The results from this study showed that it may be valuable to evaluate the BCF or BAF of CECs in earthworms over time, as there may bean increased potential for biomagnification at a given time point. However, more research is needed to determine if these differences persist for a longer time scale and if they occur in different soils or for other CECs. Out of four major metabolites examined in this study only N4- acetylsulfamethoxazole was quantifiable in the earthworm tissues. N4- acetylsulfamethoxazole was seen to increase to a peak concentration of 4.39 ± 0.4 ng g-1 at 3 d and then decreased to 2.62 ± 0.01 ng g-1 by the end of the 21 d incubation . Further, N4-acetylsulfamethoxazole was also detected in the earthworm soil with the highest concentration observed at 7 d , indicating that earthworms was capable of metabolizing these CECs and excreting the transformation product into its surrounding environment. N4-acetylsulfamethoxazole is the primary metabolite of sulfamethoxazole in humans and has been previously detected in wastewater effluent, environmental samples and plant tissue . However this was, to the best of our knowledge, the first time that N4-acetylsulfamethoxazole has been observed to form in earthworms. The continued observation of the formation of N4-acetylsulfamethoxazole, an acetyl conjugate, in the environment is of considerable interest because conjugates have the potential to maintain the biological activity of the parent compound . Further, because researchers traditionally only quantify parent compounds during environmental assessments,macetas de 10 litros the formation and accumulation of conjugates implies that there may be an underestimation of environmental exposure to CECs such as pharmaceuticals and further incomplete environmental risk assessment of CECs .
This is of particular concern for antibiotics, because of the rise of antimicrobial resistance . The major metabolite of methyl paraben, p-hydroxybenzoic acid, was observed in all soil samples, including the non-treated controls . This was likely due to the endogenous p-hydroxybenzoic acid in sphagnum peat . However, the concentration of p-hydroxybenzoic acid was higher in the spiked earthworm treatments than in the blank controls or non-earthworms chemical controls indicating that E. fetida was also capable of taking up and metabolizing methyl paraben and excreting of p-hydroxybenzoic acid into the soil. This was consistent with previous contact tests in which 70% of the initial methyl paraben was found to be metabolized to p-hydroxybenzoic acid and phenol within 48 h in E. fetida . The transformation products o-desmethylnaproxen and nordiazepam were not detected in earthworm tissues, but o-desmethylnaproxen was quantifiable in earthworm-CEC treated soils during the 21 d incubation , indicating active uptake, metabolism, and excretion. The quantification of the major metabolites for naproxen, sulfamethoxazole and methyl paraben, o-desmethylnaproxen, N4- acetylsulfamethoxazole, and p-hydroxybenzoic acid suggested a trend in the capabilities of E. fetida to take up, metabolize and excrete then transformation products of some CECs in the soil environment. Activities of several vital antioxidant enzymes were determined after exposure to CECs. A significant increase in the activity of glutathione-S-transferase in the treatment samples over the controls was observed starting after 3 d into the incubation , and the GST activity continued to increase until the end of the 21 d incubation . This observation suggested that increased exposure time resulted in increased oxidative stress because glutathione is considered a critical antioxidant that acts to maintain redox homeostasis and signaling in cells . Further, GST is a crucial enzyme family for the detoxification of xenobiotics during Phase II metabolism . Thus, the observed increase in GST activity may indicate that there was a formation of additional Phase II metabolites.
However, the detection of these potential metabolites was not attempted due to a lack of authentic standards. High GST activity was also observed at 0 h for both the controls and treated samples. However, this increase in activity is likely due to the initial stress of the earthworm being transferred into the test media, and the effect dissipated within the first day of exposure. No significant difference in catalase activity was observed between the treatment and controls until the end of the exposure period . At the 21 d time point a significant increase was seen in the treatment samples , indicating that extended exposure to CECs likely resulted in increased production of hydrogen peroxide in earthworm tissue . However, an increase in the CAT activity was also found in the control at 0 h. The increase in CAT activity was, again, likely due to the initial stress of the earthworms being transferred to different environmental conditions and the difference dissipated within 24 h. A significant increase in superoxide dismutase was observed at 1 d and 3 d . However, no significant differences were observed between the treatment and controls after 3 d . This trend was in accordance with SOD has the first line of defense against reactive oxygen species . SOD acts to catalyze the superoxide radical into oxygen molecules or hydrogen peroxide . As an increase in CAT was observed at the later time point it was likely that SOD activity increased initially, resulting in an increased production of hydrogen peroxide, which was then detoxified by CAT. Previous studies examining the biochemical effects of CEC exposure in earthworms showed somewhat similar trends. For example, a study exploring the biochemical and genetic toxicity of triclosan in E. fetida showed a dose-dependent hormesis effect over time for both CAT and GST activity, with increasing activity being observed after a 2 d exposure at low doses and an inhibition of enzyme activity being observed after 14 d at high doses. Further, similar studies considering oxidative stress in E. fetida exposed to herbicides showed an increase in enzyme activities at lower concentrations and a suppression of enzyme activities at high concentrations .Currently we are experiencing a series of global trends that are creating unique challenges for the future of sustainable development. These trends include shifting precipitations patterns, rising temperatures, growing human populations, and rapid urbanization. In order to meet these challenges, traditionally under-utilized resources, such as treated wastewater and bio solids, will have to be harnessed.
These resources are derived from wastewater treatment plants and contain biologically active, pseudo-persistent, trace chemicals referred to as contaminants of emerging concern . Land application of TWW and bio solids for agriculture and landscaping has the potential to introduce CECs into terrestrial ecosystems, from where they could accumulate, be metabolized and/or cause adverse effects in terrestrial organisms. This dissertation has described the ability of terrestrial plants and invertebrates to take up and metabolize CECs and highlighted the potential for these trace contaminants to induce biochemical changes in non-target terrestrial organisms. The findings of this project, overall conclusions, and recommendations for future work are summarized below. In arid and semi-arid areas, TWW reuse is becoming increasingly prevalent for agricultural irrigation. However, irrigation with TWW has the potential to introduce CECs including antibiotics into agroecosystems. One of the most commonly prescribed and environmentally relevant antibiotics is sulfamethoxazole. However, little is known about the fate of sulfamethoxazole in terrestrial plants. In this study, sulfamethoxazole was observed to be taken up and actively metabolized by Arabidopsis thaliana cells into six transformation products. The transformation products included oxidation of the amine group, producing Phase I metabolites, which was followed by conjugations with glutathione, glucuronic acid and leucine, producing Phase II metabolites. Phase III metabolism was assessed by determining the mass balance of 14C-sulfamethoxazole in A. thaliana cells and cucumber seedlings. Non-extractable 14C-sulfamethoxazole increased over time in both A. thaliana cells and cucumber seedlings, indicating that Phase III metabolism significantly contributed to the fate of sulfamethoxazole in A. thaliana cells and cucumbers. Further,macetas de 7 litros in A. thaliana cells and cucumber seedlings, the mass balances were calculated to range from 80-120% and 80-94%, suggesting a minor role of mineralization. The results from this study highlighted the potential of terrestrial plants to transform pharmaceuticals, forming both bio-active Phase I metabolites and Phase II conjugates, and store them as in the form of bound residues as Phase III metabolism. Plant uptake of CECs from TWW reuse and bio-solid application has been documented in agroecosystems. Previous studies suggested that plants were capable of metabolizing CECs after uptake. However, these studies often reported different results even with the same CECs, likely due to the use of different plant species and/or different laboratory conditions. In this study, the metabolism of the benzodiazepine diazepam was explored in three different plant species, A. thaliana, cucumber , and radish . The plants were exposed to diazepam in laboratory under three different laboratory exposure conditions that included a 6 d cell culture, an acute /high concentration hydroponic cultivation, and a chronic /low concentration hydroponic cultivation. 14C-Diazepam was incubated concurrently with non-labeled diazepam to determine the fractions of extractable and non-extractable radioactivity to quantify Phase III metabolism. Diazepam was taken up and metabolized in all plant species under the different exposure conditions. A. thaliana cells actively transformed diazepam into temazepam and nordiazepam via Phase I metabolism.
This metabolism mimicked human metabolism, as temazepam and nordiazepam are the minor and major metabolites, respectively, formed during human metabolism of diazepam. Intriguingly, both of these metabolites are bio-active and prescribed pharmaceuticals in their own right, alluding to a potential for increased risk from consumption not considered in previous studies. The fraction of non-extractable residue increased over the 6 d incubation, indicating extensive Phase III metabolism over time in A. thaliana cells. In cucumber and radish seedlings, a similar Phase I metabolism pattern was observed, with nordiazepam being the most prevalent metabolite at the end of the 7 d and 28 d cultivations. However, significant differences in phase III metabolism were observed between the radish and cucumber plants. For example, after the acute exposure, diazepam mass balance was 99.3% for cucumber seedlings but only 58.1% for radish seedlings, indicating increased mineralization in the radish system. Diazepam induced changes in the regulation of glycosyltransferase activity in both cucumber and radish seedlings, indicating the formation of Phase II metabolites. The results from this study showed that exposure conditions and plant species can influence the metabolism of diazepam, and formation of bio-active transformation intermediates and different phases of metabolism should be considered in order to achieve a comprehensive understanding of risks of CECs in agroecosystems.Exposure of terrestrial invertebrates to CECs will likely increase with increasing TWW reuse and bio-solid application. However, currently there is limited information on the fate and effects of CECs in terrestrial organisms. In this study, the earthworm E. fetida was exposed to three pharmaceuticals, i.e., sulfamethoxazole, diazepam, and naproxen, and one preservative, i.e., methyl paraben, for 21 d in an artificial soil. Methyl paraben did not accumulate in the earthworm tissue, likely due to its rapid degradation in the soil. The other CECs showed an accumulation in earthworm tissues from the soil/soil porewater. The presence of E. fetida did not significantly affect the adsorption of these CECs to the soil. The presence of primary metabolites in the treated soil suggested that E. fetida were capable of actively metabolizing the three pharmaceuticals and excreting the metabolites. However, the metabolism was chemical-specific, and only N4- acetylsulfamethoxazole was detected in earthworm tissues. Exposure to the four CECs also resulted in the up-regulation of several antioxidant enzymes, including glutathione-S-transferase, superoxide dismutase, and catalase, and an increase in malondialdehyde, indicating oxidative stress in the exposed E. fetida. Results from this study highlighted the need to consider the role of, and effects on terrestrial invertebrates when understanding risks of CECs in agroecosystems. Our findings illuminate the complexity of the interactions between contaminants of emerging concern and terrestrial organisms. The dissertation highlights the ability of terrestrial organisms to take up and transform CECs through metabolism, which results in both bio-activation and detoxification of the target contaminants. This project also demonstrates the ability of CECs to alter the biochemistry of the studied terrestrial organisms by changing the regulation of enzymes associated with oxidative stress and metabolism. The use of cell cultivations, hydroponic studies, and artificial soil allowed us to examine the metabolism and effects of CECs in terrestrial organisms with limited confounding factors. However, it is highly likely that similar studies conducted in soils may show low rates of uptake and different patterns in metabolism. Our research suggests that scientifically sound understanding of fate of, and risks from, CECs in the environment cannot solely rely on the assessment of the parent compound and/or only consider the potential for human exposure to CECs.