Chlorate Toxicity Research Articles

Overlooked Cytotoxicity and Genotoxicity to Mammalian Cells Caused by the Oxidant Peroxymonosulfate during Wastewater Treatment Compared with the Sulfate Radical-Based Ultraviolet/Peroxymonosulfate Process.

Byproduct formation (chlorate, bromate, organic halogen, etc.) during sulfate radical (SO4•-)-based processes like ultraviolet/peroxymonosulfate (UV/PMS) has aroused widespread concern. However, hypohalous acid (HOCl and HOBr) can form via two-electron transfer directly from PMS, thus leading to the formation of organic halogenated byproducts as well. This study found both PMS alone and UV/PMS can increase the toxicity to mammalian cells of wastewater, while the UV/H2O2 decreased the toxicity. Cytotoxicity of two wastewater samples increased from 5.6-8.3 to 15.7-29.9 mg-phenol/L, and genotoxicity increased from 2.8-3.1 to 5.8-12.8 μg 4-NQO/L after PMS treatment because of organic halogen formation. Organic halogen formation from bromide rather than chloride was found to dominate the toxicity increase. The SO4•--based process UV/PMS led to the formation of both organic halogen and inorganic bromate and chlorate. However, because of the very low concentration (<20 μg/L) and relatively low toxicity of bromate and chlorate, contributions of inorganic byproducts to toxicity increase were negligible. PMS would not form chlorate and bromate, but it generated a higher concentration of total organic halogen, thus leading to a more toxic treated wastewater than UV/PMS. UV/PMS formed less organic halogen and toxicity because of the destruction of byproducts by UV irradiation and the removal of byproduct precursors. Currently, many studies focused on the byproducts bromate and chlorate during SO4•--based oxidation processes. This work revealed that the oxidant PMS even needs more attention because it caused higher toxicity due to more organic halogen formation.

Methionine oxidation under anaerobic conditions in Escherichia coli.

Repairing oxidative-targeted macromolecules is a central mechanism necessary for living organisms to adapt to oxidative stress. Reactive oxygen and chlorine species preferentially oxidize sulfur-containing amino acids in proteins. Among these amino acids, methionine can be converted into methionine sulfoxide. This post-translational oxidation can be reversed by methionine sulfoxide reductases, Msr enzymes. In Gram-negative bacteria, the antioxidant MsrPQ system is involved in the repair of periplasmic oxidized proteins. Surprisingly, in this study, we observed in Escherichia coli that msrPQ was highly expressed in the absence of oxygen. We have demonstrated that the anaerobic induction of msrPQ was due to chlorate (ClO3 - ) contamination of the Casamino Acids. Molecular investigation led us to determine that the reduction of chlorate to the toxic oxidizing agent chlorite (ClO2 - ) by the three nitrate reductases (NarA, NarZ, and Nap) led to methionine oxidation of periplasmic proteins. In response to this stress, the E. coli HprSR two-component system was activated, leading to the over-production of MsrPQ. This study, therefore, supports the idea that methionine oxidation in proteins is part of chlorate toxicity, and that MsrPQ can be considered as an anti-chlorate/chlorite defense system in bacteria. Finally, this study challenges the traditional view of the absence of Met-oxidation during anaerobiosis.

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Detection of chlorite, chlorate and perchlorate in ozonated saline.

Medical ozone is used to treat various diseases, including numerous pathologies associated with chronic pain. Chronic pain may be treated by systemic administration of ozone, with ozonated autohemotherapy (OAH) being the commonly used method. In the clinic, intravenous infusion of ozonized saline has been used to treat various diseases. Compared with OAH, ozonized saline infusion is less technically demanding and causes minimal damage to veins. However, it has been indicated that ozone may oxidize saline and generate toxic substances, and therefore, the safety of ozone treatment has been questioned. In the present study, the potential chemical compounds produced from ozone and saline, including chlorite, chlorate and perchlorate, were examined at various time-points with ion chromatography-mass spectrometry (IC-MS). A control group (pure oxygen group) and an ozone group were included in the present study. Two subgroups were included within each group: A saline bottle (made from polypropylene) subgroup and an ozone-resistant blood transfusion bag [made from medical polyvinyl chloride, di(2-ethyl) hexyl phthalate plasticized] subgroup. For the ozone group, 100 ml saline and 100 ml medical ozone at various concentrations (20, 40 or 60 µg/ml in pure oxygen) were injected into the saline bottle or blood bag, and for the control group, 100 ml of pure oxygen was injected into the saline bottle or blood bag. The presence and the content of chlorite, chlorate and perchlorate were determined at different time-points (3, 6 and 15 days after mixing) by IC-MS. Chlorate was detected in the ozone groups at three time-points and its content increased as the ozone concentration and the reaction time increased. Under the same conditions (the same ozone concentration and the same incubation time), the chlorate content (0.90±0.14-7.69±0.48 µg/l) in the blood bag subgroup was significantly lower than that in the saline bottle subgroup (45.23±6.14-207.6±15.63 µg/l). However, chlorite and perchlorate were not detected at any time-point in the two groups. In addition, in the control group (pure oxygen group), chlorite, chlorate and perchlorate were not detected at any time-point. These results indicate that ozone reacts with saline to produce chlorate. Ozone may also react with the polypropylene saline bottle to increase the chlorate content in the bottled solution. Due to a lack of toxicology studies of chlorate in blood, it remains elusive whether ozonated saline and chlorate at the range of 0.90±0.14-7.69±0.48 µg/l has any toxic effects. The potential toxicity of chlorate should be considered when ozonated saline is used for clinical infusions.

Acute renal toxicity of sodium chlorate: Redox imbalance, enhanced DNA damage, metabolic alterations and inhibition of brush border membrane enzymes in rats.

Sodium chlorate (NaClO3 ) is widely used in paper and pulp industries and as a non-selective herbicide. Humans can be exposed to NaClO3 through contaminated drinking water due to its improper and unchecked usage in industries and as herbicide. NaClO3 is also present as a major stable by-product in drinking water that has been disinfected with chlorine dioxide. In this study, we have investigated the effect of a single acute oral dose of NaClO3 on rat kidney. Adult male Wistar rats were divided into one control and four NaClO3 treated groups that were orally given different doses of NaClO3 and euthanized 24 hr after the treatment. Oral administration of NaClO3 resulted in increased hydrogen peroxide levels, lipid, and protein oxidation while thiol and glutathione content and activities of brush border membrane enzymes were decreased in kidney in a NaClO3 dose-dependent manner. Significant alterations in the activities of enzymes involved in carbohydrate metabolism and antioxidant defense were also observed. Administration of NaClO3 induced DNA fragmentation and increased DNA-protein cross-linking. Histological studies showed marked damage in kidney from NaClO3 treated animals. These results strongly suggest that NaClO3 induces nephrotoxicity via redox imbalance that results in DNA and membrane damage, metabolic alterations and brush border membrane enzyme dysfunction.

Differential Chlorate Inhibition of Chaetomium globosum Germination, Hyphal Growth, and Perithecia Synthesis

Chaetomium globosum Kunze:Fr is a dermatophytic, dematiaceous fungus that is ubiquitous in soils, grows readily on cellulolytic materials, and is commonly found on water-damaged building materials. Chlorate affects nitrogen metabolism in fungi and is used to study compatibility among anamorphic fungi by inducing nit mutants. The effect of chlorate toxicity on C. globosum was investigated by amending a modified malt extract agar (MEA), oat agar, and carboxymethyl cellulose agar (CMC) with various levels of potassium chlorate (KClO(3)). C. globosum perithecia production was almost completely inhibited (90-100 %) at low levels of KClO(3) (0.1 mM) in amended MEA. Inhibition of perithecia production was also observed on oat agar and CMC at 1 and 10 mM, respectively. However, hyphal growth in MEA was only inhibited 20 % by 0.1-100 mM KClO(3) concentrations. Hyphal growth was never completely inhibited at the highest levels tested (200 mM). Higher levels of KClO(3) were needed on gypsum board to inhibit perithecia synthesis. In additional experiments, KClO(3) did not inhibit C. globosum, Fusarium oxysporum, Aspergillus niger, Penicillum expansum, and airborne fungal spore germination. The various fungal spores were not inhibited by KClO(3) at 1-100 mM levels. These results suggest that C. globosum perithecia synthesis is more sensitive to chlorate toxicity than are hyphal growth and spore germination. This research provides basic information that furthers our understanding about perithecia formation and may help in developing control methods for fungal growth on building materials.

Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in Aspergillus niger

We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV), 1, 2, 7, 8-Diepoxyoctane (DEO), phenols mixture (Phx)) and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS) strain, which may specify nitrite specific transporter) and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system). The most relevant and innovative findings of this research work involve the isolation of the RR ( cbrn) mutants (a new type of nitrate assimilation defective mutants), that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments) were of the brn type, whereas the remaining percentage (6.76%) was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains) were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants) with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants` yield of Aspergillus niger mutant strains representing nine gene loci ( niaD, cnx-6 loci , nrt and nirA) were selected on the bases of chlorate (600 mM) toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM). The niaD mutants showed the highest percentage (73.97%) of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9%) of niaD mutants, whereas, the least (61%) was obtained with Phx treatment. The highest percentage of cnx mutants (32%) was obtained with Phx treatment. The DEO treatment as compared to other tested treatments was the best to use for obtaining the highest ratios of either nrt (13.8%) mutants or nirA (1.9%) mutants.

Isolation and growth characterization of chlorate and/or bromate resistant mutants generated by spontaneous and induced foreword mutations at several gene loci in aspergillus niger

We aimed her mainly to evaluate the contribution of newly employed bromate selection system, in obtaining new Aspergillus niger nitrate/nitrite assimilation defective mutants, through Ultraviolet treatment (UV), 1, 2, 7, 8-Diepoxyoctane (DEO), phenols mixture (Phx)) and spontaneous treatments. The newly employed bromate selection system was able to specify only two putative novel mutant types designated brn (bromate resistant but chlorate sensitive (RS) strain, which may specify nitrite specific transporter) and cbrn mutants (bromate resistant and chlorate resistant strain, which may specify nitrate/nitrite bispecific system). The most relevant and innovative findings of this research work involve the isolation of the RR ( cbrn) mutants (a new type of nitrate assimilation defective mutants), that could be useful for studying the bispecific nitrate /nitrite transporter system. The majority of obtained bromate resistant mutants (93.3% of the total mutants obtained by all treatments) were of the brn type, whereas the remaining percentage (6.76%) was given to cbrn strains. The highest percentages of brn mutant strains (48% and 58.6% of the total RS strains) were obtained with UA after spontaneous and Phx treatment, whereas Trp has generated 29% and 42% of RS strains after UV and DEO treatments, respectively. The obtained ratios of cbrn mutants were higher (i.e. in the range of 8.4%-11.64% of the total bromate mutants) with chemical treatments, especially when U.A or Pro was serving as sole N-sources at 25ºC rather than 37ºC. A 69% mutants` yield of Aspergillus niger mutant strains representing nine gene loci ( niaD, cnx-6 loci , nrt and nirA) were selected on the bases of chlorate (600 mM) toxicity. All chlorate resistant mutants were completely sensitive to bromate (250 mM). The niaD mutants showed the highest percentage (73.97%) of chlorate resistant mutants obtained with all tested treatments. The UV treatment has generated the highest ratio (86.9%) of niaD mutants, whereas, the least (61%) was obtained with Phx treatment. The highest percentage of cnx mutants (32%) was obtained with Phx treatment. The DEO treatment as compared to other tested treatments was the best to use for obtaining the highest ratios of either nrt (13.8%) mutants or nirA (1.9%) mutants.

Nitrate reduces the detrimental effect of potassium chlorate on longan ( Dimocarpus Longan Lour.) trees

A recent finding that potassium chlorate induces longan ( Dimocarpus Longan Lour.) flowering at any seasons has led to wide use of this chemical in longan industry for off-season harvest, despite the risk of injury to the tree. In this paper, we examined the influences of pretreatment of nitrate by foliar spray with 3% potassium nitrate on the responses of potted longan ( Dimocarpus longan Lour.) cv. Shixia (a subtropical ecotype) trees to soil applications of potassium chlorate at 10 and 20 g plant −1. Chlorate treatments caused severe leaf drop and chlorophyll breakdown and suppressed budbreak, flowering and shoot growth. Pretreatment of potassium nitrate reduced the severity of leaf drop and chlorophyll breakdown caused by chlorate but the extend was less significant in high dosage chlorate (20 g plant −1) treatment than low dosage (10 g plant −1) chlorate treatment. Potassium nitrate did not alleviate the suppression effect of chlorate on budbreak, flowering and shoot growth, although it promoted these processes per se. Chlorate treatment resulted in only a transient accumulation of chlorate in the leaves, which peaked around 14 days after chlorate treatment (DACT), while chloride accumulated constantly within 28 DACT. Pretreatment of potassium nitrate tended to promote chlorate accumulation but did not cause a significant change in chloride accumulation. Severity of leaf drop was not significantly correlated to chlorate accumulation but significantly to chloride accumulation, suggesting that toxicity of chlorate to longan tree was realized during the chloride-producing process of chlorate reduction. Nitrate influenced the correlations between severity of leaf drop and chloride accumulation by reducing the slopes of the regressed lines of leaf drop versus chloride, indicating that nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during chlorate reduction. The results indicate that the application rates were excessive for potted trees. The potential benefit of potassium nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees.

Effects of experimental chlorate preparations as feed and water supplements on Escherichia coli colonization and contamination of beef cattle and carcasses

The effects of feed or water administration of experimental chlorate preparations on recovery of generic Escherichia coli and E. coli O157:H7 from the gut, hide and carcasses of feedlot cattle were tested. Sixty-four naturally colonized cattle were randomly assigned to one of four feed treatments with or without a 12 h chlorate-containing water treatment. An analysis of variance revealed a main effect of feed treatment ( P = 0.002 ) on generic E. coli concentrations in feces collected before shipment to slaughter. Main effect means were 5.57, 4.75, 5.00 and 4.08 log 10 cfu/g for animals fed an experimental chlorate product at 0, 0.01% in last meal, 0.01% for last 5 d and 0.05% of body weight in last meal, respectively. A main effect of feed treatment was observed ( P = 0.041 ) on generic E. coli concentrations in feces collected at slaughter (means for the respective treatments were 4.92, 3.57, 3.98 and 3.20 log 10 cfu/g) as well as on numbers of generic E. coli recovered ( P = 0.034 ) from hide swabs collected at the rump (means for the respective treatments were 4.86, 3.92, 3.87 and 4.06 log 10 cfu/swab). A main effect of water treatment was observed ( P < 0.016 ) on generic E. coli concentrations in rumen contents (3.44 vs. 2.72 cfu log 10 cfu/g for animals administered 0 or 2500 ppm active chlorate ion, respectively). Logistic regression analysis revealed a main effect of feed treatment ( P < 0.001 ) on the incidence of E. coli O157:H7 recovered from feces collected at slaughter (75%, 33%, 20% and 25% for animals fed an experimental chlorate product at 0%, 0.01% in last meal, 0.01% for last 5 d and 0.05% of body weight in last meal, respectively). Animals exhibited no symptoms of chlorate toxicity and negative effects on feed or water intake or animal performance were not observed.

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit(-), unable to grow in nitrate, and 28 Nit(+), able to grow in nitrate). All the Nit(-) strains lacked MoCo activity. Diploid complementation of Nit(-), MoCo(-) strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit(+) strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c(+)) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c(+) seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.

Ultrastructural, physiological and biochemical analyses of chlorate toxicity on rice seedlings

Chlorates are oxidizing substances considered phytotoxic to all green plant parts. In this study the physiological, biochemical and ultrastructural effects of chlorate treatments were studied in rice seedlings grown at two different temperatures (28 and 6 °C). Visual analyses demonstrated that the chlorate treatment provoked severe damage to plant leaves only at 28 °C. The electron microscopy of the chloroplast revealed several changes in the organelle organization, whose integrity of outer membrane and the internal network of thylakoid membranes were almost completely lost. A high level of lipid peroxidation was observed in 28 °C-chlorate-treated leaves in comparison to plants exposed to 6 °C-chlorate treatment. Photochemical efficiency of photosynthesis ( F v/ F m) was drastically affected by the 28 °C-chlorate treatment but, at low temperature, chlorate-treated plants suffered a discrete reduction in F v/ F m. Root structure and organization were analyzed by light microscopy (LM) and scanning electron microscopy (SEM). The 28 °C-chlorate treatment provoked severe damages to the exodermis, endodermis, cortex cells and root hairs. In the 6 °C-chlorate treatment, damage to root cells was also observed, but at a lower intensity. Nitrate reductase (NR) activity was analyzed and revealed the involvement of this enzyme in the chlorate action, even at a low temperature.

Chlorate resistance as a tool to study the effect of nitrate reductase antisense gene in wheat

The herbicide chlorate has been used extensively for isolation of mutants or for selection of transgenic plants defective in nitrate reduction. Chlorate, acting as an analogue of nitrate, is substrate of the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Plants being lack of NR function are resistant to chlorate. In this work chlorate resistance was used to control the efficiency of gene transfer cnxl antisense in wheat. Cnxl was transferred with cotransformation of pAHC25 containing bar and gus into immature embryo-derived calli of a spring wheat genotype (CY-45) by microprojectile bombardment The nitrate - chlorate interaction was investigated via nitrate reductase activity (NRA) assay and necrotic symptoms of chlorate toxicity. The optimal conditions of chlorate resistance test signalling the decrease of NRA were determined, and found to be 0.4 mM nitrate for NR induction, and 7 mM chlorate content for evoking greatest differences in toxic symptoms. We succeeded to regenerate of 10 plantlets resistant to phosphinothricine (PPT) indicating the expression of bar, and 6 of them were fertile producing 6 PPT resistant transgenic lines. Among the T 1 progenies of PPT transgenics, two (designated as 117 and 124) showed a significant reduction of NRA. Though T 2 progenies had a less decreased NRA in the mean of the population, but a higher chlorate resistance as compared to that of the control was still experienced.

Eukaryotic Molybdopterin Synthase: BIOCHEMICAL AND MOLECULAR STUDIES OF ASPERGILLUS NIDULANS cnxG AND cnxH MUTANTS

We describe the primary structure of eukaryotic molybdopterin synthase small and large subunits and compare the sequences of the lower eukaryote, Aspergillus nidulans, and a higher eukaryote, Homo sapiens. Mutants in the A. nidulans cnxG (encoding small subunit) and cnxH (large subunit) genes have been analyzed at the biochemical and molecular level. Chlorate-sensitive mutants, all the result of amino acid substitutions, were shown to produce low levels of molybdopterin, and growth tests suggest that they have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Thus on the basis of chlorate toxicity, it is possible to distinguish between amino acid substitutions that permit a low level of molybdopterin production and those mutations that completely abolish molybdopterin synthesis, most likely reflecting molybdopterin synthase activity per se. Residues have been identified that are essential for function including the C-terminal Gly of the small subunit (CnxG), which is thought to be crucial for the sulfur transfer process during the formation of molybdopterin. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity suggesting that this residue resides in a region important for correct folding of the fungal protein. Many years ago it was proposed, from data showing that temperature-sensitive cnxH mutants had thermolabile nitrate reductase, that CnxH is an integral part of the molybdoenzyme nitrate reductase (MacDonald, D. W., and Cove, D. J. (1974) Eur. J. Biochem. 47, 107-110). Studies of temperature-sensitive cnxH mutants isolated in the course of this study do not support this hypothesis. Homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans as discussed in this article. In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae. Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzymes.

Toxicity of Chlorate and Chlorite to Selected Species of Algae, Bacteria, and Fungi

The present study confirms that chlorate is toxic only to brown algae and not to species of other ecologically relevant taxa. The brown algaEctocarpus variabilisexhibited a LOEC of 0.005 mM (0.4 mg ClO−3/liter) and an LC50of 0.012 mM, when cultured with nitrate as a sole source of nitrogen. The toxicity to species other than brown algae as measured in growth inhibition tests ranged from 0.75 mM (96-h NOEC) forSelenastrum capricornutumto ≥7.48 mM (48-h NOEC) for the fungusTrichoderma hamatum. The nitrogen source, nitrate or ammonium, did not significantly influence the toxicity to the nonsensitive species. The tests on brown algae found that as compared with ammonium, the toxicity to nitrate-grown cultures is higher by a factor of about 10. This confirms the hypothesis that nitrate reductase is involved in the toxic effects of chlorate on brown algae. Chlorite, tested as a potential toxic metabolite of chlorate, demonstrated high toxicity to many of the taxa tested and only low toxicity toE. variabilis. It may be concluded that brown algae are exceptionally sensitive to chlorate. It may also be concluded that various nitrogen sources could not induce toxicity in nonsensitive species. From these experiments no conclusions could be drawn as to the potential role of chlorite in chlorate toxicity. Furthermore it may be concluded thatE. variabilisis a suitable laboratory test species for further investigations into the mechanism of chlorate toxicity to brown algae.

Toxicity of chlorate to marine microalgae

Recent concern over the release of chlorate into the aquatic environment has focused on its occurrence in industrial effluents and its presence in hypochlorite solutions used in drinking water disinfection. While algae are known to be more sensitive than invertebrates and fish to chlorate, very little information on chlorate toxicity to marine microalgae is available. In particular, the mechanism of toxicity of chlorate and the effect of ambient nitrate levels on chlorate uptake into algae has not been well studied. The effect of chlorate on growth and nitrate reductase activity in two species of marine microalgae Nitzschia closterium and Dunaliella tertiolecta was investigated as part of a risk assessment for the construction of a new bleached eucalypt kraft (BEK) pulp mill on the south-east coast of Australia. Nitrate inhibited chlorate uptake and toxicity in both species. Under nitrate-limiting conditions typically found in oligotrophic waters (<0.005 mg l −1), 72-h EC 50 values for chlorate were 1.9 and 11 mg l −1 for Nitzschia and Dunaliella respectively. At high nitrate concentrations both algae were insensitive to chlorate, with 72-h EC 50 values greater than 500 and 1000 mg l −1 respectively. In Dunaliella only, when chlorate was in excess of nitrate (on a molar basis), nitrate uptake was reduced due to inhibition of nitrate reductase activity. It is possible that in Dunaliella, as in higher plants, chlorate is converted by the enzyme nitrate reductase to chlorite which is the toxic agent. In Nitzschia, however, nitrate reductase activity was low even in the absence of chlorate. At chlorate concentrations inhibitory to cell division, there was no effect on photosynthesis, ATP or cellular thiols in Nitzschia. A small decrease in dehydrogenase activity was found after a 24-h exposure to chlorate, but this was not coupled to a decrease in ATP. It appears that chlorate toxicity to Nitzschia at low nitrate concentrations does not depend on chlorite as an intermediate. This study confirms that chlorate is toxic to marine microalgae, particularly in nitrate limited waters. Sensitivity to chlorate and its mechanism of toxicity appear to differ between species.

Responses of NADH and NADPH nitrate reductase activities and nitrate uptake to chlorate supply in maize seedlings

Summary Experiments were conducted to examine the responses of NADH and NADPH specific nitrate reductase (NR) activities from various organs of maize seedlings to chlorate, with a view to understand the mechanism of chlorate toxicity and to ellucidate the significance of NAD(P)H: NR under toxic conditions. Supply of 1, 5, 10 or 20 mmol/L KCIO 3 under inducing conditions (10 mmol/L nitrate) to 4-5-day-old seedlings grown without nitrogen for 24 h in light (67 W m −2 ) at 25°C inhibited NADH:NR activity in the shoots and scutellum, and to some extent in the roots also. On the other hand, NADPH: NR activity was increased substantially in the scutellum and decreased (above 1 mmol/L chlorate) in roots by chlorate supply. Total protein content of the shoot or scutellum was inhibited by 20 mmol/L chlorate and was almost unaffected at lower concentrations in these organs; it increased slightly at all concentrations of chlorate in the roots. Nitrate acquisition by the seedlings was inhibited to an almost similar magnitude at each concentration of chlorate used. In another experiment involving short term (3h) supply of 5 mmol/L chlorate to the seedlings under inducing (+nitrate) or non-inducing (−nitrate) conditions, NADH:NR activity was usually inhibited and NADPH:NR activity was increased in scutellum or unaffected in roots. During in vitro incubation of enzyme preparations for 1 h at 4°C with 5 mmol/L chlorate, 10 mmol/L nitrate or chlorate + nitrate, NADH:NR activity increased in shoots and to some extent in roots also, but had variable effects in scutellum. Root NADPH:NR activity also increased by incubation with either of these ions, while in the scutellum it was drastically reduced. The experiments demonstrate an organ specific and isoform specific response of NR to chlorate and also that chlorate was acting directly on enzyme molecules under in vitro conditions.

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii.

Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wild-type strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit- mutants of C. reinhardtii, but failed to produce His+ revertants or Arar mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three hcr loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit+ and nit- chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.

Chlorate Discharges from Pulp Mills: An Examination of Effects on River Algal Communities

Abstract To avoid the formation of organochlorine compounds in pulp products and effluents discharged into the environment, there is a trend in the North American pulping industry to change from molecular chlorine to chlorine dioxide bleaching. However, chlorate (ClO3−) formed during chlorine dioxide bleaching is known to have toxic effects in some marine algae and has been implicated in environmental problems in the Baltic Sea. This study examined the potential effects of chlorate on natural freshwater, riverine diatom communities characteristic of many rivers in western Canada. Under low ambient river nitrate concentrations (ca. 10 µg•L−1 NO3−N), chlorate additions of up to 500 µg•L1 ClO3 did not reduce the specific growth rates (µ) or change the taxonomic composition of the attached riverine diatom community. The lack of chlorate toxicity to freshwater diatoms suggests that the nitrate uptake and/or reduction mechanism(s) of freshwater diatoms have much higher affinities for NO3 than ClO3 compared to many marine macroalgae. These results indicate that chlorate discharged from new or upgraded pulpmills into freshwater river environments will not cause environmental changes to the dominant algal producers such as diatoms.

Factors Affecting Chlorate Sensitivity in Callus Cultures of Medicago sativa

Summary Tolerance to toxic levels of chlorate was induced in callus cultures of Medicago sativa (alfalfa)by incuding succinate buffer in Murashige and Skoog (1962) basal media. This phenomenon was not due to the effects of succinate itself but, rather, appeared to be due to its buffering effects as other buffers had a similar effect. Added nitrogen in the form of amino acids, also had a protective effect regarding chlorate toxicity. These protective treatments were not associated with a lack of in vivo nitrate reductase activity. Another species in the Medicago genus, M. falcata, also showed the succinate effect but species of other genera in the Leguminosae (Trifolium and Onobrychis) and Solanaceae (Solanum, Nicotiana, Petunia) did not show chlorate tolerance in the presence of succinate.