Pseudoephedrine hydrochloride causes hyperactivity in zebrafish via modulation of the serotonin pathway.

This study aimed to explore behavioral changes of embryonic and larval zebrafish caused by pseudoephedrine hydrochloride (PSE) and its underlying mechanism. Zebrafish embryos were exposed to 0.5 µM, 2 µM, and 8 µM PSE at 4 h post-fertilization (4 hpf) or 22-23 hpf. Mortality, hatching rate, coiling frequency, heart rate, behavior changes, and related gene expression were observed at different developmental stages. PSE below 8 µM did not affect zebrafish mortality, hatching rate, and heart rate compared with the control group. For embryos, PSE caused an increase at 16-32 hpf in zebrafish coiling frequency which could be rescued by serotonin antagonist WAY100635. Similarly, PSE caused an increase in the swimming distance of zebrafish larvae at 120 hpf. PSE also elevated the expression of serotonin (5-HT)-related genes 5-htr1ab and tph2 and dopamine-related gene dbh. Behavioral changes in zebrafish embryos and larvae caused by PSE may be closely associated with increased expression of 5-HT and dopamine-related genes. This may be reflected that the behavioral changes in zebrafish are a possible PSE monitoring indicator.

Selenium Nanoparticles (SeNPs) Immunomodulation Is More Than Redox Improvement: Serum Proteomics and Transcriptomic Analyses.

Selenium nanoparticles (SeNPs) are a novel elemental form selenium and often reported to possess beneficial bioactivities such as anticancer, promoting bone growth and immunomodulation. Our previous study demonstrated that chitosan-stabilized SeNPs have strong activity in immunomodulation. However, the mechanism underlying the immunomodulation of SeNPs is still unknown. The aim of this study is to identify the molecular mechanisms involved in SeNP-induced immunomodulation. Using zebrafish, as a common immunological animal model with a highly conserved molecular mechanism with other vertebrates, we conducted serum proteomic and tissue transcriptome analyses on individuals fed with SeNP in healthy or disease conditions. We also compared differences between SeNPs and an exogenous antioxidant Trolox in immune activity and redox regulation. Our results suggest that the immunomodulation activity was highly related to antioxidant activity and lipid metabolism. Interestingly, the biological functions enhanced by SeNP were almost identical in the healthy and disease conditions. However, while the SeNP was suppressing ROS in healthy individuals, it promoted ROS formation during disease condition. This might be related to the defense mechanism against pathogens. SOD and NFkß appeared to be the key molecular switch changing effect of SeNPs when individuals undergo infection, indicating the close relationship between immune and redox regulation.

Effects of ferulic acid on muscle development and intestinal microbiota of zebrafish.

Ferulic acid (FA) is one of a common ingredients in Chinese herbal medicine. FA has the interesting property of promoting growth and improving meat quality in livestock, but the mechanism is not understood. This study evaluated both safety and mechanism of efficacy in zebrafish model. At 15 µg/mL or above, FA led to pericardial oedema and delayed growth in zebrafish embryos. Dietary FA promoted growth and feed assimilation in male adult zebrafish. Genes related to myogenic development (myod1, myog and myf5) were significantly upregulated by FA and muscle fibre width in skeletal muscle was increased. At 20 µg/g, FA significantly increased number of goblet cells in zebrafish intestinal tissue, and gut microbiota composition also changed. Based on 16s rRNA gene sequences, 20 µg/g FA decreased Firmicutes and increased Bacteroides. 20 µg/g FA also stimulated the expression of PPAR-α, a gene associated with fat metabolism, and decreased the expression of PPAR-ß and PPAR-γ. These gene expression changes were beneficial to fatty acid synthesis and metabolism and decreased fat deposition. Our overall results indicated that FA can be a safe growth promotor in fish particularly in skeletal muscles.

Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates.

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPsEf and Stx-SSPsEf). Five selected toxic dinoflagellates, Alexandrium minutum, A. lusitanicum, A. tamarense, Gymnodinium catenatum, and Karenia mikimotoi, were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPsEf and Stx-SSPsEf in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

Tilapia dsRNA-activated protein kinase R (PKR): An interferon-induced antiviral effector with translation inhibition activity.

The dsRNA-activated protein kinase R (PKR) is one of key antiviral effectors induced by interferons (IFNs), and its functions are largely unknown in tilapia, an important commercial fish species suffering from several viral infectious diseases. In the present study, a PKR gene named On-PKR was identified and cloned from Nile tilapia, Oreochromis niloticus. On-PKR gene was constitutively expressed in all tissues examined, with the highest expression level observed in head kidney and liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). Importantly, the expression of On-PKR is induced by group I and group II IFNs with distinct induction kinetics in vivo: group I IFN elicits a relative delayed but sustained induction of On-PKR, whereas group II IFN triggers a rapid and transient expression of On-PKR. Moreover, the overexpression of On-PKR has been proven to inhibit the protein translation and virus replication in fish cells. The present study thus contributes to a better understanding of the functions of antiviral effectors in tilapia, and may provide clues for the prevention and therapy of viral diseases in fish.

Aconitine disrupts serotonin neurotransmission via 5-hydroxytryptamine receptor in zebrafish embryo.

Medicinal plants of the genus Aconitum are one of the most commonly used herbs in traditional medicine in East Asia to treat conditions related to the heart, pain, or inflammation. However, these herbs are also dangerous as accidental poisoning due to misuse is a recurring issue. These plants contain a number of diester-diterpenoid alkaloid compounds and aconitine is the most abundant and active one. This study investigated neurotoxicity of aconitine to zebrafish embryos in early development in relation to serotonin regulation. Experimental results showed that aconitine exposure (1, 10, and 100 µM) increased frequency of coiling behavior in zebrafish embryos in a dose-dependent manner and this effect can be triggered by either exposure to 5-hydroxytryptamine 1A (5-HT1A) receptor agonist (±)-8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) or overexpression of serotonin receptor 5-htr1ab. At the same time, coiling behavior caused by aconitine exposure could be rescued by co-exposure to 5-HT1A receptor antagonist WAY-100635 Maleate (WAY100635) and knockdown of 5-htr1ab using morpholino. Exposure to aconitine also significantly increased serotonin receptor 5-htr1ab and 5-htr1bd gene expression at 24 h post fertilization (hpf), but decreased their expression and protein expression of the serotonin receptor at 96 hpf with the high dose. These results suggest that neurotoxicity caused by aconitine is mediated through the 5-HT receptor.

Sunscreens containing zinc oxide nanoparticles can trigger oxidative stress and toxicity to the marine copepod Tigriopus japonicus.

The study, for the first time, evaluated the leaching rate of zinc oxide nanoparticles (nZnO) from human skins which were applied with three commercial sunscreens containing nZnO as an active ingredient. The leaching rate of nZnO varied greatly among the sunscreens, with a range of 8-72% (mean ± SD: 45% ± 33%). We further investigated their toxicities to the marine copepod Tigriopus japonicus. We found that 96-h median lethal concentrations of the three sunscreens to T. japonicus were > 5000, 230.6, and 43.0 mg chemical L-1, respectively, equivalent to Zn2+ concentrations at >82.5, 3.2, and 1.2 mg Zn L-1, respectively. Exposure to the individual sunscreens at environmentally realistic concentrations for 96 h led to up-regulation of antioxidant genes in T. japonicus, while they triggered the release of reactive oxygen species based on the results of in vivo assays. Evidently, these nZnO-included sunscreens can cause oxidative stress and hence pose risk to marine organisms.

In vivo toxicities of nine engineered nano metal oxides to the marine diatom Skeletonema costatum and rotifer Brachionus koreanus.

This study compared in vivo acute toxicities of nine engineered nano metal oxides to the marine diatom Skeletonema costatum and rotifer Brachionus koreanus. The sequence of their toxicities to S. costatum, based on growth inhibition, was: nano zinc oxide (nZnO) > nTiO2 (rutile) > nMgO > Annealed nMgO > nTiO2 (anatase) > Î³-nAl2O3 > nIn2O3 > α-nAl2O3 > nSnO2. Similarly, nZnO was also the most toxic to B. koreanus, but the other nano metal oxides were non-lethal. nMgO and nZnO were confirmed to trigger reactive oxygen species (ROS) mediated toxicity to the two marine organisms, while nTiO2 (both anatase and rutile forms) likely induced oxidative stress as shown by their acellular ROS production. nZnO may also cause damage in the endocrine system of B. koreanus, as indicated by the increased transcription of retinoid X receptor. Annealed nMgO reduces its toxicity via removal of O2- and impurities from its surface.

Maternal dietary exposure to selenium nanoparticle led to malformation in offspring.

Selenium (Se) is an essential element and its biological activity is related to its speciation. It is also well-known that in excess it can cause teratogenesis in fish and birds. In this study we compared dietary toxicity of elemental selenium nanoparticles (SeNPs) with selenite and selenomethionine (Se-Met). Japanese medaka (Oryzias latipes) was used as a laboratory model to determine Se effects on adults and their offspring. Adult females were individually exposed using a dry diet fortified with 0, 10 or 20 µg/g of the three Se species for 7 days and then allowed to breed for 3 days. Fertilization rate and the proportion of malformed offspring were examined. The three Se diets led to significant increase in maternal tissue Se concentration in the order of Se-Met >>selenite > SeNP. However, in terms of proportion of malformed offspring, the effect of Se-Met = selenite > SeNP. The malformations included pericardial edema and craniofacial changes, which were typical for Se toxicity. The mismatch of maternal ovary Se concentration and proportion of malformed offspring suggested total Se concentration is a poor predictor of toxicity and teratogenesis. Comparing expression of four genes related to oxidative stress in maternal tissue also showed that there were significant differences in expression patterns between three Se diets in the order of selenite = SeNP > Se-Met. Our results showed that SeNPs cause similar toxicity as other Se species but require further study to elucidate the underlying mechanism.

Influences of temperature and salinity on physicochemical properties and toxicity of zinc oxide nanoparticles to the marine diatom Thalassiosira pseudonana.

Climate change is predicted to result in rising average temperature of seawater with more extreme thermal events, and frequent rainfalls in some coastal regions. It is imperative to understand how naturally mediated changes in temperature and salinity can modulate toxicity of chemical contaminants to marine life. Thus, this study investigated combined effects of temperature and salinity on toxicity of zinc oxide nanoparticles (ZnO-NPs) to the marine diatom Thalassiosira pseudonana. Because ZnO-NPs formed larger aggregations and released less zinc ions (Zn2+) at greater temperature and salinity, toxicity of ZnO-NPs to T. pseudonana was less at 25 °C than at 10 °C and less at 32 than 12 PSU. However, toxicity of ZnO-NPs was significantly greater at 30 °C, since T. pseudonana was near its upper thermal limit. Three test compounds, ZnO, ZnO-NPs and ZnSO4, displayed different toxic potencies and resulted in different profiles of expression of genes in T. pseudonana. This indicated that ZnO-NPs caused toxicity via different pathways compared to ZnSO4. Mechanisms of toxic action of the three compounds were also dependent on temperature and salinity. These results provide insights into molecular mechanisms underlying the responses of the diatom to ZnO-NPs and Zn2+ under various regimes of temperature and salinity.

Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation.

Silver nanoparticles (AgNPs) have been increasingly commercialized and their release into the environment is imminent. Toxicity of AgNP has been studied with a wide spectrum of organisms, yet the mechanism of toxicity remains largely unknown. This study systematically compared toxicity of 10 AgNPs of different particle diameters and coatings to Japanese medaka (Oryzias latipes) larvae to understand how characteristics of AgNP relate to toxicity. Dissolution of AgNPs was largely dependent on particle size, but their aggregation behavior and toxicity were more dependent on coating materials. 96 h lethal concentration 50% (LC50) values correlated with AgNP aggregate size rather than size of individual nanoparticles. Of the AgNPs studied, the dissolved Ag concentration in the test suspensions did not account for all of the observed toxicity, indicating the role of NP-specific characteristics in resultant toxicity. Exposure to AgNP led to decrease of sodium concentration in the tissue and increased expression of Na(+)/K(+ )ATPase. Gene expression patterns also suggested that toxicity was related to disruption of sodium regulation and not to oxidative stress.

Parental dietary seleno-L-methionine exposure and resultant offspring developmental toxicity.

Selenium (Se) leaches into water from agricultural soils and from storage sites for coal fly ash. Se toxicity causes population and community level effects in fishes and birds. We used the laboratory aquarium model fish, Japanese medaka (Oryzias latipes), an asynchronous breeder, to determine aspects of uptake in adults and resultant developmental toxicity in their offspring. The superior imaging properties of the model enabled detailed descriptions of phenotypic alterations not commonly reported in the existing Se literature. Adult males and females in treatment groups were exposed, separately and together, to a dry diet spiked with 0, 12.5, 25, or 50 µg/g (dry weight) seleno-L-methionine (SeMet) for 6 days, and their embryo progeny collected for 5 days, maintained under controlled conditions and observed daily for hatchability, mortality and/or developmental toxicity. Sites of alteration included: craniofacial, pericardium and abdomen (Pc/Ab), notochord, gall bladder, spleen, blood, and swim bladder. Next, adult tissue Se concentrations (liver, skeletal muscle, ovary and testis) were determined and compared in treatment groups of bred and unbred individuals. No significant difference was found across treatment groups at the various SeMet concentrations; and, subsequent analysis compared exposed vs. control in each of the treatment groups at 10 dpf. Increased embryo mortality was observed in all treatment groups, compared to controls, and embryos had a decreased hatching rate when both parents were exposed. Exposure resulted in significantly more total altered phenotypes than controls. When altered phenotypes following exposure of both parents were higher than maternal only exposure, a male role was suggested. The comparisons between treatment groups revealed that particular types of phenotypic change may be driven by the sex of the exposed parent. Additionally, breeding reduced Se concentrations in some adult tissues, specifically the liver of exposed females and skeletal muscle of exposed males. Detailed phenotypic analysis of progeny from SeMet exposed parents should inform investigations of later life stages in an effort to determine consequences of early life exposure.

Salinity-dependent toxicities of zinc oxide nanoparticles to the marine diatom Thalassiosira pseudonana.

This study comprehensively investigated the influences of salinity, exposure concentration and time on the aggregate size, surface charge and dissolution of zinc oxide nanoparticles (ZnO-NPs; 20nm) in seawater, and examined the interacting effect of salinity and waterborne exposure of ZnO-NPs on the marine diatom Thalassiosira pseudonana for 96h. We found that aggregate sizes of ZnO-NPs significantly increased with increasing salinity, but generally decreased with increasing exposure concentration. Ion release decreased with increasing salinity, whereas the surface charge of the particles was not affected by salinity. The increased aggregate size and decreased ion release with increasing salinity, and consequently lower concentration of bioavailable zinc ions, resulted in decreased toxicity of ZnO-NPs at higher salinity in general in terms of growth inhibition (IC50) and chlorophyll fluorescence (EC50 - ФPo and EC50 - Ф2). However, IC50s and EC50s of ZnO-NPs were smaller than those of Zn(2+) (from ZnO-NPs ultrafiltrate and ZnCl2), indicating that dissolved Zn(2+) can only partially explain the toxicity of ZnO-NPs. SEM images showed that ZnO-NPs attached on the diatom frustule surface, suggesting that the interaction between the nanoparticles and the cell surface may acerbate the toxicity of ZnO-NPs. Our results linked the physicochemical characteristics of ZnO-NPs in seawater with their toxicities to the marine diatom and highlighted the importance of salinity as an influential environmental factor governing the aggregation, dissolution and the toxicity of ZnO-NPs.

Developmental toxicity and DNA damage from exposure to parking lot runoff retention pond samples in the Japanese medaka (Oryzias latipes).

Parking lot runoff retention ponds (PLRRP) receive significant chemical input, but the biological effects of parking lot runoff are not well understood. We used the Japanese medaka (Oryzias latipes) as a model to study the toxicity of water and sediment samples from a PLRRP in Morehead City, NC. Medaka exposed in ovo to a dilution series of PLRRP water had increased odds of death before hatching, but not teratogenesis or delayed hatching. Next, we adapted a long-amplicon quantitative PCR (LA-QPCR) assay for DNA damage for use with the Japanese medaka. We employed LA-QPCR to test the hypotheses that PLRRP water and sediments would cause nuclear and mitochondrial DNA damage with and without full-spectrum, natural solar radiation. Fluoranthene with and without natural sunlight was a positive control for phototoxic polycyclic aromatic hydrocarbon-induced DNA damage. Fluoranthene exposure did not result in detectable DNA damage by itself, but in combination with sunlight caused significant DNA damage to both genomes. PLRRP samples caused DNA damage to both genomes, and this was not increased by sunlight exposure, suggesting the DNA damage was unlikely the result of PAH phototoxicity. We report for the first time that PLRRP-associated pollutants cause both nuclear and mitochondrial DNA damage, and that fluoranthene-mediated phototoxicity results in similar levels of damage to the nuclear and mitochondrial genomes. These effects may be especially significant in sensitive marine ecosystems.

The difference between temperate and tropical saltwater species' acute sensitivity to chemicals is relatively small.

Due to a lack of saltwater toxicity data in tropical regions, toxicity data generated from temperate or cold water species endemic to North America and Europe are often adopted to derive water quality guidelines (WQG) for protecting tropical saltwater species. If chemical toxicity to most saltwater organisms increases with water temperature, the use of temperate species data and associated WQG may result in under-protection to tropical species. Given the differences in species composition and environmental attributes between tropical and temperate saltwater ecosystems, there are conceivable uncertainties in such 'temperate-to-tropic' extrapolations. This study aims to compare temperate and tropical saltwater species' acute sensitivity to 11 chemicals through a comprehensive meta-analysis, by comparing species sensitivity distributions (SSDs) between the two groups. A 10 percentile hazardous concentration (HC10) is derived from each SSD, and then a temperate-to-tropic HC10 ratio is computed for each chemical. Our results demonstrate that temperate and tropical saltwater species display significantly different sensitivity towards all test chemicals except cadmium, although such differences are small with the HC10 ratios ranging from 0.094 (un-ionised ammonia) to 2.190 (pentachlorophenol) only. Temperate species are more sensitive to un-ionised ammonia, chromium, lead, nickel and tributyltin, whereas tropical species are more sensitive to copper, mercury, zinc, phenol and pentachlorophenol. Through comparison of a limited number of taxon-specific SSDs, we observe that there is a general decline in chemical sensitivity from algae to crustaceans, molluscs and then fishes. Following a statistical analysis of the results, we recommend an extrapolation factor of two for deriving tropical WQG from temperate information.

Sediment quality guidelines: challenges and opportunities for improving sediment management.

During the International Conference on Deriving Environmental Quality Standards for the Protection of Aquatic Ecosystems held in Hong Kong in December 2011, an expert group, comprising scientists, government officials, and consultants from four continents, was formed to discuss the important scientific and regulatory challenges with developing sediment quality guidelines (SQGs). We identified the problems associated with SQG development and made a series of recommendations to ensure that the methods being applied were scientifically defensible and internationally applicable. This document summarizes the key findings from the expert group. To enable evaluation of current SQG derivation and application systems, a feedback mechanism is required to communicate confounding factors and effects in differing environments, while field validation is necessary to gauge the effectiveness of SQG values in sediment quality assessments. International collaboration is instrumental to knowledge exchange and method advancement, as well as promotion of 'best practices'. Since the paucity of sediment toxicity data poses the largest obstacle to improving current SQGs and deriving new SQGs, a standardized international database should be established as an information resource for sediment toxicity testing and monitoring data. We also identify several areas of scientific research that are needed to improve sediment quality assessment, including determining the importance of dietary exposure in sediment toxicity, mixture toxicity studies, toxicity screening of emerging chemicals, how climate change influence sediments and its biota, and possible use of new toxicity study approaches such as high throughput omic-based toxicity screenings.

Using whole mount in situ hybridization to examine thyroid hormone deiodinase expression in embryonic and larval zebrafish: a tool for examining OH-BDE toxicity to early life stages.

Polybrominated diphenyl ethers (PBDEs) and their oxidative metabolites (hydroxylated PBDEs; OH-BDEs) are known endocrine disrupting contaminants that have been shown to disrupt thyroid hormone regulation both in mammals and in fish. The purpose of this study was to determine the precise organ and tissue locations that express genes critical to thyroid hormone regulation in developing zebrafish (Danio rerio), and to determine the effects of an OH-BDE on their expression. While RT-PCR can provide quantitative data on gene expression, it lacks spatial sensitivity to examine localized gene expression; and, isolation of organs from zebrafish embryos is technically difficult, if not impossible. For this reason, the present study used whole mount in situ hybridization to simultaneously localize and quantify gene expression in vivo. While PBDEs and OH-BDEs have been shown to inhibit the activity and expression of deiodionases, a family of enzymes that regulate thyroid hormone concentrations intracellularly, it is unclear whether or not they can affect regional expression of the different isoforms during early development. In this study we investigated deiodinase 1 (Dio1), deiodinase 2 (Dio2), and deiodinase 3 (Dio3) mRNA expression at the following life stages (2, 8, and 1k-cells; 50%-epiboly, 6 and 18-somites, 22, 24, 48, 72 hpf and/or 10 dpf) in zebrafish and found life stage specific expression of these genes that were highly localized. To demonstrate the use of this technique for investigating potential endocrine disrupting effects, zebrafish embryos were exposed to 1, 10 and 100nM 6-OH-BDE-47. Significant increases in mean intensity of Dio1 and Dio3 expression in the periventricular zone of brain and pronephric duct, respectively (quantified by measuring intensity of coloration using ImageJ analysis software) were observed, suggesting localized response at the HPT axis with the possibility of impacting neurodevelopment. Our results demonstrate effects of OH-BDEs on thyroid regulating gene expression and provide more insight into potential sites of injury during early life stages.

Uptake of silver nanoparticles and toxicity to early life stages of Japanese medaka (Oryzias latipes): effect of coating materials.

Silver nanoparticles (AgNPs) with antimicrobial properties are perhaps the most deployed engineered nanomaterials in consumer products. Almost all AgNPs are coated with organic materials to enhance their dispersion in water. Contributions of coatings to the toxicity of NPs have received little attention. Studies using AgNPs with one of three different coating materials (citrate (Cit), gum arabic (GA), and polyvinylpyrrolidone (PVP)) showed significantly different toxicity. GA AgNP proved to be the most toxic, while PVP and Cit AgNP exhibited similar and lower toxicity. However, all AgNPs were about three to ten times less toxic than AgNO(3) when their toxicities were compared on a mass-concentration basis. Evidence for NP-specific toxicity was observed with longer time for initiation of toxicity and increased incidence of resultant spinal flexure of medaka exposed to AgNPs, compared to AgNO(3). Hyperspectral imaging of 6 µm paraffin sections of fish exposed to AgNPs revealed AgNPs and their aggregates in tissues of fish. Gill distribution was ubiquitous, while small amounts were found in other organs, including the liver and brain. AgNPs were observed regularly in the gut lumen, but rarely in mural elements and mesentery. These results suggest that while ingestion was common, gills were the principal sites of AgNP uptake. In conclusion, AgNPs is a source of toxic Ag ions, while itself contribute partially to its toxicity to fish, and which interact with skin surface and were taken up via the gills.

Chronic toxicity of double-walled carbon nanotubes to three marine organisms: influence of different dispersion methods.

AIM: Double-walled carbon nanotubes (DWNTs) are found in a variety of consumer products, but there are no ecotoxicity data of DWNTs into marine organisms. MATERIALS & METHODS: Chronic toxicity of DWNTs was investigated with the diatom Thalassiosira pseudonana, copepod Tigriopus japonicus and medaka Oryzias melastigma. DWNTs were dispersed using sonication (so-DWNTs) and stirring (st-DWNTs) for comparison. RESULTS: The median aggregation size (0.89 microm(2)) of so-DWNTs was smaller than that of st-DWNTs (21.8 microm(2)). Exposure to DWNTs led to growth inhibition of T. pseudonana with EC(50)s of 1.86 and 22.7 mg/l for so- and st-DWNTs, respectively. Population growth of T. japonicus was reduced to 0.1 mg/l for so-DWNTs and 10 mg/l for st-DWNTs. Growth inhibition in O. melastigma was observed at 10 mg/l for so-DWNTs but not for st-DWNTs. CONCLUSION: Given that so-DWNTs are consistently significantly more toxic than st-DWNTs, dispersion method and size of aggregations should be considered in DWNT toxicity testing.

Integrated stochastic environmental risk assessment of the harbour area treatment scheme (HATS) in Hong Kong.

Submarine ocean outfalls are commonly used for the disposal of partially treated effluents in coastal cities. Typically, the greatest environmental risk caused by toxic substances occurs in the near field of the outfall discharge. The ecological impact of the effluent varies greatly under different discharge and environmental conditions that are characterized by both regular and stochastic variations. For a comprehensive environmental risk assessment of a coastal discharge, it is necessary to determine both the likelihood and severity of the adverse effects on the biological community. We present the first integrated stochastic (Monte Carlo) environmental risk assessment of a major coastal sewage outfall discharge--the Stonecutters Island outfall of the Harbour Area Treatment Scheme (HATS) in Hong Kong. Unionized ammonia (NH3) is used as the target pollutant. To accurately envisage the ambient concentrations of NH3, a Lagrangian jet model (JETLAG/VISJET) is used to analyze pollutant concentrations in the nearfield of the outfall. The environmental conditions are simulated from 3D hydrodynamic model simulations over a 4 month period for typical wet and dry seasons. Statistical characteristics of the effluent discharge and receiving water temperature are derived from field data. The probability distribution of predicted exposure concentrations (EC) is generated from this integrated simulation. A species sensitivity distribution, which represents a statistical distribution of threshold sublethal effects levels or benchmark concentrations (BC) for various marine organisms is constructed using available chronic toxicity data. The environmental risk of NH3 on the marine community is characterized by computing statistical distributions of Hazard Quotient (HO = EC/BC) using Monte Carlo simulation. It is found that the probability of HO > for HATS Stage 1 (1.6 million m3/day sewage treated with chemically enhanced primary treatment) is around 0.11 for wet season but just about 0.06 for the dry season. The risk increases by about 10% to 0.08-0.13 with additional sewage loads of 0.8 million m3/day at the same level of treatment (HATS Stage 2A). With an upgrade to secondary treatment (HATS Stage 2B), the probability will be reduced to 0.03-0.05. Compared to the use of "worst case" scenarios or point pollution threshold estimates, the present method offers a more holistic ecological assessment, and is much less sensitive to arbitrary choice of model parameters. The present risk assessment approach can be readily extended to the accurate determination of mixing zones based on statistical evaluation of ecological risks.