Is Nontarget Analysis Ready for Regulatory Application? Influence of Peak-Picking Algorithms on Data Analysis.

The use of peak-picking algorithms is an essential step in all nontarget analysis (NTA) workflows. However, algorithm choice may influence reliability and reproducibility of results. Using a real-world data set, the aim of this study was to investigate how different peak-picking algorithms influence NTA results when exploring temporal and/or spatial trends. For this, drinking water catchment monitoring data, using passive samplers collected twice per year across Southeast Queensland, Australia (n = 18 sites) between 2014 and 2019, was investigated. Data were acquired using liquid chromatography coupled to high-resolution mass spectrometry. Peak picking was performed using five different programs/algorithms (SCIEX OS, MSDial, self-adjusting-feature-detection, two algorithms within MarkerView), keeping parameters identical whenever possible. The resulting feature lists revealed low overlap: 7.2% of features were picked by >3 algorithms, while 74% of features were only picked by a single algorithm. Trend evaluation of the data, using principal component analysis, showed significant variability between the approaches, with only one temporal and no spatial trend being identified by all algorithms. Manual evaluation of features of interest (p-value <0.01, log fold change >2) for one sampling site revealed high rates of incorrectly picked peaks (>70%) for three algorithms. Lower rates (<30%) were observed for the other algorithms, but with the caveat of not successfully picking all internal standards used as quality control. The choice is therefore currently between comprehensive and strict peak picking, either resulting in increased noise or missed peaks, respectively. Reproducibility of NTA results remains challenging when applied for regulatory frameworks.

Decay of Cryptosporidium parvfum DNA in cowpats in subtropical environments determined using qPCR.

Cryptosporidium oocysts pose a significant threat to public health due to its ability to contaminate environmental waters, leading to outbreaks of waterborne diseases and emphasizing the crucial need for effective water treatment and monitoring systems. This study aimed to investigate the decay of Cryptosporidium oocyst DNA in cow fecal matter under different environmental conditions prevalent in sub-tropical Southeast Queensland (SEQ) during summer and winter seasons. The effects of ambient sunlight and shaded conditions on the decay rates of C. parvum DNA in cow fecal samples were evaluated. The results showed that measurable levels of C. parvum DNA were observed for up to 60 days during the summer experiments, with a slower decay rate on the surface (k = -0.029) and sub-surface (k = -0.043) of the cowpat under shaded conditions than those on the surface (k = -0.064) and sub-surface (k = -0.079) under sunlight conditions. The decay rates of C. parvum DNA on the surface and sub-surface of the cowpat under shaded conditions were significantly slower (p = 0.004; p = 0.004) than those on the surface and sub-surface under sunlight conditions during summer experiments. During the winter treatments, measurable levels of C. parvum DNA were observed for up to 90 days, and the decay rates were slower on the surface (k = -0.036) and sub-surface (k = -0.034) of the cowpat under shaded conditions than those under sunlight conditions (k = -0.067 for surface and k = -0.057 for sub-surface). The decay rates of C. parvum DNA on the surface and sub-surface of the cowpat under shaded conditions were significantly slower than those on the surface (p = 0.009) and sub-surface (p = 0.041) under sunlight conditions during winter experiments. Moreover, the decay rate in the summer sunlight surface treatment (k = -0.064) was significantly faster from those in the winter shaded surface (k = -0.036; p = 0.018) and sub-surface (k = -0.034; p = 0.011) treatments. Similar results were also observed for summer sunlight sub-surface (k = -0.079), which was significantly faster than winter shaded surface (k = -0.036; p = 0.0008) and sub-surface (k = -0.034; p = 0.0005) treatments. Overall, these findings are important to enhance our understanding on the degradation of C. parvum DNA in cow fecal matter in SEQ, particularly in relation to seasonal variations and environmental conditions.

Passive-Sampler-Derived PCB and OCP Concentrations in the Waters of the World─First Results from the AQUA-GAPS/MONET Network.

Persistent organic pollutants (POPs) are recognized as pollutants of global concern, but so far, information on the trends of legacy POPs in the waters of the world has been missing due to logistical, analytical, and financial reasons. Passive samplers have emerged as an attractive alternative to active water sampling methods as they accumulate POPs, represent time-weighted average concentrations, and can easily be shipped and deployed. As part of the AQUA-GAPS/MONET, passive samplers were deployed at 40 globally distributed sites between 2016 and 2020, for a total of 21 freshwater and 40 marine deployments. Results from silicone passive samplers showed α-hexachlorocyclohexane (HCH) and γ-HCH displaying the greatest concentrations in the northern latitudes/Arctic Ocean, in stark contrast to the more persistent penta (PeCB)- and hexachlorobenzene (HCB), which approached equilibrium across sampling sites. Geospatial patterns of polychlorinated biphenyl (PCB) aqueous concentrations closely matched original estimates of production and use, implying limited global transport. Positive correlations between log-transformed concentrations of Σ7PCB, ΣDDTs, Σendosulfan, and Σchlordane, but not ΣHCH, and the log of population density (p < 0.05) within 5 and 10 km of the sampling sites also supported limited transport from used sites. These results help to understand the extent of global distribution, and eventually time-trends, of organic pollutants in aquatic systems, such as across freshwaters and oceans. Future deployments will aim to establish time-trends at selected sites while adding to the geographical coverage.

Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA.

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals' faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1-V3, V3-V4, and V5-V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3-V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3-V4 (n = 11) and V5-V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.

Comparative decay of culturable faecal indicator bacteria, microbial source tracking marker genes, and enteric pathogens in laboratory microcosms that mimic a sub-tropical environment.

Enteric pathogens can be present in drinking water catchments due to several point and non-point sources of faecal contamination. Pathogen and contaminant signatures will decay due to environmental stresses, such as temperature, Ultra Violet (UV) radiation, salinity, and predation. In this study, we determined the decay of the culturable faecal indicator bacterium (FIB) Escherichia coli (E. coli), two sewage-associated marker genes (Bacteroides HF183 and crAssphage CPQ_056), and enteric pathogens (Campylobacter spp., human adenovirus 40/41, and Cryptosporidium parvum) in two freshwater laboratory microcosms using culture-based, quantitative PCR (qPCR) and vital dye (determine the fraction of viable Cryptosporidium oocysts) assays. Freshwater samples from the Lake Wappa and Lake Wivenhoe (Australia) were seeded with untreated sewage and C. parvum oocysts, and their declining concentrations were measured over a 28-day period. Moreover, 16S rRNA amplicon sequencing was also undertaken to determine the change/shift in sewage-associated bacterial communities using SourceTracker. Overall, culturable E. coli and the HF183 marker gene decayed significantly (p < 0.05) faster than did the qPCR measured enteric pathogens suggesting that the absence of culturable FIB or qPCR HF183 in water samples may not indicate the absence of pathogens. The decay of crAssphage was similar to that of HAdV 40/41 and other pathogens tested, suggesting crAssphage may be a better surrogate for enteric viruses in sub-tropical catchment waters. The decay rates were greater at 25 °C compared to 15 °C, suggesting that FIB and pathogens persist longer in the winter season compared to summer. Overall decay rates of the tested microorganisms in this microcosm study suggest that sub-tropical conditions, especially temperature, have a negative impact on the persistence of tested microorganisms. Sewage-associated bacterial communities also showed similar patterns. Based on the results, which showed differences in simulated summer and winter temperatures for pathogen decay, corresponding management options and treatment need to be adjusted accordingly to minimize human health risks effectively.

The first environmental assessment of hexa(methoxymethyl)melamine and co-occurring cyclic amines in Australian waterways.

Hexa(methoxymethyl)melamine (HMMM) is commonly used as a cross-linking agent in coatings and as a vulcaniser in tyre production to increase the durability of tyres. Early reports of elevated aquatic concentrations of HMMM and a range of co-occurring cyclic amines have been linked to toxicity and mortality events of aquatic organisms. There are currently only few studies reporting environmental concentrations of HMMM and the co-occurring cyclic amines, and this study reports the first environmental assessment in Australian surface waters. Archive passive water samples from 40 rivers, creeks and lakes in South East Queensland, Australia, and covering five years of biannual sampling, were analysed to determine spatial and temporal trends. Concentrations of HMMM and cyclic amines in Australian surface waters (<5-46 and

Compositional and temporal stability of fecal taxon libraries for use with SourceTracker in sub-tropical catchments.

Characterization of microbial communities using high-throughput amplicon sequencing is an emerging approach for microbial source tracking of fecal pollution. This study used SourceTracker software to examine temporal and geographical variability of fecal bacterial community profiles to identify pollutant sources in three freshwater catchments in sub-tropical Australia. Fecal bacterial communities from 10 animal species, humans, and composite wastewater samples from six sewage treatment plants were characterized and compared to freshwater samples using Illumina amplicon sequencing of the V5-V6 regions of the 16S rRNA gene. Source contributions were calculated in SourceTracker using new fecal taxon libraries as well as previously generated libraries to determine the effects of geographic and temporal variability on source assignments. SourceTracker determined 16S rRNA bacterial communites within freshwater samples, shared taxonomic similarities to that of wastewater at low levels (typically <3%). SourceTraker also predicted occasional fecal detection of deer and flying fox sources in the water samples. No significant differences in source contributions were observed within sequences from current and previously characterized fecal samples (P ≥ 0.107). However, significant differences were observed between previously characterized and newly characterized source communities (ANOSIM P ≤ 0.001), which shared <15% community composition. Results suggest temporal instability of fecal taxon libraries among tested sources and highlight continual evaluation of community-based MST using confirmatory qPCR analyses of marker genes.

Shallow-water wave lensing in coral reefs: a physical and biological case study.

Wave lensing produces the highest level of transient solar irradiances found in nature, ranging in intensity over several orders of magnitude in just a few tens of milliseconds. Shallow coral reefs can be exposed to wave lensing during light-wind, clear-sky conditions, which have been implicated as a secondary cause of mass coral bleaching through light stress. Management strategies to protect small areas of high-value reef from wave-lensed light stress were tested using seawater irrigation sprinklers to negate wave lensing by breaking up the water surface. A series of field and tank experiments investigated the physical and photophysiological response of the shallow-water species Stylophora pistillata and Favites abdita to wave lensing and sprinkler conditions. Results show that the sprinkler treatment only slightly reduces the total downwelling photosynthetically active and ultraviolet irradiance (∼5.0%), whereas it dramatically reduces, by 460%, the irradiance variability caused by wave lensing. Despite this large reduction in variability and modest reduction in downwelling irradiance, there was no detectable difference in photophysiological response of the corals between control and sprinkler treatments under two thermal regimes of ambient (27°C) and heated treatment (31°C). This study suggests that shallow-water coral species are not negatively affected by the strong flashes that occur under wave-lensing conditions.