Effects of Modification of Light Parameters on the Production of Cryptophycin, Cyanotoxin with Potent Anticancer Activity, in Nostoc sp.

Cryptophycin-1 is a cyanotoxin produced by filamentous cyanobacteria. It has been evaluated as an anticancer agent with great potential. However, its synthesis provides insufficient yield for industrial use. An alternative solution for metabolite efficient production is to stress cyanobacteria by modifying the environmental conditions of the culture (Nostoc sp. ATCC 53789). Here, we examined the effects of light photoperiod, wavelength, and intensity. In light photoperiod, photoperiods 24:0 and 16:8 (light:dark) were tested while in wavelength, orange-red light was compared with blue. Medium, high, and very high light intensity experiments were performed to test the effect of light stress. For a 10-day period, growth was measured, metabolite concentration was calculated through HPLC, and the related curves were drawn. The differentiation of light wavelength had a major effect on the culture, as orange-red filter contributed to noticeable increase in both growth and doubled the cyanotoxin concentration in comparison to blue light. Remarkably, constant light provides higher cryptophycin yield, but slightly lower growth rate. Lastly, the microorganism prefers medium light intensities for both growth and metabolite expression. The combination of these optimal conditions would contribute to the further exploitation of cryptophycin.

Comparative ecophysiology of Dinophysis acuminata and D. acuta (DINOPHYCEAE, DINOPHYSIALES): effect of light intensity and quality on growth, cellular toxin content, and photosynthesis.

Dinoflagellates of the genus Dinophysis are the most persistent producers of lipophilic shellfish toxins in Western Europe. Their mixotrophic nutrition requires a food chain of cryptophytes and plastid-bearing ciliates for sustained growth and photosynthesis. In this study, cultures of D. acuminata and D. acuta, their ciliate prey Mesodinium rubrum and the cryptophyte, Teleaulax amphioxeia, were subject to three experimental settings to study their physiological response to different combinations of light intensity and quality. Growth rates, pigment analyses (HPLC), photosynthetic parameters (PAM-fluorometry), and cellular toxin content (LC-MS) were determined. Specific differences in photosynthetic parameters were observed in Dinophysis exposed to different photon fluxes (10-650 µmol photons · m-2  · s-1 ), light quality (white, blue and green), and shifts in light regime. Dinophysis acuta was more susceptible to photodamage under high light intensities (370-650 µmol photons · m-2  · s-1 ) than D. acuminata but survived better with low light (10 µmol photons · m-2  · s-1 ) and to a prolonged period (28 d) of darkness. Mesodinium rubrum and T. amphioxeia showed their maximal growth rate and yield under white and high light whereas Dinophysis seemed better adapted to grow under green and blue light. Toxin analyses in Dinophysis showed maximal toxin per cell under high light after prey depletion at the late exponential-plateau phase. Changes observed in photosynthetic light curves of D. acuminata cultures after shifting light conditions from low intensity-blue light to high intensity-white light seemed compatible with photoacclimation in this species. Results obtained here are discussed in relation to different spatiotemporal distributions observed in field populations of D. acuminata and D. acuta in northwestern Iberia.

Pulsed light reduces the toxicity of the algal toxin okadaic acid to freshwater crustacean Daphnia pulex.

This constitutes the first study to report on the reduction in toxicity of the dinoflagellate algal toxin okadaic acid after novel pulsed light (PL) treatments where ecotoxicological assessment was performed using a miniaturised format of the conventional in vivo freshwater crustacean Daphnia sp. acute toxicity test. Bivalves accumulate this toxin, which can then enter the human food chain causing deleterious health effects such as diarrhetic shellfish poisoning. This miniaturised toxicological bioassay used substantially less sample volume and chemical reagents. Findings revealed a 24-h EC50 of 25.87 µg/L for PL-treated okadaic acid at a UV dose of 12.98 µJ/cm2 compared to a 24-h EC50 of 1.68 µg/L for the untreated okadaic acid control, suggesting a 15-fold reduction in toxicity to Daphnia pulex. The bioassay was validated in this study and correlated well with the "classic" ISO format (r = 0.98) using the traditional reference chemical potassium dichromate (K2Cr2O7). Reduction by up to 65% in PL-treated okadaic acid concentration was confirmed by LC-MS/MS analysis. Findings from this study have positive ecological, societal and enterprise implications, such as the development of PL technology for the prevention or reduce algal contamination of fisheries and aquaculture industries.

Degradation mechanism of cyanobacterial toxin cylindrospermopsin by hydroxyl radicals in homogeneous UV/H2O2 process.

The degradation of cylindrospermopsin (CYN), a widely distributed and highly toxic cyanobacterial toxin (cyanotoxin), remains poorly elucidated. In this study, the mechanism of CYN destruction by UV-254 nm/H2O2 advanced oxidation process (AOP) was investigated by mass spectrometry. Various byproducts identified indicated three common reaction pathways: hydroxyl addition (+16 Da), alcoholic oxidation or dehydrogenation (-2 Da), and elimination of sulfate (-80 Da). The initiation of the degradation was observed at the hydroxymethyl uracil and tricyclic guanidine groups; uracil moiety cleavage/fragmentation and further ring-opening of the alkaloid were also noted at an extended reaction time or higher UV fluence. The degradation rates of CYN decreased and less byproducts (species) were detected using natural water matrices; however, CYN was effectively eliminated under extended UV irradiation. This study demonstrates the efficiency of CYN degradation and provides a better understanding of the mechanism of CYN degradation by hydroxyl radical, a reactive oxygen species that can be generated by most AOPs and is present in natural water environment.

Hydroxyl radical oxidation of cylindrospermopsin (cyanobacterial toxin) and its role in the photochemical transformation.

Cylindrospermopsin (CYN), an alkaloid guanidinium sulfated toxin, is produced by a number of cyanobacteria regularly found in lakes, rivers, and reservoirs. Steady-state and time-resolved radiolysis methods were used to determine reaction pathways and kinetic parameters for the reactions of hydroxyl radical with CYN. The absolute bimolecular reaction rate constant for the reaction of hydroxyl radical with CYN is (5.08 ± 0.16) × 10(9) M(-1) s(-1). Comparison of the overall reaction rate of CYN with hydroxyl radical with the individual reaction rate for addition to the uracil ring in CYN indicate the majority of the hydroxyl radicals (84%) react at the uracil functionality of CYN. Product analyses using liquid chromatography-mass spectrometry indicate the major products from the reaction of hydroxyl radical with CYN involve attack of hydroxyl radical at the uracil ring and hydrogen abstraction from the hydroxy-methine bridge linking the uracil ring to the tricyclic guanidine functionality. The role of hydroxyl radical initiated pathways in the natural organic matter (NOM) photosensitized transformation of CYN were evaluated. Scavenger and trapping experiments indicate that hydroxyl radical mediated transformations account for approximately ~70% of CYN destruction in surface waters under solar irradiation in the presence of NOM. The absence of solvent isotope effect indicates singlet oxygen does not play a significant role in the NOM sensitized transformation of CYN. The primary degradation pathways for HO• mediated and NOM photosensitized destruction of CYN involve destruction of the uracil ring. The fundamental kinetic parameters determined from these studies are critical for the accurate evaluation of hydroxyl-radical based technologies for the remediation of this problematic cyanotoxin in drinking water and important in the assessment of the environmental oxidative transformation of uracil based compounds.

Transformation of paralytic shellfish poisoning toxins in Crassostrea gigas and Pecten maximus reference materials.

Matrix reference materials are an important requirement for the assessment of method performance characteristics and for routine quality control. In the field of marine toxin testing where biological assays have been used and where modern analytical testing methods are now becoming available, this requirement has become an urgent one. Various approaches are utilised for preparation of such materials in the absence of available naturally occurring toxic shellfish samples. Toxin-free shellfish may be artificially fortified through the addition of cultured toxic phytoplankton or shellfish may be incurred through natural feeding on toxic algae in a laboratory environment. Both of these approaches may be potentially affected by issues relating to the degradation or transformation of toxin analytes, so studies were conducted to assess these effects within our laboratory. A range of PSP-toxic shellfish tissues were prepared using the two approaches, in both Pacific oyster (Crassostrea gigas) and king scallops (Pecten maximus). Additionally, sub-samples of incurred Pacific oyster tissue were further treated, through addition of artificial chemical stabilisers and gamma irradiation. Two separate month-long stability trials were conducted at +4 °C on each material. Results highlighted clear evidence for improved stability of materials following shellfish feeding experiments in comparison with the tissues which had been spiked with plankton. In addition, there were clear differences in stability of toxins between the two shellfish species studied. There was evidence for good stability of C1&2 toxins in both the incurred tissues and improved stability of some toxins in tissues which had been subjected to either gamma irradiation or treatment with chemical additives. The results therefore highlighted the benefits of conducting shellfish feeding if suitable stable reference materials are to be prepared containing a full range of PSP toxin analytes. The study also highlighted the benefits of post-production treatment to prolong the stability of the materials. Work is ongoing to assess the full characteristics of candidate reference materials prepared with these approaches with the aim of producing a homogenous and stable PSP reference material in Pacific oysters.

Kinetic and mechanistic study of microcystin-LR degradation by nitrous acid under ultraviolet irradiation.

Degradation of microcystin-LR (MC-LR) in the presence of nitrous acid (HNO(2)) under irradiation of 365nm ultraviolet (UV) was studied for the first time. The influence of initial conditions including pH value, NaNO(2) concentration, MC-LR concentration and UV intensity were studied. MC-LR was degraded in the presence of HNO(2); enhanced degradation of MC-LR was observed with 365nm UV irradiation, caused by the generation of hydroxyl radicals through the photolysis of HNO(2). The degradation processes of MC-LR could well fit the pseudo-first-order kinetics. Mass spectrometry was applied for identification of the byproducts and the analysis of degradation mechanisms. Major degradation pathways were proposed according to the results of LC-MS analysis. The degradation of MC-LR was initiated via three major pathways: attack of hydroxyl radicals on the conjugated carbon double bonds of Adda, attack of hydroxyl radicals on the benzene ring of Adda, and attack of nitrosonium ion on the benzene ring of Adda.

Efficient removal of microcystin-LR by UV-C/H2O2 in synthetic and natural water samples.

The destruction of the commonly found cyanobacterial toxin, microcystin-LR (MC-LR), in surface waters by UV-C/H(2)O(2) advanced oxidation process (AOP) was studied. Experiments were carried out in a bench scale photochemical apparatus with low pressure mercury vapor germicidal lamps emitting at 253.7 nm. The degradation of MC-LR was a function of UV fluence. A 93.9% removal with an initial MC-LR concentration of 1 µM was achieved with a UV fluence of 80 mJ/cm(2) and an initial H(2)O(2) concentration of 882 µM. When increasing the concentration of MC-LR only, the UV fluence-based pseudo-first order reaction rate constant generally decreased, which was probably due to the competition between by-products and MC-LR for hydroxyl radicals. An increase in H(2)O(2) concentration led to higher removal efficiency; however, the effect of HO scavenging by H(2)O(2) became significant for high H(2)O(2) concentrations. The impact of water quality parameters, such as pH, alkalinity and the presence of natural organic matter (NOM), was also studied. Field water samples from Lake Erie, Michigan and St. Johns River, Florida were employed to evaluate the potential application of this process for the degradation of MC-LR. Results showed that the presence of both alkalinity (as 89.6-117.8 mg CaCO(3)/L) and NOM (as ∼2 to ∼9.5 mg/L TOC) contributed to a significant decrease in the destruction rate of MC-LR. However, a final concentration of MC-LR bellow the guideline value of 1 µg/L was still achievable under current experimental conditions when an initial MC-LR concentration of 2.5 µg/L was spiked into those real water samples.

UV and solar TiO(2) photocatalysis of brevetoxins (PbTxs).

Karenia brevis, the harmful alga associated with red tide, produces brevetoxins (PbTxs). Exposure to these toxins can have a negative impact on marine wildlife and serious human health consequences. The elimination of PbTxs is critical to protect the marine environment and human health. TiO(2) photocatalysis under 350 nm and solar irradiation leads to significant degradation of PbTxs via first order kinetics. ELISA results demonstrate TiO(2) photocatalysis leads to a significant decrease in the bioactivity of PbTxs as a function of treatment time. Experiments conducted in the presence of synthetic seawater, humic material and a hydroxyl scavenger showed decreased degradation. PbTxs are highly hydrophobic and partition to organic microlayer on the ocean surface. Acetonitrile was employed to probe the influence of an organic media on the TiO(2) photocatalysis of PbTxs. Our results indicate TiO(2) photocatalysis may be applicable for the degradation of PbTxs.

Cyanobacterial toxin removal in drinking water treatment processes and recreational waters.

Although federal drinking water regulations determine the quality of potable water, many specifics influence how each utility chooses to treatment water. Some of the specifics include source water quality, storage capacity, existing unit process, and space. An overview of the US recreational and drinking water regulations were discussed in context of cyanobacterial toxin removal and inactivation by ancillary as well as auxiliary treatment practices. Ancillary practice refers to the removal or inactivation of algal toxins by standard daily operational procedures where auxiliary treatment practice refers to intentional treatment. An example of auxiliary treatment would be the addition of powder activated carbon to remove taste and odor compounds. The implementation of new technologies as such ultraviolet disinfection and membrane filtration, to meet current and purposed regulations, can greatly affect the algal toxin removal and inactivation efficiencies. A discussion on meeting the current regulations by altering chemical disinfection, ozone, chlorine, chloramines and chlorine dioxide included their ancillary effects on the protection against algal toxins. Although much of the research has been on the efficiency of the removal and inactivation of microcystin LR and several microcystin variants, the discussion included other algal toxins: anatoxin-a, saxitoxins, and cyclindrospermopsin.

LC/MS/MS structure elucidation of reaction intermediates formed during the TiO(2) photocatalysis of microcystin-LR.

Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO(2) photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pH(sq)=5.7). Eleven new [M+H](+) were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO(2) nanoparticles at acidic conditions (pH=4.0). Investigating the effects of pH (for 3.0

Mesoporous nitrogen-doped TiO2 for the photocatalytic destruction of the cyanobacterial toxin microcystin-LR under visible light irradiation.

The presence of the harmful cyanobacterial toxins in water resources worldwide drives the development of an innovative and practical water treatment technology with great urgency. This study deals with two important aspects: the fabrication of mesoporous nitrogen-doped TiO2 (N-TiO2) photocatalysts and their environmental application for the destruction of microcystin-LR (MC-LR) under visible light. In a nanotechnological sol-gel synthesis method, a nitrogen-containing surfactant (dodecylammonium chloride) was introduced as a pore templating material for tailor-designing the structural properties of TiO2 and as a nitrogen dopant for its visible light response. The resulting N-TiO2 exhibited significantly enhanced structural properties including 2-8 nm mesoporous structure (porosity 44%) and high surface area of 150 m2/g. Red shift in light absorbance up to 468 nm, 0.9 eV lower binding energy of electrons in Ti 2p state, and reduced interplanar distance of crystal lattices proved nitrogen doping in the TiO2 lattice. Due to its narrow band gap at 2.65 eV, N-TiO2 efficiently degraded MC-LR under visible spectrum above 420 nm. Acidic condition (pH 3.5) was more favorable for the adsorption and photocatalytic degradation of MC-LR on N-TiO2 due to electrostatic attraction forces between negatively charged MC-LR and +6.5 mV charged N-TiO2. Even under UV light, MC-LR was decomposed 3-4 times faster using N-TiO2 than control TiO2. The degradation pathways and reaction intermediates of MC-LR were not directly related to the energy source for TiO2 activation (UV and visible) and nature of TiO2 (neat and nitrogen-doped). This study implies a strong possibility for the in situ photocatalytic remediation of contaminated water with cyanobacterial toxins and other toxic compounds using solar light, a sustainable source of energy.

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation.

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.

Degradation of the cyanobacterial hepatotoxin, nodularin, under light and dark conditions.

The stability of the cyanobacterial hepatotoxin, nodularin, was determined during the incubation of purified toxin, and in nodularin-containing cell-free extracts and whole filaments of the nodularin-producer, Nodularia spumigena in sunlight and darkness. Levels of purified nodularin in aqueous solution remained approximately constant throughout the 9-day trials under all conditions, but decreased in cell-free extracts and whole filaments when incubated under all conditions, with losses being greatest in full sunlight, intermediate in sunlight minus ultraviolet wavelengths and lowest in continuous darkness. Photodegradation and detoxification in Artemia salina bioassays occurred when purified nodularin was irradiated with ultraviolet wavelengths using a laboratory lamp.

Stability studies on the cyanobacterial nicotinic alkaloid anatoxin-A.

Anatoxin-a (ANTX-a) is a potent nicotinic cholinergic bicyclic secondary amine produced by some toxigenic strains of Anabaena spp. cyanobacteria (LD50 = 0.20-0.25 mg/kg i.p., mouse). Studies were undertaken to examine the effects of sunlight, pH, oxygen, and copper and iron, known catalysts of nitrogen oxidation, on the stability of ANTX-a. Photolysis of this extremely potent toxin was demonstrated under conditions resembling those which occur naturally. First-order decay kinetics of ANTX-a in sunlight was both pH and light intensity dependent. In the solutions examined, which represented expected biological conditions, the half-life of ANTX-a was on the order of 1-2 hr. This compares to half-lives on the order of several days in the absence of sunlight, even in the presence of metal ions. Mouse bioassays indicate that the breakdown products of ANTX-a, by both mechanisms, are inactive. Sunlight photolysis is concluded to be a possible important detoxification route of ANTX-a.