Deriving safe short-term chemical exposure values (STEV) for drinking water.

Small and brief exceedances of chemicals above their guideline values in drinking water are unlikely to cause an appreciable increased risk to human health. As a result, short-term exposure values (STEV) can be derived to help decide whether drinking water can still be supplied to consumers without adverse health risks. In this study, three approaches were applied to calculate and compare STEV for pesticides. The three approaches included basing a STEV on the acute reference dose (ARfD) (Approach 1), removing conventional attribution rates and uncertainty factors from current guideline values (Approach 2) and extrapolating 1 d and 7 d no observed adverse effect levels (NOAEL) from existing toxicity data using a log-linear regression (Approach 3). Despite being very different methods, the three approaches produced comparable STEV generally within an order of magnitude, which often overlapped with other existing short-term exposure values such as short-term no adverse response levels (SNARL) and health advisories (HA). The results show that adjusting the current guideline value using standard extrapolation factors (Approach 2) often produced the most conservative values. Approach 2 was then applied to two other chemical classes, disinfection by-products (DBPs) and cyanotoxins, demonstrating the wider applicability of the approach.

An internal standard approach for homogeneous TR-FRET immunoassays facilitates the detection of bacteria, biomarkers, and toxins in complex matrices.

The recent development of a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) immunoassay enables one-step, rapid (minutes), and direct detection compared to the multistep, time-consuming (hours), heterogeneous ELISA-type immunoassays. The use of the time-resolved effect of a donor lanthanide complex with a delay time of microseconds and large Stokes shift enables the separation of positive signals from the background autofluorescence of the sample. However, this study shows that the sample matrices directly interfere with donor fluorescence and that interference cannot be eliminated by time-resolved settings alone. Moreover, the reduction in donor emission did not appear to be equivalent to the reduction in acceptor emission, resulting in incorrect FRET signal measurements. To overcome this limitation, an internal standard approach was developed using an isotype control antibody. This new approach was used to develop TR-FRET assays for rapid detection (15-30 min) of Bacillus anthracis spores and botulinum toxin (type E) in beverages, which are major concerns in bioterrorism involving deliberate food contamination. Additionally, we demonstrate the detection of B. anthracis-secreted protective antigen (PA) and the Yersinia pestis-secreted markers F1 and LcrV in blood cultures, which are early markers of bacteremia in infected hosts following a possible bioterror attack. The use of an internal standard enables the calculation of correct ΔF values without the need for an external standard. Thus, the use of the internal standard approach in homogeneous immunoassays facilitates the examination of any sample regardless of its origin, and therefore expands the applicability of TR-FRET assays for complex matrices.

Feasibility study on production of a matrix reference material for cyanobacterial toxins.

The worldwide increase in cyanobacterial contamination of freshwater lakes and rivers is of great concern as many cyanobacteria produce potent hepatotoxins and neurotoxins (cyanotoxins). Such toxins pose a threat to aquatic ecosystems, livestock, and drinking water supplies. In addition, dietary supplements prepared from cyanobacteria can pose a risk to consumers if they contain toxins. Analytical monitoring for toxins in the environment and in consumer products is essential for the protection of public health. Reference materials (RMs) are an essential tool for the development and validation of analytical methods and are necessary for ongoing quality control of monitoring operations. Since the availability of appropriate RMs for cyanotoxins has been very limited, the present study was undertaken to examine the feasibility of producing a cyanobacterial matrix RM containing various cyanotoxins. The first step was large-scale culturing of various cyanobacterial cultures that produce anatoxins, microcystins, and cylindrospermopsins. After harvesting, the biomass was lyophilized, blended, homogenized, milled, and bottled. The moisture content and physical characteristics were assessed in order to evaluate the effectiveness of the production process. Toxin levels were measured by liquid chromatography with tandem mass spectrometry and ultraviolet detection. The reference material was found to be homogeneous for toxin content. Stability studies showed no significant degradation of target toxins over a period of 310 days at temperatures up to +40 °C except for the anatoxin-a, which showed some degradation at +40 °C. These results show that a fit-for-purpose matrix RM for cyanotoxins can be prepared using the processes and techniques applied in this work.

Bioassays for evaluation of medical products derived from bacterial toxins.

Bioassays play central role in evaluation of biological products and those derived from bacterial toxins often rely exclusively on in vivo models for assurance of safety and potency. This chapter reviews existing regulatory approved methods designed to provide information on potency and safety of complex biological medicines with an insight into strategies considered for alternative procedures.

[Cyanobacterial toxins in bank filtrate. Under which conditions is their elimination reliable?]. / Cyanobakterientoxine bei der Uferfiltration : Unter welchen Umständen ist ihre Elimination sicher?

Cyanobacterial toxins are substances produced by cyanobacteria or blue-green algae. They can occur in surface waters worldwide and have to be reliably removed when using affected surface waters as a drinking water source. Bank filtration has been used for 150 years for drinking water (pre-)treatment. It utilizes natural elimination processes like sorption and degradation in the sub-surface. Retention of cells on the sediment surface is the most prominent process for eliminating these primarily cell-bound toxins. Middle to coarse grained sands eliminated more than 99.9 % of intracellular toxins within the first 10 cm of flow path. Elimination of extracellular microcystin during underground passage is mainly due to biodegradation. Reversible adsorption processes do not reduce the total load but lead to longer contact times for extended biodegradation. Laboratory experiments showed that the sediment structure, i.e. high clay/silt and organic content, is crucial for maximum adsorption. However, redox conditions play an important role for degradation rates: under aerobic conditions half-lives of less than one day occurred frequently, whereas anoxic conditions resulted in lag phases of one day and more, as well as in half lives of more than 25 days. Field experiments showed that temperature is crucial for degradation velocity under natural conditions. Under optimal conditions 10 d residence time are sufficient to reduce microcystin concentrations to values below the WHO guidelines value for drinking water (1 microg/L). Under sub-optimal conditions a residence time of up to 90 days may be necessary.

Determination of anatoxin-a in environmental water samples by solid-phase microextraction and gas chromatography-mass spectrometry.

In the present work, a method was developed and optimized aiming at the determination of anatoxin-a in environmental water samples. The method is based on the direct derivatization of the analyte by adding hexylchloroformate in the alkalinized sample (pH = 9.0). The derivatized anatoxin-a was extracted by a solid-phase microextraction (SPME) procedure, submersing a PDMS fiber in an amber vial for 20 min under magnetic stirring. GC-MS was used to identify and quantify the analyte in the SIM mode. Norcocaine was used as internal standard. The following ions were chosen for SIM analyses (quantification ions in italics): anatoxin-a: 191, 164, 293 and norcocaine: 195, 136, 168. The calibration curve showed linearity in the range of 2.5-200 ng/mL and the LOD was 2 ng/mL. This method of SPME and GC-MS analysis can be readily utilized to monitor anatoxin-a for water quality control.

Cyanobacterial toxins: risk management for health protection.

This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the risk management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles.

Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?

This article reviews current scientific knowledge on the toxicity and carcinogenicity of microcystins and compares this to the guidance values proposed for microcystins in water by the World Health Organization, and for blue-green algal food supplements by the Oregon State Department of Health. The basis of the risk assessment underlying these guidance values is viewed as being critical due to overt deficiencies in the data used for its generation: (i) use of one microcystin congener only (microcystin-LR), while the other presently known nearly 80 congeners are largely disregarded, (ii) new knowledge regarding potential neuro and renal toxicity of microcystins in humans and (iii) the inadequacies of assessing realistic microcystin exposures in humans and especially in children via blue-green algal food supplements. In reiterating the state-of-the-art toxicology database on microcystins and in the light of new data on the high degree of toxin contamination of algal food supplements, this review clearly demonstrates the need for improved kinetic data of microcystins in humans and for discussion concerning uncertainty factors, which may result in a lowering of the present guidance values and an increased routine control of water bodies and food supplements for toxin contamination. Similar to the approach taken previously by authorities for dioxin or PCB risk assessment, the use of a toxin equivalent approach to the risk assessment of microcystins is proposed.

Health risk assessment of cyanobacterial (blue-green algal) toxins in drinking water.

Cyanobacterial toxins have caused human poisoning in the Americas, Europe and Australia. There is accumulating evidence that they are present in treated drinking water supplies when cyanobacterial blooms occur in source waters. With increased population pressure and depleted groundwater reserves, surface water is becoming more used as a raw water source, both from rivers and lakes/reservoirs. Additional nutrients in water which arise from sewage discharge, agricultural run-off or storm water result in overabundance of cyanobacteria, described as a 'water bloom'. The majority of cyanobacterial water-blooms are of toxic species, producing a diversity of toxins. The most important toxins presenting a risk to the human population are the neurotoxic alkaloids (anatoxins and paralytic shellfish poisons), the cyclic peptide hepatotoxins (microcystins) and the cytotoxic alkaloids (cylindrospermopsins). At the present time the only cyanobacteral toxin family that have been internationally assessed for health risk by the WHO are the microcystins, which cause acute liver injury and are active tumour promoters. Based on sub-chronic studies in rodents and pigs, a provisional Guideline Level for drinking water of 1 microg/L of microcystin-LR has been determined. This has been adopted in legislation in countries in Europe, South America and Australasia. This may be revised in the light of future teratogenicity, reproductive toxicity and carcinogenicity studies. The other cyanobacterial toxin which has been proposed for detailed health risk assessment is cylindrospermopsin, a cytotoxic compound which has marked genotoxicity, probable mutagenicity, and is a potential carcinogen. This toxin has caused human poisoning from drinking water, and occurs in water supplies in the USA, Europe, Asia, Australia and South America. An initial health risk assessment is presented with a proposed drinking water Guideline Level of 1 microg/L. There is a need for both increased monitoring data for toxins in drinking water and epidemiological studies on adverse health effects in exposed populations to clarify the extent of the health risk.

A proposal for safety standards for human use of cholera toxin (or Escherichia coli heat-labile enterotoxin) derivatives as an adjuvant of nasal inactivated influenza vaccine.

Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) are not only the causative agents of diarrhea but are also strong mucosal adjuvants which enhance immune responses to mucosally coadministered bystander antigens. One of the most promising applications of these toxins would be as mucosal adjuvant of nasal influenza vaccine. In comparison to current inactivated vaccines, the nasal vaccine provides superior cross-protection by inducing production of cross-reacting anti-viral IgA antibodies in the respiratory tract even when the vaccine strain is different from the epidemic strain. On the use of the toxins as mucosal adjuvants in humans, toxicity and allergenicity of the toxins are problems which impinge on safety. To resolve these problems, various approaches have been attempted to produce less toxic and less allergenic CT (or LT) derivatives. We now propose the following standards for human use of safer CT (or LT) derivatives as an adjuvant of a nasal influenza vaccine. Thus, CT (or LT) derivatives can be administered intranasally together with a current inactivated influenza vaccine, provided they meet the following criteria. 1) A single dose of the derivatives, administered intranasally by spraying, should be around 100 Eg/adult in a volume of less than 0.5 ml. 2) CT (or LT) derivatives should retain the properties of the native CT (or LT), i. e., the ability to augment secretory IgA and serum IgG Ab responses to viral surface glycoproteins, when administered intranasally together with an inactivated influenza vaccine. 3) CT (or LT) derivatives should not induce IgE Ab responses to the vaccine, as well as to the CT (or LT) itself. 4) The CT (or LT) should be nontoxic; the toxicity of the derivatives, as determined by the Y-1 adrenal cell assay, should not exceed 1/100 EC(50) of the native CT (or 1/1000 ECi of the native CT). 5) CT (or LT) derivatives should not cause serious disease in guinea pigs when administered intranasally or intraperitoneally at the dose used in humans (around 100 Eg).

Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements.

The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.

Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C.

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.

The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells.

A microplate assay, based on the clustering effect induced by pertussis toxin (PT) in Chinese hamster ovary (CHO) cells, has been developed and standardized. Toxin titration is done directly in the culture microplate by twofold dilutions of 25 microliters of test material to which are added 10 000 freshly trypsinized cells in 200 microliters of culture medium per well. The dilution causing the clustering effect is determined by direct microscopic observation after 48 h of incubation. The method allows detection of 50-100 pg toxin per millilitre. For determination of neutralizing antibodies (antitoxin), twofold dilutions of 25 microliters of antiserum are first made directly in the culture microplate. Thereafter 25 microliters of toxin, containing four times the minimal clustering concentration, is added to each well. After three hours for neutralization at +37 degrees C, cells are added, incubated and examined as above. The assay has been found to be simple and reproducible for measuring the antibody response to PT in human and different animal sera. For titration of bacteria associated toxin, the CHO cells are seeded and incubated for 24 h before the addition of bacteria. Incubation and examination are done as described for toxin titration.

An international survey of clostridial sera and vaccines.

The papers present the results of a survey of the usage, assay and specification of veterinary clostridial sera and vaccines in 23 countries. Thirteen of the countries use up to 8 different antisera. All the countries use vaccines, which are prepared from 13 species and types of clostridia. Vaccines containing up to 8 such components are commonly employed. Criteria for the design and interpretation of assays are discussed and evidence for efficacy summarized.

[Standardization of the control technics of Clostridium perfringens vaccine. Use of a reference vaccine]. / Standardisation des techniques de contrôle des vaccins a Clostridium perfringens. Utilisation d'un vaccin de référence

Control of Cl perfringens toxins. It is possible to employ a standardized technique for the control of the toxins of Cl. perfringens. Titres of LD50 per ml for the mouse and L+/50 per ml may be expressed with a confidence limit of 1.5. The assay of toxins by L+/50 (relating to their antigenic value) permits the establishment of standards by which it is possible to obtain a satisfactory vaccine with a high degree of certainty. 10 L+/50 per dose for Cl. perfringens A 20 L+/50 per dose for Cl. perfringens B 180 L+/50 per dose for Cl. perfringens C 50 L+/50 per dose for Cl. perfringens D Control of vaccine values. Technique adopted : Determination of antibodies in the rabbit following vaccination. The confidence limit of the antitoxin titre is similarly equal to 1.5 when P = 0.05. It would not appear to be necessary to establish minimum levels of antibodies to confer protection; it seems nevertheless that the results are influenced by considerable variations attributable to the animals used for the most part and that a reference vaccine is required. Freeze-dried vaccine. There is a natural antigenic activity in the rabbit which retains a considerable part of its antigenic properties after maintenance for one week at +37 degrees C. In our view this would be suitable for a reference vaccine.

[Comparison between the official indirect method and the direct method of control of vaccines against sheep enterotoxemia]. / Comparaison entre la méthode officielle indirecte et la méthode directe de contrôle des vaccins contre les enterotoxémies du mouton

The official methods of control of vaccines against sheep enterotoxemia are indirect in two ways : on the one hand they are carried out on animals for which the vaccine is not intended, and on the other hand they test only an immunological serum reaction. Another method consisting of direct testing by intravenous injection of toxin has been carried out on mice previously vaccinated with different doses. It has been shown that it is possible to obtain a certain level of protection giving a good dose-effect relation. However, immunity provided by this direct method is weak although the vaccine utilized is considered very efficacious with regard to the official norms of the direct test. These results, which are both encouraging and disappointing, will be the subject of a more intensive study of the different parameters in question.