One-step, kit-based radiopharmaceuticals for molecular SPECT imaging: a versatile diphosphine chelator for 99mTc radiolabelling of peptides.

Radiotracers labelled with technetium-99m (99mTc) enable accessible diagnostic imaging of disease, provided that radiotracer preparation is simple. Whilst 99mTc radiopharmaceuticals for imaging perfusion are routinely prepared from kits, and regularly used in healthcare, there are no 99mTc-labelled receptor-targeted radiopharmaceuticals in widespread clinical use. This is in part due to the multistep radiosyntheses required for the latter. We demonstrate that the diphosphine, 2,3-bis(diphenylphosphino)maleic anhydride (BMA), is an excellent platform for preparation of kit-based, receptor-targeted 99mTc-labelled radiotracers: its conjugates are simple to prepare and can be easily labelled with 99mTc using one-step, kit-based protocols. Here, reaction of BMA with the αvß3-integrin receptor targeted cyclic peptide, Arg-Gly-Asp-DPhe-Lys (RGD), provided the first diphosphine-peptide conjugate, DP-RGD. DP-RGD was incorporated into a "kit", and addition of a saline solution containing 99mTcO4- to this kit, followed by heating, furnished the radiotracer [99mTcO2(DP-RGD)2]+ in consistently high radiochemical yields (>90%). The analogous [ReO2(DP-RGD)2]+ compound was prepared and characterised, revealing that both [99mTcO2(DP-RGD)2]+ and [ReO2(DP-RGD)2]+ consist of a mixture of cis and trans geometric isomers. Finally, [99mTcO2(DP-RGD)2]+ exhibited high metabolic stability, and selectively targeted αvß3-integrin receptors, enabling in vivo SPECT imaging of αvß3-integrin receptor expression in mice.

Transcriptomic Characterization of Inhalation Phosphine Toxicity in Adult Male Sprague-Dawley Rats.

Phosphine (PH3) is a highly toxic, corrosive, flammable, heavier-than-air gas that is a commonly used fumigant. When used as a fumigant, PH3 can be released from compressed gas tanks or produced from commercially available metal phosphide tablets. Although the mechanism of toxicity is unclear, PH3 is thought to be a metabolic poison. PH3 exposure induces multiorgan toxicity, and no effective antidotes or therapeutics have been identified. Current medical treatment consists largely of supportive care and maintenance of cardiovascular function. To better characterize the mechanism(s) driving PH3-induced toxicity, we have performed transcriptomic analysis on conscious adult male Sprague-Dawley rats following whole-body inhalation exposure to phosphine gas at various concentration-time products. PH3 exposure induced concentration- and time-dependent changes in gene expression across multiple tissues. These gene expression changes were mapped to pathophysiological responses using molecular pathway analysis. Toxicity pathways indicative of cardiac dysfunction, cardiac arteriopathy, and cardiac enlargement were identified. These cardiotoxic responses were linked to apelin-mediated cardiomyocyte and cardiac fibroblast signaling pathways. Evaluation of gene expression changes in blood revealed alterations in pathways associated with the uptake, transport, and utilization of iron. Altered erythropoietin signaling was also observed in the blood. Upstream regulator analysis identified several therapeutics predicted to counteract PH3-induced gene expression changes. These include antihypertensive drugs (losartan, candesartan, and prazosin) and therapeutics to reduce pathological cardiac remodeling (curcumin and TIMP3). This transcriptomics study has characterized molecular pathways involved in PH3-induced cardiotoxicity. These data will aid in elucidating a precise mechanism of toxicity for PH3 and guide the development of effective medical countermeasures for PH3-induced toxicity.

Nephrotoxicity Evaluation of Indium Phosphide Quantum Dots with Different Surface Modifications in BALB/c Mice.

InP QDs have shown a great potential as cadmium-free QDs alternatives in biomedical applications. It is essential to understand the biological fate and toxicity of InP QDs. In this study, we investigated the in vivo renal toxicity of InP/ZnS QDs terminated with different functional groups-hydroxyl (hQDs), amino (aQDs) and carboxyl (cQDs). After a single intravenous injection into BALB/c mice, blood biochemistry, QDs distribution, histopathology, inflammatory response, oxidative stress and apoptosis genes were evaluated at different predetermined times. The results showed fluorescent signals from QDs could be detected in kidneys during the observation period. No obvious changes were observed in histopathological detection or biochemistry parameters. Inflammatory response and oxidative stress were found in the renal tissues of mice exposed to the three kinds of QDs. A significant increase of KIM-1 expression was observed in hQDs and aQDs groups, suggesting hQDs and aQDs could cause renal involvement. Apoptosis-related genes (Bax, Caspase 3, 7 and 9) were up-regulated in hQDs and aQDs groups. The above results suggested InP/ZnS QDs with different surface chemical properties would cause different biological behaviors and molecular actions in vivo. The surface chemical properties of QDs should be fully considered in the design of InP/ZnS QDs for biomedical applications.

Biodistribution and acute toxicity of cadmium-free quantum dots with different surface functional groups in mice following intratracheal inhalation.

Indium phosphide/zinc sulfate (InP/ZnS) quantum dots (QDs) are presumed to be less hazardous than those that contain cadmium. However, the toxicological profile has not been established. The present study investigated the acute toxicity of InP/ZnS QDs with different surface modifications (COOH, NH2, and OH) in mice after pulmonary aerosol inhalation. InP/ZnS QDs were able to pass through the blood-gas barrier and enter the circulation, and subsequently accumulated in major organs. No obvious changes were observed in the body weight or major organ coefficients. Red blood cell counts and platelet-related indicators were in the normal range, but the proportion of white blood cells was altered. The InP/ZnS QDs caused varying degrees of changes in some serum markers, but no histopathological abnormalities related to InP/ZnS QDs treatment was observed in major organs except that hyperemia in alveolar septa was found in lung sections. These results suggested that the effects of respiratory exposure to InP/ZnS QDs on the lungs need to be fully considered in future biomedical application although the overall toxicity of quantum dots is relatively low.

Anticancer activity of a Gold(I) phosphine thioredoxin reductase inhibitor in multiple myeloma.

Multiple myeloma (MM), the second most common haematological malignancy, is a clonal plasma B-cell neoplasm that forms within the bone marrow. Despite recent advancements in treatment, MM remains an incurable disease. Auranofin, a linear gold(I) phosphine compound, has previously been shown to exert a significant anti-myeloma activity by inhibiting thioredoxin reductase (TrxR) activity. A bis-chelated tetrahedral gold(I) phosphine complex [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane) was previously designed to improve the gold(I) compound selectivity towards selenol- and thiol-containing proteins, such as TrxR. In this study, we show that [Au(d2pype)2]Cl significantly inhibited TrxR activity in both bortezomib-sensitive and resistant myeloma cells, which led to a significant reduction in cell proliferation and induction of apoptosis, both of which were dependent on ROS. In clonogenic assays, treatment with [Au(d2pype)2]Cl completely abrogated the tumourigenic capacity of MM cells, whereas auranofin was less effective. We also show that [Au(d2pype)2]Cl exerted a significant anti-myeloma activity in vivo in human RPMI8226 xenograft model in immunocompromised NOD/SCID mice. The MYC oncogene, known to drive myeloma progression, was downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. This study highlights the "proof of concept" that improved gold(I)-based compounds could potentially be used to not only treat MM but as an alternative tool to understand the role of the Trx system in the pathogenesis of this blood disease.

Near-Infrared-Light-Triggered Antimicrobial Indium Phosphide Quantum Dots.

The emergence of multidrug-resistant (MDR) pathogens represents one of the most urgent global public health crises. Light-activated quantum dots (QDs) are alternative antimicrobials, with efficient transport, low cost, and therapeutic efficacy, and they can act as antibiotic potentiators, with a mechanism of action orthogonal to small-molecule drugs. Furthermore, light-activation enhances control over the spatiotemporal release and dose of the therapeutic superoxide radicals from QDs. However, the limited deep-tissue penetration of visible light needed for QD activation, and concern over trace heavy metals, have prevented further translation. Herein, we report two indium phosphide (InP) QDs that operate in the near-infrared and deep-red light window, enabling deeper tissue penetration. These heavy-metal-free QDs eliminate MDR pathogenic bacteria, while remaining non-toxic to host human cells. This work provides a pathway for advancing QD nanotherapeutics to combat MDR superbugs.

An assessment of vegetation management practices and burrow fumigation with aluminum phosphide as tools for managing voles within perennial crop fields in California, USA.

Voles (Cricetidae) cause extensive damage to a variety of crops throughout much of the Northern Hemisphere. The removal of vegetation from crop fields at the end of the growing season, combined with a subsequent burrow fumigant application of aluminum phosphide, has the potential to substantially curtail vole activity but has not been thoroughly examined. We set up a study to test the impact of these management tools in perennial globe artichoke (Cynara cardunculus var. scolymus) fields in Monterey County, CA, during 2010 and 2011, to determine their potential utility as part of an integrated pest management (IPM) program for managing California voles (Microtus californicus). We used both chewing indices and mortality estimates derived via radiotelemetry to assess the efficacy of aboveground vegetation removal and aluminum phosphide applications on vole abundance. We determined the impact of plowing artichoke fields on vole activity as well. Both removal of vegetation and applications of aluminum phosphide substantially reduced vole presence within treated fields. Plowing also reduced vole abundance to the point of little residual activity following treatment. These management practices appear to be effective at eliminating voles from crop fields. Combining these tools with management practices designed to slow down reinvasion by neighboring vole populations (e.g., barriers, repellents, traps) has the potential to substantially reduce farmer reliance on rodenticides for vole management, although rodenticides will still be needed to curtail populations that reestablish within crop fields. Such an IPM approach should substantially benefit both farmers and agro-ecosystems.

Efficacy and Phytotoxicity of Phosphine as Fumigants for Frankliniella occidentalis (Thysanoptera: Thripidae) on Asparagus.

The insecticidal activity of phosphine (PH3) and ethyl formate (EF) toward Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) and their phytotoxicity to asparagus were evaluated. Both the PH3 and EF fumigants showed higher lethal concentration and time (LCT) values at lower temperatures. The LCT99 values of PH3 and EF at 5°C in a 12 liters desiccator for 4 h showed the following ranking: eggs (64.69 mg·h/liter for PH3 and EF indicating phytotoxicity to asparagus), nymphs (5.54 and 17.48 mg·h/liter, respectively), and adults (3.83 and 14.67 mg·h/liter, respectively). The adsorption of PH3 was approximately 11% at 2°C and 13% at 5°C, whereas the adsorption of EF increased sharply to 88% at 2°C and 97% at 5°C. The hatching rate of F. occidentalis eggs was approximately 95% at all locations (top, middle, and bottom) in the presence of 4 mg/liter PH3 at 5°C in a 0.65-m3 fumigation chamber for 24 h. However, extension of the treatment to 48 h resulted in 100% inhibition of egg hatching. The atmospheric level of PH3 decreased below the threshold limit value after 80 min, and phytotoxicity was not observed. The results revealed that EF is highly absorbed by asparagus and is not suitable as a fumigant, but PH3 is a suitable alternative to the fumigant methyl bromide for the control of western flower thrips in asparagus.

Gold(i) phosphine compounds as parasite attenuating agents for malaria vaccine and drug development.

Here, the anti-malarial activity of two gold(i) phosphine compounds auranofin and [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane), were examined to inform their use as potential drugs and malaria parasite-attenuating agents. In vitro, the gold compounds were active against Plasmodium falciparum and P. knowlesi as well as the rodent parasite P. chabaudi AS. Attenuation of the parasite was observed when mice were inoculated with P. chabaudi AS infected red blood cells treated in vitro with [Au(d2pype)2]Cl (1 or 2 µM) or auranofin (2 µM) for 2 or 3 h. Quantitative PCR data showed persistence of low levels of parasite DNA up to 8 days post inoculation. In some experiments, there was microscopically detectable parastiemia following inoculation which subsequently cleared. Following 1 or 3 doses of gold compound-treated parasitized red blood cells (pRBCs), protection was not observed when these mice were subsequently challenged with wild type P. chabaudi AS. In experiments where microscopically detectable parasites were observed following in vivo inoculation, mice were subsequently fully protected against a challenge infection with wildtype parasites. In an infect-and-treat rodent model, the gold compounds were unable to inhibit P. chabaudi AS growth in vivo when administered orally. Gold compounds act via the inhibition of antioxidant systems which are critical in the pathogen's survival from attack by the host oxidants. In vitro, they directly inhibit the parasite thioredoxin reductase, hence the observed suppressive activity. On the other hand, in vivo, the gold compounds may not be readily available for absorption and thus pharmacokinetic studies will be required to further examine drug bioavailability following administration. With structural differences in redox mechanisms of P. falciparum and the human host being identified, gold compounds can be better designed to more efficiently target and selectively inhibit the parasite.

Fatal aluminum phosphide poisonings in Tirana (Albania), 2009 - 2013.

BACKGROUND: Acute poisonings particularly through pesticides have become a major public health concern in Albania during the last decade. FINDINGS: The number of fatalities due to aluminum phosphide intoxications was more than doubled during a five year-period from 2009 to 2013, and a cluster of suicides perpetrated with Phostoxin was registered. Several factors are accountable for such a phenomenon, including the fact that aluminum phosphide agents are freely available in the Albanian market, their price is extremely low and they are sold without any legal restriction. The mass media unfortunately warranted an emulating effect to dramatic intoxications, which gained by such means the notoriety of a secure lethal weapon. CONCLUSIONS: Our experience with more than three hundred intoxications with aluminum phosphide agents in the last five years, showed that a considerable delay from the moment of exposure (mainly through ingestion) to specialized medical help seeking, created a considerable obstacle for a successful treatment of cases, and eventually for the survival of patients. The lack of a specific antidote adds further challenges to all these exposures. The need for public health policies aiming at prevention, awareness, and possibly the substitution of Phostoxin or other aluminum phosphide pesticides with less dangerous agents is formulated.

Field evaluation of phostoxin and zinc phosphide for the control of zoonotic cutaneous leishmaniasis in a hyperendemic area, central Iran.

BACKGROUND & OBJECTIVES: ZCL is a growing threat in many rural areas of Iran which involves 17 out of 31 provinces. This study was conducted from April to November 2011 for evaluation of the efficacy of phostoxin and zinc phosphide against rodents. METHODS: Rodent control operations were carried out using phostoxin and zinc phosphide. To evaluate the effect of rodent control operation on the main vector density, an entomological survey was carried out. The effects of the operation on the disease incidence were also evaluated. RESULTS: After intervention, the reduction rate of rodent burrows was 32.68% in the village treated with phostoxin and 58.14% in the village treated with zinc phosphide. The number of rodent holes in the control area showed 6.66-fold increase at the end of the study. The incidence of the disease decreased to 19.23 and 11.40 in areas treated with phostoxin and zinc phosphide, respectively. A total of 4243 adult sandflies were collected and identified. The most common and dominant species was Phlebotomus papatasi. In the village treated with phostoxin, the density of P. papatasi in outdoors was lower than indoors. Nevertheless, the density of P. papatasi in the village treated with zinc phosphide was higher in outdoors. INTERPRETATION & CONCLUSION: It is concluded that phostoxin is less effective and has low safety in comparison with zinc phosphide, so that this rodenticide can be used only in special situations such as lack or ineffective rodenticides and only in the colonies far from human and animal dwelling places in small scales.

[Survival after oral poisoning with insecticide against moles containing aluminium phosphide]. / Overlevelse efter peroral forgiftning med muldvarpebekæmpelsesmiddel indeholdende aluminiumfosfid.

A 31-year-old man presented with abdominal pain and vomiting with a smell of garlic and rotten fish. He was brought to the ER being circulatory affected, metabolic acidotic and he developed more episodes of arrhythmia. Oral poisoning with aluminium phosphide is a very serious condition due to release of the toxic phosphine gas. Treatment is symptomatic. To avoid contamination of the staff is it important to use air-tight containers for excretions and furthermore to use chemical clothing and breathing protection.

Ingestion of gallium phosphide nanowires has no adverse effect on Drosophila tissue function.

Engineered nanoparticles have been under increasing scrutiny in recent years. High aspect ratio nanoparticles such as carbon nanotubes and nanowires have raised safety concerns due to their geometrical similarity to asbestos fibers. III-V epitaxial semiconductor nanowires are expected to be utilized in devices such as LEDs and solar cells and will thus be available to the public. In addition, clean-room staff fabricating and characterizing the nanowires are at risk of exposure, emphasizing the importance of investigating their possible toxicity. Here we investigated the effects of gallium phosphide nanowires on the fruit fly Drosophila melanogaster. Drosophila larvae and/or adults were exposed to gallium phosphide nanowires by ingestion with food. The toxicity and tissue interaction of the nanowires was evaluated by investigating tissue distribution, activation of immune response, genome-wide gene expression, life span, fecundity and somatic mutation rates. Our results show that gallium phosphide nanowires applied through the diet are not taken up into Drosophila tissues, do not elicit a measurable immune response or changes in genome-wide gene expression and do not significantly affect life span or somatic mutation rate.

Inhalation of phosphine gas following a fire associated with fumigation of processed pistachio nuts.

On December 10, 2009, a fumigation stack containing aluminum phosphide became soaked with rain water and caught fire at a pistachio processing plant in Kern County, California. Untrained plant personnel responding to the fire had exposure to pyrolysis by-products, particulates, and extinguisher ingredients. Ten workers taken for medical evaluation had respiratory and nonspecific systemic symptoms consistent with exposure to phosphine gas. Six of the 10 workers had respiratory distress, indicated by chest pain, shortness of breath, elevated respiratory rate, or decreased oxygen saturation. Recommendations are made for the management of similar illnesses and prevention of similar exposures.

Protective effects of N-acetylcysteine on aluminum phosphide-induced oxidative stress in acute human poisoning.

OBJECTIVE: Aluminum phosphide is used as a fumigant. It produces phosphine gas (PH3). PH3 is a mitochondrial poison which inhibits cytochrome c oxidase, it leads to generation of reactive oxygen species; so one of the most important suggested mechanisms for its toxicity is induction of oxidative stress. In this regard, it could be proposed that a drug like N-acetylcysteine (NAC) as an antioxidant would improve the tolerance of aluminum phosphide-intoxicated cases. The objective of this study was to evaluate the protective effects of NAC on acute aluminum phosphide poisoning. METHODS: This was a prospective, randomized, controlled open-label trial. All patients received the same supportive treatments. NAC treatment group also received NAC. The blood thiobarbituric acid reactive substances as a marker of lipid peroxidation and total antioxidant capacity of plasma were analyzed. RESULTS: Mean ingested dose of aluminum phosphide in NAC treatment and control groups was 4.8 ± 0.9 g vs. 5.4 ± 3.3 g, respectively (p = 0.41). Significant increase in plasma malonyldialdehyde level in control group was observed (139 ± 28.2 vs. 149.6 ± 35.2 µmol/L, p = 0.02). NAC infusion in NAC treatment group significantly decreased malondialdehyde level (195.7 ± 67.4 vs. 174.6 ± 48.9 µmol/L, p = 0.03), duration of hospitalization (2.7 ± 1.8 days vs. 8.5 ± 8.2 days, p = 0.02), rate of intubation and ventilation (45.4% vs. 73.3%, p = 0.04). Mortality rate in NAC treatment and control groups were 36% and 60%, respectively with odds ratio 2.6 (0.7-10.1, 95% CI). CONCLUSION: NAC may have a therapeutic effect in acute aluminum phosphide poisoning.

Comparative cytotoxicity of gold-doxorubicin and InP-doxorubicin conjugates.

Direct comparisons of different types of nanoparticles for drug delivery have seldom been performed. In this study we compare the physical properties and cellular activity of doxorubicin (Dox) conjugates to gold nanoparticles (Au) and InP quantum dots of comparable diameter. Although the Au particles alone are non-toxic and InP is moderately toxic, Au-Dox is more effective than InP-Dox against the Dox-resistant B16 melanoma cell line. Light exposure does not augment the efficacy of InP-Dox, suggesting that conjugates are breaking down. Electron and confocal microscopy and atomic absorption spectroscopy reveal that over 60% of the Au-Dox conjugates reach the cell nucleus. In contrast, InP-Dox enters cell nuclei to a very limited extent, although liberated Dox from the conjugates does eventually reach the nucleus. These observations are attributed to faster Dox release from Au conjugates under endosomal conditions, greater aggregation of InP-Dox with cytoplasmic proteins, and adherence of InP to membranes. These findings have important implications for design of active drug-nanoparticle conjugates.

Evaluation of (4-[18F]Fluorophenyl)triphenylphosphonium ion. A potential myocardial blood flow agent for PET.

PURPOSE: The lipophilic cationic compound, (4-[¹8F]fluorophenyl)triphenylphosphonium ion (¹8F-FTPP) was synthesized and evaluated as a potential positron emission tomography (PET) myocardial perfusion agent. PROCEDURE: ¹8F-FTPP was prepared from (4-nitrophenyl)triphenylphosphonium nitrate and ammonium [¹8F]fluoride by nucleophilic aromatic substitution and was purified by high performance liquid chromatography before use. Biodistribution studies were performed in rats at 5, 30, 60 min (five rats per time point). Three rats were evaluated by microPET imaging after injection of ¹8F-FTPP. In addition, microPET imaging in rabbits (three) was performed before and after occlusion of the left anterior descending (LAD) artery with ¹³NH3 (111 MBq) and ¹8F-FTPP (74 MBq). RESULTS: Biodistribution data in rats showed rapid blood clearance and high levels of accumulation in the heart; 75:1 heart-to-blood ratio at 30 min. Uptake of radioactivity in the heart was 1.64% ID/G, 1.51% ID/g, and 1.57% ID/g at 5, 30, and 60 min. At 5, 30, and 60 min, lung activity was 0.69% ID/g, 0.03% ID/g, and 0.38% ID/g, and liver uptake was 0.34% ID/g, 0.18% ID/g, and 0.17% ID/g. Heart-to-lung ratios at 5, 30, and 60 min were 2, 5, and 4. Bone accumulation was minimal. MicroPET imaging in both rats and rabbits after injection of ¹8F-FTPP demonstrated an initial spike of activity in the myocardium corresponding to blood flow followed by a plateau after 1 min. Region of interest analysis of microPET images of normal and LAD-occluded rabbits with ¹³NH3 and ¹8F-FTPP indicated similar distributions of the two tracers in both normal and altered blood flow regions. CONCLUSION: The excellent heart-to-blood ratio of ¹8F-FTPP and its correlation with ¹³NH3 distribution in normal and LAD-occluded rabbits suggest that this radiopharmaceutical may have potential as a PET agent for characterizing mitochondrial damage and/or myocardial blood flow.

Pleural effects of indium phosphide in B6C3F1 mice: nonfibrous particulate induced pleural fibrosis.

The mechanism(s) by which chronic inhalation of indium phosphide (InP) particles causes pleural fibrosis is not known. Few studies of InP pleural toxicity have been conducted because of the challenges in conducting particulate inhalation exposures, and because the pleural lesions developed slowly over the 2-year inhalation study. The authors investigated whether InP (1 mg/kg) administered by a single oropharyngeal aspiration would cause pleural fibrosis in male B6C3F1 mice. By 28 days after treatment, protein and lactate dehydrogenase (LDH) were significantly increased in bronchoalveolar lavage fluid (BALF), but were unchanged in pleural lavage fluid (PLF). A pronounced pleural effusion characterized by significant increases in cytokines and a 3.7-fold increase in cell number was detected 28 days after InP treatment. Aspiration of soluble InCl(3) caused a similar delayed pleural effusion; however, other soluble metals, insoluble particles, and fibers did not. The effusion caused by InP was accompanied by areas of pleural thickening and inflammation at day 28, and by pleural fibrosis at day 98. Aspiration of InP produced pleural fibrosis that was histologically similar to lesions caused by chronic inhalation exposure, and in a shorter time period. This oropharyngeal aspiration model was used to provide an initial characterization of the progression of pleural lesions caused by InP.

Detection and characterisation of strong resistance to phosphine in Brazilian Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae).

As failure to control Rhyzopertha dominica (F.) with phosphine is a common problem in the grain-growing regions of Brazil, a study was undertaken to investigate the frequency, distribution and strength of phosphine resistance in R. dominica in Brazil. Nineteen samples of R. dominica were collected between 1991 and 2003 from central storages where phosphine fumigation had failed to control this species. Insects were cultured without selection until testing in 2005. Each sample was tested for resistance to phosphine on the basis of the response of adults to discriminating concentrations of phosphine (20 and 48 h exposures) and full dose-response assays (48 h exposure). Responses of the Brazilian R. dominica samples were compared with reference susceptible, weak-resistance and strong-resistance strains from Australia in parallel assays. All Brazilian population samples showed resistance to phosphine: five were diagnosed with weak resistance and 14 with strong resistance. Five samples showed levels of resistance similar to the reference strong-resistance strain. A representative highly resistant sample was characterised by exposing mixed-age cultures to a range of constant concentrations of phosphine for various exposure periods. Time to population extinction (TPE) and time to 99.9% suppression of population (LT(99.9)) values of this sample were generally similar to those of the reference strong-resistance strain. For example, at 0.1, 0.5 and 1.0 mg L(-1), LT(99.9) values for BR33 and the reference strong-resistance strain were respectively 21, 6.4 and 3.7 days and 17, 6.2 and 3.8 days. With both strains, doubling phosphine concentrations to 2 mg L(-1) resulted in increased LT(99.9) and TPE. High level and frequency of resistance in all population samples, some of which had been cultured without selection for up to 12 years, suggest little or no fitness deficit associated with phosphine resistance. The present research indicates that widespread phosphine resistance may be developing in Brazil. Fumigation practices should be monitored and resistance management plans implemented to alleviate further resistance development.

The rate-limiting step in anaerobic digestion in the presence of phosphine.

Methanogenesis is the most important anaerobic biodegradation process in nature, which is accomplished by three different kinds of bacteria - hydrolytic, acetogenic and methanogenic bacteria (MB). An experiment was performed to determine the rate-limiting step of methanogenesis under the influence of various phosphine concentrations (100, 300, 500, 700 and 1000 ppm). It was found that the growth of fermentative bacteria (FB) was severely affected by higher concentrations of phosphine (700 and 971 ppm), while the growth of hydrogen-producing acetogenic bacteria (HPAB) and MB was not affected severely at higher phosphine concentrations. Thus, HPAB and MB are less sensitive to phosphine compared with FB, which means that hydrolysis, and fermentation step is the rate-limiting step during methanogenesis under the influence of phosphine. It is recommended that special attention be paid to the first stage of methanogenesis under high concentrations of phosphine during anaerobic wastewater treatment.