The H295R in vitro cell line produces the majority of the steroidogenesis, for which reason it is commonly used as a screening tool for endocrine disrupting chemicals. Simultaneous determination of the precursor cholesterol and key steroid hormones could give a broad insight into the mechanistic disruption of the steroidogenesis. Steroid hormones have primarily been extracted from H295R incubation medium by means of liquid-liquid extraction (LLE) and the obtained recoveries and matrix effects have typically not been stated or assessed. In the present study a solid-phase extraction (SPE) method was developed and validated for the simultaneous extraction of cholesterol and five key steroid hormones pregnenolone, 17-hydroxyprogesterone, testosterone, cortisol and aldosterone from H295R incubation medium, and finally detected by LC-MS/MS. Cholesterol was recovered at a level of 55.7%, while steroid hormone recoveries ranged from 98.2 to 109.4%. Matrix effects varied between -0.6% and 62.8%. Intra-day precision was deemed acceptable, but the inter-day precision for pregnenolone and aldosterone exceeded the precision limit of 15% RSD. Although LLE has been the most frequently used extraction method in H295R studies, however, our investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE.

Analytical methodologies based on liquid or gas chromatography coupled to tandem mass spectrometry for the simultaneous determination of two or more endogenous steroid hormones in human and animal plasma and serum has received increased attention the last few years. Especially in the clinical setting steroid profiling is of major importance in disease diagnostics. This paper discusses recent findings in such multi-steroid hormone procedures published from 2001 to 2012. The aim was to elucidate possible relationships between chosen analytical technique and the obtained analyte sensitivity for endogenous steroid hormones. By evaluating the success, at which the currently applied techniques have been utilized, more general knowledge on the field is provided. Furthermore the evaluation provides directions in which future studies may be interesting to conduct.

This study demonstrates the addition of (14)C-cholesterol to the human cell line H295R will in-situ form radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the present study was to in-situ radiolabel steroid hormones from cell line-incorporated (14)C-cholesterol using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid metabolites of the steroidogenic pathway allows for an improved understanding of the various enzymatic mechanisms involved without necessarily being dependent on quantification. Generated radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer (FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be added just before the cells are incubated for 72h in well plates. Based on the obtained HPLC-FSA chromatograms, and confirmation of the observations by studies in the literature, a qualitative time profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells with (14)C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

Antibiotics in the environment are a potential threat to environmental ecosystems as well as human health and safety. Antibiotics are designed to have a biological effect at low doses, and the low levels detected in the environment have turned focus on the need for more research on environmental occurrence and fate, to assess the risk and requirement for future regulation. This article describes the first occurrence study of the antibiotic polyether ionophores (lasalocid, monensin, narasin, and salinomycin) in the Danish environment. Various environmental matrices (river water, sediment, and soil) have been evaluated during two different sampling campaigns carried out in July 2011 and October 2012 in an agricultural area of Zealand, Denmark. Lasalocid was not detected in any of the samples. Monensin was measured at a concentration up to 20 ng·L⁻¹ in river water and 13 µg·kg⁻¹ dry weight in the sediment as well as being the most frequently detected ionophore in the soil samples with concentrations up to 8 µg·kg⁻¹ dry weight. Narasin was measured in sediment samples at 2 µg·kg⁻¹ dry weight and in soil between 1 and 18 µg·kg⁻¹ dry weight. Salinomycin was detected in a single soil sample at a concentration of 30 µg·kg⁻¹ dry weight.

A multi-residue pressurised liquid extraction (PLE) methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in solid environmental matrices. The PLE methodology is combined with solid phase extraction as clean-up using liquid chromatography coupled to tandem mass spectrometry applying electrospray ionisation for detection. The samples were freeze-dried prior to extraction. The absolute recoveries for soil and sediment ranged from 71 to 123% (relative standard deviation (RSDs) below 16%) and in the range 94–133% (RSDs 9–35%) for poultry manure. The final method allowed for the detection of four ionophores down to a few hundred ng kg−1 in natural solid matrices with limit of quantifications (LOQs) being 0.96, 0.87, 0.98, and 0.64 μg kg−1 in soil for lasalocid, monensin, salinomycin, and narasin, respectively. Corresponding LOQs in sediment were 1.28, 1.34, 1.39, and 0.78 μg kg−1 for the respective ionophores, while in manure the LOQs were 0.98, 1.01, 1.45, and 1.01 μg kg−1.

We developed a chemical analytical procedure for sampling, extracting and determining epidermal skin cortisol concentrations (SCCs) in the harbour porpoise (Phocoena phocoena) using gas chromatography–tandem mass spectrometry. In brief, this involved a pressurized liquid extraction with a two-step solid-phase clean-up. A derivatization step was conducted prior to detection. To evaluate the new assay, cortisol was analysed in three different sample types obtained from four harbour porpoises: skin plates, dorsal fin skin plugs (with and without lidocaine) and epidermal scrapes. Skin cortisol concentrations could be measured using the new assay in the majority of the tested skin samples down to a minimal sample size of 49 mg dry weight (dw). Water content ranged from 10 to 46% in the plug samples, which had SCCs from 2.1 to 77.7 ng/g dw. Epidermal scrape samples had the highest water content (83–87%) and lower SCCs (0.6–15 ng/g dw), while the skin plates had intermediate water contents (60–66%) and SCCs of 2.6–13.0 ng/g dw. SCC was slightly higher in plugs with lidocaine than without (average values of 41 and 33 ng/g dw, respectively). Substantial within-individual variations in cortisol concentrations are also common in other matrices such as blood and hair. Some important factors behind this variation could be e.g. the animal's sex, age, body condition, reproductive stage, and the body region sampled, as well as season, moulting cycles and water temperature. Clearly, more research into SCCs is required. The findings described here represent the first critical steps towards using epidermal skin cell samples to assess chronic stress levels in cetaceans and the development of a widely applicable health-assessment tool in these species.

Granular activated carbon (GAC) filters can be used to reduce emissions of organic micropollutants via municipal wastewater, but it is still uncertain to which extent biological degradation contributes to their removal in GAC filters. ¹⁴C-labeled organic micropollutants were therefore used to distinguish degradation from adsorption in a GAC-filter media with associated biofilm. The rates and extents of biological degradation and adsorption were investigated and compared with other biofilm systems, including a moving bed biofilm reactor (MBBR) and a sand filter, by monitoring ¹⁴C activities in the liquid and gas phases. The microbial cleavage of ibuprofen, naproxen, diclofenac, and mecoprop was confirmed for all biofilms, based on the formation of ¹⁴CO₂, whereas the degradation of ¹⁴C-labeled moieties of sulfamethoxazole and carbamazepine was undetected. Higher degradation rates for diclofenac were observed for the GAC-filter media than for the other biofilms. Degradation of previously adsorbed diclofenac onto GAC could be confirmed by the anaerobic adsorption and subsequent aerobic degradation by the GAC-bound biofilm. This study demonstrates the potential use of ¹⁴C-labeled micropollutants to study interactions and determine the relative contributions of adsorption and degradation in GAC-based treatment systems.

In the MORPHEUS project we estimated the occurrence and load of pharmaceuticals in four areas of the South Baltic Sea. This included the released loads from selected WWTPs as well as the environmental occurrence of pharmaceutical substances downstream of the WWTPs in the coastal regions Skåne (Sweden), Mecklenburg (Germany),Pomerania (Poland) and Klaipėda (Lithuania).In this article, we report on some of the findings from the Swedishmodel area Skåne and Kristianstad Municipality in the north eastpart of Skåne (Scania) as an example of a local assessment.

In the MORPHEUS project three Swedish Wastewater Treatment Plants (WWTPs) were investigated for their release of pharmaceuticals into the recipient. One of these was Degeberga WWTP which discharges its treated wastewater into the Segesholmsån river, ending in the Baltic Sea. Segesholmsån river has a length of 23 km and an average flow of roughly 0.6 m3/s (Figure 1).

In this project the release of 15 pharmaceuticals from three different WWTPs into three different recipients inKristianstad Municipality, Region Skåne was investigated. All three WWTPs are situated within the borders of thefirst UNESCO Biosphere Reserve in Sweden, Kristianstads Vattenrike – “Vattenriket®”, established in 2005. Pharmaceutical included were: 1. Atenolol 2. Azithromycin 3. Carbamazepine 4. Ciprofloxacin 5. Clarithromycin 6. Diclofenac 7. Erythromycin 8. Estrone 9. Ibuprofen 10. Naproxen 11. Metoprolol 12. Propranolol 13. Oxazepam 14. Paracetamol 15. Sulfamethoxazole

Under våren 2017 togs prover på avloppsvatten från Skånes Universitetssjukhus (SUS) i Lund i samarbete mellan Region Skåne och Högskolan Kristianstad (HKR). Totalt analyserades fyraprovpunkter med avseende på förekomst av 23 läkemedel, antibiotika och andra mikroföroreningar. De fyra provpunkterna var 1. Pumpgrop för uppsamling, 2. Onkologen, 3. Infektion samt 4. Avlopp från Centralblocket. Resultaten redovisas baserat på olika behandlingsfunktion, vilketinnefattar grupperna J - Antiinfektiva medel för systemiskt bruk, C - Hjärta och Kretslopp, N -Nervsystemet, M - Rörelseapparaten samt övriga ämnen. Resultaten visar att vissa läkemedelsläpps ut från SUS i högre koncentration jämfört med utgående avloppsvatten från ettreningsverk (se rapporten ”Läkemedelsutsläpp från Skånska avloppsreningsverk 2017” nedan). Noterbara ämnen är ciprofloxacin, sulfamethoxazole och trimethoprim, samtligaantibiotika. Resultaten vi-sar också att olika avdelningar medicinerar sina patientgrupper olika och att avdelningarna därmed kan vara lokala punktkällor för vissa typer av antibiotika. Koncentrationen av läkemdelsrester i avloppsvattnet påverkas förstås också av den totala vattenanvändningen, som i sin tur är en konsekvens av antal anställda, antal patienter, antal toaletter och arbetsmetoder/vattenanvänd-ning per avloppsspunkt mm. De uppmätta halternavisar att det är önskvärt, i en uppföljande studie, att analysera och fastställa, företrädesvis för antibiotika, vilka andra preparat som används på SUS, och i vilken omfattning. Det är viktigt att poängtera att resultaten från denna undersökning kommer från ett provtagningstillfälle där provtagningen bestod av ett stickprov. En uppföljande studie behöver innehålla fler analystillfällen samt söka svar på om förekomsten av höga halter av antibiotika kan föranleda resistensutveckling i sjukhusens avloppssystem. Vidare behöver man undersöka vilka eventuella åtgärder som behöver vidtas, vid sjukhusen och/eller vid avloppsreningsverken, för att förhindra att höga halter av läkemedelsrester belastar vattenmiljön med dess djur och växtliv. Hänsyn måste då också tas inte bara till vilka ämnen som specifikt härrör från sjukhuset utan också i vilka mängder och i vilken omfattning de når reningsverket i aktiv form.

This study is the first to investigate the pharmaceutical burden from point sources affecting the UNESCO Biosphere Reserve Kristianstads Vattenrike, Sweden. The investigated Biosphere Reserve is a >1000 km(2) wetland system with inflows from lakes, rivers, leachate from landfill, and wastewater-treatment plants (WWTPs). We analysed influent and treated wastewater, leachate water, lake, river, and wetland water alongside sediment for six model pharmaceuticals. The two WWTPs investigated released pharmaceutical residues at levels close to those previously observed in Swedish monitoring exercises. Compound-dependent WWTP removal efficiencies ranging from 12 to 100 % for bendroflumethiazide, oxazepam, atenolol, carbamazepine, and diclofenac were observed. Surface-water concentrations in the most affected lake were ≥100 ng/L for the various pharmaceuticals with atenolol showing the highest levels (>300 ng/L). A small risk assessment showed that adverse single-substance toxicity on aquatic organisms within the UNESCO Biosphere Reserve is unlikely. However, the effects of combinations of a large number of known and unknown pharmaceuticals, metals, and nutrients are still unknown.

Under februari 2019 togs 7 dygnsprover på det totala utgående flödet av avloppsvatten från Skånes Universitetssjukhus (SUS) i Malmö samt på det totala inkommande flödet av avlopssvatten till Sjölunda avloppsreningsverk i Malmö, som är Malmös största reningsverk. Syftet var att få kunskap om utgående koncentrationer av läkemedel från sjukhuset i relation till de inkommande koncentrationerna till avloppsreningsverket. Proverna togs från måndag till söndag under en och samma vecka och analyserades med avseende på förekomst av 33 läkemedel, antibiotika och andra mikroföroreningar. Detta innefattade 9 st antibiotika, 19 st läkemedel samt 5 andra kemikalier.

There is not much knowledge about pharmaceutical residues in Norwegian horse manure. In this project we have further developed a method that enables the analysis of various antibiotics and anthelmintics in a single sample. The method is designed for the analysis of solid substrates, such as horse manure. Manure from treated horses and horse manure with additions of controlled amounts of medicines were composted outdoors in a windrow consisting of horse manure and freshly cut grass. In a pot trail earthworms were added to some of the manure from the treated horses to study the effect of worm action on the biodegradation of pharmaceutical residues. - At the start of the project, several veterinary medicines were considered. The developed analysis method covers several active ingredients in the most commonly used equine medicines. The antibiotic Tribrissen vet inj with the active ingredients sulfadiazine and trimethoprim, and the equine anthelmintics Panacur (active ingredient fenbendazole) and Banminth (active ingredient pyrantel embonate) were included in the entire preliminary study. In total, 70 samples of horse manure and horse manure compost were analysed. We found that dried manure from treated horses could be analysed directly, thus making it unnecessary to freeze-dry samples prior to sending them further for analysis. The method can be used to analyse all four active ingredients at the same time. - Contents of pharmaceutical residues in horse manure are highest 1-2 days after the horse has been treated, with some variation between substances. After this peak, residue contents in manure decrease rapidly. Degradation of the added medicines was slow in the composting trials. In the compost windrow, the contents of medicines decreased gradually throughout a 60-day period, with slightly different degradation curves for the various substances. Three of the tested active ingredients were still detectable in the compost after 60 days. The earthworm trials have to be improved and repeated, among other things, because many worms died in the untreated horse manure plots. - Our trials show that manure from treated horses should be kept apart from other manure for the first 1-3 days after medical treatment. Strategies for the management of such manure have to be developed so that this resource can be used safely in plant production. Additional studies are needed to assess the degradation rate of pharmaceutical residues using different composting methods. Important factors that need to be studied in greater detail under Norwegian conditions include temperature development, windrow dimensions, moisture conditions and earthworm survival. We did not study the contents of pharmaceutical residues in urine. - We performed a survey on soil and fertilizer products among 100 garden centre customers. The results showed that there is a certain interest for peat-free, locally produced soil and fertilizer products. Approximately half of the respondents replied that they are willing to pay as much as 20 % more for such products. Consumers preferred soil amendments that contain nutrients and are typically ”all-purpose”, i.e. a single product that can be used across a wide range of applications. - This project has been important for the project partners due to its development of an analysis for pharmaceutical residues in solid horse manure. Furthermore, it provided a basis for the further development of horse manure as a pharmaceutical residue-free product.

Hydrolytic and photolytic degradation were investigated for the ionophore antibiotics lasalocid, monensin, salinomycin, and narasin. The hydrolysis study was carried out by dissolving the ionophores in solutions of pH 4, 7, and 9, followed by incubation at three temperatures of 6, 22, and 28 °C for maximum 34 days. Using LC–MS/MS for chemical analysis, lasalocid was not found to hydrolyse in any of the tested environments. Monensin, salinomycin, and narasin were all stable in neutral or alkaline solution but hydrolysed in the solution with a pH of 4. Half-lives at 25 °C were calculated to be 13, 0.6, and 0.7 days for monensin, salinomycin, and narasin, respectively. Absorbance spectra from each compound indicated that only lasalocid is degraded by photolysis (half-life below 1 h) due to an absorbance maximum around 303 nm, and monensin, salinomycin, and narasin are resistant to direct photolysis because they absorb light of environmentally irrelevant wavelengths.

Antimalarial drugs commonly referred to as antimalarials, include a variety of compounds with different physicochemical properties. There is a lack of information on antimalarial distribution in the body over time after administration, e.g. the drug concentrations in whole blood, plasma, and urine, which must be improved in order to advance curing the parasitic disease malaria. A key problem also lies in that pharmacokinetic studies not always are performed in patient groups that may benefit most of the treatment such as children, pregnancy and lower-weight ethnic populations. Here we review the available sample preparation strategies combined with liquid chromatographic (LC) analysis to determine antimalarials in whole blood, plasma and urine published over the last decade. Sample preparation can be done by protein precipitation, solid-phase extraction, liquid-liquid extraction or dilution. After LC separation, the preferred detection tool is tandem mass spectrometry (MS/MS) but other detection methods have been used e.g. UV, fluorescence and electrochemical detection. Major trends for sample preparation of the different groups of antimalarials for each matrix and its detection have been summarized. Finally, the main problems that the researchers have dealt with are highlighted. This information will aid analytical chemists in the development of novel methods for determining existing antimalarials and upcoming new drugs

The constant release of pharmaceuticals into the environment can lead to pollution of water and soils which might constitute a risk to human and other living organisms. Residues of common pharmaceuticals such as painkillers, antimicrobials, antidepressants, contraceptives or antiparasitics may enter the environment during manufacturing and disposal processes. The main sources of releases, however, are human consumption and veterinary use. Due to the metabolic stability of some pharmaceuticals up to 90% of the active ingredients are excreted and end up in the wastewater system and finally in the environment – e.g. accumulated in the Baltic Sea and its living organisms.The EU and its Member States have in the past few years increasingly recognised the challenge of pharmaceuticals in the environment (PIE). This is exemplified first and foremost by the European Commission’s Strategic Approach to Pharmaceuticals in the Environment, but also initiatives at the national level aiming at closing main knowledge gaps in the field, comprehensive monitoring systems and more surveys to detect the occurrence of pharmaceuticals in surface and ground water. Moreover, the OECD has recently published a report on pharmaceutical residues in freshwater that calls for a better understanding of the effects of pharmaceuticals in the environment and policy actions.However, this has not yet led to the development of indicators and threshold values at the EU level that aid Member States in implementing systematic monitoring schemes for PIE. As long as this situation persists and as long as we still do not sufficiently understand the effects of pharmaceutical residues on the ecosystem and on human health, the question arises: What can be done about PIE at the regional level? The answer to this lies in the precautionary principle – i.e. taking action to prevent harm to the environment even without having exact knowledge of all facets of the problem.

When steroid hormones are emitted into the environment, they may have harmful effects on the reproduction system of aquatic life. Until now, research has primarily focused on human excretion, demonstrating that steroid hormones reach the aquatic environment due to insufficient removal in waste water treatment processes. However more recently, it has been revealed that agricultural practices also may add to the environmental burden of steroid hormones. So far, research activities have mainly focused on steroid estrogens, but also androgens, progestagens and glucocorticoids, expressed in the vertebrate steroidogenesis, may occur at substantial levels in animal manure and should be addressed. In agricultural practices the animal manure can be applied to the soil as raw manure, but also as a solid or liquid manure fraction, since current livestock production facilities utilizes a recently developed technology, which separates raw animal manure into a solid and a liquid fraction.This technology offers an improved handling and refined distribution of the manure nutrients to the farmlands and the possibility to reduce the environmental impact of manure nutrients, especially avoiding the surplus load of phosphorous. In the present work we investigated the distribution of 9 steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) in raw manure and manure separates from 10 to 15 different pig farms in Denmark utilizing 4 different separation technologies. Furthermore, we investigated a possible relationship between the steroid hormone concentration and the different manure fractions and separation technologies. The chemical steroid hormone analysis was done by inverse and integrated clean-up pressurized liquid extraction, and further cleaned by a two step solid-phase extraction before derivatization and finally analyzed by GC-MS/MS.It was found that the steroid hormones were predominant in the solid manure separate calling for manure management strategies to reduce the content of steroid hormones in separated manure solid fraction. This could potentially be achieved through composting or anaerobic digestion for biogas production of the solid fraction; however, the effects of these technologies on steroid hormones need to be verified.

Newly developed treatment technologies are capable of separating livestock manure into a liquid fraction and a solid fraction using sedimentation, mechanical, and/or chemical methods. These technologies offer a potential means of distributing nutrients to agricultural lands without the unwanted environmental risks associated with the release of steroid hormones to adjacent waterways. To assess the potential benefit of these technologies in reducing the level of release of steroid hormones to adjacent waterways, distribution profiles of nine steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol, and 17β-estradiol) were determined in raw swine manure, and in solid and liquid fractions separated from ten full-scale manure separation systems. Steroid hormone concentrations, normalized for nitrogen content, were significantly higher in separated solids than in liquids. If separated liquids are applied instead of raw manure, steroid hormone loading can be reduced by a factor of 2 at a constant nitrogen fertilization level.

BACKGROUND: Variation in the 10 toll-like receptor (TLR) genes has been significantly associated with allergic rhinitis (AR) in several candidate gene studies and three large genome-wide association studies. These have all investigated common variants, but no investigations for rare variants (MAF ≤ 1%) have been made in AR. The present study aims to describe the genetic variation of the promoter and coding sequences of the 10 TLR genes in 288 AR patients.

METHODS: Sanger sequencing and Ion Torrent next-generation sequencing was used to identify polymorphisms in a Swedish AR population and these were subsequently compared and evaluated using 1000Genomes and Exome Aggregation Consortium (ExAC) data.

RESULTS: The overall level of genetic variation was clearly different among the 10 TLR genes. The TLR10-TLR1-TLR6 locus was the most variable, while the TLR7-TLR8 locus was consistently showing a much lower level of variation. The AR patients had a total of 37 promoter polymorphisms with 14 rare (MAF ≤ 1%) and 14 AR-specific polymorphisms. These numbers were highly similar when comparing the AR and the European part of the 1000Genomes populations, with the exception of TLR10 where a significant (P = 0.00009) accumulation of polymorphisms were identified. The coding sequences had a total of 119 polymorphisms, 68 were rare and 43 were not present in the European part of the 1000Genomes population. Comparing the numbers of rare and AR-specific SNPs in the patients with the European part of the 1000Genomes population it was seen that the numbers were quite similar both for individual genes and for the sum of all 10 genes. However, TLR1, TLR5, TLR7 and TLR9 showed a significant excess of rare variants in the AR population when compared to the non-Finnish European part of ExAC. In particular the TLR1 S324* nonsense mutation was clearly overrepresented in the AR population.

CONCLUSIONS: Most TLR genes showed a similar level of variation between AR patients and public databases, but a significant excess of rare variants in AR patients were detected in TLR1, TLR5, TLR7, TLR9 and TLR10. This further emphasizes the frequently reproduced TLR10-TLR1-TLR6 locus as being involved in the pathogenesis of allergic rhinitis.

Wastewater treatment processes are monitored mainly in terms of biogenic substances removal efficiency. Only recently they started to be perceived as a potential sources of pharmaceutical residues to their recipients the and hot-spots for antibiotic resistance dissemination among bacteria. The scale of the problem has not been fully investigated and understood – missing data on the pharmaceuticals consumption, unidentified and dispersed point sources and non-unified sampling strategy of monitoring programs have been identified as the examples of problematic areas. I n scope of project REPHIRA (Reduction of Pharmaceutical Emissions from Dispersed Point Sources in Rural Areas), financed from the Interreg Baltic Sea Region Seed Money, the cooperation has been established between five partners located in the Baltic Sea catchment area: Poland, Germany, Denmark, Sweden and Finland. In order to fulfill the knowledge gap, a preliminary study was conducted by Polish partner on four wastewater treatment plants (WWTPs) localized in the coastal area in northern Poland. Objects differ in terms of load, people equivalent, treatment technology and the recipient of WWTP effluent. The amount of human gut related indicator organism, E. coli has been estimated in raw and treated wastewater, as well as in the receiver, using classical microbiology approach and cultivation method. Additionally, the bacterial resistance to cefotaxime – an antibiotic belonging to third-generation cephalosporin family – has been tested.

von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10^-4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.

von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, e.g. VWF, ABO, STXBP5 and CLEC4M. This study aims to screen comprehensively for CLEC4M variants and investigate their association with type 1 VWD in the Swedish population. In order to screen for CLEC4M variants, the CLEC4M gene region was re-sequenced and the polymorphic neck region was genotyped in 106 type 1 VWD patients from unrelated type 1 VWD families. Single nucleotide variants (SNV) and variable number tandem repeat (VNTR) allele and genotype frequencies were then compared with 294 individuals from the 1000Genomes project and 436 Swedish control individuals. Re-sequencing identified a total of 42 SNVs. Rare variants showed no accumulation in type 1 VWD patients and are not thought to contribute substantially to type 1 VWD. The only missense mutation (rs2277998, NP_001138379.1:p.Asp224Asn) had a higher frequency in type 1 VWD patients than in controls (4.9%). The VNTR genotypes 57 and 67 were observed at higher frequencies than expected in type 1 VWD patients (6.4% and 6.2%) and showed an increase in patients compared with controls (7.4% and 3.1%). Strong linkage disequilibrium in the CLEC4M region makes it difficult to distinguish between the effect of the missense mutation and the VNTR genotypes. In conclusion, heterozygous VNTR genotypes 57 and 67 of CLEC4M were highly enriched and are the most likely mechanism through which CLEC4M contributes to disease in the Swedish type 1 VWD population.

von Willebrand factor (VWF) level and function are influenced by genetic variation in VWF and several other genes in von Willebrand disease type 1 (VWD1) patients. This study comprehensively screened for VWF variants and investigated the presence of ABO genotypes and common and rare VWF variants in Swedish VWD1 patients. The VWF gene was resequenced using Ion Torrent and Sanger sequencing in 126 index cases historically diagnosed with VWD. Exon 7 of the ABO gene was resequenced using Sanger sequencing. Multiplex ligation-dependent probe amplification analysis was used to investigate for copy number variants. Genotyping of 98 single nucleotide variants allowed allele frequency comparisons with public databases. Seven VWD2 mutations and 36 candidate VWD1 mutations (5 deletions, 4 nonsense, 21 missense, 1 splice, and 5 synonymous mutations) were identified. Nine mutations were found in more than one family and nine VWD1 index cases carried more than one candidate mutation. The T-allele of rs1063857 (c.2385T > C, p.Y795 = ) and blood group O were both frequent findings and contributed to disease in the Swedish VWD1 population. VWD2 mutations were found in 20 and candidate VWD1 mutations in 51 index cases out of 106 (48%). VWF mutations, a VWF haplotype, and blood group O all contributed to explain disease in Swedish VWD1 patients.

BACKGROUND: F8 int1h inversions (Inv1) are detected in 1-2% of severe hemophilia A (HA) patients. Long-range polymerase chain reaction (PCR) and inverse-shifting PCR have been used to diagnose these inversions.

OBJECTIVES: To design and validate a sensitive and robust assay for detection of F8 Inv1 inversions.

METHODS: Archival DNA samples were investigated using mile-post assays and droplet digital PCR.

RESULTS: Mile-post assays for Inv1 showing high specificities and sensitivities were designed and optimized. Analysis of four patients, two carrier mothers and 40 healthy controls showed concordance with known mutation status with one exception. One patient had a duplication involving exons 2-22 of the F8 gene instead of an Inv1 mutation. DNA mixtures with different proportions of wild type and Inv1 DNA correlated well with the observed relative linkage for both wild type and Inv1 assays and estimated the limit of detection of these assays to 2% of the rare chromosome.

CONCLUSIONS: The mile-post strategy has several inherent control systems. The absolute counting of target molecules by both assays enables determination of template quantity, detection of copy number variants and rare variants occurring in primer and probe annealing sites and estimation of DNA integrity through the observed linkage. The presented Inv1 mile-post analysis offers sensitive and robust detection and quantification of the F8 int1h inversions and other rearrangements involving intron 1 in patients and their mothers.

Background: The occurrence of mosaicism in hemophilia A (HA) has been investigated in several studies using different detection methods. Objectives: To characterize and compare the ability of AmpliSeq/Ion Torrent sequencing and droplet digital polymerase chain reaction (ddPCR) for mosaic detection in HA. Methods: Ion Torrent sequencing and ddPCR were used to analyze 20 healthy males and 16 mothers of sporadic HA patients. Results: An error-rate map over all coding positions and all positions reported as mutated in the F8-specific mutation database was produced. The sequencing produced a mean read depth of >1500X where >97% of positions were covered by >100 reads. Higher error frequencies were observed in positions with A or T as reference allele and in positions surrounded on both sides with C or G. Seventeen of 9319 positions had a mean substitution error frequency >1%. The ability to identify low-level mosaicism was determined primarily by read depth and error rate of each specific position. Limit of detection (LOD) was <1% for 97% of positions with substitutions and 90% of indel positions. The positions with LOD >1% require repeated testing and mononucleotide repeats with more than four repeat units need an alternative analysis strategy. Mosaicism was detected in 1 of 16 mothers and confirmed using ddPCR. Conclusions: Deep sequencing using an AmpliSeq/Ion Torrent strategy allows for simultaneous identification of disease-causing mutations in patients and mosaicism in mothers. ddPCR has high sensitivity but is hampered by the need for mutationspecific design.

Many newly diagnosed Swedish severe haemophilia A (HA) patients are sporadic cases. Some genotypically non-carrier mothers have gone on to have two descendants with the same mutation, presumably because of mosaicism.

AIMS: To define the origin of mutation in sporadic cases of HA, reveal possible sex-specific differences in mutagenesis and identify potential mosaics among non-carrier mothers.

METHOD: Sanger sequencing characterized the mutations and microsatellite haplotyping determined the origin of the X chromosome carrying the mutation in 3 generations of 45 families with sporadic severe HA. Droplet digital polymerase chain reaction (ddPCR) was used in five cases to reveal that mosaicism mutations are not found on conventional DNA sequencing.

RESULTS: In 23 out of 45 families, the mother carried the mutation and in 5 out of 28 families, the grandmother was also a carrier. The X chromosome was of grandpaternal origin in 17 out of 23 cases. One of five tested mothers was a mosaic with a mutation frequency of 7%.

CONCLUSION: In 40 out of 45 families, the sporadic case resulted from a mutation in the last two generations. In 82% (23/28), the carrier mothers had a de novo mutation where the X chromosome was of paternal origin in 74% (17/23). ddPCR is a potentially powerful and promising analysis for mosaicism in HA.

Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC50, strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.

Poor quality antibiotic medicines in circulation in Sub-Saharan Africa continue to be a burden. Pharmaceutical trade in substandard and counterfeit medicines is on the rise. The chemical quality of antibiotics dispensed in health facilities and recognised drug outlets in Ghana, when compromised, could be a major drawback to efforts made in fighting antibiotic resistance globally. To improve on antibiotic drug quality monitoring, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology, which is capable of quantifying thirteen antibiotics in drug products, was developed and validated in present work. The methodology was applied to various drug products including tablets, capsules, suspensions, syrups, intravenous and injection solutions as well as ear and eye droplets used as essential medicines in a Sub-Saharan country, Ghana.

In this study we evaluate and demonstrate the occurrence of nine natural and one synthetic steroid hormone, including estrogens, androgens and progestagens in biogas final digestate byproduct (digestion liquid) commonly used as an agricultural fertilizer. We investigated two biogas sites that utilize different anaerobic digestion technologies (mesophilic and thermophilic) from swine manure and other organic wastes. Individual hormone concentration levels were observed up to 1478 ng g−1 dry weight or 22.5 mg kg−1 N with estrone and progesterone reaching highest concentration levels. Evaluation of the potential environmental burden through the application in agriculture was also assessed on the basis of predicted environmental concentrations. This study indicates that the biogas digestion process does not completely remove steroid hormones from livestock manure and use of final digestate byproduct on croplands contributes to the environmental emission of hormones.

The polar bear (Ursus maritimus) is threatened by climate changes and also from persistent organic pollutants affecting polar bear endocrinology governing growth and reproduction. To provide further insight into basic polar bear endocrinology, we determined the levels of steroids in multiple tissues and plasma from East Greenland polar bears. Tissue samples from 10 polar bears, 5 males (2 adults, 3 juveniles) and 5 females (all juveniles) were obtained from the Inuit hunt in Scoresby Sound during springtime. Eleven steroids: pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17 alpha-estradiol and 17 beta-estradiol were determined in brain, adrenal cortex, testis, testicular vein, plasma, and ovary using GC-MS/MS. In brain tissue, the neuroactive progestagen pregnenolone (11.9 +/- 4.4 ng/g ww) and dehydroepiandrosterone (2.26 +/- 0.43 ng/g ww) were found in high concentrations. Very high levels of testosterone and androstenedione were observed in testes (> 100 ng/g ww) and plasma from testicular vein (testosterone: 108 +/- 41 ng/ml; androstenedione: 35.2 +/- 11.1 ng/ml). Additionally, a strong correlation was found between the levels of steroids in testes and testicular vein plasma. Progestagens were found in very high levels in ovaries from juvenile females (> 100 ng/g ww). Finally, our study indicates that polar bears synthesize androstenedione via the a dagger-4 pathway. The present study adds new insight to our knowledge on polar bear endocrinology, which may be used in future studies on polar bear ecology and studies on some of the threats from pollution and climate changes that these animals are facing.

Sample preparation represents about two-thirds of the cost of analysis and often presents logistical bottlenecks in analytical and environmental chemistry laboratories, so reducing our capacity and preparedness to quantify organic pollutants rapidly and accurately. Selective pressurized liquid extraction (SPLE) is an analytical technique that builds upon PLE by incorporating matrix-compound (i.e., interference) retainers into the extraction step, so reducing sample-preparation steps and increasing sample throughput. SPLE methods offer distinct advantages over traditional methods, namely reduction in the costs intrinsic to sample preparation (i.e., time, solvents, labor, laboratory space, training, and potential loss of analytes). The ability to analyze and to evaluate rapidly a large number of samples directly increases the analytical capacity and preparedness of a laboratory for certain situations (e.g., large-scale studies or environmental emergencies). We review the analytical improvements via SPLE and its wide-ranging applications.

Over the past 30 years a vast number of studies have demonstrated the presence of pharmaceutical residues in the environment. But still knowledge is scarce regarding the interaction of these emerging pollutants with various matrices in nature. A chromatographic system with on-line detection was developed to perform a sorption study of six selected pharmaceuticals to four natural sediments and dewatered digested sewage treatment plant sludge with differing physicochemical characteristics. Sorption effects, measured as asymmetry factors and recoveries, differed pronouncedly among the pharmaceuticals and between the matrices, which could be explained by basic physicochemical properties of the investigated compounds in relation to matrix characteristics. Protonated and deprotonated molecular properties had the greatest importance for sorbate–sorbent interactions. Atenolol, with cationic properties, showed the highest degree of sorption regardless of the matrix studied. Diclofenac and furosemide, both acids, showed the least tendency towards interactions to natural matrices. Among the neutral compounds bendroflumethiazide, carbamazepine and oxazepam, weaker forces, such as van der Waals, aromatic electron donor–acceptor interactions, and hydrogen forces, seemed more important to determine sorption differences. Results revealed that sorption of pharmaceuticals on natural sediments decreased in the order: atenolol (+) > bendroflumethiazide > oxazepam > carbamazepine > diclofenac (−) > furosemide(–). The matrix content of organic matter measured as total organic carbon (TOC) clearly dictated drug sorption. Beside from studying matrix interaction, these results and the developed technique and methodology might find use in the development of new removal processes of pharmaceuticals from wastewater based on improved knowledge concerning chemical interactions to filter materials.

Two green chemistry extraction systems, an in-house stainless-steel column Pressurized Hot Water Extraction system (PHWE) and a commercially available Espresso machine were applied for analysing 23 active pharmaceutical ingredients (APIs) in sewage sludge. Final analysis was performed on UPLC-MS/MS using two different chromatographic methods: acid and basic. When analysing all 23 APIs in sewage sludge both extraction methods showed good repeatability. The PHWE method allowed for a more complete extraction of APIs that were more tightly bound to the matrix, as exemplified by much higher concentrations of e.g., ketoconazole, citalopram and ciprofloxacin. In total, 19 out of 23 investigated APIs were quantified in sewage sludge, and with a few exceptions the PHWE method was more exhaustive. Mean absolute recoveries of 7 spiked labelled APIs were lower for the PHWE method than the Espresso method. Under acid chromatographic conditions mean recoveries were 16% and 24%, respectively, but increased to 24% and 37% under basic conditions. The difference between the PHWE method and the Espresso method might be interpreted as the Espresso method giving higher extraction efficiency; however, TIC scans of extracts revealed a much higher matrix co-extraction for the PHWE method. Attempts were made to correlate occurrence of compounds in sewage sludge with chemical properties of the 23 APIs and there are strong indications that both the number of aromatic rings and the presence of a positive charge is important for the sorption processes to sewage sludge.

By using an innovative, positive pressure sample loading technique in combination with an in-line filter of finely ground sand the bottleneck of solid phase extraction (SPE) can be reduced. Recently published work by us has shown the proof of concept of the technique. In this work, emphasis is put on the SPE flow rate and method validation for 26 compounds of emerging environmental concern, mainly from the 1st and 2nd EU Watch List, with various physicochemical properties. The mean absolute recoveries in % and relative standard deviations (RSD) in % for the investigated compounds from spiked pure water samples at the three investigated flow rates of 10, 20, and 40 mL/min were 63.2% (3.2%), 66.9% (3.3%), and 69.0% (4.0%), respectively. All three flow rates produced highly repeatable results, and this allowed a flow rate increase of up to 40 mL/min for a 200 mg, 6 mL, reversed phase SPE cartridge without compromising the recoveries. This figure is more than four times the maximum flow rate recommended by manufacturers. It was indicated that some compounds, especially pronounced for the investigated macrolide molecules, might suffer when long contact times with the sample glass bottle occurs. A reduced contact time somewhat decreases this complication. A very good repeatability also held true for experiments on both spiked matrix-rich pond water (high and low concentrations) and recipient waters (river and wastewater) applying 40 mL/min. This work has shown that, for a large number of compounds of widely differing physicochemical properties, there is a generous flow rate window from 10 to 40 mL/min where sample loading can be conducted. A sample volume of 0.5 L, which at the recommended maximum flow rate speed of 10 mL/min, would previously take 50 min, can now be processed in 12 min using a flow rate of 40 mL/min. This saves 38 min per processed sample. This low-cost technology allows the sample to be transferred to the SPE-column, closer to the sample location and by the person taking the sample. This further means that only the sample cartridge would need to be sent to the laboratory, instead of the whole water sample, like today's procedure.

In this work the habitual behaviour of low pH in environmental organic trace analysis is challenged by investigating the full potential of building a multi-component UHPLC-ESI-MS/MS method adapted to cover common emerging contaminants of many different polarities, minimizing the elements of compromise in the performance of the final analytical separation and detection. Contributes have been made by taking advantage of common commercially available technology in understanding the impact from solvent components and the ionization of analytes which can facilitate future development of robust, sensitive and precise UHPLC-MS/MS methods. All contaminants were evaluated and optimized without prejudices regarding historical residence in terms of chromatographic conditions and ESI mode; increasing multi-method's flexibility that can be implemented in routine analysis in response to new requests as well as to emerging contaminants yet to be discovered. Our data strongly supports the questioning of the assumption that equilibrium concentrations of ions in solution reflect those produced during the electrospray process. ESI responses of [M+H](+) and limits of detection were comparable, or often better at high pH compared to acidic eluents. Presence of nitrogen basic groups such as tertiary and secondary amines in a compound increased the intensity of the ESI+ signal, and was even further elevated in basic eluent. The proton affinity probably changes for many nitrogen-containing compounds during the ionization process, making the gas-phase processes very important in generation of these ions by ESI+. There were also an unexpected large number of compounds showing their highest response at pH 7 and weak ionic strength. A flow optimized, buffert free, neutral UHPLC-MS/MS method enhanced the sensitivity for the environmental important synthetic hormone ethinyl estradiol significantly.

Läkemedelsverket gav i september 2015 ut en rapport med titeln ”Miljöindikatorer inom ramen för nationella läkemedelsstrategin (NLS)”. Rapporten indikerar bland annat stora kvalitetsbrister i tidigare utförda analyser av läkemedel i miljön under åren 2002-2013, men betonar också vikten av att samordna framtida nationella läkemedelsanalyser för att bättre använda våra offentliga resurser. I rapporten skriver man följande: ”Allra högst prioriterad ansåg arbetsgruppen att indikatorn ”mäta halter av läkemedelssubstanser i miljö” vara. Detta beroende på att det utöver att det är av stor vikt att följa utvecklingen av läkemedelsrester i miljön över tid för att utvärdera effekten av genomförda insatser, så ansåg arbetsgruppen att det finns stora möjligheter att optimera användningen av de offentliga resurserna genom en bättre samordning av mätningar i miljön. Många mätningar har gjorts historiskt av olika offentliga aktörer utan någon samordning.” Mot bakgrund av detta, och som svar på ett nationellt behov, åtog sig Högskolan Kristianstad (HKR) genom Ola Svahn och Erland Björklund, båda verksamma vid MoLab, Krinova Incubator and Science Park i Kristianstad, att leda denna interkalibreringsstudie för läkemedelsanalys i miljö-vattenprover av olika typ. Arbetet har skett på uppdrag av Havs- och Vattenmyndigheten som finansierat arbetet via bidrag ur havs- och vattenmiljöanslaget och i samarbete med fyra andra analyslaboratorier vid Umeå Universitet (UU), Sveriges Lantbruksuniversitet Uppsala (SLU), Svenska miljöinstitutet Stockholm (IVL) samt Aarhus Universitet Danmark (AU). Total deltog därmed 5 laboratorier, alla med tidigare erfarenhet av läkemedelsanalys i miljöprover.

I projektet LUSKA (Läkemedelsutsläpp från Skånska Avloppsreningsverk) deltog 6 olika reningsverksorganisationer,    geografiskt fördelade över hela Skåne; Höganäs, Klippan, Höör/Hörby, Svedala, Kristianstad och Simrishamn. Provtagning utfördes i april 2017 på fyra platser vid varje reningsverk. Tre av dessa platser valdes enligt förslag från Länsstyrelsen Skånes tillsynsvägledning; uppströms, nedströms och utgående vatten från reningsverket. Dessutom ingick en fjärde provpunkt bestående av inkommande vatten till reningsverken. I studien analyserades total 21 av 22 läkemedel enligt Läkemedelsverkets föreslagna lista på ämnen från 2015. Analysresultaten från de 8 avloppsreningsverken, samt tillhörande recipient i form av åar och sjöar visar tydligt att stora mängder läkemedel hamnar i våra omgivande skånska vatten varje år. Detta sker som en konsekvens av att reningsverken inte förmår rena läkemedelsutsläpp med befintlig teknik baserad på aktivt slam. Studien visade att de 8 reningsverken släpper ut minst 71 kg läkemedel varje år till skånska vatten enbart av dessa 21 ämnen. Den stora bulken av de uppmätta ämnena utgjordes av blodtryckssänkaren metoprolol och smärtstillaren diklofenak. Men även ämnen som karbamazepin, losartan, naproxen och oxazepam förekom i betydande koncentrationer i avloppsvattnet. Dessa ämnen innefattar flera läkemedelstyper och representerar tre generella och relativt vanliga sjukdomstillstånd som högt blodtryck, inflammation och smärta, samt depression och ångest. Baserat på resultaten i LUSKA-projektet kan man uppskatta att när ett reningsverk behandlar en miljon kubikmeter (1 000 000 m3) avloppsvatten passerar det samtidigt ut ca 4 kg av de 21 läkemedel som Läkemedelsverket tagit upp på sin övervakningslista. Enligt en grov beräkning, innefattande en majoritet av de skånska reningsverken, skulle detta innebära att nästan 600 kg kilo läkemedel läcker ut varje år från skånska reningsverk av Läkemedelsverkets föreslagna ämnen. Samtidigt måste man beakta att dessa 21 ämnen endast utgör en liten del av flera hundra läkemedelssubstanser som används för behandling av sjukdomar. Med stor sannolikhet läcker ett till flera ton läkemedel ut årligen i skånsk recipient. Mätningar i åar och sjöar visar att även om koncentrationerna sjunker nedströms verken som  en konsekvens av företrädesvis utspädning så finns det lokaler där koncentrationerna är an-märkningsvärt höga utifrån ett hållbarhetsperspektiv.

The project LUSKA (a Swedish acronym meaning ‘to figure out’ formed from the name LäkemedelsUtsläpp från SKånska Avloppsreningsverk - Pharmaceutical emissions from Scanian wastewater treatment plants) comprised six different participating wastewater treatment organisations, geographically distributed throughout the whole of the Scania region in Sweden (Skåne): Höganäs, Klippan, Höör/Hörby, Svedala, Kristianstad and Simrishamn. Sampling was done in April 2017 in four locations at each treatment plant. Three of these locations were chosen as suggested by the County Administrative Board of Skåne’s supervisory guide: upstream, downstream and outlet water from the treatment plant. In addition, a fourth sample point was included consisting of inlet water to the treatment plants. In the study, a total of 21 out of 22 pharmaceuticals were analysed according to the Swedish Medical Products Agency’s proposed substance watchlist from 2015. The analysis results from the eight treatment plants and associated recipients in the form of streams and lakes clearly shows that large amounts of pharmaceuticals end up in our surrounding Scanian waters every year. This takes place as a consequence of the wastewater treatment plants not being able to separate pharmaceutical emissions with existing technology based on activated sludge. The study showed that the eight treatment plants release at least 71 kg of pharmaceuticals every year to Scanian waters of these 21 substances alone. The major bulk of the measured substances were comprised of the blood pressure lowering drug metoprolol and the analgesic diclofenac. But even such substances as carbamazepine, losartan, naproxen and oxazepam occurred in significant concentrations in the wastewater. These substances include several pharmaceutical types and represent three general and relatively common illnesses: high blood pressure, inflammation and pain, and depression and anxiety. Based on the results in the LUSKA project, it can be estimated that when a treatment plant treats one million cubic metres (1,000,000 m3) of wastewater, at the same time approximately 4 kg pass of the 21 drugs that the Swedish Medical Products Agency included on its watchlist. According to a rough estimate, including a majority of the Scanian wastewater treatment plants, this would mean that nearly 600 kg of drugs on the Swedish Medical Products Agency’s proposed substances watchlist leak out from Scanian treatment plants every year. At the same time, it must be taken into account that these 21 substances only comprise a small part of the several hundred pharmaceutical substances used for the treatment of diseases. In all likelihood, one or more tonnes of drugs leak out into Scanian recipients annually. Measurements in lakes and streams show that even if the concentrations drop downstream of the plants, probably as a consequence of dilution, there are locales where the concentrations are remarkably high from a sustainability perspective.

In environmental trace analysis there is often a need to enrich the compounds of interest from a large sample volume, where the use of solid phase extraction (SPE) is more or less the standard technique. The presence of the sample matrix can cause clogging of the SPE-column, especially at the end of a sample load. Swedish surface waters are often humic rich making the use of traditional sample loading by a vacuum manifold very limited. This obstacle forced the development of a different sample loading technique, based on compressed air and sand as an in-line-filter, designed to load larger sample volumes as needed in trace level analysis of hormones in surface water. The developed technique, combined with a UPLC MS/MS method, showed promising reproducibility and accuracy, and enabled increased sensitivity for the analysis of hormones in humic rich surface water.

Thermal degradation of antibiotics has been studied for decades in a broad range of disciplines including food production, agriculture and analytical chemistry. Yet, there is a lack of thermal stability data for many antibiotics. Here we systematically investigated the thermal stability of ten commonly prescribed antibiotics applying a laborsaving automated inhouse pressurized dynamic flow-through system. The design of the system allowed a fast access to a large number of data at medium to subcritical water temperatures, ranging from 50-250 °C. The five ß-lactams cefadroxil, cefuroxime, amoxicillin, penicillin V, and penicillin G showed a high degree of stability with a maximum degradation of less than 30 % at 150 °C. The two quinolones ciprofloxacin and norfloxacin showed a very high thermal stability up to 200 °C, as did trimethoprim andsulfamethoxazole. At 250 °C all antibiotics were either partly of fully removed. The tetracycline doxycycline showed a specific removal pattern probably involving both binding to metal surfaces at lower temperatures as well as degradation at increased temperatures.

This paper presents the development and evaluation of a methodology for extraction, clean-up and analysis of three key corticosteroids (aldosterone, cortisol and corticosterone) in polar bear hair. Such a methodology can be used to monitor stress biomarkers in polar bears and may provide as a useful tool for long-term and retrospective information. We developed a combined pressurized liquid extraction (PLE)-solid phase extraction (SPE) procedure for corticosteroid extraction and clean-up followed by high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis. This procedure allows for the simultaneous determination of multiple steroids, which is in contrast to previous polar bear studies based on ELISA techniques. Absolute method recoveries were 81%, 75% and 60% for cortisol, corticosterone and aldosterone, respectively. We applied the developed method on a hair sample pooled from four East Greenland polar bears. Herein cortisol and corticosterone were successfully determined in levels of 0.32 +/- 0.02 ng/g hair and 0.13 +/- 0.02 ng/g hair, respectively. Aldosterone was below limit of detection (LOD<0.17 ng/g). The cortisol hair concentration found in these East Greenland polar bears was consistent with cortisol levels previously determined in the Southern Hudson Bay and James Bay in Canada using ELISA kits. (C) 2016 Elsevier B.V. All rights reserved.