Bromoform Toxicity to Marine Microalgae: Studies using the Diatom Chaetoceros lorenzianus as a Model Organism.

Bromoform is the most prominent, relatively long-lived chlorination by-product in condenser effluents from seawater-based power plant cooling systems. There are few reports on the potential toxicity of this trihalomethane to marine phytoplankton. We investigated this using a marine diatom, Chaetoceros lorenzianus as the model organism. The study was conducted by exposing the diatom to bromoform concentrations 0, 50, 100, 150, 250, 500 and 1000 µg/L for exposure time of 3 and 24 h. The mode of action of bromoform was examined using endpoints which include chlorophyll a fluorescence, cell viability by SYTOX® green stain and genotoxicity by comet assay. The relative fluorescence unit and percent viability changed significantly at all concentrations in duration of study. The 24-h IC50 for viability and chlorophyll was estimated to be 255.6 µg/L and 343.5 µg/L, respectively. The tail DNA of 5-20% observed by comet assay indicated low-level DNA damage. Bromoform manages to target cell membrane and internal machinery, DNA and chlorophyll molecule of cell, leading to cause damage at multiple physiological levels. Based on the present data, the current discharge levels of bromoform 50-250 µg/L cause significant impact on the phytoplankton under investigation. However, the impact can be limited under actual field conditions wherein mixing of cooling water with natural water bodies is considered. Nevertheless, more studies are required to understand the toxicological response of organisms to bromoform, so that discharge levels can be continued to be kept within safe levels.

Variations in trihalomethane formation potential of Microcystis aeruginosa under different growth conditions: Phenomenon and mechanism.

Cyanobacteria and their metabolites are one of the primary precursors of disinfection by-products (DBPs) in natural water environments. However, few studies have investigated whether the production of DBPs by cyanobacteria changes under complex environmental conditions and possible mechanisms underlying these changes. Therefore, we investigated the effects of algal growth phase, water temperature, pH, illumination and nutrients on the production of trihalomethane formation potential (THMFPs) by Microcystis aeruginosa in four algal metabolic fractions, that is, hydrophilic extracellular organic matter (HPI-EOM), hydrophobic EOM (HPO-EOM), hydrophilic intracellular organic matter (HPI-IOM) and hydrophobic IOM (HPO-IOM). Additionally, correlations between THMFPs and some typical algal metabolite surrogates were analyzed. The results showed that the productivity of THMFPs by M. aeruginosa in EOM could be affected significantly by the algal growth phase and incubation conditions, while the IOM productivity varied insignificantly. M. aeruginosa in the death phase could secrete more EOM and have a higher THMFP productivity than those in the exponential or stationary phases. Cyanobacteria grown under harsh conditions could have increased THMFP productivity in EOM by increasing the reactivity of algal metabolites with chlorine, for example, under low pH conditions, and secreting more metabolites in EOM, for example, under low temperature or nutrient limitation conditions. Polysaccharides were responsible for the enhanced THMFP productivity in HPI-EOM fraction, and a significant linear correlation was found between the concentration of polysaccharides and THMFPs (r = 0.8307). However, THMFPs in HPO-EOM did not correlate with dissolved organic carbon (DOC), ultraviolet absorbance at 254 nm (UV254), specific UV absorbance (SUVA) and cell density. Thus, we could not specify the kind of algal metabolites that contribute to the increased THMFPs in the HPO-EOM fraction under harsh growth conditions. Compared with the case in EOM, the THMFPs in IOM were more stable and correlated with the cell density and total amount of IOM. The results implied that the THMFPs in the EOM were sensitive to growth conditions and were independent of algal density. Considering the fact that traditional water treatment plants cannot remove dissolved organics as efficiently as algal cells, the increased THMFP productivity in EOM by M. aeruginosa under harsh growth conditions could be a potentially serious threat to the safety of the water supply.

Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends Insights into Seaweed Reactive Oxygen Species Enzymology.

Marine macroalgae, seaweeds, are exceptionally prolific producers of halogenated natural products. Biosynthesis of halogenated molecules in seaweeds is inextricably linked to reactive oxygen species (ROS) signaling as hydrogen peroxide serves as a substrate for haloperoxidase enzymes that participate in the construction these halogenated molecules. Here, using red macroalga Asparagopsis taxiformis, a prolific producer of the ozone depleting molecule bromoform, we provide the discovery and biochemical characterization of a ROS-producing NAD(P)H oxidase from seaweeds. This discovery was enabled by our sequencing of Asparagopsis genomes, in which we find the gene encoding the ROS-producing enzyme to be clustered with genes encoding bromoform-producing haloperoxidases. Biochemical reconstitution of haloperoxidase activities establishes that fatty acid biosynthesis can provide viable hydrocarbon substrates for bromoform production. The ROS production haloperoxidase enzymology that we describe here advances seaweed biology and biochemistry by providing the molecular basis for decades worth of physiological observations in ROS and halogenated natural product biosyntheses.

Exposure to disinfection byproducts and risk of type 2 diabetes: a nested case-control study in the HUNT and Lifelines cohorts.

INTRODUCTION: Environmental chemicals acting as metabolic disruptors have been implicated with diabetogenesis, but evidence is weak among short-lived chemicals, such as disinfection byproducts (trihalomethanes, THM composed of chloroform, TCM and brominated trihalomethanes, BrTHM). OBJECTIVES: We assessed whether THM were associated with type 2 diabetes (T2D) and we explored alterations in metabolic profiles due to THM exposures or T2D status. METHODS: A prospective 1:1 matched case-control study (n = 430) and a cross-sectional 1:1 matched case-control study (n = 362) nested within the HUNT cohort (Norway) and the Lifelines cohort (Netherlands), respectively, were set up. Urinary biomarkers of THM exposure and mass spectrometry-based serum metabolomics were measured. Associations between THM, clinical markers, metabolites and disease status were evaluated using logistic regressions with Least Absolute Shrinkage and Selection Operator procedure. RESULTS: Low median THM exposures (ng/g, IQR) were measured in both cohorts (cases and controls of HUNT and Lifelines, respectively, 193 (76, 470), 208 (77, 502) and 292 (162, 595), 342 (180, 602). Neither BrTHM (OR = 0.87; 95% CI: 0.67, 1.11 | OR = 1.09; 95% CI: 0.73, 1.61), nor TCM (OR = 1.03; 95% CI: 0.88, 1.2 | OR = 1.03; 95% CI: 0.79, 1.35) were associated with incident or prevalent T2D, respectively. Metabolomics showed 48 metabolites associated with incident T2D after adjusting for sex, age and BMI, whereas a total of 244 metabolites were associated with prevalent T2D. A total of 34 metabolites were associated with the progression of T2D. In data driven logistic regression, novel biomarkers, such as cinnamoylglycine or 1-methylurate, being protective of T2D were identified. The incident T2D risk prediction model (HUNT) predicted well incident Lifelines cases (AUC = 0.845; 95% CI: 0.72, 0.97). CONCLUSION: Such exposome-based approaches in cohort-nested studies are warranted to better understand the environmental origins of diabetogenesis.

Production of Dibromomethane and Changes in the Bacterial Community in Bromoform-Enriched Seawater.

The responses of bacterial communities to halocarbon were examined using a 28-d incubation of bromoform- and methanol-enriched subarctic surface seawater. Significant increases were observed in dibromomethane concentrations and bacterial 16S rRNA gene copy numbers in the treated substrates incubated for 13 d. The accumulated bacterial community was investigated by denaturing gradient gel electrophoresis and amplicon analyses. The dominant genotypes corresponded to the genera Roseobacter, Lentibacter, and Amylibacter; the family Flavobacteriaceae; and the phylum Planctomycetes, including methylotrophs of the genus Methylophaga and the family Methylophilaceae. Therefore, various phylotypes responded along with the dehalogenation processes in subarctic seawater.

Algal removal from cyanobacteria-rich waters by preoxidation-assisted coagulation-flotation: Effect of algogenic organic matter release on algal removal and trihalomethane formation.

The cyanobacteria-bloom in raw waters frequently causes an unpredictable chemical dosing of preoxidation and coagulation for an effective removal of algal cells in water treatment plants. This study investigated the effects of preoxidation with NaOCl and ClO2 on the coagulation-flotation effectiveness in the removal of two commonly blooming cyanobacteria species, Microcystis aeruginosa (MA) and Cylindrospermopsis raciborskii (CR), and their corresponding trihalomethane (THM) formation potential. The results showed that dual dosing with NaOCl plus ClO2 was more effective in enhancing the deformation of cyanobacterial cells compared to single dosing with NaOCl, especially for CR-rich water. Both preoxidation approaches for CR-rich water effectively reduced the CR cell count with less remained dissolved organic carbon (DOC), which benefited subsequent coagulation-flotation. However, preoxidation led to an adverse release of algogenic organic matter (AOM) in the case of MA-rich water. The release of AOM resulted in a poor removal in MA cells and a large amount of THM formation after oxidation-assisted coagulation-flotation process. The reduction in THM formation potential of CR-rich waters is responsible for effective algae and DOC removal by alum coagulation. It is concluded that the species-specific characteristic of cyanobacteria and their AOM released during chlorination significantly influences the performance of coagulation-flotation for AOM removal and corresponding THM formation.

Time of the day dictates the variability of biomarkers of exposure to disinfection byproducts.

Non-persistent environmental chemicals (NOPEC) are xenobiotics with short half-lives of elimination (<7h). Similar to chronopharmacokinetics, NOPEC metabolism may follow diurnal patterns of cytochrome P450 activity. The role of circadian liver clock in shaping NOPEC metabolism and their concomitant measurements of biomarkers of exposure and effect remains poorly understood in real-life human settings. Metabolic activation (toxication) by CYP2E1 converts trihalomethanes (THM) to harmful metabolites. We investigated the diurnal variation of urinary THM exposures and their metabolism patterns as catalyzed by CYP2E1 redox activity, using the surrogate marker of 4-hydroxynonenal (4HNE). We implemented three time-series trials with adult volunteers conducting specific household cleaning activities at predefined times of a day. Circadia variation of 4HNE was assessed with a cosinor model and its mesor levels increased with THM exposure. The time of exposure within the day dictated the magnitude of urinary THM levels and their toxication effect; in all three trials and relative to urinary THM levels before the activity, lower and higher median THM were measured right after the activity in morning and afternoon/night, respectively. This is consistent with higher reported CYP2E1 redox activity in light/active phase. Population health studies should incorporate time-stamped biomarker data to improve the understanding of chronic disease processes.

Simulation of Ligand Binding to Membrane Proteins.

Membrane proteins are involved in a large variety of functions. Most of these protein functions are regulated by ligand binding with diverse modes of action: agonists, partial agonists, antagonists, and allosteric modulators, potentiators and inhibitors. From the pharmacological point of view, membrane proteins are one if not the major target for drug development. However, experimental structure determination of membrane proteins in complex or in free form still represents a great challenge. Molecular dynamics (MD) simulations commonly reach the microsecond scale on membrane systems. This numerical tool is mature enough to predict and add molecular details on the different ligand-binding modes. In the present chapter, I will present the different steps to design, simulate, and analyze a MD simulation system containing a protein embedded in a membrane and surrounded by water and ligand. As an illustration, the simulation of the ligand-gated ion channel γ-aminobutyric acid type A receptor (GABAAR) surrounded by one of its allosteric potentiators, bromoform, will be presented and discussed.

Microbial reductive dehalogenation of trihalomethanes by a Dehalobacter-containing co-culture.

Trihalomethanes such as chloroform and bromoform, although well-known as a prominent class of disinfection by-products, are ubiquitously distributed in the environment due to widespread industrial usage in the past decades. Chloroform and bromoform are particularly concerning, of high concentrations detected and with long half-lives up to several hundred days in soils and groundwater. In this study, we report a Dehalobacter- and Desulfovibrio-containing co-culture that exhibits dehalogenation of chloroform (~0.61 mM) to dichloromethane and bromoform (~0.67 mM) to dibromomethane within 10-15 days. This co-culture was further found to dechlorinate 1,1,1-trichloroethane (1,1,1-TCA) (~0.65 mM) to 1,1-dichloroethane within 12 days. The Dehalobacter species present in this co-culture, designated Dehalobacter sp. THM1, was found to couple growth with dehalogenation of chloroform, bromoform, and 1,1,1-TCA. Strain THM1 harbors a newly identified reductive dehalogenase (RDase), ThmA, which catalyzes chloroform, bromoform, and 1,1,1-TCA dehalogenation. Additionally, based on the sequences of thmA and other identified chloroform RDase genes, ctrA, cfrA, and tmrA, a pair of chloroform RDase gene-specific primers were designed and successfully applied to investigate the chloroform dechlorinating potential of microbial communities. The comparative analysis of chloroform RDases with tetrachloroethene RDases suggests a possible approach in predicting the substrate specificity of uncharacterized RDases in the future.

Water quality following extensive beetle-induced tree mortality: Interplay of aromatic carbon loading, disinfection byproducts, and hydrologic drivers.

The recent bark beetle epidemic across western North America may impact water quality as a result of elevated organic carbon release and hydrologic shifts associated with extensive tree dieback. Analysis of quarterly municipal monitoring data from 2004 to 2014 with discretization of six water treatment facilities in the Rocky Mountains by extent of beetle impact revealed a significant increasing trend in total organic carbon (TOC) and total trihalomethane (TTHM) production within high (≳50% areal infestation) beetle-impacted watersheds while no or insignificant trends were found in watersheds with lower impact levels. Alarmingly, the TTHM concentration trend in the high impact sites exceeded regulatory maximum contaminant levels during the most recent two years of analysis (2013-14). To evaluate seasonal differences, explore the interplay of water quality and hydrologic processes, and eliminate variability associated with municipal reporting, these treatment facilities were targeted for more detailed surface water sampling and characterization. Surface water samples collected from high impact watersheds exhibited significantly higher TOC, aromatic signatures, and disinfection byproduct (DBP) formation potential than watersheds with lower infestation levels. Spectroscopic analyses of surface water samples indicated that these heightened DBP precursor levels are a function of both elevated TOC loading and increased aromatic character. This association was heightened during precipitation and runoff events in high impact sites, supporting the hypothesis that altered hydrologic flow paths resulting from tree mortality mobilize organic carbon and elevate DBP formation potential for several months after runoff ceases. The historical trends found here likely underestimate the full extent of TTHM shifts due to monitoring biases with the extended seasonal release of DBP precursors increasing the potential for human exposure. Collectively, our analysis suggests that while water quality impacts continue to rise nearly one decade after infestation, significant increases in TOC mobilization and DBP precursors are limited to watersheds that experience extensive tree mortality.

Environmental and personal determinants of the uptake of disinfection by-products during swimming.

BACKGROUND: Trihalomethanes (THMs) in exhaled breath and trichloroacetic acid (TCAA) in urine are internal dose biomarkers of exposure to disinfection by-products (DBPs) in swimming pools. OBJECTIVE: We assessed how these biomarkers reflect the levels of a battery of DBPs in pool water and trichloramine in air, and evaluated personal determinants. METHODS: A total of 116 adults swam during 40min in a chlorinated indoor pool. We measured chloroform, bromodichloromethane, dibromochloromethane and bromoform in exhaled breath and TCAA in urine before and after swimming, trichloramine in air and several DBPs in water. Personal determinants included sex, age, body mass index (BMI), distance swum, energy expenditure, heart rate and 12 polymorphisms in GSTT1, GSTZ1 and CYP2E1 genes. RESULTS: Median level of exhaled total THMs and creatinine adjusted urine TCAA increased from 0.5 to 14.4µg/m(3) and from 2.5 to 5.8µmol/mol after swimming, respectively. The increase in exhaled brominated THMs was correlated with brominated THMs, haloacetic acids, haloacetonitriles, haloketones, chloramines, total organic carbon and total organic halogen in water and trichloramine in air. Such correlations were not detected for exhaled chloroform, total THMs or urine TCAA. Exhaled THM increased more in men, urine TCAA increased more in women, and both were affected by exercise intensity. Genetic variants were associated with differential increases in exposure biomarkers. CONCLUSION: Our findings suggest that, although affected by sex, physical activity and polymorphisms in key metabolizing enzymes, brominated THMs in exhaled breath could be used as a non-invasive DBP exposure biomarker in swimming pools with bromide-containing source waters. This warrants confirmation with new studies.

Sites of Anesthetic Inhibitory Action on a Cationic Ligand-Gated Ion Channel.

Pentameric ligand-gated ion channels have been identified as the principal target of general anesthetics (GA), whose molecular mechanism of action remains poorly understood. Bacterial homologs, such as the Gloeobacter violaceus receptor (GLIC), have been shown to be valid functional models of GA action. The GA bromoform inhibits GLIC at submillimolar concentration. We characterize bromoform binding by crystallography and molecular dynamics (MD) simulations. GLIC's open form structure identified three intra-subunit binding sites. We crystallized the locally closed form with an additional bromoform molecule in the channel pore. We systematically compare binding with the multiple potential sites of allosteric channel regulation in the open, locally closed, and resting forms. MD simulations reveal differential exchange pathways between sites from one form to the other. GAs predominantly access the receptor from the lipid bilayer in all cases. Differential binding affinity among the channel forms is observed; the pore site markedly stabilizes the inactive versus active state.

NKT cell modulates NAFLD potentiation of metabolic oxidative stress-induced mesangial cell activation and proximal tubular toxicity.

Obesity and nonalcoholic fatty liver disease (NAFLD) are associated with the development and progression of chronic kidney disease. We recently showed that NAFLD induces liver-specific cytochrome P-450 (CYP)2E1-mediated metabolic oxidative stress after administration of the CYP2E1 substrate bromodichloromethane (BDCM) (Seth RK, Das S, Kumar A, Chanda A, Kadiiska MB, Michelotti G, Manautou J, Diehl AM, Chatterjee S. Toxicol Appl Pharmacol 274: 42-54, 2014; Seth RK, Kumar A, Das S, Kadiiska MB, Michelotti G, Diehl AM, Chatterjee S. Toxicol Sci 134:291-303, 2013). The present study examined the effects of CYP2E1-mediated oxidative stress in NAFLD leading to kidney toxicity. Mice were fed a high-fat diet for 12 wk to induce NAFLD. NAFLD mice were exposed to BDCM, a CYP2E1 substrate, for 4 wk. NAFLD + BDCM increased CYP2E1-mediated lipid peroxidation in proximal tubular cells compared with mice with NAFLD alone or BDCM-treated lean mice, thus ruling out the exclusive role of BDCM. Lipid peroxidation increased IL-1ß, TNF-α, and interferon-γ. In parallel, mesangial cell activation was observed by increased α-smooth muscle actin and transforming growth factor-ß, which was blocked by the CYP2E1 inhibitor diallyl sulphide both in vivo and in vitro. Mice lacking natural killer T cells (CD1d knockout mice) showed elevated (>4-fold) proinflammatory mediator release, increased Toll-like receptor (TLR)4 and PDGF2 mRNA, and mesangial cell activation in the kidney. Finally, NAFLD CD1D knockout mice treated with BDCM exhibited increased high mobility group box 1 and Fas ligand levels and TUNEL-positive nuclei, indicating that higher cell death was attenuated in TLR4 knockout mice. Tubular cells showed increased cell death and cytokine release when incubated with activated mesangial cells. In summary, an underlying condition of progressive NAFLD causes renal immunotoxicity and aberrant glomerular function possibly through high mobility group box 1-dependent TLR4 signaling and mesangial cell activation, which, in turn, is modulated by intrinsic CD1D-dependent natural killer T cells.

Removal of trihalomethane from chlorinated seawater using gamma radiation.

Chlorine addition as a biocide in seawater results in the formation of chlorination by-products such as trihalomethanes (THMs). Removal of THMs is of importance as they are potential mutagenic and carcinogenic agents. In this context, a study was conducted that used ionizing radiation to remove THMs from chlorinated (1, 3, and 5 mg/L) seawater by applying various dosages (0.4-5.0 kGy) of gamma radiation. Bromoform (BF) showed a faster rate of degradation as compared to other halocarbons such as bromodichloromethane (BDCM) and dibromochloromethane (DBCM). In chlorine-dosed seawater, total irradiation dose of 0.4 to 5 kGy caused percentage reduction in the range of 6.9 to 76.7%, 2.3 to 99.6%, and 45.7 to 98.3% for BDCM, DBCM, and BF, respectively. During the irradiation process, pH of the chlorinated seawater decreased with increase in the absorbed dose; however, no change in total organic carbon (TOC) was observed. The results show that gamma dose of 2.5 kGy was adequate for maximum degradation of THM; but for complete mineralization, higher dose would be required.

Trihalomethane exposure and biomonitoring for the liver injury indicator, alanine aminotransferase, in the United States population (NHANES 1999-2006).

Exposure to trihalomethanes (or THMs: chloroform, bromoform, bromodichloromethane, and dibromochloromethane [DBCM]) formed via drinking water disinfection has been associated with adverse reproductive outcomes and cancers of the digestive or genitourinary organs. However, few studies have examined potential associations between THMs and liver injury in humans, even though experimental studies suggest that these agents exert hepatotoxic effects, particularly among obese individuals. This study examined participants in the National Health and Nutrition Examination Survey (1999-2006, N=2781) to test the hypothesis that THMs are associated with liver injury as assessed by alanine aminotransferase (ALT) activity in circulation. Effect modification by body mass index (BMI) or alcohol consumption also was examined. Associations between blood THM concentrations and ALT activity were assessed using unconditional multiple logistic regression to calculate prevalence odds ratios (ORs) with 95% confidence intervals (CIs) for exposure among cases with elevated ALT activity (men: >40IU/L, women: >30IU/L) relative to those with normal ALT, after adjustment for variables that may confound the relationship between ALT and THMs. Compared to controls, cases were 1.35 times more likely (95% CI: 1.02, 1.79) to have circulating DBCM concentrations exceeding median values in the study population. There was little evidence for effect modification by BMI, although the association varied by alcohol consumption. Among non-drinkers, cases were more likely than controls to be exposed to DBCM (OR: 3.30, 95% CI: 1.37, 7.90), bromoform (OR: 2.88, 95% CI: 1.21, 6.81), or brominated THMs (OR: 4.00, 95% CI: 1.31, 12.1), but no association was observed among participants with low, or moderate to heavy alcohol consumption. Total THM levels exceeding benchmark exposure limits continue to be reported both in the United States and globally. Results from this study suggest a need for further characterization of ALT activity and possibly other hepatic or metabolic diseases in populations with elevated drinking water THM concentrations.

Preferential expression of a bromoperoxidase in sporophytes of a red alga, Pyropia yezoensis.

A 2,158 bp cDNA (PyBPO1) encoding a bromoperoxidase (BPO) of 625 amino acids was isolated from Pyropia yezoensis. Phylogenetic analysis using amino acid sequences of BPOs suggested that P. yezoensis and cyanobacteria were grouped in the same clade and separated from brown algae. Genomic Southern blot analysis suggested that PyBPO1 existed as a single copy per haploid genome. RT-PCR revealed that PyBPO1 was actively expressed in filamentous sporophytes but repressed in leafy gametophytes under normal growth conditions. High expression levels of PyBPO1 in sporophytes were observed when sporophytes were grown under gametophyte conditions, suggesting that preferential expression of PyBPO1 occurs during the sporophyte phase. BPO activity of cell-free extracts from sporophytes and gametophytes was examined by activity staining on native PAGE gel using o-dianisidine. One activity band was detected in sporophyte sample, but not in gametophyte sample. In addition, we found that bromide and iodide were effective substrate, but chloride was not. BPO activity was observed-likely in chloroplasts-when sporophyte cells were incubated with o-dianisidine and hydrogen peroxide. Cellular BPO staining showed the same halogen preference identified by in-gel BPO staining. Based on GS-MS analysis, bromoform was detected in medium containing sporophytes. Bromoform was not detected under dark culture conditions but was detected in the culture exposed to low light intensity (5 µmol m(-2) s(-1)) and increased under a moderate light intensity (30 µmol m(-2) s(-1)).

Effect of chlorine dioxide on cyanobacterial cell integrity, toxin degradation and disinfection by-product formation.

Bench scale tests were conducted to study the effect of chlorine dioxide (ClO2) oxidation on cell integrity, toxin degradation and disinfection by-product formation of Microcystis aeruginosa. The simulated cyanobacterial suspension was prepared at a concentration of 1.0×10(6)cells/mL and the cell integrity was measured with flow cytometry. Results indicated that ClO2 can inhibit the photosynthetic capacity of M. aeruginosa cells and almost no integral cells were left after oxidation at a ClO2 dose of 1.0mg/L. The total toxin was degraded more rapidly with the ClO2 dosage increasing from 0.1mg/L to 1.0mg/L. Moreover, the damage on cell structure after oxidation resulted in released intracellular organic matter, which contributed to the formation of trihalomethanes (THMs) and haloacetic acids (HAAs) as disinfection by-products. Therefore, the use of ClO2 as an oxidant for treating algal-rich water should be carefully considered.

Simple growth patterns can create complex trajectories for the ontogeny of constitutive chemical defences in seaweeds.

All of the theory and most of the data on the ecology and evolution of chemical defences derive from terrestrial plants, which have considerable capacity for internal movement of resources. In contrast, most macroalgae--seaweeds--have no or very limited capacity for resource translocation, meaning that trade-offs between growth and defence, for example, should be localised rather than systemic. This may change the predictions of chemical defence theories for seaweeds. We developed a model that mimicked the simple growth pattern of the red seaweed Asparagopsis armata which is composed of repeating clusters of somatic cells and cells which contain deterrent secondary chemicals (gland cells). To do this we created a distinct growth curve for the somatic cells and another for the gland cells using empirical data. The somatic growth function was linked to the growth function for defence via differential equations modelling, which effectively generated a trade-off between growth and defence as these neighbouring cells develop. By treating growth and defence as separate functions we were also able to model a trade-off in growth of 2-3% under most circumstances. However, we found contrasting evidence for this trade-off in the empirical relationships between growth and defence, depending on the light level under which the alga was cultured. After developing a model that incorporated both branching and cell division rates, we formally demonstrated that positive correlations between growth and defence are predicted in many circumstances and also that allocation costs, if they exist, will be constrained by the intrinsic growth patterns of the seaweed. Growth patterns could therefore explain contrasting evidence for cost of constitutive chemical defence in many studies, highlighting the need to consider the fundamental biology and ontogeny of organisms when assessing the allocation theories for defence.

Microbial transformation of dissolved organic matter from different sources and its influence on disinfection byproduct formation potentials.

Biodegradation-induced changes in the characteristics of dissolved organic matter (DOM) and the subsequent effects on disinfection byproduct formation potentials (DBPFPs) were investigated using six different sources of DOM (algae, leaf litter, reed, compost, paddy water, and treated municipal sewage effluent). Microbial incubation of the DOM samples increased the specific ultraviolet absorbance and humic-like fluorescence but decreased the protein/tannin-like fluorescence and relative distribution of smaller-sized DOM components. Comparison of the original versus biodegraded DOM samples using resin fractionation and pyrolysis-gas chromatography/mass spectrometry revealed that the biodegradation-induced changes were highly dependent on DOM sources and exhibited no consistent trends among the different sources. Changes in DBPFPs also differed with DOM source. Vascular plant-derived DOM (leaf litter and reed) demonstrated an enhancement in specific DBPFP after biodegradation, whereas little change or even a slight decrease was observed for the other DOM sources. Correlations that were significant between specific DBPFPs and the aromatic content or humic-like fluorescence for the original DOM samples were no longer significant after microbial degradation. The relative abundance of hydrophobic to hydrophilic structures in DOM is suggested to be a general indicator for the formation potential of trihalomethanes irrespective of DOM source and the state of biodegradation.

The role of nitrobenzene on the yield of trihalomethane formation potential in aqueous solutions with Microcystis aeruginosa.

Algae are one of the most important disinfection by-product (DBP) precursors in aquatic environments. The contents of DBP precursors in algae are influenced by not only environmental factors but also some xenobiotics. Trihalomethane formation potential (THMFP) in both the separate and interactive pollution of Microcystis aeruginosa and Nitrobenzene (NB) was investigated in batch experiment to discover the effects of xenobiotics on the yield of DBP precursors in the algal solution. The results show that in the separate NB solution, NB did not react with Cl(2) to form trihalomethane (THM), whereas in the algae solution, THMFP had a significant positive linear correlation with M. aeruginosa density in both solution and extracellular organic matter (EOM). The correlation coefficients were 0.9845 (p = 3.567 × 10(-4)) and 0.9854 (p = 1.406 × 10(-4)), respectively. According to regression results, about 77.9% of the total THMFP came from the algal cells, while the rest came from EOM. When the interactive pollution of M. aeruginosa and NB occurred, the growth of algae was inhibited by NB. The density of M. aeruginosa in a high concentration NB solution (280 µg/L) was only 71.1% of that in the solution without NB after 5 days of incubation. However, THMFP in the mixture (algae and NB) and the EOM did not change significantly, and the productivity of THMFP by the algae (THMFP/10(8)cells) increased with the increase in NB concentration. There was a significant linear correlation between THMFP/10(8)cell and NB concentration (r = 0.9117, p < 0.01), which shows the contribution of the algae to THM formation was enhanced by NB. This result might be caused by the increased protein productivity and the biodegradation of NB by M. aeruginosa.