| 1. |
- Castro, Mafalda
(author)
-
Chlorinated Paraffins: improved understanding of their bioaccumulation and toxicity in Daphnia magna
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- Chlorinated paraffins (CPs) are industrial chemicals, mainly used as flame retardants, plasticizers and metal cutting fluids. Their production has reached historically high levels in the last decade, with an annual production exceeding one million tonnes. In 2017, short chain chlorinated paraffins (SCCPs) were regulated due to their Persistent, Bioaccumulative and Toxic (PBT) properties, while medium and long chain chlorinated paraffins (MC and LCCPs) were suggested as alternatives. The high hydrophobicity of CPs, which complicates bioaccumulation and aquatic toxicity testing, has hindered proper hazard identification by regulatory authorities. This project was initiated in response to the insufficient understanding of bioaccumulative and toxicological properties of these chemicals, which have even surpassed the environmental levels of legacy Persistent Organic Pollutants (POPs) in certain regions.The research presented in this thesis, contributes to filling these knowledge gaps by adapting methods for reliable bioaccumulation and aquatic toxicity assessment. In Paper I, passive dosing, traditionally used for other highly hydrophobic compounds, was adapted and validated for CPs. SC, MC and LCCPs partitioned from silicone into water and, when the crustacean Daphnia magna was introduced into the test system, the CPs were observed to be effectively taken up by the test organism. This passive-dosing approach was further used in Paper II, to investigate the bioconcentration and bioaccumulation potential in D. magna. All tested CPs were found to bioaccumulate in daphnids, including highly hydrophobic, long chained CP congeners. The two most bioaccumulative CPs in Paper II (CP-52 and Huels70C) were thereafter used in a chronic toxicity study (Paper III) and significantly decreased population growth and disrupted fatty acid metabolism of D. magna. Finally, in Paper IV, liposome-mediated delivery of chemicals to aquatic biota was adapted for the first time for organic contaminants, including CPs. This approach yielded stable body burdens of the tested chemicals in D. magna and allowed for kinetic and toxicity assessments.Overall, two alternative bioaccumulation and aquatic toxicity testing methods were successfully adapted for technically challenging (industrial) chemicals. These methods allowed the determination of endpoints of scientific and regulatory interest, such as the high bioaccumulation and toxicity potential of CPs, but were also used to demonstrate their metabolic disruption potential in small crustaceans.
|
|
| 2. |
- Ribbenstedt, Anton, 1986-
(author)
-
Toxicometabolomics and biotransformation product screening in single zebrafish embryos
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- 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.
|
|
| 3. |
- West, Johannes, 1991-
(author)
-
Degradation Pathways of Dimethylmercury in Natural Waters
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Mercury (Hg) is a naturally occurring toxic trace metal whose release into the environment has been (and still is) exacerbated by human activities. The bioavailability of Hg is tied to its chemical speciation, with monomethylmercury (MMHg) being the primary form of Hg that bioaccumulates and biomagnifies. At the top of some food webs, MMHg can reach harmful levels. In addition to MMHg, a second methylated Hg species, dimethylmercury (DMHg), is found in aquatic environments. Dimethylmercury is especially abundant in the oceans, where it could potentially act as an important source of MMHg. The stability of DMHg in natural waters and which biogeochemical processes may be important for its degradation are poorly constrained. This knowledge gap could partially be related to the extreme toxicity and the volatility of DMHg, making it challenging to handle in the laboratory.Through experimental studies, this thesis work has aimed to increase the understanding of DMHg cycling in the aquatic environment. Focusing on the stability and degradation of DMHg, abiotic pathways of DMHg degradation were explored. In Paper I, DMHg degradation via a novel sulfide-mediated pathway was demonstrated for both particulate and dissolved sulfide phases. Key aspects of the process were investigated through the manipulation of pH and DMHg:S ratios. In Paper II, the previously disputed process of aqueous photochemical decomposition of DMHg was confirmed for various natural and artificial water types. This process was studied further in Paper III by investigating the impact of dissolved organic matter and chloride on DMHg photodecomposition rates and the involvement of photochemically produced reactive intermediates. Monomethylmercury was the primary product of both sulfide-mediated decomposition and photodecomposition of DMHg. Rate calculations (Paper I and II) and comparison with MMHg photodecomposition (Paper II and III) confirm that both sulfide-mediated decomposition and aqueous photodecomposition of DMHg could be environmentally significant. Paper IV presents methods and considerations for laboratory work with DMHg, providing insights into both experimental and analytical aspects. This work demonstrates that DMHg can be analyzed together with MMHg and that significant safety risks of working with DMHg can be largely avoided. These insights are important as future work on DMHg is necessary to elucidate its role in aqueous Hg cycling in general and for MMHg cycling in particular.
|
|
| 4. |
- Martella, Giulia, 1993-
(author)
-
DNA adductomics: Method development and integration in biological effect monitoring
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species.From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with relatively high and low contamination loads based on PAH and trace metal concentrations in the sediment (Paper III). Also, significant correlations were found between DNA adducts and specific contaminants.These method developments and findings uniquely contribute to the field of environmental omics, providing tools for data processing and demonstrating the applicability of nucleic acid adductomics in environmental health research and biological effect monitoring.
|
|
| 5. |
- Gerdes, Zandra, 1989-
(author)
-
Exploring the ecotoxicity of microparticle debris
- 2021
-
Doctoral thesis (other academic/artistic)abstract
- The global distribution and anticipated increase of environmental microplastic (MP) pollution are concerning. However, while the impacts of macroplastic litter on wildlife are apparent, we know relatively little about the MP hazard potential. Moreover, the current ecotoxicological methodology is inadequate for solid waste particles and MP hazard assessment because it fails to distinguish particle and chemical effects. This thesis improves our understanding of the particle effects of MP relative to other microparticles.First, a comparative analysis of effect studies on MP and mineral particulates across different biological organisation levels revealed high similarities in responses between these materials (Paper I). At the suborganismal levels, the similarity in the effect concentrations suggests shared particle effect mechanisms. At the higher levels, however, MP induced more severe impacts, possibly due to chemical leaching. Moreover, the highly variable MP effect concentrations motivated exploring the role of polymer properties and ageing status on MP effects; these aspects were addressed in Papers II-III.In Paper II, the possibility of MP acting as a vector of contaminants was evaluated, showing enhanced transport of highly hydrophobic organic contaminants (HOC) at very high HOC and MP concentrations. However, observing it at environmentally relevant contaminant levels would be unlikely. Paper III compared behavioural and physiological responses in benthic amphipods to MP exposure using different polymers (polystyrene and polyethylene terephthalate) and clay as a non-plastic reference particle. The amphipods avoided sediments with high concentrations of the added material regardless of the material type, including aged and virgin MP and clay. Solid waste, including MP, co-occur with various suspended solids in aquatic environments; therefore, the natural solids can serve as reference material when evaluating the MP particle effect. In Paper IV, a novel method for testing MP effects in mixtures with reference particles was proposed. In the exposure experiment with daphnids, the method was used to derive hazard thresholds for the MP contribution to suspended matter conditional on the total suspended solid concentration in the water. Together, these studies add to our understanding of MP-biota interactions and suggest that similarly sized MP and natural particulates share similar particle effects. However, MP might have a higher potential as vectors of chemical contaminants, which needs to be further evaluated in environmentally relevant settings.
|
|
| 6. |
- Motiei, Asa, 1980-
(author)
-
Microbiome Of Ecotoxicity Assays
- 2021
-
Doctoral thesis (other academic/artistic)abstract
- Microorganisms are ubiquitous and present in animal microbiomes, particulates, and colonizable surfaces of test systems. From an ecotoxicological perspective, they are metabolically active biological compartments that respond to test conditions, including test substances. In exposure experiments, microorganisms can both alleviate toxicity via, for example, biotransformation, and reinforce the adverse effects via, for example, disrupted microbiome-host interactions. Acknowledging these interactions is essential for a mechanistic understanding of results in effect studies and developing assays towards more ecologically relevant hazard assessment. Therefore, there is increasing attention toward “microbiome aware ecotoxicology” in recent years, focusing mostly on test organism microbiomes. I studied how microorganisms present in systems designed for acute and chronic toxicity assays with Daphnia magna affect the test outcome. The experimental studies showed that bacteria introduced in the system intentionally (as a part of the experimental design; Papers I, II, and III) or unintentionally (with the microbiome of the test animals; Paper IV) responded to the test substances and mediated the exposure for the target species. In these studies, we employed the emerging contaminants ciprofloxacin (an antibiotic drug; Paper I) and various fossil-based polymers (microplastic; Papers II, III, and IV), representing a microbiome disrupting and a biofilm promoting type of substance respectively. In Paper I, we hypothesized that exposure to antibiotics would primarily target the daphnid microbiome with downstream effects on the host fitness. To test this hypothesis, we chronically exposed daphnids to ciprofloxacin, which resulted in decreased microbiome diversity. However, contrary to our hypothesis, there were significant stimulatory effects on the host fitness and antioxidant production due to the direct pro-oxidative ciprofloxacin effects on the host. Although the microbiome was not directly involved in the growth-related responses to the ciprofloxacin exposure, the microbiome’s alterations suggest that exposure to any antimicrobials, which – unlike ciprofloxacin – do not stimulate antioxidant production, would result in gut dysbiosis with possible adverse effects on the host. Further, we hypothesized that in assays with particulate test materials, such as microplastic, bacterial biofilms increase particle aggregation, affecting exposure levels. This hypothesis was tested using D. magna exposed to a mixture of kaolin clay and polystyrene with and without biofilm (Paper II). We found that biofilm significantly decreased the adverse effects exerted by particulates directly, most likely, by providing nutrition for the daphnids, and indirectly, by inducing particle aggregation. In Paper III, we compared biofilm communities established on the plastic (polyethylene, polypropylene, and polystyrene) vs. non-plastic (cellulose and glass) substrates. The biofilm communities on the plastic were significantly different from those on the non-plastic materials; hence, microplastic contribution to the suspended solids in the exposure can drive the biofilm community composition in the system. Finally, in Paper IV, we found that in a closed system designed to evaluate microplastic effects on D. magna, bacteria originated from the daphnid microbiome colonize particulates and affect their aggregation and animal survival. Together, these findings suggest that chemical exposure (Paper I), the microbiome of the test animal (Paper IV), the composition of the suspended solids (SS) (Papers II and IV), and their surface properties (Paper III) contribute to the diversity and abundance of the biofilm in the test system, which can affect the test outcome. Thus, the microbiome reacts to and interacts with contaminants within a test system, which calls for the appreciation of these interactions when interpreting the results as well as new developments toward standardization of the bacterial component in (eco)toxicity assays with eukaryotic test species.
|
|
