Bestimmung von prioritären organischen Substanzen in schwebstoffhaltigen Oberflächenwasser mittels Festphasenextraktionsscheiben

Die europäische Wasserrahmenrichtlinie (WRRL, Direktive 2000/60/EG) fordert eine intensive Überwachung von Oberflächengewässer auf die in der Direktive 2008/105/EG genannten prioritären und prioritär gefährlichen Stoffe. Vieler dieser Substanzen können wegen ihres hydrophoben Charakters stark an Schwebstoffen (SPM) sorbieren. Daher muss die sogenannte Gesamtwasserprobe, also die Wasserprobe einschließlich der darin befindlichen Feststoffe, untersucht werden. Die üblicherweise verwendeten Probenvorbereitungsverfahren, wie etwa die flüssig-flüssig Extraktion (LLE) oder die Festphasenextraktion (SPE), werden durch die Bildung von Emulsionen, unzureichende Extraktion der partikelgebundenen Analyten oder Verstopfungen auf Grund der SPM gestört. Folglich werden SPM und Wasserprobe häufig voneinander getrennt, z.B. durch Filtration, und separat analysiert. Dieser Ansatz ist mit einem hohen Zeit- und Arbeitsaufwand verbunden. Eine Alternative können Festphasenextraktionsscheiben (SPE disk) sein. Sie besitzen einen größeren Durchmesser als SPE Kartuschen und neigen daher seltener zu Verstopfungen. Dadurch kann eine Extraktion der Gesamtwasserprobe in einem einzigen Verfahrensschritt ermöglicht werden. Nach erstmaliger ausführlicher Untersuchung des Auftretens von residualem Wasser und seinen Auswirkungen auf die Festphasenextraktion mit SPE disks, um analytische Störungen zu reduzieren, wurde eine Multikomponentenmethode zur Spurenanalyse von 54 organischen Xenobiotika in Oberflächenwasser mittels SPE disk/Gaschromatographie-Massenspektrometrie (GC-MS) unter Berücksichtigung der WRRL und ihrer Folgerichtlinien entwickelt und validiert. Das entwickelte Verfahren ermöglicht die Bestimmung von polycyclischen aromatischen Kohlenwasserstoffen (PAK), polychlorierten Biphenylen (PCB), polybromierten Diphenylethern (PBDE), Organochlorpestiziden (OCP) und anderen Pestiziden in 1 L Wasser mit SPM-Gehalten von bis zu 1000 mg/Probe. Dazu wurde die SPE disk Methode mit zwei GC-MS Methoden kombiniert, die sich nur in ihren Injektionsmodi unterscheiden, um einen großen Konzentrationsbereich abzudecken. Der große Konzentrationsbereich ist auf die große Anzahl der untersuchten Analyten und den anvisierten Bestimmungsgrenzen (BG), welche mit den Werten der Umweltqualitätsnorm (UQN) verbunden sind, zurückzuführen. Die Jahresdurchschnittswerte der UQN für die untersuchten Analyten in Oberflächenwasser liegen zwischen 0,0005 und 2,4 µg/L. Die erreichten BG von bis zu 0,1 ng/L (S/N = 6:1) sind niedriger als die vieler in der Literatur beschriebener Methoden und erstmalig wurde eine mit SPE disk gekoppelte Large Volume Injektion/GC-MS Methode validiert. Die Gesamtanalysenzeit beträgt ca. 2,5 h/Probe, einschließlich beider GC-MS Methoden. Für 85 % der untersuchten Analyten können alle Anforderungen der WRRL mit der beschriebenen SPE disk/GC-MS Prozedur erfüllt werden. Eine Verbesserung der Bestimmungsgrenze kann in Zukunft zum Beispiel durch die Erhöhung des Probenvolumens, um 2 L oder mehr, oder durch die Verwendung sensitiverer Detektionsmethoden wie der GC-MS/MS, erreicht werden.The European Water Framework Directive (WFD, Directive 2000/60/EC) requires an extensive monitoring of surface water on priority and priority hazardous substances mentioned in Directive 2008/105/EC. Many of these substances can sorb strongly on suspended particulate matter (SPM) due to their hydrophobic character. Therefore, the so called whole water sample, the water sample including solid matter, has to be investigated. The usually used sample preparation methods, such as liquid-liquid extraction (LLE) or solid phase extraction (SPE), are affected by SPM by the formation of emulsions, insufficient extraction of particle-bound analytes or plugging. Consequently, SPM and water sample are often separated, e.g., by filtration, and analysed separately. This approach is associated with a high expenditure of time and work. An alternative may be the use of SPE disks. They have an enhanced diameter compared to SPE cartridges and therefore tend less to plugging. Therefore, an extraction of the whole water sample may become possible in one step. After a first extensive investigation of the occurrence of residual water and its effects in disk SPE to reduce analytical interferences, a multi-component trace analysis of 54 organic xenobiotics in surface water by SPE disk/gas chromatography-mass spectrometry (GC-MS) was developed and validated considering the requirements of the WFD and its following directives. The developed procedure allows the determination of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs) and other pesticides in 1 L water containing up to 1000 mg SPM/sample. The SPE disk sample preparation is combined with two GC-MS methods, differing only in their injection modes to cover a large concentration range. This large concentration range is due to the high number of investigated analytes and the targeted limits of quantification (LOQs), which are associated with the environmental quality standard (EQS) values. The annual average - EQS values in surface water are between 0.0005 and 2.4 µg/L for the investigated analytes. The reached LOQs up to 0.1 ng/L (S/N = 6:1) are lower compared to numerous methods described in literature and for the first time a SPE disk method coupled to large volume injection/GC-MS method was validated. The overall processing time is about 2.5 h/sample, including both GC-MS methods. For 85 % of the investigated analytes all requirements of WFD were fulfilled by the described SPE disk/GC-MS procedure. In future an improvement of the LOQs could be achieved for example by the increase of the sample volume of 2 L or more or by the use of more sensitive detection methods such as GC-MS/MS

Effect of ultrasonic, thermal and ozone pre-treatments on waste activated sludge solubilisation and anaerobic biodegradability

In order to enhance the efficiency of anaerobic digestion, the effects of ultrasounds, ozonation and thermal pre-treatment have been studied on waste activated sludge. The feature of this study was to carry out the comparison of the three pre-treatments in the same conditions and on the same sludge sample. Each treatment was tested in two conditions close to optimum conditions to maximise batch anaerobic sludge biodegradability. All treatments led to chemical oxygen demand and matter solubilisation and had little influence on mineral matter. In terms of solubilisation thermal pre-treatment was better than sonication or ozonation. But, in terms of batch anaerobic biodegradability, best results were obtained with ultrasounds with an energy of 6250 or 9350 kJ/kg TS and a thermal treatment at 170 or 190°C. Moreover, treatments had effects on physicochemical characteristics of sludge samples: apparent viscosity decreased after all treatments but the reduction was more important with thermal treatment. Median diameter of sludge flocs were reduced after sonication, increased after thermal treatment and did not change after ozonation. Finally, capillary suction time (CST) increased after ozonation, increased highly after sonication and was reduced after thermal treatmen

Glycogen Storage Disease Type Ia in Canines: A Model for Human Metabolic and Genetic Liver Disease

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including “lactic acidosis”, larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general