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.

Characterization of scallop midgut gland certified reference material for quantification of diarrhetic shellfish toxins.

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ±â€¯0.061) mg/kg for OA and (0.45 ±â€¯0.11) mg/kg for DTX1.

Capillary electrophoresis-tandem mass spectrometry for multiclass analysis of polar marine toxins.

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values. Graphical abstract ᅟ.

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.

Heat treatment and the use of additives to improve the stability of paralytic shellfish poisoning toxins in shellfish tissue reference materials for internal quality control and proficiency testing.

The need for homogenous reference materials stable for paralytic shellfish toxins is vital for the monitoring and quality assurance of these potent neurotoxins in shellfish. Two stabilisation techniques were investigated, heat treatment through autoclaving and the addition of preserving additives into the tissue matrix. Short and long-term stability experiments as well as homogeneity determination were conducted on materials prepared by both techniques in comparison with an untreated control using two LC-FLD methods. Both techniques improved the stability of the matrix and the PSP toxins present compared to the controls. A material was prepared using the combined techniques of heat treatment followed by spiking with additives and data is presented from this optimised reference material as used over a two year period in the Irish national monitoring program and in a development exercise as part of a proficiency testing scheme operated by QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) since 2011. The results were indicative of the long-term stability of the material as evidenced through consistent assigned values in the case of the proficiency testing scheme and a low relative standard deviation of 10.5% for total toxicity data generated over 24 months.

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

Food poisoning outbreaks linked to mussels contaminated with okadaic acid and ester dinophysistoxin-3 in France, June 2009.

In June 2009, 11 outbreaks of food poisoning occurred in France, involving 45 individuals who had consumed mussels harvested in Vilaine Bay (Northwestern France). Because the toxic dinoflagellate Dinophysis spp. had been detected in the area from mid-May, okadaic acid (OA) and dinophysistoxins were suspected to be the cause of these outbreaks, although the weekly monitoring tests by mouse bioassay had been negative. With the help of the French reporting system for food-borne disease outbreaks, the detailed data on epidemiology, mussel consumption and complete product traceback, were collected for 11 individuals involved in three reported outbreaks. The batch of mussels identified as the source of these three outbreaks contained concentrations of toxins of the okadaic acid group that were approximately eight times higher than the European regulatory limit. Moreover, based on the consumption data available for the 11 cases, a lowest observable adverse effects level (LOAEL) was deduced. The LOAEL calculated from this study, although based on a very limited number of individuals, was in the same range, i.e. approximately 50 µg OA equivalents per person, as the LOAEL established by the European Food Safety Authority in 2006.

Potential use of gamma irradiation in the production of mussel and oyster reference materials for paralytic shellfish poisoning toxins.

Bivalve shellfish samples containing paralytic shellfish poisoning toxins were subjected to gamma irradiation dosage trials in order to assess the potential suitability of the technique in the production of toxin reference materials. Two candidate reference materials of tissue homogenates, mussels (Mytilus sp.) and native oysters (Ostrea edulis), were prepared in-house. Both were subjected to gamma irradiation at four different dose levels, 3.0, 6.0, 13.0 and 18.1 kGy. Bacterial levels were shown to be eliminated in the mussels and significantly reduced in the oysters following irradiation at all four dose levels. Paralytic shellfish poisoning (PSP) toxin concentrations were not significantly reduced in any of the samples indicating the treatment had no adverse affect on the initial stability of any of the PSP toxins monitored. Chromatographic results showed near-identical profiles for treated and non-treated samples inferring that no fluorescent toxin degradation products or matrix interferences were produced during the irradiation process. Results therefore proved that gamma irradiation treatment reduced bacterial levels within paralytic shellfish poisoning reference materials without compromising analyte content, with the subsequent potential to enhance the stability of future candidate reference materials treated in this manner.

Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue.

A rapid liquid chromatographic (LC) method with postcolumn oxidation and fluorescence detection (excitation 330 nm, emission 390 nm) for the determination of paralytic shellfish toxins (PSTs) in shellfish tissue has been developed. Extracts prepared for mouse bioassay (MBA) were treated with trichloroacetic acid to precipitate protein, centrifuged, and pH-adjusted for LC analysis. Saxitoxin (STX), neoSTX (NEO), decarbamoylSTX (dcSTX), and the gonyautoxins, GTX1, GTX2, GTX3, GTX4, GTX5, dcGTX2, and dcGTX3, were separated on a polar-linked alkyl reversed-phase column using a step gradient elution; the N-sulfocarbamoyl GTXs, C1, C2, C3, and C4, were determined on a C-8 reversed-phase column in the isocratic mode. Relative toxicities were used to determine STX-dihydrochloride salt (diHCl) equivalents (STXeq). Calibration graphs were linear for all toxins studied with STX showing a correlation coefficient of 0.999 and linearity between 0.18 and 5.9 ng STX-diHCI injected (equivalent to 3.9-128 microg STXeq/100 g in tissue). Detection limits for individual toxins ranged from 0.07 microg STXeq/100 g for C1 and C3 to 4.1 microg STXeq/100 g for GTX1. Spike recoveries ranged from 76 to 112% in mussel tissue. The relative standard deviation (RSD) of repeated injections of GTX and STX working standard solutions was < 4%. Uncertainty of measurement at a level of 195 microg STXeq/100 g was 9%, and within-laboratory reproducibility expressed as RSD was 4.6% using the same material. Repeatability of a 65 microg STXeq/100 g sample was 3.0% RSD. Seventy-three samples were analyzed by the new postcolumn method and both AOAC Official Methods for PST determination: the MBA (y = 1.22x + 13.99, r2 = 0.86) and the precolumn LC oxidation method of Lawrence (y = 2.06x + 12.21, r2 = 0.82).

[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.

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins.

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis.

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four post-column derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49 mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34 mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX-5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.