Periodic Reporting for period 1 - CyTiS (Cytostatic compound fate and transport in soil and groundwater)
Reporting period: 2022-11-01 to 2024-10-31
Summary of the context and overall objectives of the project
Cytostatic compounds are used globally as chemotherapy drugs to treat cancer, and increasingly these are outpatient treatments. Up to 90% of the drug can be excreted by the patient, and can therefore enter municipal wastewater systems or, in rural areas, onsite wastewater treatment systems like septic systems. Most municipal systems and septic tanks are not equipped to degrade cytostatic compounds, and consequently these compounds can reach soils directly via leaching fields, through discharge of wastewater treatment plant effluent to holding ponds, or through application of sludge to agricultural fields. The objective of CyTiS was to explore the transport of cytostatic compounds in soil types relevent to leaching fields and agricultural soils which may impact groundwater, and to explore the impact cytostatic compounds have upon microbial soil communities. Because of their genotoxic, carcinogenic, and teratogenic effects, these compounds have the potential to damage ecosystems and people. There is no safe limit for these compounds in drinking water. Very little is known regarding the transport of this wide class of compounds in soil to groundwater, and nothing is known regarding their impact on microbial communities in soils. Healthy and diverse microbial communities are responsible for the degredation of organic contaminants in soils, and if cytostatic compounds impact either the health or diversity of these communities it may impede the remediation of many other contaminants and organic wastes.
The first aim of this project was to use sorption studies to assess the potential for cytostatic compounds to be transported in soil environments to groundwater. Sorption of chemicals to soil is a process that can slow or prevent their transport to groundwater (a drinking water resource) and therefore can increase the likelihood of degredation of a compound in soil. The only previous study to assess sorption of cytostatic compounds to soil examined cyclophosphamide and ifosfamide, two closely related alkylating agents, in soil. CyTiS examined the sorption of cyclophosphamide, methotrexate, capecitabine, and doxorubicin, four cytostatic compounds which are widely used globally to treat a wide range of cancers and each of which represents diffferent classes of cytostatic compounds. Assessing different classes of compounds increases the applicability of the findings of this project to a wider number of chemotherapy drugs, which is importnant because the lack of previous research in this area means that there is not yet a framework for assessing which classes of compounds may pose a risk for transport and which may be sequestered in the soil.
The second aim of this project was to examine the impact of cytostatic compounds on the health and diversity of microbial ecosystems in soil. No previous work has examined the effect of introducing cytostatic compounds to these communities. The importance of diverse soil communities in degrading all tyoes of organic material in waste streams makes it vital to understand how the introduciton of toxic pharmaceuticals, especially in the ever increasing amounts required for cancer treatment globally, may influence the health and productivity of these vital communities. CyTiS is the first project to have taken a preliminary survey of the potential impact that these compounds could be having, by examining the alterations to health and diversity of microbial communitites exposed to different classes of cytostatic compounds at both varied concentrations and over time.
The first aim of this project was to use sorption studies to assess the potential for cytostatic compounds to be transported in soil environments to groundwater. Sorption of chemicals to soil is a process that can slow or prevent their transport to groundwater (a drinking water resource) and therefore can increase the likelihood of degredation of a compound in soil. The only previous study to assess sorption of cytostatic compounds to soil examined cyclophosphamide and ifosfamide, two closely related alkylating agents, in soil. CyTiS examined the sorption of cyclophosphamide, methotrexate, capecitabine, and doxorubicin, four cytostatic compounds which are widely used globally to treat a wide range of cancers and each of which represents diffferent classes of cytostatic compounds. Assessing different classes of compounds increases the applicability of the findings of this project to a wider number of chemotherapy drugs, which is importnant because the lack of previous research in this area means that there is not yet a framework for assessing which classes of compounds may pose a risk for transport and which may be sequestered in the soil.
The second aim of this project was to examine the impact of cytostatic compounds on the health and diversity of microbial ecosystems in soil. No previous work has examined the effect of introducing cytostatic compounds to these communities. The importance of diverse soil communities in degrading all tyoes of organic material in waste streams makes it vital to understand how the introduciton of toxic pharmaceuticals, especially in the ever increasing amounts required for cancer treatment globally, may influence the health and productivity of these vital communities. CyTiS is the first project to have taken a preliminary survey of the potential impact that these compounds could be having, by examining the alterations to health and diversity of microbial communitites exposed to different classes of cytostatic compounds at both varied concentrations and over time.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
The sorption studies addressing the first goal of the CyTiS project were performed using mixed batch reactors to determine the sorption coefficients of four cytostatic compounds in two soil types (sand and sandy-loam). Sorption coefficients are a measure of how much of the compound attaches to the soil; a larger value indicates a greater likelihood of adsorbing. The coefficients were determined by spiking each compound to different final concentrations into pre-equilibrated batch reactors containing soil and an aqueous phase. The effect of pH, ionic strength, and competing compounds was assessed by varying the aqueous phase in the reactors; the effect of raw wastewater on the sorption coefficient of each compound was also examined by equilibrating the soil with raw settled wastewater and then spiking in the target compound. Sorption coefficients were calculated for each compound first under neutral non-competitive conditions by varying the spike concentrations, then under the varied aqueous conditions. For three of the tested compounds the sorption coefficients determined in this project are the first such reported under any conditions.
The second goal of the CyTiS project was to examine the impact of cytostatic compounds upon microbial communities in soil. For the experiments, soil samples were obtained from two different septic system leaching fields; one field from a residence which had had a cancer patient using the system and one field from a residence with no known cancer patients. The microbial communities were analyzed in grab samples immediately after sampling to ensure that living communities were present. All experiments also included ‘control’ sample groups of soil which were not exposed to cytostatic compounds. One set of experiments used mixed batch reactors to assess the immediate effect of different concentrations of either cyclophosphamide or doxorubicin upon the microbial communities. Soil from each site was mixed with synthetic wastewater spiked with the target compound and left, covered but open for air exchange, for 24 hours before the soil was collected for DNA analysis. For another set of experiments, soil was placed in small columns, through which synthetic wastewater spiked with the target compound was gravity filtered three times a week for one to four weeks. At appropriate time points samples were collected. For all soil samples in both experiment groups, DNA was extracted and sequenced using amplicon sequencing. Community analysis examined the differences in taxonomic distribution among different treatments in both soil communities. This is the first community analysis performed assessing the impact of cytostatic compounds on soil microbiomes.
The second goal of the CyTiS project was to examine the impact of cytostatic compounds upon microbial communities in soil. For the experiments, soil samples were obtained from two different septic system leaching fields; one field from a residence which had had a cancer patient using the system and one field from a residence with no known cancer patients. The microbial communities were analyzed in grab samples immediately after sampling to ensure that living communities were present. All experiments also included ‘control’ sample groups of soil which were not exposed to cytostatic compounds. One set of experiments used mixed batch reactors to assess the immediate effect of different concentrations of either cyclophosphamide or doxorubicin upon the microbial communities. Soil from each site was mixed with synthetic wastewater spiked with the target compound and left, covered but open for air exchange, for 24 hours before the soil was collected for DNA analysis. For another set of experiments, soil was placed in small columns, through which synthetic wastewater spiked with the target compound was gravity filtered three times a week for one to four weeks. At appropriate time points samples were collected. For all soil samples in both experiment groups, DNA was extracted and sequenced using amplicon sequencing. Community analysis examined the differences in taxonomic distribution among different treatments in both soil communities. This is the first community analysis performed assessing the impact of cytostatic compounds on soil microbiomes.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
The sorption coefficients, together with the other sorption data, suggest that the sorption of cytostatic compounds to soil and the potential for transport of the compounds to groundwater is strongly dependent on the class of compound. Two of the compounds have very low sorption coefficients under neutral conditions (cyclophosphamide <0.5 and capecitabine 1.5-1.6 mL/g depending on the soil) and sorption measurements under all other aqueous conditions cluster around this linear model. Both of these compounds, representing alkylating agents and antimetabolites respectively, are therefore very likely to remain in the aqueous phase and be transported through soil, because they are not likely to adsorb under any conditions. These compounds therefore pose a potential risk to groundwater if they are not subsequently degraded. As there is no safe limit for cytostatic compounds in drinking water, these findings indicate that more research on the capture of these compounds in soil environments is necessary. Additionally, groundwater studies utilizing untargeted metabolomics for contaminant screening should ensure that these classes of cytostatic compounds are included in the reference libraries. Doxorubicin in contrast has very high sorption coefficients in both soils (407-2686 mL/g) under neutral conditions. In sand, sorption measurements under all other aqueous conditions cluster tightly around the linear model. In sandy-loam it appears that sorption of doxorubicin is actually increased in the presence of raw wastewater. These data all suggest that doxorubicin is very unlikely to be transported through soil. The discharge of doxorubicin to soil therefore likely does not pose a risk to groundwater. However, doxorubicin was the chosen representative of the class of cytostatic compounds known as cytostatic antibiotics, and the fact that it is likely to be retained in soils suggests that it may have an impact upon the soil ecosystem at the point of entry. The second part of this project takes a preliminary look at this impact, but further work on degradation pathways seems advisable. Under neutral, noncompetitive conditions, the sorption coefficient of methotrexate (65-69 mL/g) suggested that methotrexate would adsorb to soil. However, an increase in pH or the presence of competing compounds (especially in the raw wastewater) drastically altered the sorption measurements of methotrexate in both soil types, dropping them well below the neutral linear model. These findings suggest that methotrexate may in fact remain in the aqueous phase in waste streams. Especially in areas with a high water table, methotrexate may have the capacity to reach groundwater. Further sorption studies may elucidate the likelihood of continued transport in groundwater with higher mineral concentrations and lower organic concentrations.
The diversity of soil microbial communities was affected to different degrees by exposure to cytostatic compounds. In the single dose tests with varied concentrations of either cyclophosphamide or doxorubicin, the taxonomic distributions varied somewhat among samples treated with different compounds, but the overall diversity did not appear to be affected by the treatment. Exposure over four weeks demonstrated more pronounced difference in taxonomic composition and overall community diversity among treated samples. These results suggest that cytostatic compounds retained in soils may have an impact on the composition of the soil microbial community, which could have reverberating impacts on the activity of these communities degrading other co-occuring organic contaminants. Further research should be conducted examining the direct impact of cytostatic compounds on microbial communities, but also on the capacity of these impacted communities to then degrade other compounds in waste streams. These preliminary results have implications for the long term effectiveness of soil microbiota to, for instance, continue to degrade domestic waste in leaching fields if once exposed to cytostatic compounds.
The diversity of soil microbial communities was affected to different degrees by exposure to cytostatic compounds. In the single dose tests with varied concentrations of either cyclophosphamide or doxorubicin, the taxonomic distributions varied somewhat among samples treated with different compounds, but the overall diversity did not appear to be affected by the treatment. Exposure over four weeks demonstrated more pronounced difference in taxonomic composition and overall community diversity among treated samples. These results suggest that cytostatic compounds retained in soils may have an impact on the composition of the soil microbial community, which could have reverberating impacts on the activity of these communities degrading other co-occuring organic contaminants. Further research should be conducted examining the direct impact of cytostatic compounds on microbial communities, but also on the capacity of these impacted communities to then degrade other compounds in waste streams. These preliminary results have implications for the long term effectiveness of soil microbiota to, for instance, continue to degrade domestic waste in leaching fields if once exposed to cytostatic compounds.