New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 mug diuron l(-1). Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 mug l(-1) defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 mug chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 mumol quanta m(-2) s(-1) with Phaeodactylum and thylakoids, respectively.

Fatal monensin toxicity in a dog after chewing a bovine intra-ruminal slow-release device

A 20kg, 10-month-old male Kelpie developed a rapid onset of profound paresis progressing to flaccid paralysis and dyspnoea, followed by death about 36 hours after chewing on a partly discharged anti-bloat capsule from a dead cow. Intoxication by monensin in the capsule was considered the cause of death. No Lodes holocyclus were found on the dog. Evidence of muscle damage was seen in clinical biochemistry assays of plasma, but consent for necropsy was not obtained. The median lethal dose for Beagle dogs of the material contained in anti-bloat capsules is 0.5-1.0g. As this represents a serious toxicity risk if dogs chew these devises, the manufacturer includes a warning on potential dog toxicity in product literature.

A framework for table driven testing of Java classes

With the advent of object-oriented languages and the portability of Java, the development and use of class libraries has become widespread. Effective class reuse depends on class reliability which in turn depends on thorough testing. This paper describes a class testing approach based on modeling each test case with a tuple and then generating large numbers of tuples to thoroughly cover an input space with many interesting combinations of values. The testing approach is supported by the Roast framework for the testing of Java classes. Roast provides automated tuple generation based on boundary values, unit operations that support driver standardization, and test case templates used for code generation. Roast produces thorough, compact test drivers with low development and maintenance cost. The framework and tool support are illustrated on a number of non-trivial classes, including a graphical user interface policy manager. Quantitative results are presented to substantiate the practicality and effectiveness of the approach. Copyright (C) 2002 John Wiley Sons, Ltd.

Possible reasons for variation in Ixodes holocyclus toxicity

Tick paralysis caused by Ixodes holocyclus affects an estimated 20,000 domestic animals each year along the eastern coast of Australia (Stone 1988). Animals are presented with clinical signs ranging from mild paresis to ascending flaccid paralysis and varying degrees of respiratory and cardiac compromise. Mortality rates are significantly increased in animals presented with respiratory compromise compared to those animals without respiratory compromise, regardless of the degree of flaccid paralysis (Atwell et al 2001). Anecdotal evidence of ticks (collected from different sites in northern New South Wales) causing different clinical signs and mortality in hyper-immune dogs used for serum production, suggests that there may be a variation in toxin production and toxin content between individual ticks (Warne, N 2002 pers comm). This literature review suggests that two possible contributing factors to toxin variation may be the genetic variation within the I. holocyclus species and the variation in the host's response to tick feeding.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Consensus neuropathological diagnosis of common dementia syndromes: testing and standardising the use of multiple diagnostic criteria

The aim of this study was to assess the variation between neuropathologists in the diagnosis of common dementia syndromes when multiple published protocols are applied. Fourteen out of 18 Australian neuropathologists participated in diagnosing 20 cases (16 cases of dementia, 4 age-matched controls) using consensus diagnostic methods. Diagnostic criteria, clinical synopses and slides from multiple brain regions were sent to participants who were asked for case diagnoses. Diagnostic sensitivity, specificity, predictive value, accuracy and variability were determined using percentage agreement and kappa statistics. Using CERAD criteria, there was a high inter-rater agreement for cases with probable and definite Alzheimer's disease but low agreement for cases with possible Alzheimer's disease. Braak staging and the application of criteria for dementia with Lewy bodies also resulted in high inter-rater agreement. There was poor agreement for the diagnosis of frontotemporal dementia and for identifying small vessel disease. Participants rarely diagnosed more than one disease in any case. To improve efficiency when applying multiple diagnostic criteria, several simplifications were proposed and tested on 5 of the original 210 cases. Inter-rater reliability for the diagnosis of Alzheimer's disease and dementia with Lewy bodies significantly improved. Further development of simple and accurate methods to identify small vessel lesions and diagnose frontotemporal dementia is warranted.

Testing landscape metrics as indicators of habitat loss and fragmentation in continuous eucalypt forests (Queensland, Australia)

Landscape metrics are widely applied in landscape ecology to quantify landscape structure. However, many are poorly tested and require rigorous validation if they are to serve as reliable indicators of habitat loss and fragmentation, such as Montreal Process Indicator 1.1e. We apply a landscape ecology theory, supported by exploratory and confirmatory statistical techniques, to empirically test landscape metrics for reporting Montreal Process Indicator 1.1e in continuous dry eucalypt forests of sub-tropical Queensland, Australia. Target biota examined included: the Yellow-bellied Glider (Petaurus australis); the diversity of nectar and sap feeding glider species including P. australis, the Sugar Glider P. breviceps, the Squirrel Glider P. norfolcensis, and the Feathertail Glider Acrobates pygmaeus; six diurnal forest birds species; total diurnal bird species diversity; and the density of nectar-feeding diurnal bird species. Two scales of influence were considered: the stand-scale (2 ha), and a series of radial landscape extents (500 m - 2 km; 78 - 1250 ha) surrounding each fauna transect. For all biota, stand-scale structural and compositional attributes were found to be more influential than landscape metrics. For the Yellow-bellied Glider, the proportion of trace habitats with a residual element of old spotted-gum/ironbark eucalypt trees was a significant landscape metric at the 2 km landscape extent. This is a measure of habitat loss rather than habitat fragmentation. For the diversity of nectar and sap feeding glider species, the proportion of trace habitats with a high coefficient of variation in patch size at the 750 m extent was a significant landscape metric. None of the landscape metrics tested was important for diurnal forest birds. We conclude that no single landscape metric adequately captures the response of the region's forest biota per se. This poses a major challenge to regional reporting of Montreal Process Indicator 1.1e, fragmentation of forest types.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Testing the link between the latitudinal gradient in species richness and rates of molecular evolution

Numerous hypotheses have been proposed to explain latitudinal gradients in species richness, but all are subject to ongoing debate. Here we examine Rohde's (1978, 1992) hypothesis, which proposes that climatic conditions at low latitudes lead to elevated rates of speciation. This hypothesis predicts that rates of molecular evolution should increase towards lower latitudes, but this prediction has never been tested. We discuss potential links between rates of molecular evolution and latitudinal diversity gradients, and present the first test of latitudinal variation in rates of molecular evolution. Using 45 phylogenetically independent, latitudinally separated pairs of bird species and higher taxa, we compare rates of evolution of two mitochondrial genes and DNA-DNA hybridization distances. We find no support for an effect of latitude on rate of molecular evolution. This result casts doubt on the generality of a key component of Rohde's hypothesis linking climate and speciation.

Caenorhabditis elegans mutants resistant to phosphine toxicity show increased longevity and cross-resistance to the synergistic action of oxygen

Phosphine (hydrogen phosphide, PH3) is the fumigant most widely used to protect stored products from pest infestation. Despite the importance of this chemical, little is known about its mode of action. We have created three phosphine-resistant lines (pre-1, pre-7, pre-33) in the model organism C. elegans, with LC50 values 2, 5, and 9 times greater than the fully susceptible parental strain. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a nonlethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. We take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, all three mutants have an extended average life expectancy of from 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Because the wild-type and mutant nematodes develop at the same rate, the longevity is unlikely to be caused by a clk-type reduction in oxidative metabolism, a potential alternative mechanism of phosphine resistance.

Tool support for testing concurrent Java components

Concurrent programs are hard to test due to the inherent nondeterminism. This paper presents a method and tool support for testing concurrent Java components. Too[ support is offered through ConAn (Concurrency Analyser), a too] for generating drivers for unit testing Java classes that are used in a multithreaded context. To obtain adequate controllability over the interactions between Java threads, the generated driver contains threads that are synchronized by a clock. The driver automatically executes the calls in the test sequence in the prescribed order and compares the outputs against the expected outputs specified in the test sequence. The method and tool are illustrated in detail on an asymmetric producer-consumer monitor. Their application to testing over 20 concurrent components, a number of which are sourced from industry and were found to contain faults, is presented and discussed.

In situ characterization of stickiness of sugar-rich foods using a linear actuator driven stickiness testing device

A stickiness testing device based on the probe tack test has been designed and tested. It was used to perform in situ characterization of drying hemispherical drops with an initial radius 3.5 mm. Tests were carried out in two drying temperatures, 63 and 95 degreesC. Moisture and temperature histories of the drying drops of fructose, honey, sucrose, maltodextrin and sucrose-maltodextrin mixtures were determined. The rates of moisture evaporation of the fructose solution was the fastest while those of the maltodextrin solution was the lowest. A profile reversal was observed when the temperature profiles of these materials were compared. Different modes of failure were observed during the stickiness tests. Pure fructose and honey solutions remained completely sticky and failed cohesively until the end of drying. Pure sucrose solution remained sticky and failed cohesively until complete crystallization occurred. The surface of the maltodextrin drops formed a skin shortly after the start of drying. It exhibited adhesive failure and reached a state of non-adhesion. Addition of maltodextrin significantly altered the stickiness of sucrose solution. (C) 2002 Elsevier Science Ltd. All rights reserved.

Biodegradation and ecotoxicity evaluation of a bionolle and starch blend and its degradation products in compost

A polymer based on a blend of starch and Bionolle(TM) has been prepared and tested for biodegradation in compost. The polymer was completely mineralised to carbon dioxide in 45 days. The potential toxicity of the polymer was tested against the earthworm Eisenia fetida using a modification of the American Standard for Testing Materials E1976-97. The earthworms were exposed to 30 g of the polymer for 28 days and changes in weight recorded. In addition, the polymer was firstly degraded by the compost and the worms exposed to the breakdown products for 28 days. Differences in weight were also recorded. In each case the production of juveniles was noted and all earthworms were examined for pathology. The results obtained were processed statistically using a t-test. The number of juveniles, produced from the breakdown products, was highly significant (P < 0.001) when compared to the earthworms added to the intact polymer. There was a definitely significant difference (P < 0.01, t = 3.25) in change in weight between the earthworms that were exposed to the polymer directly and those that were exposed to the breakdown products. There was no indication of any pathology of any earthworms. The polymer is considered safe for this species. (C) 2002 Elsevier Science Ltd. All rights reserved.