BIOTRANSFOROMICS: Bioanalysis to Engineer Understanding in Wastewater Treatment

Lead Research Organisation: University of Nottingham
Department Name: Div of Process and Environmental Eng

Abstract

The wastewater treatment process (WWTP) plays a critical role in providing clean water. However, emerging and predominately unregulated, bioactive chemicals such as steroids and pharmaceutical drugs are being increasingly detected in surface waters that receive wastewater effluent. Although present at low concentrations, their inherent bioactive nature has been linked to abnormalities in aquatic organisms and there are also water reuse and human health implications. As part of the urban water cycle, the WWTP is the gatekeeper to the surface waters e.g. rivers. Pharmaceuticals enter wastewater treatment from inappropriate disposal of unused drugs to the sink/toilet or via landfill. Prescribed or illicit drug use also has the inevitable consequence of being metabolised in the human body (to parent, Phase I / II metabolites) and excreted in urine, which subsequently enters the WWTP. Coupled with naturally produced and excreted bioactive steroids, the challenge for wastewater treatment is that it was never designed to remove these bioactive chemicals and is inefficient.

Evaluating the prevalence and fate of a steroid or pharmaceutical in the WWTP is challenging as human enzymatic metabolism causes the bioactive chemical to exist in multiple forms - parent, Phase I and Phase II metabolites. Phase II metabolites predominate urine excretion and are the starting products entering the wastewater environment. They therefore act as the precursors to the biotransformations that take place during treatment and produce the Phase I and/or parent forms of the bioactive chemical. Before treatment technologies can be developed and evaluated for pharmaceutical and steroid removal in the WWTP, our understanding needs to improve on how the different bioactive chemical forms behave, and their relationships to each other. This means identifying the biotransformations between metabolites and parent forms. To achieve this requires a move from targeted analysis - we analyse for what we expect to see - to develop methods that are non-targeted and search for Phase II metabolites and their associated Phase I / parent forms.

Drawing on inspiration from metabolomics approaches used in the biosciences, the aim of this proposal is to develop a novel non-target method to identify bioactive chemical Phase II metabolites and their biotransformation products in wastewater. Knowledge of Phase II metabolite occurrence and fate in the wastewater environment is important in assessing the impact of user behaviour, process and environmental factors or bioactive chemical parent removal. This will inform on WWTP efficiency, provide data for optimising models that predict pharmaceuticals and steroids, and evaluate environmental risk.

Planned Impact

The proposed research will accelerate understanding by providing a method to identify Phase II metabolites and their biotransformation products without bias. Application of this method will identify pharmaceuticals and steroids of concern in UK wastewater and importantly account for the relationships between the different metabolite and parent forms that exist in the wastewater environment.

Pharmaceutical manufacture is one of the UK's leading industries, with medicines accounting for 10% of the NHS budget to support the health and well-being of the population. Growing awareness of pharmaceuticals in wastewaters, surface waters and even drinking water has led to closer scrutiny and increasing regulation of pharmaceuticals and waste. Although present at low concentrations, their inherent bioactive nature has been linked to abnormalities in aquatic organisms and there are also water reuse and human health implications. The wastewater treatment process (WWTP) is a gatekeeper to maintaining the urban water cycle and providing clean water for all. The societal impact of this research is clear, to support and enhance human and environment health. Impact also extends to the pharmaceutical industry who are committed to ensuring drug manufacture, use and disposal does not adversely affect human health or the environment (e.g. Pfizer's PIE - Pharmaceuticals in the Environment).

This research is highly relevant to Water Companies in evaluating WWTP effectiveness for removing bioactive pharmaceutical drugs and steroids. In particular the UK Water Industry Research organisation who developed the Chemical Investigation Programme (CIP) in collaboration with the Environment Agency to assess the scope of requirements involved in upholding the recent European Directives - the Water Framework Directive (WFD) and the new Priority Substances Daughter Directive - put in place to regulate Environmental Quality Standards.

It will align and add value to the UK Water Industry £140M CIP2 that begins in 2015. For this, Water Companies are required to deliver an ambitious programme of sampling and analysis until 2020 to understand the impact of priority hazardous substances, specific pollutants, emerging substances and pharmaceuticals to and from wastewater treatment works on the environment. In addition the UK regulators are required to set their own standards for further potential pollutants and are aware that to respond to these obligations requires greater understanding of the effectiveness of WWTPs for removing BACs.

This proposal also impacts on the increasingly stringent EU requirements in water quality and pollution control e.g. the 2013 Watch List under the WFD that requires EU-wide monitoring of one steroid and two pharmaceuticals to support the prioritisation process in future reviews of the priority substances list. Impact also goes beyond the EU including the Global Water Research Coalition and US Environmental Protection Agency.

Publications

10 25 50
 
Description Further experience from grant writing/reviewing and on reflection, the scope for this £100k 2 year study was very ambitious, incorporating method development and validation, followed by several application routes. Each objective had wide scope, as well as several avenues not considered at the grant writing stage and later showed direct relevance to research aim. Developing and validating an analytical methodology capable of screening wastewaters to detect non-target Phase II metabolites (Objective 1) was the cornerstone of the work, followed by application (objectives 2 and 3). Objective 1 was expanded beyond the original grant scope to include biotransformation products, as it became clear that these are very relevant to the aspirations of this work for improving wastewater treatment for bioactive pollutant removal. The method also benefited from a change to the original plan and software used for data analysis. The new software enabled greater flexibility, along with a more intuitive user interface that has led to the creation of a new workflow tool that combines and integrates multiple confirmation endpoints to identify unknown precursor and biotransformation products and relationships to bioactive chemicals in environmental water matrices. These changes to the research work plan have been validated through identification of biotransformation products that are themselves bioactive, including O-desmethylvenlafaxine and O-desmethyltramadol in wastewater matrices. The decision to expand objective 1 in terms of pollutant range and software use impacted on available time for application to the wastewater treatment process and bench scale studies. However objectives 2 and 3 was always going to be a starting point to demonstrate method need and scope from a process engineering perspective, with data obtained to be used to leverage further funding for in depth investigation. From a resource viewpoint and despite Pharmacy facilities including a range of LC-MS instruments to mitigate risk, analysis was challenging with certain unforeseen delays including supplier shutdown due to pump fault, waiting on new software, balancing of multiple grants limiting access. Outcomes to date have opened up multiple avenues to proceed and the focus is now more on disseminating the findings through journals and trade, and applying for a grant to continue momentum that this research has identified, which will enable improvements to how we evaluate current WW treatment and design new technologies able to successfully remediate bioactive chemicals so water is fit for reuse.
Exploitation Route Academic routes: extend current target methods and thinking focusing on the bioactive chemical pollutant which are utilised in the UK to include untargeted precursor and biotransformation products to gain improved understanding of environmental prevalence, fate, and process impact for wastewater treatment and receiving waters. Extends to related research topics including antimicrobial resistance (commenced with NERC EVAL FARMS project which I lead for the chemicals). Extends to Universities in ODA countries to extend their current methods and investigation (commenced with UNAM on untreated wastewater reuse). Value to non-environmental/process research methods and investigation through the workflow tool developed (commenced with University Loughborough). Non-academic routes: pharmaceutical industry both UK and abroad (commenced BRITEST and ODA compliant countries, India). UK Water industry and CIPII, with CIP III starting 2019 April confirmed to also include AMR in addition to pharmaceuticals and steroids (commenced with water company). Dairy farming and agriculture for water use and reuse and chemical load, including supporting AMR (commenced with NERC EVAL FARMS). SMEs and application of treatment technologies for wastewater treatment, extending the scope of treatment capability (commenced Lindhurst Engineering and H2AD.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description European Commission consultation on the risks of pharmaceuticals in the environment, including the impact on AMR (society response)
Geographic Reach Europe 
Policy Influence Type Participation in a national consultation
Impact Both legislative and regulatory. At UK level, impact to multi million UK Water Inudstry investment in the chemical investigation programmes. Pharmaceutical manufacturers impact.
URL https://ec.europa.eu/info/consultations/public-consultation-pharmaceuticals-environment_en
 
Title CD 2.1 BIOTRANSFOROMICS workflow 
Description Development of new workflow tool in TF Compound Discoverer (small molecule identification software, version 2.1) for data analysis of both targeted and untargeted bioactive chemicals in wastewater samples. Combination of unknown metabolomics, impurities and degradants, and environmental databases for the analysis of complex data with integrated compound identification capabilities and statistical analysis. Incorporates database, data handling and control, and data analysis. • Performs retention time alignment, unknown compound detection, and compound grouping across all samples.The BIOTRANSFOROMICS workflow: • Predicts elemental compositions for all compounds • Identifies compounds using mzCloud (ddMS2). • Performs similarity search for all compounds with ddMS2 data using mzCloud. • Find and identify expected metabolites with differential analysis (sample vs control comparison, trend plot for time points). • Performs retention time alignment, detects expected metabolites, dealkylation and dearylation products and bio-transformation products with resolution aware isotope pattern matching, and groups expected compounds across all samples. • Applies FISh scoring to all expected and transformation compounds automatically for MS2 or MSn confirmation. Hides chemical background (using Blank samples). 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? No  
Impact Combines and integrates multiple confirmation endpoints to identify unknown precursor and biotransformation and relationships to bioactive chemicals. Identified for first time through untargeted metabolomics-derived approach the bioactive biotransformation product, o-desmethylvenlafaxine in effluent, which was not present in the influent. Also identified conjugate precursors e.g. N4-acetyl sulfamethoxazole. Demonstrates that reduction of bioactive chemicals through wastewater treatment need to consider how treatment deals with conjugate precursor and biotransformed products (particularly those which are bioactive themselves) or treatment will not be sufficient to mitigate entrance to receiving water environments. 
 
Title LITSoN database of innovation relevant to the UK Water Sector 
Description The need for innovation in the water sector to meet societal needs is widely accepted. The UK Water Partnership LITSoN pilot (Linking Innovation To Societal Needs) provides an overview of innovation in UK water utilities, demonstrating benefits in terms of increased innovation alignment, better market information and opportunities for capability development, ultimately contributing to economic growth. Outcomes form the 4 invited workshops with industry and academia is to produce a database of innovation relevant to the UK water sector which will be accessible to all UKWIR members - and participants. This will feed into the ISCF including EOI and funding under water. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact ISCF wave EOI 
 
Description Academic - methods and instrument 
Organisation Loughborough University
Country United Kingdom 
Sector Academic/University 
PI Contribution Training and adaption of BIOTRANSFOROMICS workflow tool for analysis of non-environmental samples.Visits between Loughborough and Nottingham (PI and postdoctoral researcher). Training to Loughborough collaborators on software and workflow tool.
Collaborator Contribution Visits between Loughborough and Nottingham. Use of Loughborough instruments for environmental samples. Support Nottingham instruments.
Impact developed knowledge base, shared methods, use of instrument to confirm validation.
Start Year 2017
 
Description 15th International Conference on Environmental Science And Technology (CEST), Greece 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Adapting methods from grant for wastewater reuse perspective as GCRF relevance. Presentation to international audience, with questions and discussion throughout 6 day conference. Reference - Andrea Garduno Jimenez, Shridharan Parthasarathy, Catherine A. Ortori, David A. Barrett, Rachel L. Gomes (2017) Development of method to quantify bioactive pollutants due to wastewater irrigation in environmental matrices, 15th International Conference on Environmental Science And Technology (CEST), Rhodes, Greece (31st Aug - 2nd Sept 2017)
Year(s) Of Engagement Activity 2006,2017
URL http://cest.gnest.org/node/2304
 
Description 16th International Conference on Chemistry and the Environment (ICCE 2017), Norway 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Shridharan Parthasarathy, Catherine A. Ortori, David A. Barrett, Rachel L. Gomes Bioanalysis to Engineer Understanding in Wastewater Treatment: Targeted and Non-Targeted Approaches for Monitoring Emerging Pollutants from the United Kingdom
Year(s) Of Engagement Activity 2017
URL http://icce2017.org/
 
Description 90th Water Environment Federation's Annual Technical Exhibition and Conference (WEFTEC), Chicago 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation to industry conference under Late Breaking Research theme. Presented to international audience, with questions and discussion throughout 6 day conference. Reference - Shridharan Parthasarathy, Catherine A. Ortori, David A. Barrett, Rachel L. Gomes (2017) Simultaneous Analysis of Emerging Contaminants in Wastewater from the United Kingdom using Non-Targeted Metabolomics Approaches by Liquid Chromatography Tandem Mass Spectrometry, 90th Water Environment Federation's Annual Technical Exhibition and Conference (WEFTEC), Chicago, USA (29 September to 4 October, 2017). http://www.werf.org/c/WEFTEC/2017/WEFTEC_2017_Tech_Sessions.aspx / http://app.core-apps.com/weftec2017/event/308fca97bf3a4511431d73e39e87f7f8
Year(s) Of Engagement Activity 2017
URL http://app.core-apps.com/weftec2017/event/308fca97bf3a4511431d73e39e87f7f8
 
Description Britest visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Britest visit to University of Nottingham ti update on BIOTRANS project and potential for application to the chemical manufacturers that the Britest organisation represent. Discussed analytics, obtaining the 'right' data and to what granularity. Offered to investigate application to the wastewater treatment plant of one chemical manufacturer. February 2017.
Year(s) Of Engagement Activity 2017
URL https://www.britest.co.uk/
 
Description Invited seminar, Centre for Water Systems, Exeter University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Invited to deliver seminar on research activities focusing on BIOTRANSFOROMICS project, January 2017. Discussed post seminar scope for linking research interests.
Year(s) Of Engagement Activity 2017
URL http://emps.exeter.ac.uk/news-events/events-colloquia/event/?semID=1945&dateID=4532
 
Description KTN ISCF Manufacturing and materials Workshop (Resource Productivity) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Develop and refine the thinking which is currently shaping the Industrial Strategy Challenge Fund (ISCF) Manufacturing & Materials theme with the aim to support and drive UK business-led innovation as effectively as possible. Mission statement - To halve the environmental impact of manufacturing processes by 2030 making UK companies more profitable and resilient to the cost and availability of energy, water and materials (through approaches such as design for manufacturing, use and end-of-life, materials substitution, remanufacturing, and additive manufacturing). Contributing from process performance and profit, and wastewater treatment as a manufacturing environment.
Year(s) Of Engagement Activity 2017
 
Description KTN Water 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact KTN Water visit which was opened to the University of Nottingham. Engineering water research shared with KTN and wider University.
• Introduction to KTN for those who are not familiar (Jonathan)
• Jonathan to provide updates on the funding landscape wrt sustainability and water including:
o ISCF both in terms of process, future challenges and what might happen to challenges that have been workshopped by not launched
o Any insight on GCRF watery/sustainability type prospects (Rachel G)
• Update Jonathan on changes at Nottingham including our new group and our work on strategic relationships (Rachel G)
• We can share our mini water capability statement and talk about recent activity and growth in that area (Rachael D and Rachel G)
• We can also talk about our National Biofilm Innovation Centre recent award (Rachel G)
Year(s) Of Engagement Activity 2017
 
Description MI WASH (water, Sanitation and Health) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Represented University of Nottingham at 3 workshops in 2017 and 2018 and hosted 2018 workshop. Sharing and developing research collaborations under WASH relevant to GCRF future funding.
Year(s) Of Engagement Activity 2012,2017,2018
URL http://www.midlandsinnovation.org.uk/midlands-innovation.aspx
 
Description MOOC AMR in the Food Chain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Invited to deliver a perspective on pharmaceutical and steroid pollutants in the environment based on the BIOTRANS project. Highlighted the need to consider chemical pollutants when considering AMR, in addition to the microorganism perspective. Need for data on pollutant fate and biotransformations, which currently unknown how inform on AMR development and spread. University of Nottingham media delivered short video for the MOOC AMR in the Food Chain. The MOOC has been nominated and shortlisted for the British Universities Film and Video Council's "Learning on Screen" awards, March 2017.
Year(s) Of Engagement Activity 2016,2017
URL https://www.futurelearn.com/courses/antimicrobial-resistance-food-chain
 
Description NERC EPSRC DST India-UK Water Quality workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Scoping workshop November 2016 in prep for the Water Quality call issued February 2017. Opportunity to meet with academic and industry practitioners from India and the UK (especially the NERC-funded side). Updated on targeted and untargeted methods for steroid and pharmaceutical pollutants and their conjugate metabolites. High level of interest in these pollutants (expected more 'traditional' pollutants to be of interest) and flagged need for analytics including training and application. India - Reddys, Unilever, Mahathma Ghandi University / UK - Bath, Newcastle, Oxford.
Year(s) Of Engagement Activity 2016,2017
URL http://www.nerc.ac.uk/latest/events/list/water-workshop/
 
Description Severn Trent Water 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Meetings with Severn Trent Water on progress and initial outcomes from method developing and application from the BIOTRANS project. Conjugates of interest in wastewaters, need for understanding fate and transformation to bioactive to inform on current technologies. Potential discussed about evaluating Packington STW pilot technologies for BIOTRANS pollutant (targeted and untargeted method). STW highlighted well placed for next AMP and CIP III where pollutants extending to include AMR which pharmaceutical antibiotics act as co-selection drivers (March 2016, October 2016, February 2017).
Year(s) Of Engagement Activity 2016,2017
 
Description Severn Trent Water Sept 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Discussion and debate on research and innovation with Severn Trent water. Head of Corporate Partnerships for University of Nottingham attending alongside 6 Severn Trent Water members.
Year(s) Of Engagement Activity 2017
 
Description Sex Drugs and Engineering, Pint of Science, Nottingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Invited to present at the first Nottingham Pint of Science, May 2016. Delivered 'Sex Drugs and Engineering' in the stream 'Designed with you in mind: Creative engineering solutions'. Talked about the challenge and need for analytics to underpin the engineering of wastewater treatment. Focused on need for cross disciplinary research to understand how bioactive chemicals are excreted and analytical approaches to determine conjugates which act as precursors to the bioactive pollutant.
Year(s) Of Engagement Activity 2016
URL https://pintofscience.co.uk/event/designed-with-you-in-mind
 
Description UK Water Partnership LITSoN Industry / Academia Workshop on research and innovation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Invited to share overview of current research and innovation projects and where and how it applies to the water sector, and rating its potential impact against UKWIR's Big Questions (also represented selected projects to showcase University of Nottingham research). To feed into the UK Water Partnership (UKWP) reviewing research and innovation activity in the UK water sector to understand how it aligns with societal needs in a project called LITSoN. An initial pilot project in 2017 identified a number of opportunities for collaboration, currently being actively pursued by multiple water companies who are funding this follow-on work to collect more detailed information. Showcase my and UoN capabilities to water companies and across the sector, as well as receiving access to the whole national database.Series of workshops to collect data on current water research and innovation projects and stimulate collaboration. Key findings and recommendations will be shared with all participants and UKWP members in April to help inform future activities and identify collaboration opportunities. Details of individual projects will be shared amongst those organisations that release them.

• Opportunities to improve impact scores through industry collaboration ahead of REF2021
• Understand future funding opportunities through the Industrial Strategy Challenge Fund
• Showcase research for potential collaboration with water companies
• Gain insight into current research and innovation in the UK water sector to identify gaps and future priorities
Year(s) Of Engagement Activity 2016,2018
URL https://www.theukwaterpartnership.org/litson-review-of-innovation-in-uk-water-utilities/
 
Description XVI International Water Research Association World Water Congress, Mexico 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation under Late Breaking Research theme. Presented to international audience, with questions and discussion throughout 6 day conference. Reference - Shridharan Parthasarathy, Catherine A. Ortori, David A. Barrett, Kris Still and Rachel L. Gomes (2017) Simultaneous Analysis including Non-Targeted Approaches for Bioactive Chemicals in Wastewater from the United Kingdom using Liquid Chromatography Tandem Mass Spectrometry, XVI World Water Congress, Cancún, Mexico (29 May to 3 June, 2017).
Year(s) Of Engagement Activity 2017
URL http://worldwatercongress.com/