Bioconcentration factors (BCFs) in rainbow trout were measured for 10 anionic surfactants with a range of alkyl chain lengths and different polar head groups. The BCFs ranged from 0.04 L kg^–1 ww (for C₁₀SO₃) to 1370 L kg^–1 ww (C₁₆SO₃). There was a strong correlation between the log BCF and log membrane lipid–water distribution ratio (D_MLW, r² = 0.96), and biotransformation was identified as the dominant elimination mechanism. The strong positive influence of D_MLW on BCF was attributed to two phenomena: (i) increased partitioning from water into the epithelial membrane of the gill, leading to more rapid diffusion across this barrier and more rapid uptake, and (ii) increased sequestration of the surfactant body burden into membranes and other body tissues, resulting in lower freely dissolved concentrations available for biotransformation. Estimated whole-body in vivo biotransformation rate constants k_B-BCF are within a factor three of rate constants estimated from S9 in vitro assays for six of the eight test chemicals for which k_B-BCF could be determined. A model-based assessment indicated that the hepatic clearance rate of freely dissolved chemicals was similar for the studied surfactants. The dataset will be useful for evaluation of in silico and in vitro methods to assess bioaccumulation. 

Toxicometabolomics and biotransformation product (bioTP) elucidation were carried out in single zebrafish (ZF) embryos exposed to carbamazepine (CBZ). Exposures were conducted in 96-well plates containing six CBZ concentrations ranging from 0.5 μg/L to 50 mg/L (n = 12 embryos per dose). In the 50 mg/L dose group, 33% of embryos developed edema during the exposure (120 hpf), while hatching was significantly delayed in three of the lower-dose groups (0.46, 3.85, and 445 μg/L) compared to the control at 48 hpf. Toxicometabolomic analysis together with random forest modeling revealed a total of 80 significantly affected metabolites (22 identified via targeted lipidomics and 58 via nontarget analysis). The wide range of doses enabled the observation of both monotonic and nonmonotonic dose responses in the metabolome, which ultimately produced a unique and comprehensive biochemical picture that aligns with existing knowledge on the mode of action of CBZ. The combination of high dose exposures and apical endpoint assessment in single embryos also enabled hypothesis generation regarding the target organ for the morphologically altering insult. In addition, two CBZ bioTPs were identified without additional exposure experiments. Overall, this work showcases the potential of toxicometabolomics and bioTP determination in single ZF embryos for rapid and comprehensive chemical hazard assessment.

Host-microbiome interactions are essential for the physiological and ecological performance of the host, yet these interactions are challenging to identify. Neurotransmitters are commonly implicated in these interactions, but we know very little about the mechanisms of their involvement, especially in invertebrates. Here, we report a peripheral catecholamine (CA) pathway involving the gut microbiome of the model species Daphnia magna. We demonstrate the following: (i) tyrosine hydroxylase and Dopa (3,4-dihydroxyphenylalanine) decarboxylase enzymes are present in the gut wall; (ii) Dopa decarboxylase gene is expressed in the gut by the host, and its expression follows the molt cycle peaking after ecdysis; (iii) biologically active _L-Dopa, but not dopamine, is present in the gut lumen; (iv) gut bacteria produce _L-Dopa in a concentration-dependent manner when provided l-tyrosine as a substrate. Impinging on gut bacteria involvement in host physiology and ecologically relevant traits, we suggest _L-Dopa as a communication agent in the host-microbiome interactions in daphnids and, possibly, other crustaceans.

IMPORTANCE Neurotransmitters are commonly implicated in host-microbiome communication, yet the molecular mechanisms of this communication remain largely elusive. We present novel evidence linking the gut microbiome to host development and growth via neurotransmitter _L-Dopa in Daphnia, the established model species in ecology and evolution. We found that both Daphnia and its gut microbiome contribute to the synthesis of the _L-Dopa in the gut. We also identified a peripheral pathway in the gut wall, with a molt stage-dependent dopamine synthesis, linking the gut microbiome to the daphnid development and growth. These findings suggest a central role of _L-Dopa in the bidirectional communication between the animal host and its gut bacteria and translating into the ecologically important host traits suitable for subsequent testing of causality by experimental studies.

A procedure was developed for rapid screening of xenobiotic biotransformation products (bioTPs) in single zebrafish (ZF; Danio rerio) embryos. Exposure was carried out from 0–120 hours post fertilization (hpf) to 6 different concentrations of the model compound propranolol (PPL). Following in-plate extraction and non-target instrumental analysis by high resolution mass spectrometry, suspected bioTPs were identified using custom data filtration scripts and matching to in silico structural predictions. A total of eight PPL bioTPs were identified (five at a level 1 confidence and one at a level 2–3 confidence). These findings supplement previously generated toxicometabolomic models derived from the same dataset, and were obtained without conducting additional exposure experiments. In addition to facilitating assessments of inter-individual variability in bioTP production in ZF embryos, we demonstrate that bioTPs can be elucidated using extremely small quantities of biomass (i.e. ∼200 μg). To the best of our knowledge, this is the first time bioTP elucidation has been carried out in single ZF embryos.

Toxicometabolomic studies involving zebrafish embryos have become increasingly popular for linking apical endpoints to biochemical perturbations as part of adverse outcome pathway determination. These experiments involve pooling embryos to generate sufficient biomass for metabolomic measurement, which adds both time and cost. To address this limitation, we developed a high-throughput toxicometabolomic assay involving single zebrafish embryos. Incubation, microscopy, embryo extraction, and instrumental metabolomic analysis were all performed in the same 96-well plate, following acquisition of conventional toxicological endpoints. The total time for the assay (including testing of 6 doses/n = 12 embryos per dose plus positive and negative controls, assessing conventional endpoints, instrumental analysis, data processing and multivariate statistics) is <14 days. Metabolomic perturbations at low dose were linked statistically to those observed at high dose and in the presence of an adverse effect, thereby contextualizing omic data amongst apical endpoints. Overall, this assay enables collection of high resolution metabolomic data in a high throughput manner, suitable for mode of action hypothesis generation in the context of pharmaceutical or toxicological screening.

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the final multivariate statistical models, obscuring their outputs and prediction capabilities. Once developed, the workflow allowed us to detect several probable modes-of-action of propranolol in zebrafish, and link them to apical endpoints in the embryos, which were then confirmed through thorough literature searches. The final output from the models was ultimately used to determine a benchmarking dose based on metabolomics endpoints for the first time. In Paper III, the data processing workflow from Paper II was modified to capture propranolol biotransformation products. A total of 7 structures were identified, of which 4 were confirmed with authentic standards, all from the datasets generated in Paper II. In Paper IV we combined the workflows from Papers I, II and III and applied them to the pharmaceutical carbamazepine, which occurs at high concentrations in wastewater treatment plant effluents. Through this approach we determined several modes-of-action for carbamazepine in zebrafish embryos and measured biotransformation products in both embryos and exposure water. Overall, this thesis demonstrated the possibilities of high-throughput chemical mode-of-action determination in single zebrafish embryos using targeted and non-targeted liquid chromatography mass spectrometry, data filtering scripts and multivariate statistics while simultaneously screening for biotransformation products.

The antidepressant amitriptyline is a widely used selective serotonin reuptake inhibitor that is found in the aquatic environment. The present study investigates alterations in the brain and the liver metabolome of gilt-head bream (Sparus aurata) after exposure at an environmentally relevant concentration (0.2 mu g/L) of amitriptyline for 7 d. Analysis of variance-simultaneous component analysis is used to identify metabolites that distinguish exposed from control animals. Overall, alterations in lipid metabolism suggest the occurrence of oxidative stress in both the brain and the liver-a common adverse effect of xenobiotics. However, alterations in the amino acid arginine are also observed. These are likely related to the nitric oxide system that is known to be associated with the mechanism of action of antidepressants. In addition, changes in asparagine and methionine levels in the brain and pantothenate, uric acid, and formylisoglutamine/N-formimino-L-glutamate levels in the liver could indicate variation of amino acid metabolism in both tissues; and the perturbation of glutamate in the liver implies that the energy metabolism is also affected. These results reveal that environmentally relevant concentrations of amitriptyline perturb a fraction of the metabolome that is not typically associated with antidepressant exposure in fish.

Adults are mainly exposed to per- and polyfluoroalkyl substances (PFASs) via ingestion of food, inhalation of air and ingestion of dust, whereas for children the exposure to PFASs is largely unknown. This study aimed to reconstruct the serum concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) in children after infancy up to 10.5 years of age and to test if dietary intake is the major exposure pathway for children to PFOA, PFOS and PFHxS after infancy. For this work, a dataset from a Finnish child cohort study was available, which comprised serum concentrations of the studied perfluoroalkyl acids (PFAAs) and PFAS concentration measurements in dust and air samples from the children's bedrooms. The calculated PFAA intakes were used in a pharmacokinetic model to reconstruct the PFAA serum concentrations from 1 to 10.5 years of age. The calculated PFOA and PFOS intakes were close to current regulatory intake thresholds and diet was the major exposure medium for the 10.5 year-olds. The one-compartment PK model reconstructed median PFOA and PFOS serum concentrations well compared to corresponding measured median serum concentrations, while the modelled PFHxS serum concentrations showed a constant underestimation. The results imply that children's exposure to PFOA and PFOS after breastfeeding and with increasing age resembles the exposure of adults. Further, the children in the Finnish cohort experienced a rather constant exposure to PFOA and PFOS between 1 and 10.5 years of age. The PFHxS exposure sources and respective pharmacokinetic parameter estimations need further investigation.

Methylmercury (MeHg) and perfluoro-octanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

The quantity and composition of fibers released from functional textiles during accelerated washing were investigated using the GyroWash method. Two fabrics [polyamide (PA) and polyester/cotton (PES/CO)] were selected and coated with perfluorohexane-based side-chain fluorinated polymers. Fibers released during washing ranged from similar to 10 to 500 mu with a similar distribution for the two textile types. The PA-based fabric released considerably more fibers >20 mu m in length compared to the PES/CO-based fabric (>1000/GyroWash for PA vs similar to 200/GyroWash fibers for PES/CO). After one GyroWash (2-15 domestic washes), fibers that contained approximately 240 and 1300 mu g total fluorine per square meter (mu g F/m(2)) were released from the PA and PES/CO fabrics, respectively. Current understanding of the fate of microplastic fibers suggests that a large fraction of these fibers reach the environment either in effluent wastewater or sewage sludge applied to land. In the environment, the fluorinated side chains will be slowly cleaved from the backbone of the side-chain fluorinated polymers coated on the fibers and then transformed into short-chain perfluoroalkyl acids. On the European scale, emissions of up to similar to 0.7 t of fluorotelomer alcohol (6:2 FTOH) per year were estimated for outdoor rain jackets treated with fluorotelomer-based side-chain fluorinated polymers.