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Dissertation, RWTH Aachen University, 2023; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2022). = Dissertation, RWTH Aachen University, 2023, Pollinators are important key stone species for the health of ecosystems, providing the ecosystem service of pollination for wild flowers as well as for food crops. But their numbers have been declining for years, resulting in pollinator shortages and lack of pollination. Bees are probably the most publically known pollinators. They have inspired many groups from the public to take action and call for more protection, especially in agricultural practices. In the scientific community, the Western honeybee Apis mellifera is most often used as surrogate species in research regarding pollinators. Several studies have reported, that A. mellifera is not a suitable surrogate species to cover the more than 200,000 wild living bee species, as it is highly domesticated, living in hives of several thousand individuals and can thus buffer small scale effects by sheer numbers of individuals. But so far, data on other bee species such as solitary bees, is limited to non-existent. In this study we wanted to test the native bumblebee species Bombus terrestris as model species for holistic biomonitoring projects, measuring different classes of anthropogenic pollutants. B. terrestris is covered in dense fur, has a small flight range (~800 m), is ubiquitously spread throughout Europe and can be bred commercially, making it an ideal species for small-resolution biomonitoring. The project focussed on two main questions: 1) To which anthropogenic pollutants are pollinators exposed in typical land-use scenarios and in which concentrations? 2) Do these field-realistic concentrations cause adverse effects in bumblebees? In a two-year field study, we placed commercial bumblebee hives in urban and agrarian locations around Aachen, Germany. Foragers, pupae and honey were collected, extracted using a modified QuEChERS approach and measured for 25 target pesticides. In addition, foragers were measured for 12 target metals. In total, residues of 15 different pesticides were found. The most often found pesticides were the herbicide prosulfocarb (36.4% of foragers) and the fungicide flutolanil (15.9% of all foragers). 33% of foragers carried mixtures of pesticides (>1 pesticide per insect). Often observed mixtures in foragers were prosulfocarb-difenoconazole, boscalid-azoxystrobin and azoxystrobin-difenoconazole. In general, pupae had lower pesticide concentrations than foragers with mean cumulative concentrations of 6.0 ng/individual for foragers and 3.3 ng/individual for pupae in agricultural areas. Only four active substances were detected in honey samples. Mean cumulative concentrations of 258.6 ± 65.1 mg/kg dw and 383.6 ± 59.3 mg/kg dw were measured for heavy metals and half-metals in April and July, respectively. Significant differences between the land-use types were only found for boron and cadmium. A maximum concentration of 0.94 mg/kg dw Cd (0.07 µg/bee) was measured. No land-use specific pollution profiles could be identified for either pesticides, (half-)metals or their combination. The results show that pesticides occur in all investigated location, no matter the land-use type. But they seem to be rather individual occurrences instead of mass poisonings. For the following toxicity assays we investigated three priority pollutants: cadmium, terbuthylazine and difenoconazole, all detected in bumblebees in our study. They were tested as single substances and binary mixtures in a standardised oral toxicity assay (OECD Guideline 247). Terbuthylazine and difenoconazole did not cause mortality in the tested concentration ranges (NOED ≥22 µg/bee and ≥903 μg/bee, respectively). Cadmium caused mortality with a LD50 of 6.70 µg Cd/bee and effects at ED50= 1.92 µg Cd/bee after 48 h. In the binary mixtures with pesticides, the toxicity of cadmium increased to LD50= 2.84 µg Cd/bee with terbuthylazine and 3.92 µg Cd/bee with difenoconazole (synergism). We developed a novel assay to assess mobility of individuals using a flight arena, which improved the sensitivity of the effect detection compared to oral mortality tests. Significant differences between mobility of test and control groups were measured at a dose of 0.59 µg Cd/bee. The results of the oral tests highlight that it is important to consider pollutant mixtures when assessing the adverse effects in real field situations. The results from the mobility test indicate that adverse effects on bumblebee mortality and thus colony survival could already occur at concentrations measured in the field. In conclusion, B. terrestris has proven to be a suitable species for biomonitoring approaches, as it is easy to handle, resistant to different temperatures, its handling is less laborious than honeybee beekeeping and comparatively cheap. As B. terrestris can be used to monitor field concentrations as well as assess their toxicity, direct connections between exposure and effect can be drawn without the need to extrapolate e.g. on the species level. The data gathered in this study can be used as a baseline to establish a national/global monitoring scheme. We showed that wild living pollinators are exposed to mixtures of different substances, spanning different substance categories. Effects of these mixtures cannot be reliably predicted so far. Therefore, it is important that mixture effects are integrated into current regulations and monitoring schemes., Published by RWTH Aachen University, Aachen

Dissertation, RWTH Aachen University, 2022; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2023). = Dissertation, RWTH Aachen University, 2022, Sediment risk assessment of pesticides was controversially discussed within the scientific and regulatory community in the last years. There are different water-sediment studies with respective purposes. On the one hand, the ecotoxicological potential of pesticides on non-target organism such as sediment dwellers can be investigated. On the other hand, determination of pesticide transformation and degradation could be the purpose of water-sediment studies. The European Food Safety Authority published a scientific opinion on sediment risk assessment, which contained amongst other a lack of knowledge concerning the assessment of ecotoxicological studies. The determined toxicological endpoints are based on the assumption, that test organism and test compounds were evenly distributed within the sediment. However, test organism such as the sediment dwelling larvae of Chironomus riparius reside preferably in the upper sediment layer, due to favorable living conditions. Furthermore, it is well understood that test compounds with lipophile properties tend to adsorb on the upper layer of the sediment, after application into the overlying water. Correspondingly, the spatial concentration differences are also not considered in water-sediment studies investigating the transformation. The distribution of transformation products and their conditions of formation are not respected.Goal of the present Dissertation was to elucidate the spatial distribution of three model compounds in different water sediment test systems by employing a special developed core sampling methodology. Experimental results were compared with simulated data by a developed Toxic Substances in Surface Water (TOXSWA) model contributing to a mechanistic understanding of the model compounds transport process within the sediment. By this comparison, the influence of different adsorption properties of the model compounds and organic mass of the sediment on diffusion should be elaborated. The model compounds were selected according to their adsorption properties, namely bixafen (KfOM: 2244 L kg-1), fluopyram (KfOM: 161 L kg-1) and N,N-dimethylsulfamide (KfOM: 0 L kg-1). The core sampling methodology was used for water-sediment test systems according to OECD 218 (Sediment-Water Chironomid Toxicity Using Spiked Sediment), OECD 219 (Sediment-Water Chironomid Toxicity Using Spiked Water) and OECD 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems) for examining the spatiotemporal behaviors of the model compounds with respect to ‘spiked water’ and ‘spiked sediment’ studies and different water: sediment ratios. Each of the water-sediment studies was implemented with artificial sediment, but as well with two natural water-sediments, respectively. The artificial sediment consisted of ca. 3% organic mass and was classified as sandy loam. The natural sediment from the Wiehltalsperre provided 13% organic mass and was identified as silt-loam, while the other natural sediment from Anglersee had a poor organic mass content (0.3%) and was sandy. In the OECD 218 (‘spiked sediment’) studies, the results of N,N-dimethylsulfamide showed that the majority (50 - 80%) the of the polar model compound migrated from all employed sediments into the overlying water within few days. Due to the moderate adsorption properties of fluopyram, the mass transfer from sediment to overlying water was performed more slowly and differences between the employed sediment could be observed. In the studies using artificial and silt loamy sediment, the fluopyram concentration within the sediment revealed a step-like distribution pattern with increasing concentrations from top to the bottom layer (e.g.: total concentrations of 91.6, 96.4 and 100.2 µg L-1). As the sandy sediment provided a poor amount of organic mass, the major part of fluopyram (50 – 60%) was recovered in the overlying water resulting in an even distribution within the sediment. Consideration of the strong adsorptive bixafen revealed no pronounced mass transfer from the sediment into the overlying water in the studies with artificial and silt-loamy sediment, while it was more pronounced within the sandy sediment studies resulting in an uneven distribution (total concentrations from top to bottom layer: 134.9, 155.3 and 166.3 µg L-1). The impact of adsorption properties of the model compounds and organic mass of the sediment was also revealed in water-sediment studies with ‘spiked water’ (OECD 219&308). N,N-dimethylsulfamide migrated unhindered through all sediments, while fluopyram and bixafen tended to adsorb on the sediment surface, respectively the top layer of the sediment. Governed by the organic mass content, the resulting concentration gradient of fluopyram was maintained in the artificial and silt loamy sediment (e.g.: total concentrations from top to bottom layer 9.3, 6.1 and 3.1 µg L-1), while it did not persist within the sandy sediment over the whole test duration. The strong adsorption of bixafen caused that the model compound remained unevenly distributed within all employed sediments. Besides, the dissipation of the model compounds was also governed by their respective adsorption properties and the organic mass of the sediment. Correspondingly, the dissipation of bixafen into the silt-loamy sediment provided the shortest dissipation half-live. Because of the smaller overlying water volume, the dissipation half-lives provided a shorter period within the OECD 219 water-sediment systems as compared to the respective OECD 308 systems. Experimental and simulated results showed good agreements. The core sampling methodology as well as a TOXSWA model can be used in frame of sediment risk assessment to determine the exposure concentration of a test compound on sediment dwelling organism. It was possible to follow the spatial-temporal behavior of the test compounds by employment of these measures and it was shown that diffusion retarded by adsorption is the relevant transport process within the sediment. Further, the TOXSWA model could be used to determine the dissipation half-lives of the model compounds within the different water-sediment systems. Consequently, a mechanistic explanation for biphasic dissipation kinetics was also revealed., Published by RWTH Aachen University, Aachen

Two main scenarios were investigated regarding the formation of non-extractable residues and their characteristics of the herbicide pendimethalin in soil. First, the incubation in soil alone was examined and second, the incubation in soil amended with 20 weight-% compost. Incubation of pendimethalin in a LUFA 2.2 soil (loamy fine sand, USDA) at an application rate of 5 mg kg-1 soil led to a mineralization of 26.2 % AR and a formation of non-extractable residues of 32.0 % after 400 days (end of study), hence moderate amounts of non-extractable residues were found. The amount of non-extractable residues increased over time and 14CO2-development was observed over the whole time. 14C-pendimethalin was degraded to several metabolites of which one could be identified as M455H001 (2-methyl-3,5-dinitro-4-(pentan-3-ylamino) benzoic acid) amounting to 5 % AR after 400 days of incubation. Amendment with compost (incubation time until 217 days) to the soil led to a slightly higher formation of non-extractable residues (38.2 % AR, 217 days) and reduced the mineralization almost completely (1.7 % AR after 217 days of incubation). Also in this study, metabolite M455H001 was identified but detected in minor amounts (2.4 % AR after 217 days, highest amount detected). A tenfold application of pendimethalin to soil led to a delayed formation of non-extractable residues and the degradation of pendimethalin occurred at a lower rate, too, but this might have been due to the incorrect water content which was not adjusted the whole time. Humic matter fractionation revealed that in soil alone, the radioactivity was almost evenly distributed among the fractions of fulvic acids, humic acids and humins at all sampling dates investigated. When soil was amended with compost, the major part of the radioactivity in the residues was found in the fraction of humins. The purified fulvic acid fraction was analyzed with size-exclusion chromatography. The major part of the injected radioactivity of the fulvic acid fraction eluted rather early, together with the fulvic acids themselves. This amount increased with time. It amounted to 92 % of the radioactivity in the fulvic acids fraction or 9.4 % of applied radioactivity after 400 days of incubation. A minor portion of the radioactivity eluted at a later time point from the SEC column. This was assigned to radioactivity not associated firmly with fulvic acids. This portion amounted to 0.4 % AR (400 days of incubation). Comparing with the retention times of the parent pendimethalin and the metabolite M455H001, this portion of radioactivity was tentatively assigned to the metabolite M455H001 whereas no pendimethalin could be identified. When soil was amended with compost, the amount of radioactivity eluting in the range of radioactivity not associated firmly with fulvic acids was higher. A silylation reaction was conducted on extracted soil to evaluate whether the non-extractable residues were covalently bound to or rather sequestered in pores of the soil matrix. From soil alone (study B), ~8 - 20 % (or ~1.5 - 3.6 %AR) of the non-extractable residues could be released. Considering the amount released by chloroform treatment of the soil, the amount released by silylation alone would have been even lower. The amount of non-extractable radioactivity released by silylation decreased with increasing incubation time, hence suggesting a firmer association with the soil matrix. It is concluded that the major portion of the bound residues is indeed bound covalently to the soil matrix and only a minor portion was present rather in a sequestered form. The analysis of the obtained extracts additionally revealed that only part of the released (thus sequestered) radioactivity was present as parent pendimethalin. HPLC of a sample suggested the presence of metabolite M455H001 and radioactive residues associated with soil organic matter. In contrast to incubation in soil alone, silylation could release more radioactivity of the non-extractable residues formed in compost amended soil. However, higher amounts of radioactivity released might not be linked to silylation itself but to the initial treatment with CHCl3 prior to the silylation process. These leads to the suggestion that the methanolic extraction method suitable for extraction of pendimethalin and its metabolites from soil alone was not suitable for exhaustive extraction of pendimethalin and metabolites from soil with compost addition. The higher amount of organic matter resulted probably in the higher amount of pendimethalin (equivalents) being sorbed to the soil matrix due to the higher amount of possible adsorption locations in compost amended soil. In an additional experiment, the remobilization experiment assessed the potential of non-extractable residues to be released in the future. Small quantities were biodegraded, indicated by minor portions degraded completely to 14CO2 and minor amounts becoming extractable after incubation in fresh soil. It was concluded that only minor amounts could become bioavailable with time, provided that the organic matter matrix is thoroughly altered by degradation or oxidation. All in all, with the results of this investigation, the non-extractable residues formed by pendimethalin in this soil are considered to be mainly covalently bound to the soil matrix. Compost amendment enhanced the formation of non-extractable residues and thus, the herbicide was less bioavailable and was degraded to a lesser degree than in soil alone. The methods used in the present dissertation are a tool to investigate the formation and characteristics of xenobiotic non-extractable residues and to distinguish those being sequestered (type I) and covalently bound (type II).

Dissertation, RWTH Aachen University, 2019; Aachen 1 Online-Ressource (XVIII, 184 Seiten) : Illustrationen, Diagramme (2019). = Dissertation, RWTH Aachen University, 2019, Fish are highly dependent on olfaction since a variety of indispensable behaviors are mediated via this sensory modality. Situated in cavities at the dorsal part of the cranium, the fish olfactory epithelium (OE), which contains the olfactory sensory neurons (OSNs) responsible for odorant detection, is in almost direct contact to the ambient water and thus readily accessible for both odorants and dissolved contaminants. In the last decades, an increasing number of studies demonstrated that numerous pollutants, among them metals and pesticides, are able to impair olfaction in environmentally relevant concentrations, thereby disrupting pivotal behaviors. However, the toxic mechanisms underlying olfactory dysfunction largely remain unknown and despite the paramount significance of olfaction for fish, olfactory endpoints have not yet been incorporated into the environmental risk assessment of chemicals. The objective of the present Ph.D. thesis was to investigate the impact of three commonly used pesticides (chlorpyrifos, linuron, and permethrin) and cadmium (Cd) on the olfactory system of zebrafish (Danio rerio). In doing so, this thesis further aimed at contributing to the establishment of zebrafish as a model for evaluating olfactory toxicity. In order to enhance the understanding of the toxic mechanisms underlying olfactory dysfunctions, the effect of contaminants on the olfactory system was analyzed at different levels of biological organization. Employing quantitative real time polymerase chain reaction, it was shown that Cd, chlorpyrifos, and permethrin differentially alter the expression of OSN marker genes in the OE. Furthermore, Cd and chlorpyrifos induced genes associated with stress in the OE, but only Cd increased the expression of such genes in the olfactory bulb (OB). The assessment of second messengers with central roles in olfactory signal transduction showed that linuron decreased the level of cyclic adenosine monophosphate (cAMP) in the OE. The influence of the pollutants on the olfactory mediated endocrine response of male zebrafish to the pheromone prostaglandin F2α (PGF2α) was examined by analyzing 11 ketotestosterone (11 KT) plasma levels. The pesticides did not cause a statistically significant impairment of this endocrine response, whereas male zebrafish exposed to Cd displayed a downward trend of 11 KT levels. The assessment of the antipredator response following treatment with the respective contaminant demonstrated that Cd and chlorpyrifos significantly disrupted this crucial behavior. While it recovered from chlorpyrifos induced deficits following 48 h of depuration, the antipredator response did not recover in fish previously exposed to Cd. The suitability of zebrafish early life stages as alternative models for olfactory toxicity assessments was examined by studying contaminant induced effects on OSN marker gene expression, cell death in the olfactory placode, and olfactory evoked swimming activity. In this process, no statistically significant alterations were detected. Since water chemistry is known to markedly influence acute metal toxicity, its impact on Cd induced olfactory impairment was investigated, thereby showing that hardness, pH, and dissolved organic carbon substantially modified the latter. The research presented in the thesis at hand demonstrated that chlorpyrifos and Cd disrupted the antipredator response, an olfactory mediated behavior essential for fish survival. The results from the gene expression study strongly supported the assumption that Cd is taken up into the OE and OB, causing oxidative stress in these organs. By contrast, oxidative stress may not be a predominant factor of chlorpyrifos induced olfactory dysfunction. The decreasing trend observed for the endocrine response to PGF2α following Cd treatment may further point at a potential impact on reproduction and needs to be subject of additional research. Zebrafish proved to be an excellent model for the assessment of olfactory toxicity, in particular with respect to the investigation of mechanisms underlying an olfactory dysfunction. However, the suitability of its early life stages as alternatives for olfactory toxicity testing could not be confirmed based on the data obtained in this thesis. Overall, the work contributed to the establishment of zebrafish as a model for the study of olfactory toxicity and underlined the importance of the incorporation of olfactory endpoints into the environmental risk assessment of chemicals., Published by Aachen

Biodegradation of organic pollutants in soil generally results in the formation of metabolites, microbial biomass, mineralisation products and bound or non-extractable residues (NER). It is speculated that NER can pose a risk for humans due to the remobilisation and further distribution of active parent compounds or their metabolites over the food web (BARRACLOUGH ET AL., 2005). Although there are many studies on NER formation available, their chemical structures are still unknown. However, without this knowledge, a proper risk assessment for the pollutant and the related NER during its transformation in soil is impossible. Part of the NER may be biogenic, since the pollutant-derived C and CO2 released from its mineralisation are assimilated by microorganisms into their cellular components [e.g. fatty acids (FA) and amino acids (AA)], which are subsequently incorporated into soil organic matter (SOM) after cell death. In order to study the microbial biomass contribution to NER formation, soil was incubated with either 13C-labelled 2,4-Dichlorophenoxy acetic acid (2,4-D) or ibuprofen (Ibu) for 64 and 90 days, respectively. At different sampling dates, the soil was analysed for the presence of the 13C label in FA and AA. The 13C label assimilated in biomolecules was determined in both the living and the non-living SOM fractions. Moreover, for investigating the relevance of heterotrophic CO2 fixation for the incorporation of 13C into biogenic NER in soil and to distinguish between formations of NER directly from 2,4-D and via CO2, an experiment with unlabelled 2,4-D and labelled CO2 was conducted. In addition, to understand both the processes of the biogenic NER formation and the incorporation of 13C label into FA and AA from either 13C6-2,4-D or 13CO2 in the complex soil environment, a simple biological model system with the known 2,4-D degrader (Cupriavidus necator JMP 134), was studied. After 64 days of 13C6-2,4-D incubation in soil, the total contents of 13C detected in AA in SOM indicated that 44% of the initially applied 13C6-2,4-D equivalents had been converted to microbial biomass and finally to biogenic residues. The intermediate maximum of 13C-FA in SOM indicated a 20% conversion of 13C6-2,4-D to biomass. However, 13C-FA in the non-living SOM fraction decreased to 50% indicating their metabolisation and their further distribution within the food web. Contrary to the 13C-FA, 13C-AA in non-living SOM pool were surprisingly stable. The soil experiment with 13CO2 showed that after 16 days of incubation, the heterotrophic CO2 fixation was relevant in the assimilation of 13C label into biomass components, in particular FA. In addition, the liquid culture experiment with C. necator JMP 134 demonstrated the high importance of CO2 fixation in the incorporation of 13C label into the biomass components in the later phase of incubation. In this study about 4% of 13C derived from 13C6-2,4-D was assimilated in the biomass of this bacterial strain via CO2 fixation. A total content of biogenic NER of 54% of 13C6-ibu equivalents at the end of the 13C6-ibu soil experiment was indicated by the amount of 13C found in AA in SOM. From the maximum content of 13C-FA detected in this experiment, at least 24% (of 13C6-ibu equivalents) biomass must have been formed. Contrary to the 13C6-2,4-D soil incubation experiment, the 13C-FA remained stable until the end. However, the formation of NER in the soil incubated with 13C6-ibu started much later than that in the 13C6-2,4-D study, due to the longer lag phase and the interruption of the incubation before the destabilisation of biogenic NER. The 13C-AA in SOM remained also stable as found in 13C6-2,4-D experiment. The results from these soil biodegradation experiments provide the first evidence that nearly all NER both from 2,4-D and Ibu are biogenic, containing only natural microbial biomass components stabilised in SOM. However, biogenic residues are formed if the respective organic contaminant is readily degraded by microorganisms under significant formation of CO2. Depending on the yield coefficients of C conversion into biomass, we expect ratios of biomass plus biogenic residues to CO2 of about 0.2 to 1. In the 13C6-2,4-D study, the ratio was ~ 0.8 and in the 13C6-ibu was 1.2. Biogenic residues are clearly excluded from the definition of NER according to IUPAC. However, they are usually not analysed separately. The potential risk of NER from readily metabolised organic contaminants in soils is thus highly overestimated in many cases due to biogenic residue formation. Therefore, the formation of biogenic residues must be taken into consideration, when determining the mass balances of contaminants during their microbial degradation in soil.