Pakistan-US Science and Technology Cooperation Program Phase 7 (2017 Deadline) Endocrine disrupting chemicals in Kabul and Swat rivers and their impact on fish populations and rural community livelihoods US Partner: Linda Lee, Purdue University Pakistan Partner: Bushra Khan, University of Peshawar   Project summary The presence of endocrine disrupting compounds (EDCs) in surface waters from industrial and municipal wastewater discharge has emerged as a serious issue globally. EDCs interfere with hormone systems in humans and animals, causing serious health and environmental problems. This project will evaluate the EDC types and concentrations in the Kabul and Swat Rivers; identify factors impacting EDC loads in this watershed; evaluate aquatic ecosystem health; assess local communities’ perceptions of water quality and river ecosystem health, their relationships to their lives and livelihoods, and their willingness to engage in conservation practices; and provide recommendations to reduce the impact of EDCs on river health for use by policymakers, researchers and regulatory agencies. Progress Reports 2020: Pakistan is one of the most water-stressed countries in the world, which is likely to worsen due to high population growth. River Kabul and its tributaries including River Swat are major freshwater sources in the KP province of Pakistan that serve the water needs of most of the Northern mountain and Northwestern plain areas of the province. These rivers also serve as a rich source of various fish species, which local populations depend on for their livelihood as well as the tourism industry. Untreated effluent discharges of more than 100 small and large-scale industrial units enter the Kabul and Swat rivers directly or indirectly. For more than a decade, there have been complaints about the declining water quality of these rivers and reduced crop production. There has also been a substantial decline in fish numbers as well as reports of mysterious fish kills, ultimately affecting the livelihoods of rural communities. The presence of endocrine disrupting compounds in surface waters from discharges from industrial and municipal wastewater has emerged as a serious issue globally. These chemicals interfere with the hormone system in humans and animals. Exposure to these chemicals even at low concentrations has been linked to numerous human health and ecosystem health problems including birth defects and other various developmental disorders. A United Nations report declared such compounds to be a ‘global threat’ to wildlife and humans, particularly infants and children. Nearly 1300 chemicals are known or suspected to be endocrine disrupting compounds This project is the first study we know targeted at determining the levels of endocrine disrupting chemicals in surface waters of Pakistan and assess their correlation to native fish biodiversity and the subsequent impact on rural population socioeconomic conditions. This project is highly collaborative with each project aim involving collaboration and mentoring between the US and Pakistani teams including PIs, co-PIs, and four Pakistani and three USA graduate student research scholars. Although our work focuses mostly on the SWAT and Kabul Rivers in the KP province, results from this study will be helpful in conducting other such studies on other Pakistani Rivers in the Punjab, Sindh and Baluchistan provinces. The ultimate long-term goal of this study is to aid in the design of a National River cleaning strategy for Pakistan and identify future research priorities. Project activities during the second project year focused on continued accomplishments of four of our six our project objectives 1-3 and 5. Objective (1) Evaluate the EDC types and concentrations in the Kabul and Swat Rivers. Objective (2) Identify factors impacting EDC loads in this watershed. Objective (3) Evaluate aquatic ecosystem health with a focus on fish diversity and selected reproductive endpoints. Objective (4) Correlate fish diversity/health with contaminant concentrations. Objective (5) Assess local communities’ perceptions of water quality and river ecosystem health, their relationships to their lives and livelihoods, and their willing to engage in conservation practices to improve the situation. Objective (6) Provide recommendations to reduce the impact of EDCs on river health for use by policy makers, researchers and regulatory agencies. We finished processing our quality assurance and quality control samples for the water and sediment samples followed by tabulating all the chemical data and sharing it via a google drive with all members on the team. Different team members were tasked with using and synthesizing the data for targeted publications (listed in publications as targeted publications) that we will be working on over the rest of 2020. Maps of quantitative data were made with ArcGIS Pro software for use in documentary and manuscripts. Maps were able to demonstrate differences between low flow and high flow seasons for ∑phthalates and ∑pharmaceutical concentrations at different sampling locations. Our chemical data in the water and sediment sampled from the Kabul and Swat river systems at three different times during our project of which two were during low flow conditions and one during high flow conditions were synthesized. High flows were in the order of 4.4-9.3 km/h in Swat and 1.6-5.2 km/h in Kabul. Low flows were in the order of 0.7 – 3.7 hm/h for the Kabul and <1.6 km/h for the Swat. Chemical data synthesized included quantitative data for 68 organic compounds as well as our results from additional screening to identify other organic compounds not captured in our initial screening and quantification analyses. We identified and quantified the occurrence of numerous organic compounds along both river systems. In general, seasonal differences were typically observed with compound concentrations being lower under high river flow conditions. High flow conditions resulting snowmelt tend to dilute chemical concentrations since these sources are unaffected by anthropogenic activity. Heavy rains during the monsoon seasons could bring in additional chemicals such as herbicides and other chemicals used in agricultural from soil being eroded into the river, but this may not lead to an observable increase in water concentrations due to the massive amounts of water. The concentration so of the various compounds detected varied at different river locations and seasonally. A group of endocrine disrupting compounds known as plasticizers called phthalates were found at all locations along both river systems. Bisphenol A, another plasticizer known to disrupt the endocrine system, was found in most samples but at lower concentrations than the phthalates. Several pharmaceuticals, including sulfamethoxazole, diclofenac, ibuprofen, acetaminophen, and naproxen were detected at some locations, but consistently at low concentrations. Pesticides frequently detected and quantified included DEET, which is commonly used in insect repellent, and butachlor, which is an herbicide commonly used in Pakistan. In the Swat River, total phthalate concentrations ranged from 100-500 ppt during high flow and 500-4300 ppt during low flow. Total pharmaceutical concentrations ranged from nondetect to 20 ppt during high flow and none detected to 820 ppt during low flow. In the Kabul River, total phthalate concentrations ranged from 200 - 900 ppt during high flow and 400 -1000 ppt during low flow. The total pharmaceutical concentrations ranged from nondetect to 10 ppt during low flow and up to 800 ppt during low flow. In the Kabul River, seasonal differences in phthalate concentrations were only observed at a few sampling points. Whereas, for pharmaceuticals, seasonal differences were consistently observed with pharmaceuticals being primarily detected only under low flow conditions. Bisphenol A was only quantified in the Swat and Kabul Rivers during low flow. In contrast, the pesticide butachlor was only quantified in the Swat River during the high-flow summer growing season since its use is agricultural. No seasonal effect was observed for the insecticide DEET. Differences in upstream and downstream concentrations were most clear under low flow conditions in the Swat River where concentrations downstream were typically higher than upstream. However, in the Kabul River, no clear trends were obvious between upstream and downstream concentrations of phthalates and pharmaceuticals. Of the chemicals that were found in the rivers, the phthalate class of compounds consistently occurred in both river systems and at the highest concentration by far of all the chemicals found. Phthalates are known to leach from plastics including labware; however, phthalates that may have leached during extraction of the water and sediment samples were accounted for in our quality assurance and quality control samples. Phthalates are also known to cause endocrine disruption, thus can alter fish heath and fish diversity. Although both the Kabul and Swat rivers had considerable amounts of phthalates, river Kabul had comparatively greater amounts. Phthalate concentrations were highest during the summer season. Phthalates are known to mimic hormones thus disrupt the endocrine system that controls sex differentiation in fish as well as several other health indices. Based on data from the United States Environmental Protection Agency Knowledgebase, the phthalates and bisphenol A concentrations water samples from both the Kabul and Swat rivers are below levels expected to produce an observable effect in fish. However, these phthalates were also the organic chemicals present at the highest concentrations in the sediments. Total phthalate concentrations in the sediments approached 60 ppm, which is up to 5 orders of magnitude greater than in the water samples. In addition, several other compounds were found in both the water and the sediment samples, albeit at much lower concentrations than the phthalates. However, the complex mixture of compounds may have additive effects on fish communities, but this is complex and hard to assess. High concentrations of phthalates in the river system are likely coming from solid wastes that enter the river or water that has passed through uncovered solid waste piles. In addition, several small pieces of plastics were consistently observed in the sediment samples, thus potentially serving as a continuous exposure source of phthalates to the benthic organisms. Also several fish species lay their eggs in the shallow fast moving sediment and rocky bottom of the rivers, thus phthalates desorbing form the sediments or plastic pieces in the sediment can lead to phthalate exposure to early life stages of aquatic organisms, which may lead to impacts on number and diversity of the adult fish communities. Fish Health Component: Our original objectives were to 1) Evaluate aquatic ecosystem health with a focus on fish diversity and selected reproductive endpoints, and 2) Correlate fish diversity/health with contaminant concentrations in Rivers Swat and Kabul. Between 2018 and 2019, a total of 121 fish belonging to 6 species were sampled and processed. Sites in River Swat included Madyan, Panjigram and Landakay and sites from Kabul River included Warsak Dam, and Akbarpura. Sites were selected a priori in relation to their location to urban and industrial sites since no chemical data was available at the time. Each fish was measured, weighed, and general health and reproductive assessment measured using a combination of blood parameters and overall body condition including reproductive condition. Because potential effects of contaminants are known to vary widely across species, comparisons of fish health between sites needs to be done by species. Unfortunately, we did not collect the same fish species from all sites within each River system, therefore we could not compare directly across sites and no conclusions related to the potential impact of chemicals on fish populations can be made at this point. We have processed fillets from all of these fish and extracts will be chemically analyzed in the next month. It is worth noting that some chemical concentrations in sediments exceeded threshold values known to cause negative impacts on fish and other aquatic organisms. We also quantified several water quality parameters and noted that poor water and habitat quality are likely major drivers of fish population dynamics in these sites. More studies need to be conducted in these River systems to determine the ultimate cause of fish population declines. Social Science Component: Overall, we completed 25 semi-structured interviews with community leaders and members in the research area to understand how water quality and quantity impact livelihoods of water users. We obtained over 900 minutes of interview recording as our qualitative data. Using results from these interviews, we developed a survey questionnaire and completed 445 household surveys. Through this data collection, 1 US graduate student and 6 Pakistani graduate students were trained in social science research methods. This data shows that livelihoods in this area are significantly shaped by changes in water quality and quantity. Specifically, there was nearly universal agreement that water quality in both the Swat and Kabul Rivers had decreased in the past ten years. Further, nearly all respondents reported a substantial decrease in the fish numbers and size. While households continue to rely heavily on the water system for irrigation, fishing, tourism, and domestic supply, decreasing quality and quantity has driven the need to adapt to these social-ecological changes. For example, respondents reported diversifying their livelihoods, changing their water source, and increasing their agricultural inputs in response to increasing social-ecological change and water stress. Respondents report that these adaptations often require time, money, information, and support from others in the household, therefore not all households are able to adapt in ways that would improve their water supply. Together, our research provides insights on strategies that can be used to support local communities’ capacity to adapt in ways that improve their food security, livelihoods, and well-being. We also developed a survey and interviewed 15 leaders in the water sector. This was to be followed by an interactive workshop, but due to the COIVD-19 situation, we were unable to hold the workshop. However, we did draft a documentary with a summary of the findings from this work coupled with thoughts, concerns and/or frustrations voiced by some of the leaders who also want to promote behaviors that will improve water quality. We will continue to work over the next few months to revise our documentary and to submit our work in peer-reviewed journals. Acknowledgments: The grantees are thankful to the Higher Education Commission and Pakistan-U.S. Science and Technology Cooperation Program (HEC/ R&D/ PAK-US/ 2017/ 783), Pakistan for providing financial support for this research collaboration. 2019: Pakistan is one of the most water-stressed countries in the world, which is likely to worsen due to high population growth. River Kabul and its tributaries including River Swat are major freshwater sources in the KP province of Pakistan that serve the water needs of most of the Northern mountain and Northwestern plain areas of the province. These rivers also serve as a rich source of various fish species, which local populations depend on for their livelihood as well as the tourism industry. Untreated effluent discharges of more than 100 small and large-scale industrial units enter the Kabul and Swat rivers directly or indirectly. For more than a decade, there have been complaints about the declining water quality of these rivers and reduced crop production. There has also been a substantial decline in fish numbers as well as reports of mysterious fish kills, ultimately affecting the livelihoods of rural communities. The presence of endocrine disrupting compounds in surface waters from discharges from industrial and municipal wastewater has emerged as a serious issue globally. These chemicals interfere with the hormone system in humans and animals. Exposure to these chemicals even at low concentrations has been linked to numerous human health and ecosystem health problems including birth defects and other various developmental disorders. A United Nations report declared such compounds to be a ‘global threat’ to wildlife and humans, particularly infants and children. Nearly 1300 chemicals are known or suspected to be endocrine disrupting compounds This project is the first study we know targeted at determining the levels of endocrine disrupting chemicals in surface waters of Pakistan and assess their correlation to native fish biodiversity and the subsequent impact on rural population socioeconomic conditions. This project is highly collaborative with each project aim involving collaboration and mentoring between the US and Pakistani teams including PIs, co-PIs, and four Pakistani and three USA graduate student research scholars. Although our work focuses mostly on the SWAT and Kabul Rivers in the KP province, results from this study will be helpful in conducting other such studies on other Pakistani Rivers in the Punjab, Sindh and Baluchistan provinces. The ultimate long-term goal of this study is to aid in the design of a National River cleaning strategy for Pakistan and identify future research priorities. Project activities during the second project year focused on continued accomplishments of four of our six our project objectives 1-3 and 5. Objective (1) Evaluate the EDC types and concentrations in the Kabul and Swat Rivers. Objective (2) Identify factors impacting EDC loads in this watershed. Objective (3) Evaluate aquatic ecosystem health with a focus on fish diversity and selected reproductive endpoints. Objective (4) Correlate fish diversity/health with contaminant concentrations. Objective (5) Assess local communities’ perceptions of water quality and river ecosystem health, their relationships to their lives and livelihoods, and their willing to engage in conservation practices to improve the situation. Objective (6) Provide recommendations to reduce the impact of EDCs on river health for use by policy makers, researchers and regulatory agencies. Our accomplishments in Year 2 continued to focus completing Objectives 1-3 and 5. In evaluating our progress, we have generally achieved or exceeded our Year 2 Milestones. We conducted a second round of sampling of the Kabul and Swat rivers, conducted sediment sampling of a subset of sites on the Kabul and Swat rivers, performed extraction and chemical analysis of all water and sediment samples taken in Years 1 and 2, conducted a second round of fish sampling for the River Kabul and analyzed all fish data from Years 2 and 3, developed an IRB-approved community survey and completed surveys of three communities (Jehangira, Madyan, Landakay) considered representative of communities that directly benefit or may be impacted by river health.. We also nearly completed additional quality assurance and quality control studies on the in-field active sampling method. In January 2018, the Pakistan team sampled river samples from the SWAT River (2 sites) and the Kabul river (4 sites), which were processed and sent to Purdue to analyze as a first step of identifying the type of endocrine disrupting chemicals present in the rivers. Several pharmaceuticals and compounds released from personal care products along with a few hormones and pesticides were found. This was followed up with additional and more extensive sampling of both rivers (10 from Swat River and 9 from Kabul River) by the USA and Pakistani team together in June 2018 using a solar-powered battery-operated metering pump on-site that allowed the capture of chemicals from a large volume of water within one hour. Additional water samples were collected from the Swat and Kabul rivers in February 2019 at the same locations sampled in June 2018 to determine how low-flow influences the contaminant load in the rivers. Water samples from the Swat and Kabul rivers from January 2018, June 2018, and February 2019 were extracted and analyzed at Purdue University. Consistent results across river water and river sediment samples from each sampling event indicate that particulary phthalates, a class of many different chemicals utilized in plastics, cosmetics, and other everyday items, are the predominant compounds present in both rivers. Phthalates and bisphenol A are known endocrine disruptors which have been shown in scientific studies to cause harmful effects on fish development and fish communities. During the USA’s June 2018 trip to Pakistan, seven sites from the Swat and Kabul Rivers were sampled for fish health. From each site, we processed ~ 30 individual fish. For the Swat River, Swati was the fish selected, as it is common in this area and an important fish for human consumption. For the Kabul River, common carp was sampled, as Swati fish are not present in this area. Carp are also an important fish for locals and are abundant and easy to capture. In Year 2, fish were collected from only River Kabul (Warsak and Akbarpura), but using the same methods as in Year 1. Livers from the fish of a reference site exhibited normal structures while livers from the fish of polluted sites did not appear normal. The gonads (ovaries and testes) from the fish of the reference site also exhibited a normal structure whereas the gonads from the fish sampled at the polluted sites showed ovary degeneration and disorganization in structures within the testes. Surveying the local communities’ perceptions of water quality and river ecosystem health, their relationships to their livelihoods and their willingness to engage in conservation practices to improve the situation was completed. Towards Objective 5 (assess local communities’ perceptions of water quality and river ecosystem health, their relationships to their lives and livelihoods, and their willing to engage in conservation practices to improve the situation), interview data from Year 1 was completed and led to the development of a survey to increase our understanding of tradeoffs in adaptation decision making, which was implemented this year (Year 2). We completed 448 surveys with heads of households in Jehangira, Madyan, and Landaky during spring of 2019. The survey gathered information on household demographics and livelihoods strategies, perceptions of stress on their agricultural and domestic water supplies, adaptation strategies they have employed, and finally, on the factors that influence their decision to employ or not employ specific adaptation strategies. From the preliminary analysis of the interview and survey data revealed environmental management and livelihood diversification as predominate adaptation strategies. For example, respondents repeatedly report decreasing or exiting fishing as a livelihood strategy due to diminishing fish population and increasing agricultural inputs in response to decreasing yields. 2018: Pakistan is one of the most water-stressed countries in the world, which is likely to worsen due to high population growth. River Kabul and its tributaries including River Swat are major freshwater sources in the Khyber Pakhtunkhwa (KP) province of Pakistan that serve the water needs of most of the Northern mountain and Northwestern plain areas of the province. These rivers also serve as a rich source of various fish species, which local populations depend on for their livelihood as well as for the tourism industry. Untreated effluent discharges of more than 100 small and large-scale industrial units enter the Kabul and Swat rivers directly or indirectly. For more than a decade, there have been complaints about the declining water quality of these rivers and reduced crop production. There has also been a substantial decline in fish numbers as well as reports of mysterious fish kills, ultimately affecting the livelihoods of rural communities. The presence of endocrine disrupting compounds (EDCs) in surface waters from discharges from industrial and municipal wastewater has emerged as a serious issue globally. These chemicals interfere with the hormone system in humans and animals. Exposure to these chemicals even at low concentrations has been linked to numerous human health and ecosystem health problems including birth defects and other various developmental disorders. A United Nations report declared such compounds to be a ‘global threat’ to wildlife and humans, particularly infants and children. Nearly 1300 chemicals are known or suspected to be EDCs. To our knowledge, ours is the first study targeted at determining the levels of EDCs in surface waters of Pakistan, assessing their relationship with native fish biodiversity and the subsequent impact on the socioeconomic conditions of rural populations. This project is highly collaborative with each project aim involving collaboration and mentoring between the US and Pakistani teams including PIs, co-PIs, and four Pakistani and three USA graduate student research scholars. Although our work focuses mostly on the Swat and Kabul Rivers in the KP province, results from this study will be helpful in conducting other such studies on other Pakistani Rivers in the Punjab, Sindh and Baluchistan provinces. The ultimate long-term goal of this study is to aid in the design of a National River cleaning strategy for Pakistan and to identify future research priorities. Our activities during our first project year focused on accomplishments under 1-3 and 5 of our 6 following project objectives: 1) Evaluate the EDC types and concentrations in the Kabul and Swat Rivers 2) Identify factors impacting EDC loads in this watershed 3) Evaluate aquatic ecosystem health with a focus on fish diversity and selected reproductive endpoints 4) Correlate fish diversity and health with contaminant concentrations 5) Assess local communities’ perceptions of water quality and river ecosystem health, their relationships to their lives and livelihoods, and their willing to engage in conservation practices, and 6) Provide recommendations to reduce the impact of EDCs on river health for use by policy makers, researchers and regulatory agencies In evaluating our progress, we have met and exceeded our proposed Year 1 Milestones for these objectives, which were facilitated by having an extremely collaborative research team and a revision on the method used to sample the river water. We collaboratively prepared maps of the area around the Swat and Kabul rivers annotated with discharges and activities in the region that affect water quality and quantity. Pakistan colleagues surveyed the rivers to facilitate identifying key sources of discharge points and input prior to the USA team visit. Pakistan colleagues also helped the teams access other types of data such as river discharge data, land use data and soil maps, which are needed for use in an assessment tool to quantify the impact of land management practices in large complex watersheds. In January 2018, the Pakistan team sampled river samples from the Swat River (two sites) and the Kabul river (four sites), which were processed and sent to Purdue for analysis to begin to identify the type of EDCs present in the rivers. Several pharmaceuticals and compounds released from personal care products along with a few hormones and pesticides were found. This was followed up with more extensive sampling of both rivers (10 samples from Swat River and nine from Kabul River) by the USA and Pakistani team together in June 2018 using a solar-powered battery-operated metering pump on-site that allowed the capture of chemicals from a large volume of water within one hour. At each sampling point, general and specific environmental conditions as well as parameters regarding the discharge of rivers were also measured. During the sampling process, training of Pakistani students and faculty on sampling procedures and collection of ancillary data occurred. Samples are currently being analyzed at Purdue University. During the USA’s June 2018 trip to Pakistan, seven sites from the Swat and Kabul Rivers were sampled for fish health. From each site, we processed ~30 individual fish. For the Swat River, Swati was the fish selected, as it is common in this area and an important fish for human consumption. For the Kabul River, common carp was sampled, as Swati fish are not present in this area. Carp are also an important fish for locals and are abundant and easy to capture. Dr. Sepúlveda trained PhD student Saba Shoukat, Peshawar University, on how to assess fish health, including how to bleed, necropsy, age and collect tissues for different analyses. Several different health measurements were made on approximately 200 fish during the USA team’s trip. The first phase of assessing local communities’ perceptions of water quality and river ecosystem health, their relationships to their livelihoods and their willingness to engage in conservation practices to improve the situation was completed. This consisted of 13 interviews on Swat river and 12 interviews on Kabul river with informal and formal community leaders, farmers, and fishermen. The interview effort was led by a Purdue PhD student and supported by three University of Peshawar graduate students. Translations of the first few recorded interviews began while still in Pakistan. While in the field, we also observed relevant sites along the rivers including fisheries businesses, hydropower and industrial development areas, and waste dumping sites. Also, while in Pakistan, the USA team was able to have numerous discussions, visit other Pakistan faculty outside the project team, and enjoy the culture unique to the area. Prior to the USA team trip, the Pakistan team put on a two-day workshop on “Status of River Swat water quality and threats to native fish species” in March 2018, which helped plan and facilitate the USA team’s visit. The workshop was attended by students, technicians, faculty members, and members of the business community, political leadership, and local administration with participants from Pakistan, Canada, and the USA. The workshop was organized in collaboration with the following organizations: Higher Education Commission of Pakistan, Canadian High Commission, World Community Services, Department of Environmental Sciences, University of Peshawar, and University of Agriculture Peshawar. In addition to the US team visit to Pakistan in June 2018, the Pakistan principle investigator (PI) spent a month in July-August 2017 at Purdue University with the USA PI.