Applicability of Chronic Multiple Linear Regression Models for Predicting Zinc Toxicity in Australian and New Zealand Freshwaters.

Bioavailability models, for example, multiple linear regressions (MLRs) of water quality parameters, are increasingly being used to develop bioavailability-based water quality criteria for metals. However, models developed for the Northern Hemisphere cannot be adopted for Australia and New Zealand without first validating them against local species and local water chemistry characteristics. We investigated the applicability of zinc chronic bioavailability models to predict toxicity in a range of uncontaminated natural waters in Australia and New Zealand. Water chemistry data were compiled to guide a selection of waters with different zinc toxicity-modifying factors. Predicted toxicities using several bioavailability models were compared with observed chronic toxicities for the green alga Raphidocelis subcapitata and the native cladocerans Ceriodaphnia cf. dubia and Daphnia thomsoni. The most sensitive species to zinc in five New Zealand freshwaters was R. subcapitata (72-h growth rate), with toxicity ameliorated by high dissolved organic carbon (DOC) or low pH, and hardness having a minimal influence. Zinc toxicity to D. thomsoni (reproduction) was ameliorated by both high DOC and hardness in these same waters. No single trophic level-specific effect concentration, 10% (EC10) MLR was the best predictor of chronic toxicity to the cladocerans, and MLRs based on EC10 values both over- and under-predicted zinc toxicity. The EC50 MLRs better predicted toxicities to both the Australian and New Zealand cladocerans to within a factor of 2 of the observed toxicities in most waters. These findings suggest that existing MLRs may be useful for normalizing local ecotoxicity data to derive water quality criteria for Australia and New Zealand. The final choice of models will depend on their predictive ability, level of protection, and ease of use. Environ Toxicol Chem 2023;42:2614-2629. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Probabilistic risk assessment of mine-derived copper in the Ok Tedi/Fly River, Papua New Guinea.

The Ok Tedi mine discharges waste rock and tailings into the Ok Tedi River in Papua New Guinea. This has resulted in elevated copper concentrations throughout the Ok Tedi/Fly River system, which can potentially impact aquatic biota. Ten years of measured copper and toxicity monitoring data were used to assess the risk of chronic effects from the mine-derived copper. Cumulative probability plots of dissolved and labile copper were compared to a species sensitivity distribution (SSD) of published copper toxicity data for four regions of the river. The Cu-SSD was used to estimate the risk of chronic effects to aquatic organisms in the Ok Tedi/Fly River at a range of potential copper exposure scenarios. The risk to species at the median labile copper concentration for each region showed a gradient effect with distance downstream from the mine and only the most sensitive (0.2-11%) species were at risk. There were copper exceedances of the region-specific guideline values (GV) and default guideline value (DGV) 88% and 74% of the time, respectively, in the Ok Tedi region (closest to the mine) and this is considered a high risk of chronic effects. Measured copper concentrations in the middle Fly River, lower Fly River (farthest downstream of the mine) and the river at Kiunga (reference site) exceeded the region-specific GVs and DGVs less frequently to rarely and present a lower risk of chronic effects from copper. The risk was supported using toxicity tests with the local microalgal species Chlorella sp. Comparison of recent (2010-2020) and historical (1996-2004) copper monitoring data from the Ok Tedi/Fly River indicates a decrease in the labile copper concentrations (30-76%) at key sites from impacted regions and a subsequent decrease in risk. This coincides with improved mining practices aimed at reducing the copper load into the Ok Tedi/Fly River.

Metal forms and dynamics in urban stormwater runoff: New insights from diffusive gradients in thin-films (DGT) measurements.

Stormwater runoff typically contains significant quantities of metal contaminants that enter urban waterways over short durations and represent a potential risk to water quality. The origin of metals within the catchment and processes that occur over the storm can control the partitioning of metals between a range of different forms. Understanding the fraction of metals present in a form that is potentially bioavailable to aquatic organisms is useful for environmental risk assessment. To help provide this information, the forms and dynamics of metal contaminants in an urban system were assessed across a storm. Temporal patterns in the concentration of metals in dissolved and particulate (total suspended solids; TSS) forms were assessed from water samples, and diffusive gradients in thin-films (DGTs) were deployed to measure the DGT-labile time-integrated metal concentration. Results indicate that the concentrations of dissolved and TSS-associated metals increased during the storm, with the metals Al, Cd, Co, Cu, Pb and Zn representing the greatest concern relative to water quality guideline values (GVs). The portion of labile metal as measured by DGT devices indicated that during the storm a substantial fraction (∼98%) of metals were complexed and pose a lower risk of acute toxicity to aquatic organisms. Comparison of DGT results to GVs indicate that current GVs are likely quite conservative when assessing stormwater pollution risks with regards to metal contaminants. This study provides valuable insight into the forms and dynamics of metals in an urban system receiving stormwater inputs and assists with the development of improved approaches for the assessment of short-term, intermittent discharge events.

Application of Bioavailability Models to Derive Chronic Guideline Values for Nickel in Freshwaters of Australia and New Zealand.

There has been an increased emphasis on incorporating bioavailability-based approaches into freshwater guideline value derivations for metals in the Australian and New Zealand water quality guidelines. Four bioavailability models were compared: the existing European biotic ligand model (European Union BLM) and a softwater BLM, together with 2 newly developed multiple linear regressions (MLRs)-a trophic level-specific MLR and a pooled MLR. Each of the 4 models was used to normalize a nickel ecotoxicity dataset (combined tropical and temperate data) to an index condition of pH 7.5, 6 mg Ca/L, 4 mg Mg/L, (i.e., approximately 30 mg CaCO3 /L hardness), and 0.5 mg DOC/L. The trophic level-specific MLR outperformed the other 3 models, with 79% of the predicted 10% effect concentration (EC10) values within a factor of 2 of the observed EC10 values. All 4 models gave similar normalized species sensitivity distributions and similar estimates of protective concentrations (PCs). Based on the index condition water chemistry proposed as the basis of the national guideline value, a protective concentration for 95% of species (PC95) of 3 µg Ni/L was derived. This guideline value can be adjusted up and down to account for site-specific water chemistries. Predictions of PC95 values for 20 different typical water chemistries for Australia and New Zealand varied by >40-fold, which confirmed that correction for nickel bioavailability is critical for the derivation of site-specific guideline values. Environ Toxicol Chem 2021;40:100-112. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Towards Sustainable Environmental Quality: Priority Research Questions for the Australasian Region of Oceania.

Environmental challenges persist across the world, including the Australasian region of Oceania, where biodiversity hotspots and unique ecosystems such as the Great Barrier Reef are common. These systems are routinely affected by multiple stressors from anthropogenic activities, and increasingly influenced by global megatrends (e.g., the food-energy-water nexus, demographic transitions to cities) and climate change. Here we report priority research questions from the Global Horizon Scanning Project, which aimed to identify, prioritize, and advance environmental quality research needs from an Australasian perspective, within a global context. We employed a transparent and inclusive process of soliciting key questions from Australasian members of the Society of Environmental Toxicology and Chemistry. Following submission of 78 questions, 20 priority research questions were identified during an expert workshop in Nelson, New Zealand. These research questions covered a range of issues of global relevance, including research needed to more closely integrate ecotoxicology and ecology for the protection of ecosystems, increase flexibility for prioritizing chemical substances currently in commerce, understand the impacts of complex mixtures and multiple stressors, and define environmental quality and ecosystem integrity of temporary waters. Some questions have specific relevance to Australasia, particularly the uncertainties associated with using toxicity data from exotic species to protect unique indigenous species. Several related priority questions deal with the theme of how widely international ecotoxicological data and databases can be applied to regional ecosystems. Other timely questions, which focus on improving predictive chemistry and toxicology tools and techniques, will be important to answer several of the priority questions identified here. Another important question raised was how to protect local cultural and social values and maintain indigenous engagement during problem formulation and identification of ecosystem protection goals. Addressing these questions will be challenging, but doing so promises to advance environmental sustainability in Oceania and globally.

Single-molecule fluorescence quantification with a photobleached internal standard.

In cellular and molecular biology, fluorophores are employed to aid in tracking and quantifying molecules involved in cellular function. We previously developed a sensitive single-molecule quantification technique to count the number of proteins and the variation of the protein number over the population of individual subcellular organelles. However, environmental effects on the fluorescent intensity of fluorophores can make it difficult to accurately quantify proteins using these sensitive techniques. In this letter, we demonstrate the use of photobleaching to extract an accurate single-molecule calibration intensity distribution from the sample directly to avoid any differences in environment that may alter the count. Using this technique, we were able to show that goat antimouse IgG antibody labeled with Alexa Fluor 488, an environmentally insensitive fluorophore, exhibited an average fluorescence equivalent to 4.6 single fluorophores. SynaptopHluorin vesicles, which contain the environmentally sensitive green fluorescent protein, exhibited an average of 4.4 single green fluorescent proteins per vesicle.

High-throughput fluorescence-activated nanoscale subcellular sorter with single-molecule sensitivity.

Recent single-cell and single-molecule studies have shown that a variety of subpopulations exist within biological systems, such as synaptic vesicles, that have previously been overlooked in common bulk studies. By isolating and enriching these various subpopulations, detailed analysis with a variety of analytical techniques can be done to further understand the role that various subpopulations play in cellular dynamics and how alterations to these subpopulations affect the overall function of the biological system. Previous sorters lack the sensitivity, sorting speed, and efficiency to isolate synaptic vesicles and other nanoscale systems. This paper describes the development of a fluorescence-activated nanoscale subcellular sorter that can sort nearly 10 million objects per hour with single-molecule sensitivity. Utilizing a near-nanoscale channel system, we were able to achieve upward of 91% recovery of desired objects with a 99.7% purity.

Probing the interior of synaptic vesicles with internalized nanoparticles.

Synaptic vesicles are subcellular organelles that are found in the synaptic bouton and are responsible for the propagation of signals between neurons. Synaptic vesicles undergo endo- and exocytosis with the neuronal membrane to load and release neurotransmitters. Here we discuss how we utilize this property to load nanoparticles as a means of probing the interior of synaptic vesicles. To probe the intravesicular region of synaptic vesicles, we have developed a highly sensitive pH-sensing polymer dot. We feel the robust nature of the pH-sensing polymer dot will provide insight into the dynamics of proton loading into synaptic vesicles.

Determining the number of specific proteins in cellular compartments by quantitative microscopy.

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.

Synaptosomes as a platform for loading nanoparticles into synaptic vesicles.

Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ~16%, that is, ~16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters.

Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision.

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

Intracellular delivery of bioactive molecules using light-addressable nanocapsules.

This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645 nm). We demonstrate this method with the intracellular release of the second messenger IP(3) in CHO-M1 cells and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP(3) released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa O(6)-alkylguanine-DNA alkyltransferase (AGT) fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ∼8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells.

Passive secondary biological treatment systems reduce estrogens in dairy shed effluent.

Steroid estrogens are found at high concentrations in untreated dairy shed effluents. Reduction of estrogenic activity and steroid estrogen concentrations was assessed in two systems used to treat dairy shed effluents: the two-pond system and the advanced pond system. Both include anaerobic and aerobic treatment stages. Samples of effluent were collected from the systems and analyzed for free estrogens, conjugated estrogens and total estrogenic activity using E-Screen assay. Both systems showed increases of up to 8000% in aqueous free estrogens and estrogenic activity after anaerobic treatment, followed by decreases after aerobic treatment (36-83%). The complete systems decreased total steroid estrogen concentrations by 50-100% and estrogen activity by 62-100%, with little difference between systems. Removal rates were lower in winter for both systems. Final effluents from the advanced pond system contained total estrogens at <15-1400 ng/L and estrogenic activity at 3.2-43 ng/L. Final effluent from the two-pond system contained total estrogens at <15-300 ng/L and estrogenic activity at 3.3-25 ng/L. At times the final effluent EEQs exceeded guideline values for protection of freshwater fish and suggest further treatment may be required.

Steroid estrogens, conjugated estrogens and estrogenic activity in farm dairy shed effluents.

Agricultural wastes are a source of steroid estrogens and, if present, conjugated estrogens may add to the estrogen load released to soil and aquatic environments. Dairy shed effluent samples were collected from 18 farms for analysis of steroid estrogens by GC-MS, conjugated estrogens by LC-MS-MS, and estrogenic activity by E-screen in vitro bioassay. 17alpha-estradiol was found at highest concentrations (median 730 ng l(-1)), followed by estrone (100 ng l(-1)) and 17beta-estradiol (24 ng l(-1)). Conjugated estrogens (estrone-3-sulfate, 17alpha-estradiol-3-sulfate and 17beta-estradiol-3,17-disulfate) were measured in most samples (12-320 ng l(-1)). Median estrogenic activity was 46 ng l(-1) 17beta-estradiol equivalents. Conjugated estrogens contributed up to 22% of the total estrogen load from dairy farming, demonstrating their significance. Steroid estrogens dominated overall estrogenic activity measured in the samples. Significantly, 17alpha-estradiol contributed 25% of overall activity, despite potency 2% that of 17beta-estradiol, highlighting the importance in environmental risk assessments of this previously neglected compound.

Sizing subcellular organelles and nanoparticles confined within aqueous droplets.

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.