Bjarne Styrishave

Determination of pharmaceuticals in environmental and biological matrices using pressurised liquid extraction—Are we developing sound extraction methods?

Pressurised liquid extraction (PLE) is now a well established and extensively applied extraction technique in environmental analysis for pollutants such as persistent organic pollutants (POPs). During the past decade, an emerging group of environmentally interesting analytes are pharmaceuticals that are continuingly released into the environment. This class is comprised with compounds of various properties. As the field of the analysis of these compounds grows, an increasing number of PLE methods for pharmaceuticals of varying quality are developed and published. This review summarises the critical PLE parameters during PLE method development and highlight them with examples from recently published papers utilising pressurised liquid extraction for the determination of pharmaceuticals in environmental and biological matrices. These recent methods are summarised and critically discussed with the aim to provide important reflections to alleviate in future PLE development for pharmaceuticals in environmental matrices.

H295R cells as a model for steroidogenic disruption: A broader perspective using simultaneous chemical analysis of 7 key steroid hormones

The effects of three model endocrine disruptors, prochloraz, ketoconazole and genistein on steroidogenesis were tested in the adrenocortical H295R cell line to demonstrate that a broader mechanistic understanding can be achieved in one assay by applying chemical analysis to the H295R assay. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and 17β-estradiol) were analyzed using a novel and thoroughly validated GC-MS/MS method. In addition to the simultaneous quantification of 7 steroid hormones, the present method also negates the potential problems of cross-reactivity that can be encountered in some immunoassays. Although all 3 test compounds decrease the concentrations of the main sex steroids, the chemicals exerted different effects upstream in the pathway. Exposure to prochloraz resulted in increased hormone levels upstream of steroid 17 alpha-hydroxylase/17,20 lyase (P450c17) and decreases downstream. Ketoconazole inhibited the entire pathway, while exposure to genistein resulted in increased hormone levels upstream of 3-β-hydroxysteroid dehydrogenase (3β-HSD) and decreases downstream. The results demonstrate that chemical analysis combined with the H295R cell assay is an useful tool for studying the mechanisms by which endocrine disruptors interfere with the steroidogenic pathway.

Environmental risk assessment of three selective serotonin reuptake inhibitors in the aquatic environment: A case study including a cocktail scenario

We present an environmental risk assessment of three selective serotonin reuptake inhibitors (SSRIs; citalopram, sertraline, and fluoxetine) in the aquatic environment based on two case scenarios. Abiotic and biotic degradation experiments and sorption estimates were used to predict environmental concentrations of three SSRIs from the wastewater of two psychiatric hospitals, the primary sector, and wastewater entering and leaving wastewater treatment plants (WWTPs). Assuming a sewage treatment retention time of 8 h, abiotic degradation was low, for all three SSRIs inhibitors, ranging between 0 and 2% for hydrolysis and 0 and 6% for photolysis. The biodegradation was also slow, ranging from 0 to 3% within an 8-h period. In untreated sewage, citalopram (CIT) and sertraline (SER) concentrations may be high enough to exert effects on the aquatic biota (CIT: 0.19-10.3 µg/L; SER: 0.14-17.1 µg/L). Removal of the pharmaceuticals is due primarily to sorption in the WWTP. Sertraline was estimated to have the highest concentrations in the sewage effluents, 4.4 and 19.9 ng/L for the two cases, respectively. In treated wastewater, individual SSRI concentrations are probably too low to exert effects on biota. By using concentration addition, a cocktail exposure scenario was estimated. The predicted concentration in the biota calculated from the cocktail effect was 0.05 and 0.16 nmol/g for the two cases, respectively, and SER was found to give the highest contribution to this cocktail effect. The results indicate that the concentrations in the wastewater effluents are one to two orders of magnitude lower than the concentrations likely to cause an effect in the aquatic biota.

Hydroxylated polychlorinated biphenyls decrease circulating steroids in female polar bears (Ursus maritimus)

As a top predator in the Arctic food chain, polar bears (Ursus maritimus) are exposed to high levels of persistent organic pollutants (POPs). Because several of these compounds have been reported to alter endocrine pathways, such as the steroidogenesis, potential disruption of the sex steroid synthesis by POPs may cause implications for reproduction by interfering with ovulation, implantation and fertility. Blood samples were collected from 15 female polar bears in Svalbard (Norway) in April 2008. The concentrations of nine circulating steroid hormones; dehydroepiandrosterone (DHEA), androstenedione (AN), testosterone (TS), dihydrotestosterone (DHT), estrone (E1), 17α-estradiol (αE2), 17β-estradiol (βE2), pregnenolone (PRE) and progesterone (PRO) were determined. The aim of the study was to investigate associations among circulating levels of specific POP compounds and POP-metabolites (hydroxylated PCBs [OH-PCBs] and hydroxylated PBDEs [OH-PBDEs]), steroid hormones, biological and capture variables in female polar bears. Inverse correlations were found between circulating levels of PRE and AN, and circulating levels of OH-PCBs. There were no significant relationships between the steroid concentrations and other analyzed POPs or the variables capture date and capture location (latitude and longitude), lipid content, condition and body mass. Although statistical associations do not necessarily represent direct cause-effect relationships, the present study indicate that OH-PCBs may affect the circulating levels of AN and PRE in female polar bears and that OH-PCBs thus may interfere with the steroid homeostasis. Increase in PRO and a decrease in AN concentrations suggest that the enzyme CYP17 may be a potential target for OH-PCBs. In combination with natural stressors, ongoing climate change and contaminant exposure, it is possible that OH-PCBs may disturb the reproductive potential of polar bears.

Analysis and environmental concentrations of the herbicide dichlobenil and its main metabolite 2,6-dichlorobenzamide (BAM): a review.

Dichlobenil is an herbicide which has been applied in many countries for weed control in non-agricultural areas such as railroads, car parks and private gardens. In the aquatic environment it has been used for control of floating aquatic weeds. Dichlobenil is relatively persistent in the environment, and primarily bound to solid matrices. Of great concern is its main degradation product 2,6-dichlorobenzamide which is water soluble and therefore transported downward in aquifers, contaminating groundwater resources. It is often found in concentrations exceeding 0.1 μg/L, which is the maximum allowed concentration of pesticides in groundwater set by the European Commission. In many countries, the usage of dichlobenil and the problems associated with groundwater contamination by 2,6-dichlorobenzamide have resulted in intensive research and monitoring of these compounds. This review gives the first overview of analytical strategies available for determining dichlobenil and 2,6-dichlorobenzamide in environmental matrices. It also summarizes studies presenting measured environmental concentrations of dichlobenil and 2,6-dichlorobenzamide identified in the literature during the past two decades. Thereby a preliminary picture of the distribution of dichlobenil and 2,6-dichlorobenzamide in the environment can be outlined for the first time.

Cytochrome P450 expression is moult stage specific and regulated by ecdysteroids and xenobiotics in the crab Carcinus maenas.

The shore crab Carcinus maenas has a high capacity for metabolizing polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP) enzymes are involved in this metabolism and also have a role in development and reproduction. This investigation is a systematic gene expression analysis of six CYPs in C. maenas. Expression of CYP2 and CYP3-like genes was predominant in hepatopancreas, while expression of CYP4-like genes was predominant in gills and epidermis. Expression of all six CYP genes fluctuated over the moult cycle in the hepatopancreas and structurally related genes were regulated coordinately. The study suggests that hepatopancreas is a major site of CYP gene expression in C. maenas confirming previous biochemical studies showing that this tissue is the major compartment for CYP mediated xenobiotic metabolism in crustaceans. In addition, the data show that CYP2 and CYP3 related genes respond to ecdysteroid and xenobiotic treatment, while those related to CYP4 genes do not and likely are involved in a more general physiological function such as fatty acid metabolism. The developmental variations of CYP expression suggest a molecular mechanism for the stage specific susceptibility of crabs exposed to environmental pollutants.

SPATIAL AND TEMPORAL DISTRIBUTION OF SHORE CRABS CARCINUS MAENAS IN A SMALL TIDAL ESTUARY(LOOE ESTUARY, CORNWALL, ENGLAND)

Abstract Tidal, diel, and spatial variations in numbers, sex, size, and colour morphology of shore crabs Carcinus maenas caught in baited drop nets during tidal periods, at neap tide and spring tide, were studied in the strongly tidal Looe Estuary, Cornwall, Southwest England. Depth, salinity, temperature, pH, and oxygen tensions were measured simultaneously. High numbers of both genders were caught in the estuary. In total, 61% of adult crabs caught were females. However, the sex ratio (males over females) in the catches significantly increased (P < 0.05) from approximately 0.2 at the station nearest to the mouth of the estuary to approximately 4 at the innermost station. Due to the well-established relationship between carapace colouration and intermoult duration, catches were analysed with regard to red and green colour forms, besides for sex and size. Green crabs were caught throughout the estuary and constituted 79% of total catches. Green males dominated the shallow stations, whereas green females dominated the deep stations. The catch per unit effort (CPUE) was related to tidal and diel phases, with most adult crabs being caught during high tide and only few during low tide. Also, more adult crabs were caught during night time than during day time. The CPUE increased with increasing depth, and crabs were never caught at salinities below 15‰ and rarely at salinities below 20‰. Oxygen tension, temperature, and pH exerted no effect on the distribution of shore crabs. Even though conclusions based on these data depend on whether catch-data analysis reflects true population abundances, sex ratios, and colour morphology compositions, the data suggest that the small size, strong current, and high salinity characteristics of the Looe Estuary allow both genders and colour forms to migrate into the estuary during high tide and to return to the shore before low tide, thereby exploiting a marginal feeding habitat.

Aniline Is Rapidly Converted Into Paracetamol Impairing Male Reproductive Development

Industrial use of aniline is increasing worldwide with production estimated to surpass 5.6 million metric tons in 2016. Exposure to aniline occurs via air, diet, and water augmenting the risk of exposing a large number of individuals. Early observations suggest that aniline is metabolized to paracetamol/acetaminophen, likely explaining the omnipresence of low concentrations of paracetamol in European populations. This is of concern as recent studies implicate paracetamol as a disrupter of reproduction. Here, we show through steroidogenic profiling that exposure to aniline led to increased levels of the Δ4 steroids, suggesting that the activity of CYP21 was decreased. By contrast, paracetamol decreased levels of androgens likely through inhibition of CYP17A1 activity. We confirm that aniline in vivo is rapidly converted to paracetamol by the liver. Intrauterine exposure to aniline and paracetamol in environmental and pharmaceutical relevant doses resulted in shortening of the anogenital distance in mice, a sensitive marker of fetal androgen levels that in humans is associated with reproductive malformations and later life reproductive disorders. In conclusion, our results provide evidence for a scenario where aniline, through its conversion into antiandrogenic paracetamol, impairs male reproductive development.

Brain region-specific perfluoroalkylated sulfonate (PFSA) and carboxylic acid (PFCA) accumulation and neurochemical biomarker responses in east Greenland polar bears (Ursus maritimus).

Perfluoroalkyl substances (PFASs) is a growing class of contaminants in the Arctic environment, and include the established perfluorinated sulfonates (PFSAs; especially perfluorooctane sulfonate (PFOS)) and carboxylic acids (PFCAs). PFSAs and PFCAs of varying chain length have been reported to bioaccumulate in lipid rich tissues of the brain among other tissues such as liver, and can reach high concentrations in top predators including the polar bear. PFCA and PFSA bioaccummulation in the brain has the potential to pose neurotoxic effects and therefore we conducted a study to investigate if variations in neurochemical transmitter systems i.e. the cholinergic, glutaminergic, dopaminergic and GABAergic, could be related to brain-specific bioaccumulation of PFASs in East Greenland polar bears. Nine brain regions from nine polar bears were analyzed for enzyme activity (monoamine oxidase (MAO), acetylcholinesterase (AChE) and glutamine synthetase (GS)) and receptor density (dopamine-2 (D2), muscarinic cholinergic (mAChR) and gamma-butyric acid type A (GABA-A)) along with PFSA and PFCA concentrations. Average brain ∑PFSA concentration was 25ng/g ww where PFOS accounted for 91%. Average ∑PFCA concentration was 88ng/g ww where PFUnDA, PFDoDA and PFTrDA combined accounted for 79%. The highest concentrations of PFASs were measured in brain stem, cerebellum and hippocampus. Correlative analyses were performed both across and within brain regions. Significant positive correlations were found between PFASs and MAO activity in occipital lobe (e.g. ∑PFCA; rp=0.83, p=0.041, n=6) and across brain regions (e.g. ∑PFCA; rp=0.47, p=0.001, ∑PFSA; rp=0.44, p>0.001; n=50). GABA-A receptor density was positively correlated with two PFASs across brain regions (PFOS; rp=0.33, p=0.02 and PFDoDA; rp=0.34, p=0.014; n=52). Significant negative correlations were found between mAChR density and PFASs in cerebellum (e.g. ∑PFCA; rp=-0.95, p=0.013, n=5) and across brain regions (e.g. ∑PFCA; rp=-0.40, p=0.003, ∑PFSA; rp=-0.37, p=0.007; n=52). AChE activity and D2 density were negatively correlated with single PFCAs in several brain regions, whereas GS activity was positively correlated with PFASs primarily in occipital lobe. Results from the present study support the hypothesis that PFAS concentrations in polar bears from East Greenland have exceeded the threshold limits for neurochemical alterations. It is not known whether the observed alterations in neurochemical signaling are currently having negative effects on neurochemistry in East Greenland polar bears. However given the importance of these systems in cognitive processes and motor function, the present results indicate an urgent need for a better understanding of neurochemical effects of PFAS exposure to wildlife.

Steroid hormones in blood plasma from Greenland sledge dogs (Canis familiaris) dietary exposed to organohalogen polluted minke whale (Balaenoptera acuterostrata) blubber

Persistent organic pollutants such as polychlorinated biphenyls (PCB) are known to disrupt steroidogenesis and subsequent concentrations of circulating endogenous hormones. This is also suspected to occur in Arctic predatory species, such as polar bears (Ursus maritimus) and a study was therefore conducted in Greenland sledge dogs (Canis familiaris) as a sentinel species for adverse effects on steroid homeostasis. The control and exposed groups were composed of four sister-bitches all fed pork fat (Suis suis) and organohalogen contaminated minke whale (Balaenoptera acutorostrata) blubber, respectively, for 321–576 days. In the exposed group, this constituted a mean daily intake of 128 μg ∑PCBs (5 μg/kg/day). At the end of the study, organohalogen contaminant (OHC) were measured in adipose tissue and hormone levels in blood of sledge dogs. The hormones included 11 products of the steroidogenesis, which were ∑Androgen (dehydroepiandrosterone, androstenedione, testosterone, and dehydrotestosterone), ∑Estrogen (estrone, 17α-estradiol and 17β-estradiol), and ∑Progestagen (pregnenolone, progesterone, 17-OH-pregnenolone, and 17-OH-progesterone) concentrations. This allowed us to study the mechanisms underlying possible disruption of blood steroid concentrations. Results showed that exposed bitches had significantly higher concentrations of all OHC compounds. Unexpectedly, ∑Androgen, ∑Estrogen and ∑Progestagen concentrations were also significantly higher in exposed dogs. Data indicated that daily OHC contaminant exposure were concomitant with elevation in concentrations of steroid hormones in blood plasma of exposed sledge dogs. Based on these unexpected results, data suggest that more investigations should be focused on steroid endocrine disruption and potential health effects in Arctic top predators including domesticated dogs.