Oxygen-sensing cells release neurotransmitters, including serotonin, in response to hypoxia in the blood or surrounding air/water. This stimulates the glossopharyngeal and/or vagal nerves, triggering increased ventilation via the respiratory reflex. In the adult, they are located in the carotid body (glomus cells) and lung epithelia (pulmonary neuroendocrine cells) of amniotes, and in the epithelia of the gills and orobranchial cavity (‘neuroepithelial cells’) of anamniotes. Despite their physiological importance, little is known about the molecular mechanisms of their development, while the evolutionary relationships between the various oxygen-sensing cell types are unknown. The chromaffin cells of the mammalian adrenal medulla are hypoxia-sensitive transiently during neonatal life. Both carotid body glomus cells and adrenal chromaffin cells arise from the neural crest and require the transcription factors Phox2b and Ascl1 for their development. Given these similarities, I aimed to test the hypothesis that the same molecular mechanisms underlie their development. Expression analysis of 13 sympathoadrenal pathway genes throughout chicken carotid body development revealed striking similarities with adrenal chromaffin cell development. Analysis of mouse mutants showed that the transcription factors Hand2, Sox4 and Sox11 are required for carotid body development. In addition, loss of the receptor tyrosine kinase Ret or the transcription factor AP-2β, which significantly affects sympathetic ganglion but not adrenal chromaffin cell development, has no effect on the carotid body. Adrenal chromaffin cells differentiate from neurons that migrate into the adrenal gland from ‘primary’ sympathetic ganglia at the dorsal aorta. Carotid body glomus cells were previously proposed to arise from neuronal “émigrés” from neighbouring ganglia: the superior cervical ganglion in mammals and the nodose ganglion in the chick. However, nodose neurons are considered to be nodose placode-derived. Using electroporation and grafting in the chick, I confirmed that the nodose placode does not contribute to the carotid body, identified a small population of autonomic neural crest-derived neurons in the nodose ganglion, and confirmed the existence of “bridges” of neurons between the nodose ganglion and the carotid body. My data suggest that, like adrenal chromaffin cells, carotid body glomus cells differentiate from autonomic neural crest-derived neurons in nearby ganglia, which migrate into the carotid body primordium and down-regulate neuronal markers. The proposed evolutionary relationship between the carotid body glomus cells and the serotonin-positive neuroepithelial cells of anamniote gills has never been tested. Using vital dye labelling, neural fold grafts, genetic lineage-tracing in zebrafish and analysis of zebrafish mutants lacking all neural crest cells, I found that serotonin-positive cells in the gills and orobranchial epithelia of lamprey (jawless fish), zebrafish (ray-finned bony fish) and frog (anamniote tetrapod) are not neural crest-derived, and hence are not homologous to carotid body glomus cells. Genetic lineage-tracing in mouse and neural fold grafts in chick also confirmed that serotonin-positive neuroendocrine cells in the lung are not neural crest- derived, hence must have an endodermal origin (since the lungs are out-pocketings of the gut). My results suggest that the neuroepithelial cells of anamniotes are not related to carotid body glomus cells, but rather are homologous to the oxygen-sensing cells of the lung. Consistent with this hypothesis, I found that many genes expressed during carotid body development are not expressed by the epithelia of either chick lungs or lamprey gills. Taken together, my data suggest that as air-breathing evolved, gut endoderm- derived cells that originally responded to hypoxia in water were maintained in the lungs to monitor oxygen levels in air, while a population of neural crest-derived chromaffin cells near the pharyngeal arch arteries was recruited to monitor oxygen levels in blood.

Background: Use of pharmaceuticals can lead to unchanged or metabolite residues in surface water that may result in negative environmental effects. Sweden has adopted the Generational goal defining direction and changes needed to become a sustainable nation, these align with the UN Sustainable Development Goals (SDGs). Sweden collects and analyzes samples for pharmaceuticals and other contaminants in surface water. Aim: To estimate risks connected to pharmaceuticals in complex mixtures, exemplified by nonsteroidal anti-inflammatory drugs (NSAIDs), and discuss how this can be used to influence the actions needed to reach the Generational goal and the SDGs of Agenda 2030. Methods: Here, measured environmental concentrations (MECs) of the NSAIDs diclofenac, ibuprofen, ketoprofen, and naproxen in Swedish surface waters and in Uppsala’s Fyris River were accessed from a database and used in conjunction with predicted no-effect concentrations (PNECs) from the literature to derive risk quotients(RQ=MEC/PNEC). For all drugs a standardized PNEC derived from OECD guideline base-set tests were found, and for diclofenac and ibuprofen also non-traditional guideline PNECs were identified. Risk cups applied by summation of MEC/PNEC-risk quotients are considered safe if the sum of RQ <1, and as proposed inSOU 2019:45, if one chemical adds more risk than 0.1 to the risk cup it would be better to substitute it for another, if possible. Results and Discussion: Standardized PNECs derived from OECD guideline base-set tests were more than 60-fold greater than non-traditional PNECs for diclofenac and ibuprofen, affecting their individual RQ contribution and total sum of RQ. Based on the non-traditional PNECs, the sum of RQ were more than or near 1 in some cases in Fyris River and elsewhere, thus indicating risk to biota especially in 2010. Diclofenac and ibuprofen typically contributed more to Risk cups than did ketoprofen and naproxen. Especially diclofenac should be considered for substitution, if possible. Swedish sales data indicate at least one more NSAID compound suitable for analysis. In addition, more than 70 pharmaceuticals were identified in Fyris River, adding to pressure on environment from NSAIDs. Risk cups are conservative and require sparse data relative to other methods, and thus can be used to prioritize further efforts. A difficulty is to find relevant ecotoxicological data for pharmaceuticals and therefore an open access database would be of value, preferably complemented with sales data for APIs. However, since a default RQ-value of 0.1 was suggested in SOU 2019:45, a lack of data would not hinder action. Use of risk cups makes it possible to work toward e.g., sustainable production practices benefiting SDG 12. Inaction after identifying a problem conflicts with SDGs 6 and 12, since it would lead to less clean water and more sanitation issues and non-sustainable consumption and production. Conclusion: Risk cups as applied here are suitable as a first tier of pharmaceutical mixture risk estimation since they are quick to perform and demand less data than other methods. Because of their dependence on PNECs, it is important to use a relevant effect test, with results preferably published in an open access database. Diclofenac’s non-traditional risk quotient indicate that the ecological status of the Fyris River is at risk, supporting the official moderate ecological status classification. This thesis suggests an additional NSAID, etoricoxib, as a possible candidate for future studies, based on the number of other NSAIDs on the market and sales numbers, pointing at the usefulness of sales data for a better understanding of risk. In addition to the NSAID group, other pharmaceuticals, active metabolites, and non-pharmaceutical chemicals add to the pressure on the environment. Data on the risk cups and risk quotients can be used as a basis for improvements at sewage treatment plants and factories as well as for launching informative campaigns to physicians and the general public, actions which all may lead to a more sustainable future.

Recently, the presence of illicit drugs in effluents from Wastewater Treatment Plants (WWTPs) and the aquatic environment has raised concern over their possible negative effects on aquatic organisms. In this study, therefore ecotoxicological data was retrieved through a literature survey and by using the software ECOSAR. Predicted No Effect Concentrations (PNEC) of five types of illicit drugs and their metabolites including 1. Cocaine (COC) and its metabolite Benzoylecgonine (BE), 2. Amphetamine (AMPH), 3. Methamphetamine (METH), 4. Cannabis ((delta 9 tetrahydrocannabinol (THC), 11-Nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-CCOH)) and 5. 3,4-Methylenedioxymethamphetamine (MDMA) for species of three trophic levels in aquatic ecosystems derived. Predicted Environmental Concentrations (PEC) of above-mentioned drugs in twelve WWTPs’ influent and two effluents in Halland County have been measured by Swedish Toxicology Research Center (SWETOX co). Acute and chronic Risk Quotients (RQ) of the mixture of illicit drugs based upon two novel approaches calculated for the effluents of two WWTPs. Wastewater treatment plant in Ängstorp found with better removal efficiency of above- mentioned illicit drugs and the lower total RQs of (0.01<MRQacute<0.1; MRQchronic = 0.13) in comparison with that of Västra Stranden with the total RQs of (0.1<MRQacute <1; MRQchronic = 1.4). Although the RQ of WWTPs’ effluent was higher than 1 in Västra Stranden, there was no potential of risk on aquatic organisms in surface waters receiving that effluent. AMPH and Cannabis found as the most degradable substances through the WWTPs with (90-100%) of removal efficiency. Moreover, the results revealed that Cannabis (THC-COOH) was the most hazardous illicit drug on aquatic species in case of acute and chronic effects while, in terms of genotoxicology, a mixture of Cocaine metabolites found the most dangerous mixture of illicit drugs on zebrafish embryos.