)O along with ß-hematin (synthetic hemozoin or malaria pigment) were analyzed in the solid state using resonance Raman spectroscopy. The critical finding was that from the model compounds investigated, all except [Fe(OEP)](2)O exhibited the enhancement of the totally symmetric mode ν(4) when exciting the molecules with 782 and 830 nm laser lines. Through a detailed comparison of X-ray crystallographic structures, it is proposed that intermolecular noncovalent interactions play an integral role in enabling excitonic interactions to occur in these heme supramolecular systems. Comparison of the solid- and solution-phase electronic spectra in the near-IR region indicated more absorbance in the solid state between 800 and 900 nm. The electronic spectrum of [Fe(OEP)](2)O shows minimal absorbance in this region compared to that of the other compounds. All heme derivatives investigated have similar structure with a five-coordinate high-spin iron(III) ion. The crystallographic data indicate no significant differences in porphyrin geometry between TPP and OEP derivatives studied. However, [Fe(OEP)](2)O contains less supramolecular interactions in comparison to the other species. The supramolecular bonding enhances the probability of through-space interactions between the transition dipoles from electronic transitions of extended π systems. Our results indicate that the intensity of ν(4) is in part strongly affected by C-H···X hydrogen bonding interactions when X is an electron-donating entity. Such information may have important implications in the design and monitoring of antimalarial drugs that specifically interfere with hemozoin formation.

Crystal nucleation, growth, and morphology of the synthetic malaria pigment beta-hematin and the effect thereon by quinoline additives: the malaria pigment as a target of various antimalarial drugs.

The morphology of micrometer-sized beta-hematin crystals (synthetic malaria pigment) was determined by TEM images and diffraction, and by grazing incidence synchrotron X-ray diffraction at the air-water interface. The needle-like crystals are bounded by sharp {100} and {010} side faces, and capped by {011} and, to a lesser extent, by {001} end faces, in agreement with hemozoin (malaria pigment) crystals. The beta-hematin crystals grown in the presence of 10% chloroquine or quinine took appreciably longer to precipitate and tended to be symmetrically tapered toward both ends of the needle, due to stereoselective additive binding to {001} or {011} ledges. Evidence, but marginal, is presented that additives reduce crystal mosaic domain size along the needle axis, based on X-ray powder diffraction data. Coherent grazing exit X-ray diffraction suggests that the mosaic domains are smaller and less structurally stable than in pure crystals. IR-ATR and Raman spectra indicate molecular based differences due to a modification of surface and bulk propionic acid groups, following additive binding and a molecular rearrangement in the environment of the bulk sites poisoned by occluded quinoline. These results provided incentive to examine computationally whether hemozoin may be a target of antimalarial drugs diethylamino-alkoxyxanthones and artemisinin. A variation in activity of the former as a function of the alkoxy chain length is correlated with computed binding energy to {001} and {011} faces of beta-hematin. A model is proposed for artemisinin activity involving hemozoin nucleation inhibition via artemisinin-beta-hematin adducts bound to the principal crystal faces. Regarding nucleation of hemozoin inside the digestive vacuole of the malaria parasite, nucleation via the vacuole's membranous surface is proposed, based on a reported hemozoin alignment. As a test, a dibehenoyl-phosphatidylcholine monolayer transferred onto OTS-Si wafer nucleated far more beta-hematin crystals, albeit randomly oriented, than a reference OTS-Si.