Trishuli Assessment Tool Field Manual A Standardized Methodology for Freshwater Aquatic Biodiversity Sampling and Long-Term Monitoring for Hydropower Projects in the Himalayan Region IN PARTNERSHIP WITH About IFC IFC—a member of the World Bank Group—is the largest global development institution focused on the private sector in emerging markets. We work in more than 100 countries, using our capital, expertise, and influence to create markets and opportunities in developing countries. In fiscal year 2021, IFC committed a record $31.5 billion to private companies and financial institutions in developing countries, leveraging the power of the private sector to end extreme poverty and boost shared prosperity as economies grapple with the impacts of the COVID-19 pandemic. For more information, visit www.ifc.org. © International Finance Corporation. First printing, December 2021. All rights reserved. 2121 Pennsylvania Avenue, N.W. 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Additionally, “International Finance Corporation” and “IFC” are registered trademarks of IFC and are protected under international law. All other product names, trademarks and registered trademarks are property of their respective owners. All photographs: ©IFC unless otherwise stated Citation: IFC. 2021. Trishuli Assessment Tool Field Manual. International Finance Corporation. Washington, D.C., USA. Table of Contents Forewords |5 Acknowledgments |7 Acronyms and Abbreviations |9 List of Boxes, Figures, and Tables | 10 1. INTRODUCTION | 14 1.1 Overview and Applications of the Trishuli Assessment Tool | 14 1.2 Trishuli Assessment Tool at a Glance | 15 1.3 Why Sample and Monitor Freshwater Aquatic Biodiversity | 16 1.4 Questions Addressed with the Trishuli Assessment Tool | 17 2. FIELD METHODOLOGY | 19 2.1 Sampling Design for Environmental Impact Assessment | 19 2.1.1 What to Sample for the EIA—Aquatic Biodiversity Indicators | 19 2.1.2 Where to Sample for the EIA—Sampling Sites | 20 2.1.3 When to Sample for the EIA—Seasonality | 22 2.2 Sampling Design for Long-Term Monitoring | 22 2.2.1 What to Sample for Long-Term Monitoring—Aquatic Biodiversity Indicators | 23 2.2.2 Where to Sample for Long-Term Monitoring—Sampling Sites | 23 2.2.3 When to Sample for Long-Term Monitoring—Seasonality | 24 2.3 How to Sample for the EIA and Long-Term Monitoring | 25 2.3.1 Preparation for Field Sampling | 25 2.3.2 Field Team | 26 2.3.3 Site Sampling Design | 26 2.3.4 Habitat Descriptions | 26 2.3.5 Associated Data to Collect | 27 2.4 Fish Field Sampling Methods | 27 2.4.1 Field-Method Selection | 27 2.4.2 Sampling Effort for Each Field Method | 28 2.4.3 Specifics of Fish Field Sampling Methods | 29 Backpack Electrofishing | 29 Cast Net | 31 Dip Net | 32 Underwater Video | 33 Environmental DNA | 34 2.4.4 Monitoring Fish Movement through a Fish Ladder | 36 2.4.5 How to Record Fish Data | 37 2.4.6 How to Process the Fish Collections | 38 2.5 Field Sampling Method for Macroinvertebrates | 39 2.5.1 Multihabitat Sampling Using Kick Net | 41 2.6 Field Sampling Method for Periphyton | 44 3 3. DATA ANALYSIS AND PRESENTATION | 47 3.1 Introduction | 47 3.2 Fish Metrics | 47 Metric 1: Fish Species Richness | 50 Metric 2: Species Composition | 51 Metric 3: Proportion of Individuals of Each Fish Species | 54 Metric 4: Distribution of Target Fish Species | 55 Metric 5: Relative Abundance of Target Fish Species | 57 Metric 6: Recruitment of Target Fish Species (Relative Abundance of Juveniles) | 59 Metric 7: Length of Target Fish Species | 62 3.3 Macroinvertebrate Metrics | 65 Metric 1: Macroinvertebrate Taxa Richness and Proportion | 66 Metric 2: EPT Index | 67 Metric 3: Relative Abundance of Functional Feeding Groups | 69 3.4 Periphyton Metrics | 71 3.5 Preliminary Assessment of No Net Loss or Net Gain for International Lenders | 72 4. REPORTING | 75 4.1 Overview | 75 4.2 Sample ESIA Report—Aquatic Biodiversity Baseline Chapter | 75 4.3 Sample BMEP Report for Monitoring Aquatic Biodiversity for a Hydropower Project | 76 5. REFERENCES | 79 6. APPENDIXES | 82 Appendix A Field Data Sheet for Fish Data Recording | 82 Appendix B Field Data Sheet for Recording Site and Habitat Characteristics | 83 Appendix C Data Sheets for Macroinvertebrate Field Data Recording | 84 Appendix C.1 Site Information Sheet | 84 Appendix C.2 Habitat Estimation Sheet | 85 Appendix D Data Sheet for Periphyton Sampling Field Data | 86 Appendix E Sample Macroinvertebrate Field Data for the Trishuli River | 87 Appendix F Detailed Instructions for Conducting Backpack Electrofishing | 90 Appendix G Best Practice Manual for Backpack Electrofishing | 91 4 Foreword Hydropower projects can be transformational in nature for a variety of reasons. They may produce a step change in electricity supply that supports electrification; they may back the integration of variable renewable energy (VRE); they may bring multipurpose benefits such as flood control or climate change mitigation; or they may support regional integration. It is this transformational nature of hydropower projects that often make them both complex and rewarding to pursue. If planned sustainably, they can provide benefits to local communities. Hydropower projects have always faced a range of environmental and social problems, but today, it is recognized that the knowledge base and tools are in place to ensure that projects are implemented sustainably and responsibly, following best practices. In some countries like Nepal, the transformational nature of a project can therefore be in demonstrating this good practice and building capacity to hold future projects to an agreed standard. Making sure that Nepal’s rich biodiversity is conserved while developing large infrastructures such as hydropower projects and dams will be of paramount importance. Aquatic biodiversity preservation needs even more support. Recent studies found that most hydropower projects are not adequately considering their impacts on the environment, particularly Nepal’s important freshwater resources and threatened aquatic species. Basin planning based on strategic environmental and social impacts is often missing. Hydropower EIAs need to more robustly assess aquatic resources and biodiversity to properly assess impacts and develop mitigation to help maintain freshwater resources while developing hydropower in Nepal. To this end, the Trishuli Assessment Tool provides a standardized approach that will enhance hydropower project EIAs and promote monitoring of aquatic resources, helping in aquatic biodiversity conservation. The World Bank and IFC encourage hydropower projects to consider adopting an approach such as that offered in this field manual to adequately assess and monitor aquatic biodiversity. Robust environmental and social assessment is the first step in ensuring good practice for planning and implementing sustainable hydropower that will benefit Nepal’s people while safeguarding its natural environment. Pravin Karki Global Lead for Hydropower & Dams The World Bank Group, USA 5 Foreword Wisdom on freshwater resource management of the Himalayan region is crucial for sustainable development in most Trans-Himalayan countries. Developing hydropower in the region faces many challenges, including climate change and the preservation of globally threatened fish species. In recent years, decommissioning of hydropower dams due to safety, law, policy, economy, and ecology has even become a trend in other parts of the world. Yet, hydropower development, if done properly by taking into account a deeper understanding of fish migration patterns and ecosystem services in mid-hill rivers, could facilitate sustainable energy production. Despite a proliferation of hydropower projects in the Himalayas, knowledge of fish behaviors in high-altitude areas remains rudimentary. The water basins of the mid-hills with many endemic fishes are also highly feasible areas for hydropower projects. Thus, caution, along with wisdom, is required to protect endemic and migratory fish species. It is a matter of great acknowledgment that many international lenders, such as IFC and the World Bank, require hydropower projects to avoid a net loss of biodiversity values for critical habitats. Such wisdom should be adopted by other lenders and institutions for sustainable hydropower development. Recent studies have highlighted the importance of the development of fish sanctuaries as well as declaration of national parks for conserving rare, vulnerable, endemic, and key fish species close to hydropower locations. The Trishuli Assessment Tool—developed following a workshop held in Nuwakot—shares new information and approaches for conducting proper environmental impact assessments (EIAs) of hydropower projects. Designed to collect and analyze field data, the tool provides a standardized approach to enhance the hydropower EIAs for monitoring aquatic biodiversity focusing on fish, macroinvertebrates, and periphyton. The tool describes sampling and interpretation methods in simple, precise, and clear language, which should be highly useful and practical for those who need to perform such EIAs. I would like to congratulate all associated authors and IFC for bringing this important publication to fruition. Tek Bahadur Gurung Adjunct Professor, Fisheries Program, Agriculture and Forestry University Former Executive Director, Nepal Agricultural Research Council 6 Acknowledgments This field manual is based on the Trishuli Assessment Tool, which was developed at a workshop in November 2019 by more than 30 international and Nepalese aquatic biology specialists, government scientists, and hydropower environmental staff, as part of IFC’s Nepal Hydropower Environmental and Social Advisory Program in Nepal. The tool was field tested on the Trishuli River in February 2020. The project was led by a team of aquatic biodiversity specialist consultants hired by IFC: • William Beaumont, Electric Fishing Technical Services Ltd., United Kingdom • Julie Claussen, Fisheries Conservation Foundation, USA • Dibesh Karmacharya, Center for Molecular Dynamics Nepal • David Philipp, Fisheries Conservation Foundation, USA • Adrian Pinder, Bournemouth University, United Kingdom • Adarsh Man Sherchan, Center for Molecular Dynamics Nepal • Deep Narayan Shah, Central Department of Environmental Science, Tribhuvan University, Nepal • Ram Devi Tachamo Shah, Aquatic Ecology Centre, Kathmandu University, Nepal • Gina Walsh, Independent Consultant, South Africa The Trishuli Assessment Tool and preparation of this field manual were initiated and coordinated by IFC Biodiversity Consultant Leeanne E. Alonso as part of IFC’s Nepal Hydropower Environmental and Social Advisory Program, led by Senior Asia Environmental, Social, and Governance Advisory Lead Kate Lazarus. Many thanks to Adrian Pinder for assisting with the data analysis section, Upasana Shrestha for logistical support, Helen Ho Yan Luk for editing, and Martinus Johannes for design. We are grateful to William Beaumont for providing electrofishing guidance in Appendixes F and G as well as numerous reviewers for their helpful comments. We would also like to thank the governments of Australia, Norway, and Japan for their support. Special thanks to all participants of the Trishuli Assessment Tool workshop including: • Nurendra Aryal, Department of National Parks and Wildlife Conservation, Ministry of Forests and Environment, Nepal • Ashok Baniya, Nepal Water and Environment Development Corporation Upper Trishuli – 1 Hydropower Project, Nepal • Auras Bhandari, Nepal Water and Environment Development Corporation Upper Trishuli – 1 Hydropower Project, Nepal • Tara Datt Bhatt, Nepal Electricity Authority Training Center • Milan Dhungana, Ministry of Forests and Environment, Nepal • Prakash Gaudel, Nepal Electricity Authority • Tek Bahadur Gurung, Nepal Agricultural Research Council • Bibhuti Ranjan Jha, Kathmandu University, Nepal • Janak Kumar Jha, Water and Energy Commission Secretariat, Nepal • Baburaja Maharjan, Nepal Electricity Authority • Raj Kapur Napit, Freelance Consultant, Nepal 7 • Umesh Pathak, Upper Sanjen Hydropower Project, Nepal • Mark Pedersen, IFC, USA • Nikita Pradhan, Center for Molecular Dynamics Nepal • Shankar Pyakurel, Trishuli Hydropower Company, Nepal • Asha Raymajhi, Fisheries Research Division, Ministry of Agriculture and Livestock Development, Nepal • Subodh Sharma, Kathmandu University, Nepal • Anjana Shrestha, Center for Molecular Dynamics Nepal • Rabindra Timilsina, Swet Ganga Hydropower & Construction Ltd., Nepal • Suresh Wagle, Program on Aquatic Natural Resources Improvement, United States Agency for International Development, Nepal • Rakesh Yadav, Freelance Consultant, Nepal Environmental and Scientific Services, Nepal Participants at the November 2019 workshop where the Trishuli Assessment Tool was developed. 8 Acronyms and Abbreviations AC Alternating current APHA American Public Health Association Inc. BMEP Biodiversity monitoring and evaluation program/plan CPR Cardiopulmonary resuscitation CMDN Center for Molecular Dynamics Nepal CPUE Catch per unit effort CSBI Cross-Sector Biodiversity Initiative DC Direct current eDNA Environmental DNA EDTA Disodium ethylenediaminetetraacetic acid EFlow Environmental flow EIA Environmental impact assessment EPT Ephemeroptera, Plecoptera, and Trichoptera ESIA Environmental and social impact assessment FRTC Forest Research and Training Center GBIF Global Biodiversity Information Facility GIS Geographic information system GPS Global positioning system HPP Hydropower project IFC International Finance Corporation IUCN International Union for Conservation of Nature MoFE Ministry of Forests and Environment NNL No net loss RTU Recognizable taxonomic unit Units cm centimeter Hz hertz M molarity (moles per liter) m meter m² square meters ml milliliter mm millimeter mm² square millimeters MW megawatt µm micrometer 9 LIST OF BOXES 22 | Box 2.1 Key Elements of Sampling Design for the EIA 25 | Box 2.2 Key Elements of Sampling Design for Long-Term Monitoring 30 | Box 2.3 Electrofishing Equipment 31 | Box 2.4 Cast-Net Equipment 32 | Box 2.5 Dip-Net Equipment 34 | Box 2.6 Underwater Video Equipment 43 | Box 2.7 Macroinvertebrate Sampling Equipment 45 | Box 2.8 Periphyton Sampling Equipment 57 | Box 3.1 CPUE Definition LIST OF FIGURES 20 | Figure 2.1 Aquatic Habitats within a River 21 | Figure 2.2 Example Satellite Image Showing Different Habitat Types in the Main Stem River and a Tributary within the Area of Impact of an HPP 21 | Figure 2.3 Recommended Sampling Design to Collect Aquatic Data for an EIA Baseline or Long-Term Monitoring of an HPP 24 | Figure 2.4 Example of Sampling Design for Monitoring HPP Impacts on Aquatic Biodiversity 30 | Figure 2.5 Backpack Electrofisher and its use in the Rocky Streams of the Trishuli River Basin 31 | Figure 2.6 Fisher Throwing Cast Net in the Trishuli River 32 | Figure 2.7 Two types of dip nets 33 | Figure 2.8 Researcher Holding Video Camera Underwater in a Tributary 34 | Figure 2.9 Examples of GoPro Waterproof Video Cameras 35 | Figure 2.10 Environmental DNA Process 38 | Figure 2.11 Example of Fish Field Sampling Data Sheet 40 | Figure 2.12 Macroinvertebrate Orders and Sensitivity to Pollutants in River Basin 41 | Figure 2.13 Sampling Process for Benthic Macroinvertebrates 42 | Figure 2.14 Categorization of Aquatic Habitat Types for Multihabitat Field Sampling Using a Kick Net 50 | Figure 3.1 Number of Fish Species Recorded Per Site 53 | Figure 3.2 Important Fish Species 10 54 | Figure 3.3 Pie Charts Showing Species and Percentages of Fish Recorded in Various Sampling Regions 56 | Figure 3.4 Schizothorax richardsonii Distribution Map across the Sampling Sites 56 | Figure 3.5 Schizothorax progastus Distribution Map across the Sampling Sites 58 | Figure 3.6 CPUE of S. richardsonii by Electrofishing Upstream of Dam, Spring 2021 59 | Figure 3.7 Bar Charts Presenting Spring Survey Field Data (Electrofishing) 61 | Figure 3.8a Bar Charts of CPUE and Density for Five Tributary Sites 61 | Figure 3.8b Density of Juveniles per 100 m² for Five Tributary Sites in Spring Surveys over Three Years 63 | Figure 3.9 Mean Fish Length at Four Tributary Sites, Spring 2021 64 | Figure 3.10 Mean Fish Length at Four Tributaries over Four Years 66 | Figure 3.11 Number of Macroinvertebrate Genera per Site 67 | Figure 3.12 Images of EPT Taxa 69 | Figure 3.13 EPT Index at Three Different Sites 69 | Figure 3.14 EPT Taxa as Percentage of All Taxa at Three Different Sites 70 | Figure 3.15 Relative Abundance of Each Functional Feeding Group at Three Sites, Spring 2021 70 | Figure 3.16 Relative Abundance of Each Functional Feeding Group at Site UCH over Three Years 73 | Figure 3.17 CPUE of Schizothorax richardsonii for One Site over Nine Annual Surveys 73 | Figure 3.18 CPUE of Schizothorax richardsonii for One Site over 12 Annual Surveys LIST OF TABLES 19 | Table 2.1 Key Aspects of Monitoring the Three Target Groups 27 | Table 2.2 Comparison of Fish Catch Using Cast Nets and Electrofishing in the Trishuli River Tributaries in February 2020 28 | Table 2.3 Field Methods for Each of the Fish Indicators to Be Used in Each Habitat Type 28 | Table 2.4 Sampling Effort Per Site for Each of the Fish Field Sampling Methods 37 | Table 2.5 Comparison of Fish-Ladder Automated Monitoring Techniques 39 | Table 2.6 Functional Feeding Groups and Food Resources of Benthic Macroinvertebrates 48 | Table 3.1 Recommended Metrics for Fish Data Analysis 48 | Table 3.2 Sample Field Data Presentation for EIA and Monitoring Reports 50 | Table 3.3 Example Summary Data: Number of Fish Species Recorded per Site 11 52 | Table 3.4 Species Recorded by All Sampling Methods in Spring 2021 53 | Table 3.5 Presence or Absence of Fish Species Upstream of Dam with All Methods Combined 54 | Table 3.6 Number of Fish Recorded Upstream of Dam (Six Sites)—All Methods Combined 57 | Table 3.7 Sample CPUE Conversion 58 | Table 3.8 Electrofishing Field Data, Spring 2021 58 | Table 3.9 Summary Data for Spring Survey Field Data (Electrofishing) 60 | Table 3.10 Schizothorax richardsonii Juveniles (Electrofishing) 60 | Table 3.11 S. richardsonii Juveniles (Cast Nets) 61 | Table 3.12 S. richardsonii Juveniles (Targeted Electrofishing in Selected 100 m² Areas in Spring Sampling) 63 | Table 3.13 Fork Length Measurements for S. richardsonii at Four Tributary Sites 64 | Table 3.14 Mean Fish Length at Four Tributaries over Four Years 65 | Table 3.15 Summary of Recommended Macroinvertebrate Metrics 66 | Table 3.16 Number of Macroinvertebrate Genera per Site 68 | Table 3.17 Number of Macroinvertebrate Genera for All Sites at the Trushuli River 69 | Table 3.18 Number of EPT Taxa per Site 70 | Table 3.19 Number of Individuals for Each Functional Feeding Group at Three Sites, Spring 2021 70 | Table 3.20 Number of Individuals for Each Functional Feeding Group at Site UCH over Three Years 71 | Table 3.21 Periphyton Metric 73 | Table 3.22 Adaptive Management Thresholds 12 � Introduction 1.1 Overview and Applications For fish, the field methods include cast nets, which are typically used to collect freshwater fish for of the Trishuli Assessment Tool hydropower EIAs in Nepal. Studies have shown that cast nets alone are only moderately effective The Trishuli Assessment Tool is a standardized for catching fish, thus many fish species are methodology for sampling freshwater aquatic missed. The tool adds the method of electrofishing, biodiversity in hydropower projects. This tool which is highly effective for sampling fish but was developed to: 1) strengthen the collection requires training and can only be used in low-flow of aquatic biodiversity data for environmental and clear waters, such as tributaries. Additional impact assessments (EIAs)1 and international-level methods of dip nets and underwater video add environmental and social impact assessments data for tributaries. The emerging technology of (ESIAs) and 2) provide a simple yet standardized environmental DNA (eDNA) is also part of the method for the long-term monitoring of aquatic field methodology, as it can be extremely effective biodiversity in relation to hydropower projects. at detecting species that are not captured by other The Trishuli Assessment Tool project is a field methods. follow-up to the cumulative impact assessment This field manual is ideal for use by environmental of the Trishuli River Basin led by IFC (2020), staff, consultants, researchers, academics, and which identified the need for more robust and government agencies to collect robust data standardized sampling of aquatic biodiversity for EIAs and monitor aquatic habitats and when planning hydropower projects. The tool biodiversity to evaluate impacts of hydropower was developed by a group of 30 international projects and the success of mitigation measures. and Nepalese aquatic scientists at a workshop The data analyses presented in this manual in 2019 and tested during a field survey in 2020 allow hydropower projects to track changes in (Philipp et al. 2020; IFC 2021). It provides a field specific indicators between the pre-construction sampling methodology for three focal groups of baseline and the construction and operational aquatic biodiversity: fish, macroinvertebrates, stages. This can help demonstrate if a hydropower and periphyton as indicators of overall aquatic project’s mitigation measures successfully biodiversity. The collected data document maintain aquatic biodiversity, resulting in no net species richness and relative abundance of fish loss or even a net gain of biodiversity values to and macroinvertebrates as well as provide a comply with government and international lender measure of the status and health of the aquatic requirements.2 ecosystem. For fish, the group evaluated and field tested many aquatic sampling methods and This manual is applicable to all types of concluded that the following methods are best for hydropower projects (HPPs), from small run-of- assessing and monitoring fish in the Himalayan river to larger peaking projects because all of them region: backpack electrofishing, cast nets, dip have some impact on the aquatic environment. nets, underwater video, and environmental DNA Evaluating and monitoring aquatic biodiversity (eDNA). This field manual provides guidance for before, during, and after construction of an HPP implementing the Trishuli Assessment Tool in the provides essential data to guide the project on how rivers of Nepal and other Himalayan regions. to reduce its impacts on the aquatic environment. 1 For the purpose of this manual, EIAs cover both national-level and international-level EIAs. Where used, an EIA refers to international lender requirements and Good International Industry Practice. 2 International lenders include institutions such as the World Bank, IFC, Asian Development Bank, Asian Infrastructure Investment Bank, Korea Exim Bank, CDC Group, and Japan International Cooperation Agency. 14 1.2 Trishuli Assessment Tool at a Glance Objectives and Standardized field methodology to: application • Collect robust baseline of aquatic biodiversity for hydropower EIA • Monitor a set of aquatic indicators over time to assess: • Changes in target groups during HPP cycle: ∙ Fish ∙ Macroinvertebrates ∙ Periphyton • Success of hydropower project mitigation measures • No net loss or net gain of biodiversity (international lenders) EIA sampling Three sampling regions, each with multiple sampling sites: regions and • Upstream of dam (including reservoir, main stem, and spawning tributaries) sites • Diversion reach between dam and powerhouse • Downstream of powerhouse (main stem and tributaries) EIA sampling EIA baseline sampling should be done as often as possible. Three seasons listed below should be seasons sampled as minimum: • Fall (post-monsoon): October to November • Winter (post-monsoon): January to February • Spring (pre-monsoon): March to May Long-term Long-term monitoring sampling sites should be selected based on the EIA results to track important monitoring biodiversity indicators in locations where project impacts are expected and mitigation measures are sampling sites implemented. Long-term Long-term monitoring should include at least two seasons per year: monitoring • Fall (post-monsoon): October to November sampling seasons • Winter (post-monsoon): January to February A third season should be included when possible, especially for fish migrations: • Spring (pre-monsoon): March to May Target taxa • Fish (all species and target fish species) • Macroinvertebrates • Periphyton Field sampling Fish field sampling methods that should be used, where feasible, for each site: methods • Backpack electrofishing • Dip net • Cast net • Environmental DNA (eDNA) • Underwater video Macroinvertebrates and periphyton standardized field sampling method developed for Nepal by Tachamo Shah et al. (2020a): • Macroinvertebrates: multihabitat sampling using kick net • Periphyton: stone scrubbing Data analysis Fish metrics: metrics 1. Species richness 5. Relative abundance of target fish species 2. Species composition 6. Recruitment of target fish species 3. Proportion of species 7. Length of target fish species 4. Species distribution Macroinvertebrate metrics: 1. Taxa richness 2. Ephemeroptera, Plecoptera, and Trichoptera (EPT) index 3. Proportion of functional feeding groups Periphyton metric: 1. Dry biomass Field sampling Sampling team should include (as appropriate): team • Fish specialist(s) trained and experienced in electrofishing • Fish specialist(s) with expertise in identification of Himalayan fish species • Macroinvertebrate specialist capable of identification of species • Students or field assistants experienced with sampling and processing of fish or macroinvertebrates • Local fishermen with expertise in cast-netting 15 1.3 Why Sample and of 50 hydropower projects in Nepal (Shah et al. 2020) found that none of them have conducted Monitor Freshwater Aquatic any monitoring of the aquatic ecosystem and Biodiversity biodiversity to evaluate project impact or the success of their mitigation actions. An Asian Hydropower projects have significant impacts Development Bank study (ADB 2018) of the on the aquatic ecosystem and the organisms potential impacts of damming of rivers in Nepal living in the river basin. HPPs change the river on aquatic biodiversity revealed inadequate flow, quantity, timing, water chemistry, and assessment and monitoring. water temperature as well as create blockages to migrating fish and other organisms moving While most EIAs of hydropower projects in upstream and downstream in the river. Altering Nepal include some field sampling of aquatic flows change the aquatic habitats and often create biodiversity, the geographical coverage, taxonomic conditions for predators, invasive fish, plants, groups, sampling effort, field methodology, and and other organisms to flourish in the new flow data analysis vary greatly across project EIAs and conditions above the dam, in the diversion reach, are usually minimal (Shah et al. 2020). There is a and below the powerhouse. clear need for a field methodology that promotes the use of robust and standardized methods “Biodiversity monitoring is the process of to document aquatic biodiversity for Nepal’s determining the status of and tracking changes Himalayan rivers. in living organisms and the ecological complexes of which they are a part. Biodiversity monitoring As more HPPs are built on Nepal’s rivers, is important because it provides a basis for monitoring of the impacts and changes in the evaluating the integrity of ecosystems, their aquatic ecosystem are essential not only for responses to disturbances, and the success of the survival of the aquatic species but also for actions taken to conserve or recover biodiversity. ensuring good water quality and healthy aquatic Research addresses questions and tests hypotheses ecosystems for future generations of Nepalese. about how these ecosystems function and change and how they interact with stressors,” according to the Canadian Biodiversity Ecosystem Status and Trends 2010 report (Federal, Provincial and Territorial Governments of Canada 2010). By Nepalese government regulations and laws as well as international lenders’ standards, hydropower projects are required to implement mitigation actions to avoid or reduce project impacts on the environment, particularly on aquatic species and habitats, to protect aquatic animals—Aquatic Animal Protection Act, 2017 (1960)— and support biodiversity. Mitigation actions typically include: 1) releasing an environmental flow (EFlow) at all times to ensure sufficient water is available in the river for aquatic species, 2) building a fish ladder to allow migratory fish to pass the dam, and 3) captive breeding of native fish species and stocking. Other mitigations may include aquatic habitat restoration or modifications, regulations on fishing in the reservoir, trapping and trucking of fish upstream, and measures to ensure safe fish passage downstream over or through the dam (Adeva- Bustos et al. 2021). Government agencies and international lenders require long-term monitoring to demonstrate successful implementation of the mitigation measures and the sustainability of the aquatic ecosystem during the construction and operational phases of an HPP. A 2020 World Bank review 16 1.4 Questions Addressed with To answer these questions, the following are assessed with field data: the Trishuli Assessment Tool 1. How do fish and macroinvertebrate The Trishuli Assessment Tool focuses on species vary between the pre-construction, data collection and analysis for fish, and construction, and operational phases? macroinvertebrates, and periphyton to answer the a. Number of species following questions: b. Community composition (including For an EIA baseline (pre-construction phase): presence of invasive species) c. Relative abundance of all species 1. Which species are there? (richness and composition) d. Distribution of species a. Species lists e. Relative abundance of target fish species such as: b. Number of species i. Mahseer species 2. How many individuals are there? (relative ii. Snow trout species abundance) f. Recruitment of target fish species a. Number of individuals per species collected 2. How do indicators of aquatic ecosystem status 3. Where are the species and individuals located? and health vary over time? (distribution) a. Macroinvertebrate indexes a. Map of species distributions b. Periphyton biomass b. Map of relative abundance 4. Recruitment (reproductive success) a. Relative abundance of juveniles b. Fish sizes 5. Aquatic ecosystem health and water quality a. Macroinvertebrate indexes b. Periphyton biomass For long-term monitoring of a hydropower project during the construction and operational phases, questions that can be addressed with the Trishuli Assessment Tool include: 1. What impact is the hydropower project having on aquatic biodiversity? 2. Are the project mitigation measures working to reduce project impacts? 3. Is no net loss or net gain achievable for the aquatic biodiversity indicators? 17 � Field Methodology 2.1 Sampling Design for in the aquatic ecosystem in Nepal’s Himalayan rivers, and provide ecosystem services such Environmental Impact as food to local people. They serve as good Assessment indicators for monitoring due to their sensitivity to specific changes within the aquatic ecosystem. 2.1.1 What to Sample for the EIA— Selection of target species within these groups is Aquatic Biodiversity Indicators recommended to focus on species of conservation concern or those that may be at higher risk The Trishuli Assessment Tool focuses on sampling from project impacts. Within the fish group, two three crucial elements of aquatic biodiversity: target species that are globally threatened and • Fish distributed throughout the Himalayan river basins are recommended: the mahseer species, including • Macroinvertebrates the golden mahseer (Tor putitora) and Tor tor, • Periphyton and snow trout species, particularly the common These three aquatic biodiversity groups were snow trout (Schizothorax richardsonii). Table 2.1 selected because they are abundant, play key roles outlines the key aspects of these groups. Table 2.1 Key Aspects of Monitoring the Three Target Groups Taxon Description and importance Sensitive to changes in Aspect to monitor Fish Prominent aquatic vertebrates and • River flow rate and depth • Species richness top predators, including threatened • Water temperature • Species composition species; serve as a commercially • Habitat for spawning • Relative abundance, important food source • Connectivity maturity stage, and • Food availability distribution of selected fish species: • Mahseer species • Snow trout species Macroinvertebrates Aquatic invertebrates larger than • River flow rate and depth • Community 500 micrometer (µm), including • Water temperature composition insects, crustaceans, mollusks, and • Sediments • Relative abundance of annelids that serve as food sources • Riverbed substrate key taxa: for fish, birds, and other animals; • Organic matter • Ephemeroptera serve important functions within the • Plecoptera aquatic ecosystem, such as breaking • Trichoptera down organic matter as well as • Composition of filtering and cleaning the water functional feeding groups Periphyton Blue-green algae, fungi, microbes, • River flow rate and depth Dry biomass bacteria, plant detritus, and animals • Water temperature that cling to rocks and other • Sediments substrates; serve as the basis of the • Rocks aquatic ecosystem food chain 19 2.1.2 Where to Sample for the EIA— • Key aquatic habitats for macroinvertebrates (for Sampling Sites example, diverse riverbed habitats including different flow types) Sampling Regions Selecting Sampling Sites An aquatic survey for an EIA of a hydropower project should include sites where impacts from Sampling sites should be selected by first the project may occur. These sites are generally evaluating and mapping all the main aquatic located in three sampling regions: habitats in the project area using available • Upstream of the HPP, including the reservoir satellite imagery and field reconnaissance. Access area and safety are important considerations for site • Diversion reach between the dam and the selection. The aquatic habitats (Figures 2.1 and powerhouse (for HPPs with a diversion reach) 2.2) include: • Downstream of the powerhouse, especially for • Rapids—fast-flowing and turbulent areas where peaking projects water flows over rocks • Riffles—similar to rapids but with a less intense Sampling Sites and lower flow rate • Runs—areas where water flows are uninhibited Within each of these regions, sampling sites should • Pools—still water areas within the river channel, include: usually deeper than other areas • The main stem river • Backwater—still or low-flowing water created • Large tributaries at least 300 meters (m) by natural channel migration along the site of from the confluence with the main river; also the river upstream if tributary is not affected by other • Braided channels—a network of river channels dams separated by small sand bars • Small tributaries at least 300 meters from the confluence with the main river • Key sites for fish spawning and larval nursing grounds, often upstream in the tributaries and at the end of the tributary just before it meets the main river Figure 2.1 Aquatic Habitats within a River 20 Figure 2.2 Example Satellite Image Showing Different Habitat Types in the Main Stem River and a Tributary within the Area of Impact of an HPP Source: Google Earth. Number of Sampling Sites each region, covering both the main stem river and tributaries. Additional sampling sites should be The aquatic survey should include multiple included to cover more habitats or important sites. (replicate) sampling sites in each region to capture Additional sites upstream should be included to the natural variation between sites within the effectively cover the movement range of migratory region. This natural variation is high in Himalayan fish species. Likewise, sites further downstream river basins and can vary even within a few may be needed to assess changes in the river meters. For an EIA baseline, as many replicate ecology due to alterations in water or sediments sites as possible should be sampled in each region. flows. Figure 2.3 illustrates an ideal sampling At a minimum, two to six sites should be sampled design. in each of the major aquatic habitats identified in Figure 2.3 Recommended Sampling Design to Collect Aquatic Data for an EIA Baseline or Long-Term Monitoring of an HPP Reservoir Note: Dots signify sampling site replicates within each of the three sampling regions. Dots with “T” are on the tributaries or at the confluence of the main river with a tributary. Other dots are along the main stem river. 21 2.1.3 When to Sample for the EIA— construction to document a yearly cycle for the Seasonality EIA. Two years of baseline data would provide a robust baseline. A full year of sampling data Field sampling for the EIA baseline of a provides information on the lifecycle of target hydropower project must be conducted in all of species that will serve as a solid baseline against the seasons relevant for aquatic biodiversity. In which to measure changes and evaluate if no net the Nepal Himalaya, the onset of the monsoon loss or net gain has been achieved. Each sampling season in May or June (pre-monsoon) is the survey should include all of the sampling sites trigger for many migratory fish to start moving and dedicate sufficient time at each site to fully upstream to their spawning sites. Similarly, many implement the field methods. This usually requires migratory fish species start moving downstream one to two days per sampling site. Extra care must for overwintering in October or November at be taken when sampling during the wet season due the end of the monsoon season (post-monsoon). to strong river flows. During the winter season (December to March), fish may reside under rocks as the water level Minimum Sampling Schedule and temperature drop. Many macroinvertebrates that are insects spend only part of their lives in Hydropower project budgets and field access water and complete their life cycle mostly within often limit the number of pre-construction a year. Sampling in all seasons allows the capture sampling surveys conducted for an EIA. of a wide range of macroinvertebrates at mature However, a minimum number of seasonal larval stages. Similarly, water levels in the river sampling surveys is essential to obtaining a affect the distribution and abundance of fish solid understanding of the aquatic biodiversity. and macroinvertebrates; therefore, sampling in Field sampling for the EIA baseline should be multiple seasons is essential to establishing a conducted in at least three seasons: robust baseline. • Fall (post-monsoon): October to November • Winter (post-monsoon): January to February For an EIA baseline, sampling should be conducted as often as possible to document • Spring (pre-monsoon): March to May the variation between seasons and months. When possible, sampling during a fourth Seasonal sampling provides a strong baseline of season—May to June (pre-monsoon)—is also information about where and when the fish and recommended, particularly for migratory fish macroinvertebrates are found in the project area species. In Nepal, four seasons are often sampled and the watershed prior to construction of an for an EIA. HPP. This information is important for assessing project impacts and developing mitigation actions to maintain aquatic biodiversity. See Box 2.1 for key elements of sampling design for an EIA. 2.2 Sampling Design for Long- Term Monitoring Ideal Sampling Schedule The first step in long-term biodiversity monitoring A sampling survey should ideally be carried is to clearly define the objectives of the monitoring out monthly for at least a year prior to HPP program and the questions that will be answered Box 2.1 Key Elements of Sampling Design for the EIA 1. Use satellite imagery and field visits to identify and map all aquatic habitats in three regions: • Upstream of HPP project • Diversion reach (if applicable) • Downstream of powerhouse 2. Select at least two to six sampling sites in each of the three regions to represent all habitat types. 3. Conduct field surveys during three seasons for one to two years prior to HPP construction: • Fall (post-monsoon): October to November • Winter (post-monsoon): January to February • Spring (pre-monsoon): March to May 22 with the monitoring results. Section 1.4 outlines Project Impact and Mitigation-Specific some key questions that can be answered using Sampling Sites the Trishuli Assessment Tool for long-term aquatic biodiversity monitoring related to hydropower. Long-term monitoring for a hydropower project often focuses on assessing specific measures The sampling design for long-term monitoring is designed to reduce project impacts on aquatic developed based on information obtained during biodiversity. Sampling sites must be located the EIA field surveys. Sampling sites and field appropriately in order to evaluate the success methods will usually be a subset of those used for of such measures. Some examples of mitigation the EIA baseline, with some exceptions. measures and associated sampling sites are presented in Figure 2.4. If the EIA concludes that an HPP has no impacts on a sampling region (for 2.2.1 What to Sample for Long-Term example, downstream of the powerhouse), the Monitoring—Aquatic Biodiversity number of monitoring sites in that region may be Indicators reduced or eliminated. Long-term monitoring using the Trishuli Control Sites Assessment Tool focuses on the same three groups Some biodiversity monitoring programs, such of aquatic biodiversity: as the “Before-After-Control-Impact” approach • Fish (Green 1979), include control sites that are not • Macroinvertebrates affected by a hydropower project for comparison • Periphyton to sites within the project impact areas. For HPPs in the Himalayas, it is often challenging to find true control sites that are equivalent to 2.2.2 Where to Sample for Long-Term the pre-project conditions of the impact sites. This challenge is due to the cumulative impacts Monitoring—Sampling Sites of hydropower projects and other developments, Selecting Sampling Sites such as road construction, fishing pressures, and water mills. In addition, the natural variation in In contrast to an EIA, for which field sampling water flow rate, temperature, and substrate is must be done at many sites to obtain a robust high within the river basin, resulting in seemingly understanding of the aquatic biodiversity, similar sites with different habitats, conditions, sampling sites for long-term monitoring should be and species. In Figure 2.4, control sites may be on selected based on the objectives of the monitoring tributaries downstream of the powerhouse as an program. Such a program is normally used to evaluation of fish spawning outside of the project evaluate if an HPP’s mitigation measures are impact areas. successful in maintaining aquatic biodiversity during its construction and operational phases. Control sites can be used to compare what is happening in another part of the river over time. Thus, long-term monitoring sites should include: In this case, each control site should be analyzed • Sites with predicted impacts from an HPP over time independently to evaluate changes at (derived from the EIA) that site. The trajectory of changes at a control site can be compared to that at impact sites. See • Sites where HPP mitigation measures will Section 3 for suggested analysis using control sites. be implemented (from the EIA) • Sites important for aquatic biodiversity (for Number of Sampling Sites example, migratory routes, spawning sites, feeding grounds, nursing grounds, areas of The number of sampling sites for long-term high biodiversity, or unique habitats) monitoring will depend on the extent of HPP • Control sites outside of the HPP’s area impacts and the number of species of conservation of impact concern or important habitats documented in the aquatic ecosystem. For long-term monitoring, Long-term monitoring sites are usually selected sampling sites should be surveyed within each of from those surveyed for the EIA. However, the three regions to evaluate the project impact sometimes the EIA study reveals additional sites and success of mitigation measures at different that may be important for monitoring, particularly HPPs (Figure 2.4). Preferably, two to six sites if threatened species or unique habitats are will be monitored in each region in order to documented, or if there are site-specific consider natural variations and different habitats. project impacts. 23 Figure 2.4 Example of Sampling Design for Monitoring HPP Impacts on Aquatic Biodiversity Reservoir In addition, one to two control sites outside of • Monitoring should be done with the same field the project impact area should be surveyed for methods and sampling effort for each sampling comparison of trends over time. site and survey period. If sampling effort is not equivalent, it can be standardized using the catch per unit effort in order to make comparisons 2.2.3 When to Sample for Long-Term between survey periods or years (see Section 3). Monitoring—Seasonality • Field sampling data must always be compared between the same season and not between Field sampling for long-term monitoring should different seasons (see Section 3). For example, be conducted two to three times a year in the same data can be compared between spring 2020 seasons for the EIA sampling: and spring 2021 field surveys but not between • Fall (post-monsoon): October to November spring 2020 and fall 2020 field surveys. • Winter (post-monsoon): January to February Additional seasons should be included when How Long to Monitor possible: The length of the long-term monitoring program • Spring dry season (pre-monsoon): should be determined by the program objectives March to May and questions. Monitoring to evaluate the • Spring (pre-monsoon): May to June success of mitigation measures and maintenance of aquatic biodiversity indicators usually takes Essential Caveats on Long-Term Monitoring several years before changes become apparent or target thresholds are met (see Section 3). • Monitoring must be conducted as close to the same date during the same time frame (such as Long-term monitoring should be conducted season or month) each year. for at least one year, prefereably two, prior to • Monitoring should be conducted under the construction and during all years of construction same weather and river conditions each year of an HPP. Monitoring should continue during to minimize changes caused by changing operation until the data indicate that the project weather or river conditions. Sampling should is not having negative impacts on the aquatic be avoided in rain or flooding when flow environment and all parties (HPP operator, and turbidity are not normal or typical for government, and funding agencies) agree that the season. If rain is reported in the basin or monitoring is no longer needed. See Box 2.2 for water is turbid, survey must not begin until key elements of sampling design for long-term turbidity normalizes. monitoring. 24 In general, long-term monitoring should be Several key points are highlighted below as conducted: essential preparation for the field surveys: • Necessary permissions for sampling from • Pre-construction phase: One to two years all relevant government departments and • Construction phase: Throughout all years authorities must be obtained before leaving of construction for the field. This includes permits to conduct • Operations phase: research in national parks or other protected Minimum: Three years areas, permits to collect fish, macroinvertebrate, Robust: 10 years and periphyton samples, and permits for Ideal: Life of project electrofishing and eDNA. • An accurate weather forecast of the study area should be reviewed to identify expected 2.3 How to Sample for the EIA extreme weather conditions that can compromise the ability of an expert or an and Long-Term Monitoring observer to perform field activities. Surveys should be rescheduled to alternate days if 2.3.1 Preparation for Field Sampling extreme weather conditions, such as cold temperatures, rain, flood, and high wind, This field manual assumes that users of the are expected. Trishuli Assessment Tool are familiar with the • The field team must have all the necessary basics of field work and sampling in Nepal. Thus, personal protective equipment, including first- the tool does not cover all information needed to aid box, life jackets, working communication conduct a field survey. Additional information can devices, and safety boots or shoes. Electrofishing be found in Nepal’s Hydropower Environmental requires additional safety gear. Impact Assessment Manual (MoFE 2018) and the Freshwater Ecosystem Assessment Handbook • All equipment must be in good working (FRTC/MoFE 2022). condition, which should be checked by the field team leader. Box 2.2 Key Elements of Sampling Design for Long-Term Monitoring 1. Select sampling sites to include: • Sites with predicted HPP impacts (derived from the EIA) • Sites where HPP mitigation measures will be implemented (from the EIA) • Sites important for aquatic biodiversity (for example, migratory routes, spawning sites, areas of high biodiversity, or unique habitats) • Control sites outside of the HPP’s area of impact 2. Select two to six sampling sites in each of the three regions to cover all habitat types and one to two control sites outside of the HPP’s area of impact. 3. Conduct field surveys during at least two (ideally three) seasons for each year: • Fall (post-monsoon): October to November • Winter (post-monsoon): January to February • Spring (pre-monsoon): March to May (if applicable) 4. Sample fish, macroinvertebrates, and periphyton 5. Field surveys should be conducted: • One to two years prior to construction • Throughout the HPP’s construction • Three to 10 years during the HPP’s operations (ideally throughout the life of the project) 25 2.3.2 Field Team • Backup support of geographic information system (GIS) and other data management as Implementation of the Trishuli Assessment Tool well as logistic and emergency management requires a team of qualified biologists who from their home organization (consulting firm, are trained in field methods with field-work university, or research institute) experience in Nepal. The team should include: 1. A field team leader with demonstrated proficiency in the field sampling methods and 2.3.3 Site Sampling Design field team management as well as experience or knowledge of the survey areas; experience with The following steps should be followed to set up report preparation and data analyses is also the sampling design for each sampling site: required for the team leader 1. At each sampling site, select a 400 m section of 2. One to two fish researchers with qualifications river that contains a variety of aquatic habitats and experience or training to identify local such as rapids, riffles, runs, pools, backwater, and regional fish species in the field and in and braided channels. the laboratory preferably with fish taxonomy • Tributaries: select a section that is more than training; they must also have experience with 300 m above its confluence with the main the fish sampling methods of the Trishuli stem river or larger stream Assessment Tool • Main stem river: select a section with 3. One to two fish researchers trained in the use appropriate shallow, low-flow areas that are of electrofishing and its safety measures safe for sampling, such as near the confluence 4. One to two macroinvertebrate researchers with tributaries, river bends, and backwaters qualified and trained in the field sampling 2. Mark the midpoint of the 400 m sampling methods with experience in sorting and stretch with a permanent mark (such as paint identifying macroinvertebrates in the field on a rock) or select a landmark like a bridge 5. A data recorder trained in the data recording or other marker. Record the global positioning methodology of the Trishuli Assessment Tool system (GPS) coordinates of the midpoint. 6. Two to four field assistants who may be 3. Mark and record GPS coordinates of the students, consultants, or trained local boundaries of the sampling site: community members to assist with fish and • 200 m downstream of the midpoint macroinvertebrate sampling • 200 m upstream of the midpoint 7. One to two local fishermen proficient in cast 4. Within the 400 m stretch, identify the best netting for fish areas for each sampling method so that each A laboratory or analysis team may also be method has its specific sampling locations and required. These may include (as appropriate): does not overlap (if possible). 8. Laboratory macroinvertebrate researchers (one 5. Record and describe in detail the specific areas expert and one assistant) qualified and trained delineated for each method so that sampling to sort and identify macroinvertebrate samples, during future monitoring surveys will be able preferably an aquatic insect taxonomist to find the exact sampling locations. 9. An ecological statistics data analyst to assist 6. Start sampling downstream and work upstream with data analysis (if needed) to avoid disturbing the riverbed and causing sediments to flow to downstream sites. 10. A genetics laboratory collaborator to analyze eDNA The field team must have the following resources 2.3.4 Habitat Descriptions and training: • Training to use and maintain the sampling The aquatic habitat should be described and equipment in the field, data collection, documented in a data sheet (see Appendix B) specimen preservation, and data recording before sampling begins: and keeping 1. Describe the stretches of the river or stream in • Ability to swim in deep water the sampling site to include information on: • Willingness to follow the directions of the field • Description of upstream, midpoint, team leader and to wear a life jacket and other and downstream boundaries plus personal protective equipment as necessary length (in meters) 26 • Wetted width and total width (in meters) in 2.4 Fish Field Sampling upstream, midpoint, and downstream areas Methods • Flow conditions (high, medium, or low) • Percentage of aquatic habitats as below for The sampling methods for fish aim to collect data upstream and downstream areas: for: Rapids = high turbulence, high flow with • All fish species steep vertical drop over rocks or boulders • Target fish species, such as mahseer (Tor spp.) Riffles = less turbulence, high flow over and snow trout (Schizothorax spp.) smoother substrate, shallower than 0.5 m Runs = low turbulence, high flow over smoother substrate, deeper than 0.5 m 2.4.1 Field Method Selection Pools = low turbulence, low flow, deeper than 1 m The Trishuli Assessment Tool comprises the following set of fish field sampling methods: Shallow slacks = low turbulence, low flow, shallower than 1 m • Backpack electrofishing Backwater = low turbulence, low flow, • Cast nets connected to but off from the main flow • Dip nets 2. Draw a map of the study site with details of • Underwater video the boundaries, easily identifiable habitats, • Environmental DNA (eDNA) location of water types (such as pools, riffles, and rapids), and sites where sampling was These methods and others were field tested in conducted. Use a field notebook. February 2020 on the Trishuli River. Electrofishing was found to be the most effective method for collecting fish in the tributaries, documenting 2.3.5 Associated Data to Collect two to four times as many fish as were collected by cast nets (Philipp et al. 2020; see Table 2.2). In addition to data on target organisms, data Gill nets were evaluated but excluded as a on the location (GPS coordinates), habitat, recommended method due to its harmful effects weather conditions, flow rate, and water depth on the captured fish (Philipp et al. 2020). should be recorded at each sampling site. See As many of these field methods should be used at data sheet in Appendix B for additional data each sampling site as possible, but not all methods that need to be recorded. are suitable for all sampling sites. Methods will need to be selected based on the target indicator to be sampled, habitat type, and feasibility (see Table 2.3). Feasibility will include access to sampling site; availability of experienced field personnel and necessary equipment; depth of the water and ability of researchers to walk and wade in the river; river flow rate, turbidity, and depth; as well as weather conditions. Table 2.2 Comparison of Fish Catch Using Cast Nets and Electrofishing in the Trishuli River Tributaries in February 2020 Cast net Electrofishing Site Total Sample CPUE No. of Total Sample CPUE No. of Site code no. of time species no. of time species fish (min.) fish (min.) TAD Tadi Khola 20 57 21.1 4 106 32 199 15 MAI Mailung Khola 26 445 34.7 1 44 35 75.4 4 LCH Lower Chilime Khola 22 55 24 1 80 15 320 2 SAK Salankhu Khola 5 26 11.5 3 99 34 175 7 Source: Philipp et al. 2020 Note: no. = number; min. = minutes; CPUE = catch per unit effort (see Section 3.1) 27 Table 2.3 Field Methods for Each of the Fish Indicators to Be Used in Each Habitat Type Fish indicators Tributaries Main stem channel Main stem shore* All fish species • Backpack electrofishing • Cast nets • Backpack electrofishing • Cast nets • eDNA • Underwater video • Underwater video • Dip nets • Dip nets • eDNA Snow trout and mahseer • Backpack electrofishing • Cast nets • Backpack electrofishing adults • Cast nets • eDNA • eDNA • eDNA Snow trout and mahseer • Backpack electrofishing • Cast nets • Backpack electrofishing juveniles • Cast nets • Dip nets • Underwater video • Dip nets Note: *Along the shore of the main stem river in areas with lower flow and low turbidity (clear water) that are suitable for wading 2.4.2 Sampling Effort for Each Field As with all field sampling, circumstances may Method arise that prevent the full implementation of the recommended sampling effort. For example, The Trishuli Assessment Tool’s recommended weather conditions may change and halt field sampling effort for each fish sampling method is sampling, or the river may become turbid as a shown in Table 2.4. This standard protocol was result of upstream sand mining, thus affecting tested in the Trishuli River in February 2020 and the effectiveness of electrofishing. The sampling was found to provide a robust assessment of the effort for each field method at each site should fish biodiversity at each site (Philipp et al. 2020). be carefully recorded, including minutes spent Table 2.4 - Sampling Effort Per Site for Each of the Fish Field Sampling Methods Approximate Number of units per Field method Units of sampling effort sampling time Personnel site* per site** Core methods Backpack Time (minutes) sampling with 40 minutes sampling: 40 minutes** 3 people electrofishing electrofisher current on 20 minutes downstream 20 minutes upstream Cast nets Number of cast-net throws 100 cast-net throws: ~60 minutes 2 people 50 throws downstream 50 throws upstream Underwater video Time spent recording per set 12 sets of 5 min. each: 60 minutes 1 person 6 sets downstream (plus 1 for 6 sets upstream safety) Dip nets Number of dip-net emersions 10 dip-net samples: ~30 minutes 1 person 5 samples downstream 5 samples upstream eDNA Number of 2-liter water samples Six 2-liter water samples ~120 minutes 2–4 people Note: *Sampling design at each site includes a 400 m river stretch marked at a midpoint; **record the time spent on actual sampling for every method, subtracting travel or setup time; “downstream” refers to sampling 200 m downstream of the midpoint; “upstream” refers to sampling 200 m upstream of the midpoint. 28 electrofishing or underwater video recording as Training and Safety well as the number of cast-net throws and dip-net When done properly, electrofishing can be very samples (Table 2.4). The sampling effort at each safe and effective for capturing fish. However, it site can be standardized using catch-per-unit- can also be highly dangerous if the operator is not effort (CPUE) transformation in order to draw familiar with the electrofisher and safety features. comparisons between sites and sampling periods All members of the team must wear electrically (see more on CPUE in Box 3.1 in Section 3). insulating chest waders and rubber boots and be careful not to touch water during sampling. The electrofisher operator must obtain training from a 2.4.3 Specifics of Fish Field Sampling certified professional prior to using the equipment. Methods The electrofisher must have adequate safety systems, such as immersion cutout and emergency shut-off button. Backpack Electrofishing Target Organisms and Habitat Overview All fish species of various sizes and ages can Electrofishing using a backpack electrofisher be collected with electrofishing. Backpack delivers a low-voltage electrical field into the electrofishing is only possible in shallow areas water, which temporarily incapacitates fish so suitable for wading with low flow and low that they float to the surface of the stream and turbidity (clear water). Thus, this method is best can be collected with a net. It is the most effective suited for tributaries and at the confluence of the method for sampling and documenting fish; thus, main stem with tributaries (mouth of the tributary, it should be implemented whenever conditions are where fish spawning often occurs). suitable. The Trishuli Assessment Tool protocol recommends electrofishing for a total of 40 Seasonality minutes at each sampling site: 20 minutes within Electrofishing is most effective during the dry the 200 m downstream of the midpoint and season when water has low flow and low turbidity another 20 minutes within a second area in the (clear). Electrofishing cannot be used in high-flow 200 m upstream of the midpoint. During each of (monsoon season) or turbid waters. these 20-minute periods, the team should sample the full range of representative habitats that can be Personnel safely surveyed in each of the six habitats—rapids, Three people are needed: 1) an “operator” who runs, riffles, pools, slack water, and backwater— will operate the electrofisher and collect the fish on a percentage of time basis that is representative with a net, 2) a “bucket” person to carry the of the amount of such habitats in the upstream bucket for the fish collections and to assist the and downstream locations. operator if needed, and 3) a “recorder” to keep Advantages track of the time and record data as well as to ensure that safety precautions are observed. Since • Extremely effective in sampling large numbers the electrofisher is heavy (15 kg), the team may and high levels of species or size diversity choose to rotate the duties if all team members are • Requires little time for actual in-water sampling trained in the use of the equipment. The operator • Can sample in shallow water (slow or fast) must be trained by a professional in the use of effectively the electrofisher and be able to carry it for an • Can sample in complex, rocky habitats very extended period of time in the cold, rocky streams effectively of the Himalayan region. Challenges Time • Specialized and expensive backpack electrofisher The Trishuli Assessment Tool recommends a total (US$3,000–US$10,000) of 40 minutes of electrofishing at each sampling site: 20 minutes within the 200 m downstream • Training and practice required of the midpoint and 20 minutes within a second • Heavy equipment area in the 200 m upstream of the midpoint for • Requires a three-person team best results. Sometimes, conditions do not allow • Safety concerns and precautions for the recommended sampling time; for example, changing weather may halt sampling or upstream • Special permits from government needed sand mining may cause turbidity, thus affecting • Requires shallow and clear water (tributaries electrofishing effectiveness. The time spent on and backwater as well as side channels) 29 electrofishing should be recorded and standardized the correct power settings for the safest and using CPUE to allow for comparisons between most effective fish collection are described in sites with different sampling efforts (see Section 3). Appendix F: Detailed Instructions for Conducting Backpack Electrofishing. See Box 2.3 for Sampling Process equipment needed for electrofishing. An operator carries a backpack electrofishing In addition, and very importantly, prior to using unit on his or her back, holding the pole with this equipment, all members of the team should the electric node in one hand and a long handle read and understand the information presented in dip net in the other hand while walking slowly Appendix G: Best Practice Manual for Backpack through the water—ready to catch any fish Electrofishing. floating to the surface (Figure 2.5). The electric node must be underwater when operating. Specimen Collection and Processing A second “bucket” person should accompany the operator and carry the bucket (perhaps The collected fish will be held alive in buckets an additional net) for collecting fish. A third of fresh water for processing at the end of each “recorder” will watch the other members carefully 20-minute period (see Section 2.4.4). If lots to ensure the safety of the team, keep track of the of fish are caught, it is best to process them time, and record information from the operator. immediately and keep them together in a large tub until sampling is complete. All fish that are Specific details of the procedures for operating not kept as voucher specimens will be returned to the electrofishing equipment and for choosing the river alive. Figure 2.5 Backpack Electrofisher and its use in the Rocky Streams of the Trishuli River Basin Box 2.3 Electrofishing Equipment • Backpack electrofisher with battery and electrodes (Smith-Root LR-24 backpack electrofisher recommended) • Long-handled dip net with electrically insulated handle (for collecting fish) • Two pairs of chest waders with built-in electrically insulated boots (for the operator and the fish collector) • One pair of rubber boots (for the recorder) • Three to four buckets • GPS • Data notebook and pencil • Camera or cellphone to photograph habitats and fish 30 Data Management Training and Safety Fish collections from downstream and upstream The caster must be experienced with throwing the should be recorded and kept separately. Data cast net (Figure 2.6). A local fisher should be hired will be recorded using a standardized data to use the cast net. The team must be able to swim sheet (Appendix A). Locations and durations in case they are pulled or fall into the stream. Care of sampling efforts as well as records of all fish must be taken to avoid falling into the river when captured will be documented in detail in field sampling in the main stem. notebooks, including photographs of the fish collected and the areas sampled. Figure 2.6 Fisher Throwing Cast Net in the Unit of Sampling Effort for Analysis Trishuli River Electrofishing sampling effort is measured as the time (number of minutes) spent actively electrofishing when the current is on for each site (minutes per site). Time to move between sections or adjust the equipment should be excluded. Cast Net Overview Cast-net sampling involves a recorder (or bucket person) and a net caster who will throw the cast net 100 times at each sampling site: 50 casts downstream and 50 casts upstream. The number of cast-net throws can be adjusted according to the habitat and environment. At some sites, Seasonality 25 throws may be sufficient while in other sites Cast nets can be used in both dry and wet with more diverse habitats, 200 throws may be seasons, although high monsoon would likely be needed. For comparisons over time or between too dangerous. sites, it is best to keep the number of throws the same for each site, but comparisons between Personnel different number of throws (different sampling A team of two people, including a net caster and effort) can still be done using CPUE (see Box 3.1 another to hold the collecting bucket and record and Table 3.7 in Section 3). the data, is sufficient. Advantages Equipment Requirements • Moderately effective for catching fish of small Cast nets come in many sizes and shapes. and medium size For long-term monitoring, cast nets with the • Can be used in many different habitats including exact same size of mesh, length, and diameter deep and moderately moving water must be used during every sampling survey at each sampling site. A cast-net mesh size of about • Requires only two people (caster and 25 millimeters (mm) is recommended for the bucket carrier) Trishuli Assessment Tool in order to capture • Cast nets are relatively inexpensive and available small fish, including juveniles. Cast nets can in Nepal range from 2 m to 3 m in length with a 2.5 m • The most used technique in Nepal, therefore to 5 m expanded diameter. See Box 2.4 for most compatible with previous data equipment needed for cast-net fish field sampling. Challenges • Requires skill and experience to cast the net well Box 2.4 Cast Net Equipment • Limited efficacy for sampling small benthic species (for example, Loach spp.) • Cast net(s) • Less effective in some aquatic habitats, such as • Buckets rocky substrate • GPS • Inconsistent mesh size between studies • Data notebook and pencil limits comparisons • Camera or cellphone to photograph habitats and fish 31 Time are pulled or fall into the stream. See Box 2.5 for The amount of time needed to cast 50 throws equipment needed for dip-net fish field sampling. per 200 m river stretch depends on the skill of the caster and access to the river. The time spent during the casting of the 50 throws should be Box 2.5 Dip-Net Equipment recorded so that time can be used in the data analysis if desired. • Dip net—select the appropriate net size Specimen Collection and Processing based on the depth and extent of the habitat to be sampled. A good option is All netted fish will be held alive and kept in good a 40 centimeter (cm) wide X 46 cm long condition in buckets of fresh water for processing X 20 cm deep net with 3 mm mesh and at the end of the 50 cast-netting attempts (see a telescopic pole extending up to 3 m Section 2.4.6). All fish that are not kept as voucher (see Figure 2.7) specimens will be returned to the river alive. • Buckets • GPS Data Management • Data notebook and pencil Results of fish numbers captured will be • Camera/cellphone to photograph recorded for each cast-net throw to assess habitats and fish variation in success across the site. Locations and durations of sampling efforts as well as records of all fish captured will be documented in detail in field notebooks, including photographs of the areas sampled. Figure 2.7 Two Types of Dip Nets Unit of Sampling Effort for Analysis Sampling effort is measured by the number of cast-net throws per site. Dip Net Overview For dip-net sampling, a single individual will attempt to collect larval and juvenile fish (less than 30 mm in total length) opportunistically in 10 very shallow areas using a small or micro mesh dip net of appropriate size for the sampling area. Record the total time spent sampling. Advantages • Equipment is inexpensive and easy to use • Requires little time for actual in-water sampling • A reliable method for capturing larval fish • Requires only a single operator Target Organisms and Habitats • Provides evidence of species recruitment and Dip nets are ideal for collecting larval and juvenile identifies spawning and nursery areas fish. However, they can only be used in shallow, low- flow areas, mostly in tributaries, where Challenges juvenile fish may be present. Areas in which larval • Requires spotting larval fish visually in fish can be observed swimming should be targeted shallow water preferentially, but if none can be found, then the • Extremely size selective dip netter should sample in areas where larval fish • Requires shallow and clear water may likely occur. • May result in low capture rates Seasonality Training and Safety Dip nets will work best in the dry season when flow is low and water is clear. They should not be No special training is required although used in high water season. knowledge of fish habitats is advantageous. The dip-net user must be able to swim in case they 32 Personnel Advantages One person is sufficient, with a second person • Can observe many fish and often species not nearby for safety and recording data. captured with other gear • Good for documenting fish in specific habitat Time types and to record juveniles in spawning sites Time for dip-net use will depend on the skill of • Possible to document migrating fish in the user and access to adequate sampling sites. particular habitats Sampling time is estimated to be around 30 minutes. The time spent actively using the dip net • Requires only one operator for recording and a should be recorded for each sample and added up second person for safety for a total time spent dip netting. • Minimal training • Provides permanent record Specimen Collection and Processing Netted fish will be held alive and in good Challenges condition in buckets of fresh water for processing • Equipment is minimally expensive at the end of each successful dip-netting trial (see (US$100–300) Section 2.4.6). • Data analysis requires lab-based viewing to Data Management count and identify fish Locations and durations of sampling efforts • Requires very clear water as well as records of all fish captured will be • Deployment and retrieval of equipment may documented in detail in field notebooks, including require swimming photographs of the areas sampled. • May be time consuming Unit of Sampling Effort for Analysis Training and Safety Sampling effort is measured as the number of Little training is required to operate the video “dips” or dip-net samples per site. camera. The operator must observe safety precautions and know how to swim in case they Underwater Video fall into the water while taking video. See Box 2.6 for equipment needed for fish field sampling using Overview underwater video. At each sampling site, a researcher will use an underwater video camera, such as a GoPro Target Organisms and Habitats camera, to record all fish activity for 12 sets Underwater video can capture any fish species but of five-minute recording periods (Figure 2.8). is particularly effective for documenting juvenile Video should be taken in all aquatic habitat and larval fish, which are often hard to catch with types available at the site (such as rapids, runs, other methods. Habitats should include tributaries riffles, pools, slack water, and backwater). and the confluence of the main stem river with Video recording should start at the lower end tributaries, where spawning occurs for many fish of the 400 m delineated sampling area, recording species. The 12 five-minute video segments should six sets of five-minute recording downstream be recorded in different target habitats at each site. of the midpoint and then six sets upstream of the midpoint. Sampling Process For this field method, the videographer will position himself or herself close to the edge of Figure 2.8 Researcher Holding Video Camera the water and hold the GoPro video camera Underwater in a Tributary underwater. A consistent method should be developed for all sites and surveys, such as holding the camera straight ahead to facilitate comparisons over time. Sampling sites should be selected where juvenile fish may occur. The videographer may sit or lie on rocks near the river’s edge to obtain a good position for holding the camera underwater. Each sampling period should be five minutes at a habitat. The videographer and the data recorder (second person) then move to another habitat type at the site and record another five-minute segment. A total of 12 segments should be recorded within the 400 m sampling site. 33 Seasonality Environmental DNA Underwater video can only be used in clear water, Overview so dry season is best. Environmental DNA (eDNA) is an emerging Personnel technology that documents species through A team of two people is sufficient: one to do the detection of DNA in water or soil samples. underwater video and the other to record data and The Trishuli Assessment Tool recommends using be close by for safety. eDNA when possible because this technique can detect and record species that are not Time captured with other methods. Species lists can Twelve sets of five-minute videos will be recorded grow with this technique, which is particularly for a total of 60 minutes. Additional time will useful for EIA baseline sampling to detect rare be needed to select sampling locations and move or threatened species. Its applicability for long- between them. term monitoring is still in research stages since measurement of abundance is only possible as Video Processing and Data Management a relative comparison of the amount of DNA The videos need to be downloaded onto a detected in each sample. eDNA sampling requires computer and reviewed by people who can collaboration with a genetics laboratory to identify Himalayan fish. To collect the data, sequence the DNA from the samples. the reviewer will list the species and number of individuals of each species observed in each five- Field Methodology minute video. Environmental DNA sampling involves taking samples of water from each site and filtering Unit of Sampling Effort for Analysis them to collect animal DNA from the water Sampling effort is measured as the time (minutes) (Figure 2.10). There are many approaches to spent actively recording per site. collecting and analyzing the water samples for DNA. Hydropower projects are encouraged to investigate options and decide on the best Box 2.6 Underwater Video Equipment approach and partner for their eDNA sampling needs. Some eDNA laboratories, such as Nature • Handheld, waterproof video camera, such Metrics (https://www.naturemetrics.co.uk), offer as GoPro (Figure 2.9) simple field collecting kits and resources for eDNA • Batteries and cables sampling and analysis. • Laptop computer (for reviewing the The following procedure was developed by videos) the Center for Molecular Dynamics Nepal's • GPS Fish Biodiversity Project (see http://fish.org.np/ • Data notebook and pencil background). • Camera or cellphone to photograph At each sampling site, five 2-liter water samples habitats (one each from upstream, downstream, pool, riffle, and sediment habitats) are collected in aseptic glass bottles at locations within the 400 m delineated sampling area. These water samples can then be taken to filtration stations set up on Figure 2.9 Examples of GoPro Waterproof Video the bankside safe from disturbing other activities. Cameras Those five water samples plus a separate control distilled water sample will be filtered to collect cells or DNA on a fine filter membrane (Whatman or Millipore filter with 47 mm diameter and pore size of 0.45 μm) using a hand-pump portable vacuum system. The six filters will then be preserved separately in Longmire’s solution to protect the DNA and taken back to the lab where the DNA will be extracted. Specific sequences will be amplified using polymerase chain reaction Source: gopro.com techniques, with different fish DNA samples amplified and then sequenced; by comparing sequences amplified from the eDNA water samples with known sequences from public databases like 34 GenBank, species present at or upstream from the buffer currently offers the best DNA sampling sites will be identified. Each location preservation retention), and optimal lab will be assessed for the presence or absence of protocols are constantly evolving to extract all species of fish potentially in the river at the the best genomic data site. Locations and durations of sampling efforts • Specific and bulky field equipment and supplies will be documented in detail in field notebooks, • A team of genetic specialists is required in pre- including photographs of the sites. and post-processing, especially in developing the Advantages most robust bioinformatics pipelines • Is highly effective in detecting presence of high • Expensive laboratory analysis (around US$8,000 numbers of species per set of 18 samples) • Can detect the presence of species that are very • Abundance data questionable but improving difficult to collect with other methods (relative abundance by proportion is the currently available standard) • Can be employed in almost any water conditions • False positives are possible (unless blocking • DNA samples can be kept long term for future primers are used to negate particular taxa reference studies groups that are least likely to exist in said • DNA samples can be used to target species other waters, but that increases bias) than fish by changing the target genomic code • The DNA reference databases for the Himalayan (changing base primer set) from cyprinids to region do not include all fish species and may mammals or particular species of interest include incorrectly identified DNA sequences Challenges • Requires substantial time to get final results • The method is still in a developing phase; some • Machinery sensitivity is high and multifactorial anomalies still need scientific validation elements (such as temperature, technical • Optimal collection standard in terms of type handling, and data pipeline robustness) of water (emerging research suggests shallow determine the sensitivity and specificity sediments), collection buffer (Longmire of results Figure 2.10 Environmental DNA Process 1 2 3 Note: Steps 1 and 2—filtering water samples in the field; step 3—evaluating DNA results with computer software Non–IFC photographs: ©Center for Molecular Dynamics Nepal (CMDN). Used with the permission of CMDN. Further permission required for reuse. 35 Target Organisms and Habitats 2.4.4 Monitoring Fish Movement eDNA can sample all types of organisms that through a Fish Ladder shed DNA in the water. A challenge for eDNA is that DNA travels with the water flow so that the In 2020, a World Bank study of 50 hydropower sample may not be from where it was collected. projects in Nepal revealed that 13 of them eDNA can sample all types of habitats. have constructed fish ladders to allow for fish to migrate past the dam (Shah et al. 2020). Seasonality However, only one of these fish ladders at the eDNA sampling can be implemented in all seasons. Khimti HPP has ever been studied or monitored It is recommended not to sample immediately after to evaluate its effectiveness for passing fish over heavy rainfall as silt and mudflow causes high the dam (Kaasa 2008). water turbidity causing clogging of filter papers and blocking the particle of interest (DNA) from International good practice calls for fish ladders remaining on the filter paper. to be monitored constantly through project operations to record if fish are using the ladder, Personnel which species they are, and how many fish are Field sampling requires two to three people to able to pass through. Monitoring also allows for filter the water samples. Simple filtering kits are evaluation of the design of the fish ladder so that now available that require only one researcher modifications can be made if needed. to collect the samples. A genetic specialist or Monitoring the movement of fish through a collaboration with a genetics laboratory is fish ladder requires different methods from necessary to sequence the DNA. A specialist in those included in the Trishuli Assessment Tool the taxonomic group sampled (fish in this case) and thus will not be addressed in detail in this is needed to verify the species list and interpret manual. Fish-ladder monitoring should be the results. continuous during the fish-migration periods, Time both upstream and downstream. Each fish species has its own migratory periods, so Collection of water samples takes only a few multiple periods may need to be monitored to minutes. Filtering the water from each sample evaluate all target species. Monitoring is not can be quick (less than 5 minutes) with new required when fish are not migrating. sampling kits or may take 30 minutes to an hour with traditional methods. Genetic analysis of the There are many methods hydropower projects can samples may take several months. use for the long-term monitoring of fish in their fish ladders. Some of these methods are listed as Data Management follows, from simplest to most complicated (see Data from the genetic analysis of the DNA Table 2.5 for comparison): will be a list of species with DNA sequences 1. Manual fish counts that match those found in the water sample. The species list comes from the international 2. Fish traps GenBank reference database, which may contain 3. Camera or video recording errors. A fish specialist should review the list 4. Pit-tag telemetry and evaluate the source of the GenBank samples 5. Active telemetry to verify the identifications. The data produced also include the number of DNA strands or 6. Automated underwater video with fish recognizable taxonomic units (RTUs) in the identification software sample from each species. The RTU number may possibly be used as an estimate of the relative abundance of each species. Research is ongoing to verify if RTUs can be used as relative abundance for long-term monitoring. 36 Table 2.5 Comparison of Fish-Ladder Automated Monitoring Techniques Source: https://fishbio.com/automated_monitoring 2.4.5 How to Record Fish Data • Completeness. Prepare data sheets (see Appendix B) for recording every detail, Detailed and consistent data recording is a including habitat and sampling survey fundamental part of data management (see information, names of places and details of Figure 2.11 for a sample fish field sampling data locations, methods, dates, times, and names sheet). Thus, it is important to be extremely of people involved. Examples would be site diligent in recording the data: description maps, fish-collection data sheets, • Fish data should be recorded in a standardized and sampling-method sheets. Sampling sites data sheet such as Appendix A. should have full names as well as ID codes, GPS locations, and a written description of • Habitat and location data should be recorded in the location. Another data sheet (Appendix A) a data sheet like Appendix B. should be used for fish data: species, number, • Each specimen sample must be clearly labeled length, and weight. with the sampling-site number, specimen • Organization. Store the data sheets in an number, and date. organized manner. Clearly label all samples • For data analysis, the data should be entered using easily distinguishable codes and into an Excel spreadsheet. numbering systems before storing them in a safe • Be sure to include the unit of measurement for and organized fashion. every set of data, for example, degree Celcius • Redundancy. All data should be stored in at (°C) for water temperature, gram (g) for weight, least three places or formats. For example, the and millimeter (mm) for fish length. handwritten data sheets need to be kept in a • Take photographs of all sampling habitats and secure location; photos of every sheet should be selected fish specimens. taken on a designated cell phone at the end of each day and those photos should be uploaded Considerations to the cloud for storage. • Finally, all data need to be entered into Excel The following are several important aspects spreadsheets (or a similar data storage system) to consider: that are housed in a secure site accessible by all team members who need access (read-only). 37 2.4.6 How to Process the Fish the genus and a species number (such as Collections Schizothorax sp. 1). The common name (in Nepalese or English) should also be noted along with the scientific name. Field Processing 2. Measure total length (mm) of fish from All fish captured using the sampling methods the snout to the end of tail. of the Trishuli Assessment Tool should be kept 3. Measure fork length (mm) of fish from alive and healthy in buckets of fresh water until the snout to the fork in tail. processed. All fish should be handled with care so 4. Measure weight (grams). that they can be released unharmed. 5. Record the maturity stage of sub-sample of For every fish collected, the following data should target species (for example, reproductive male be recorded (see Appendix A): with seed or female with eggs). 1. Identify the species. If the species cannot 6. Note if a photograph was taken be identified in the field, a specimen, of the specimen. photograph, and detailed description should 7. Note if a DNA fin clip was taken. be taken. Note if the identification provided 8. Note if a voucher specimen was taken. is of high, medium, or low confidence. Species identification must include the 9. Record the fish ID code. scientific name (genus and species) or 10. Include any notes on the fish collected. Figure 2.11 Example of Fish Field Sampling Data Sheet 38 Most fish will be released after each sampling 2.5 Field Sampling Method for method is completed. Macroinvertebrates For each new species of fish captured, the following additional steps should be taken: Macroinvertebrates are an important component 1. Photograph the fish. of the freshwater ecosystem, comprising the largest portion of the aquatic food web and forming a 2. One specimen should be preserved in vital link between aquatic plants, algae, and leaf 85 percent ethanol in a sampling bottle for litter to the fish species and other animals that later verification in the laboratory and as a depend on the river system, including birds. voucher specimen for the reference collection. 3. If possible and of interest, a small (5– 10 mm²) Macroinvertebrates are diverse groups of small sample of fin tissue can be removed invertebrates less than 0.5 mm that can be seen immediately (from live fish or immediately with unaided eye, including insects, annelids, after death) and preserved in a DESS solution arachnids, crustaceans, clams, and gastropods. containing 20 percent dimethyl sulfoxide, These organisms inhabit diverse habitats from 0.25 molar (M) disodium ethylene diamine flowing to still water and feed on a wide range of tetra acetic acid (EDTA), and saturated substrates, depending on their habitat preferences. sodium chloride (Yoder et al. 2006) for DNA Macroinvertebrate communities in a river’s extraction and subsequent genetic analyses. downstream reaches are linked to those in the upstream. Headwater streams harbor organisms Laboratory Processing and Deposition known as “shredders” that break coarse organic All fish specimens collected in the field will be particulate matters; the mid-rivers contain examined in a laboratory, such as a government “scrapers” that feed on algae, diatoms, and other or university fish collection, to identify the aquatic vegetation, while the lower reaches have species using fish-identification resources and the “collector-gatherers” and “collector-filterers” knowledge of fish taxonomy experts. Specimens that consume fine organic particulate matters. should be deposited in a recognized fish collection. “Predators” feed on live animals such as small In Nepal, this would include the National Fisheries invertebrates. Maintaining all these types of Research Centre Godawari of Nepal Agricultural macroinvertebrates is essential for the aquatic Research Council, Kathmandu University, and ecosystem as they help break down organic matter Tribhuvan University. and filter the water, providing clean water for humans and aquatic animals (Table 2.6). Table 2.6 Functional Feeding Groups and Food Resources of Benthic Macroinvertebrates Functional feeding Example family or order of Food resources of the functional group groups macroinvertebrates Shredders Coarse organic particulate matter, including Amphipoda; Limnocentropodidae twigs and leaves Scrapers Periphyton and diatoms Brachycentridae; Glossosomatidae; Coleoptera Collector-gatherers Diatoms, bacteria, and fine organic Trichoptera; Ephemeroptera particulate matter Collector-filterers Fine organic particulate matter Simuliidae; Chironomidae Predators Zooplankton and small invertebrates Plecoptera; Megaloptera; Odonata Source: FRTC/MoFE 2022 39 Some macroinvertebrate taxonomic groups • Live their lives partly or wholly in water or taxa (species, genera, or families) serve as • Are cosmopolitan in nature and highly diverse excellent indicators of river basin health and • Are abundantly found in river systems ecosystem change. Three major orders of aquatic insects—Ephemeroptera (mayflies), Plecoptera • Remain in a generally small area and habitat (stoneflies), and Trichoptera (caddisflies)—make Many macroinvertebrates are sensitive to changes up the EPT index, which uses the species’ presence in habitat, water quality, temperature, flow or abundance to measure water quality. Some rate, and sediments. Figure 2.12 illustrates how families of Diptera (flies), such as Chironomidae, different macroinvertebrate taxa have varying are tolerant of poor water quality and may be the levels of sensitivity to pollutants in water basin, only macroinvertebrates found in heavily modified with some taxa tolerant of poor water quality, aquatic ecosystems. Macroinvertebrates are good some moderately tolerant, and some that can only indicators for assessing the health of the aquatic live in good quality water. ecosystem because they: Figure 2.12 Macroinvertebrate Orders and Sensitivity to Pollutants in River Basin Source: Tachamo Shah et al. 2020a 40 2.5.1 Multihabitat Sampling Using 3. Move, mix, or rub the river-bottom Kick Net substrates manually for a minute to dislodge organisms and substrates so that they flow The macroinvertebrate sampling method for the into the kick net. Trishuli Assessment Tool follows the standardized 4. Rub and wash rocks and other substrates methodology of multihabitat sampling using kick for a minute to collect additional net (Tachamo Shah et al. 2020a). macroinvertebrates. Macroinvertebrate sampling can only be done in 5. Keep and store each sample separately. relatively shallow and low-flow waters, such as in 6. Transfer each sample into a white tray tributaries, at the confluence of tributaries with and inspect it for macroinvertebrates of the main stem river (at the mouth of the tributary), rare or high conservation value, such as and along the banks of the main stem. the threatened Himalayan relict dragonfly (Epiophlebia laidlawi). Overview of the Sampling Process 7. Remove large organic debris and stones from At each sampling site, 20 macroinvertebrate the sample. samples are collected within a 100 m river 8. Transfer the rest of the remaining samples into stretch that contains a variety of aquatic a plastic bucket filled halfway with water. habitats. The samples cover a total area of around 1.25 m² of stream bottom. Sampling is 9. Stir the sample and pass it through a hand net done using a standard kick net with a square of mesh size 500 μm. metallic frame (25 cm × 25 cm) and mesh size 10. Repeat this rinsing process until only mineral of 0.5 mm. substrates remain in the bucket. 11. Visually inspect the sample to pick out any The field process for the multihabitat remaining macroinvertebrates. sampling using a kick net is as follows (see also Figure 2.13): 12. Transfer the collected macroinvertebrates (from the hand net) to a sample container 1. Start sampling from downstream to upstream or bottle with 95 percent ethanol for later at each sampling site. identification in the laboratory. 2. Place the kick net at the river bottom against the flow of the river. Figure 2.13 Sampling Process for Benthic Macroinvertebrates 1 2 3 4 Non–IFC photographs: ©R.D. Tachamo Shah. Used with the permission of R.D. Tachamo Shah. Further permission required for reuse. Note: Step 1 = using a standard kick net in a sampling site; step 2 = sorting macroinvertebrates in the field; step 3 = sorting and identifying specimens in the laboratory; step 4 = macroinvertebrate specimens in petri dishes 41 Selecting the Sampling Sites Challenges Before sampling at each sampling site, the diversity • Samples can only be taken from relatively of aquatic habitats should be assessed within shallow and low-flowing waters the selected 100 m stretch of river (within the • Expertise in identification of macroinvertebrate 400 m Trishuli Assessment Tool sampling area). groups required The percentage coverage of each habitat type within the 50 m downstream stretch below the Training and Safety midpoint and the 50 m upstream stretch should be Field sampling does not require much training. estimated and recorded on the Habitat Data Sheet Safety precautions must be taken when sampling (Appendix C). Macroinvertebrate samples should in the water: life jacket is recommended, be selected from this information to ensure that all particularly in sites with high river discharge microhabitats, substrates, water depths, and flow and large rivers. Training in macroinvertebrate velocities are included in the sample (Figure 2.14). identification is required for sorting and identifying the specimens in the laboratory. Advantages of Macroinvertebrates for Sampling See Box 2.7 for a list of equipment needed for and Monitoring macroinvertebrate sampling. • Occur in high abundance and relatively easy to sample Target Organisms and Habitats • Relatively larger body size, easier to identify Macroinvertebrates can serve as indicators of • Highly diverse taxonomically and ecologically water quality and health of the river basin. Larva and nymph stages of benthic macroinvertebrates • Live from a few months to years so they are included in the assessment as they spend integrate short- and long-term pollution and their entire lives in water. All representative disturbance exposures riverbed habitats including flow types—rapid, • Limited mobility preventing them from escaping riffle, run, and pool—should be sampled for from occasional pollutions macroinvertebrates. • Many taxa are highly sensitive to changes in water quality, flow regimes, water-level fluctuations, and habitat changes Figure 2.14 Categorization of Aquatic Habitat Types for Multihabitat Field Sampling Using a Kick Net Note: Red squares are locations of the selected 20 sampling subsites that represent the diversity of aquatic habitats. 42 Box 2.7 Macroinvertebrate Sampling Equipment IN FIELD • Nonconsumables • GPS or topographic map • Camera or cellphone to photograph habitats and specimens collected • Magnifying glasses • Kick net (25 cm × 25 cm] with a square-shape metallic frame and mesh size of 0.5 mm • Hand net (circular-shaped metallic frame with mesh size of 0.5 mm) • One pair of chest waders • One pair pf half boots • One pair of rubber gloves • White trays • Wide forceps • Plastic buckets • Consumables • 99.9% ethanol • Printed methodology, pencil and sharpener, cardboard, permanent marker, cellotape, and scissors • Sample box • White transparent plastic vials (8 ml) IN LABORATORY • Hand net (circular-shaped metallic frame with mesh size of 0.5 mm) • White trays • Fine forceps • Petri dishes • Stereomicroscope • 99.9% ethanol • White transparent plastic vials (8 ml) Seasonality Specimen Collection and Processing Macroinvertebrate sampling can be done In the laboratory, each benthic sample is rinsed throughout the year except in heavy monsoon in clean water and transferred onto white trays. season. Sampling must be done in both dry and All specimens are picked out of the sediments and post-wet seasons with low and high flows to sorted into groups based on their taxonomic order. capture a diverse range of macroinvertebrates They are then identified to the highest possible in the site. taxonomic level (species, genus, and family) using available reference sources and museum Personnel collections. After sorting, the specimens are stored A team of two to three people is needed to collect in transparent plastic vials containing 95 percent the samples and sort the specimens. One to two ethanol. Each vial is labeled with a paper slip researchers are needed to identify the specimens in containing a sample code before being sealed and the laboratory. stored in a recognized invertebrate museum or collection. Use of a high-powered halogen lamp Time and sharp forceps are advised. It takes one to two hours to sample each field site. Laboratory work to sort and identify the Data Management specimens depends on the diversity and number of Data should be collected on the standardized data individuals in the sample. Usually, 10 to 12 hours sheets in Appendix C. per sample are required to completely sort and identify the specimens as well as count the number Unit of Sampling Effort for Analysis of individuals per taxon. Sampling effort for macroinvertebrates is measured as 20 kick-net samples per site. 43 2.6 Field Sampling for Data Management Periphyton Locations and durations of sampling efforts should be documented in detail in field notebooks, Periphyton are small aquatic plants, such as algae, including photographs of the sites. Periphyton that cling to rocks in the river. The dry biomass data should be recorded on the Periphyton Data of periphyton is a good indicator of the primary Sheet (Appendix D). productivity within the aquatic ecosystem, which Specimen Processing in the Laboratory forms the base of the food chain that sustains all aquatic life in the river basin. The biomass of The biomass of periphyton is determined by the periphyton supports diversity and abundance of standard ash-free dry mass method (APHA 1995). macroinvertebrates in a river. In the laboratory, the following steps should be undertaken to dry the periphyton sample: Overview of the Sampling Process 1. Weigh a clean glass-fiber filter paper. 1. Periphyton sampling should proceed from 2. Filter 100 mL of water with periphyton sample downstream to upstream at each sampling through the glass-fiber filter paper. site. Periphyton samples are to be collected 3. Dry the collected periphyton residue on across various substrates, water depths, and the filter paper at 105°C for one hour in a flow velocities. laboratory oven. 2. At each sampling site, five hand-sized stones 4. Dry the sample at 500°C for three to four (with a maximum diameter or long axis of 10– hours in a muffle furnace. 15 cm) are removed from the stream at a depth of 20–40 cm in slow-flowing areas perennially 5. Weigh the filter paper with the dried under water and from locations undisturbed by periphyton sample (known as ash). benthic sampling. The stones should be picked 6. Calculate the biomass of periphyton using the from the depth at random without the collector following formula: looking into the water at the stones. Biomass of periphyton = [(weight after 3. The stones are to be separately scrubbed in drying at 105°C – initial weight of filter a rinsed tray with a brush to scrape off all of paper) – weight after drying at 500°C]/area of the periphyton and then rinsed with 100 ml periphyton sample collection. distilled water. Unit of Sampling Effort for Analysis 4. The tray, the brush, and the funnel used Sampling effort for periphyton is measured as are rinsed thoroughly with water after each scraping five stones per site. stone is sampled, and the wash is added to the sample. 5. The periphyton collection in the tray is transferred to a 100 ml sample bottle and then 2 ml Lugol’s iodine solution is added for preservation. 6. The bottles are then labeled and stored in a dark bag for transportation. See Box 2.8 for a list of equipment needed for periphyton sampling. Selecting the Stones The dimensions of each stone are measured with a measuring tape and noted in the field data sheet in accordance with the periphyton sample labels. The longest axis or length (X), the longest horizontal axis perpendicular to X or width (Y), the longest vertical axis of the stone or thickness (Z), and circumference (C) are measured for calculating the surface area of the stone. To enhance standardized comparisons, the same person should sample the stones and process the periphyton samples for the entire length of the field trip. 44 Box 2.8 Periphyton Sampling Equipment IN FIELD • Nonconsumables • GPS or topographic map • Camera or cellphone to photograph habitats and specimens collected • Scrub brush • White trays • 100 ml sample bottles (five per site) • Funnel (for transferring sample to sample bottle) • Consumables • 99.9% ethanol • Distilled water • Lugol’s iodine solution • Paper labels for samples IN LABORATORY • Glass-fiber filter paper (with pore size of 0.45 µm) • Filter bottle • Drying oven • Muffle oven • Digital scale for fine measurements (four digits) 45 � Data Analysis and Presentation 3.1 Introduction 3.2 Fish Metrics Presentation, analysis, and reporting of the data Fish should be included in a hydropower project’s collected in the field is an extremely important but EIA and long-term monitoring program because often overlooked part of the EIA and monitoring they are prominent organisms in the aquatic process. It is essential that the data are interpreted ecosystem, with many globally or regionally and conveyed in a way that can be readily threatened and rare species that warrant understood and used by a hydropower project protection. Fish can be identified and analyzed so that it can implement changes to mitigate any at the species level. negative impacts from the project. Similarly, data analysis is needed to clearly show that the project Below are seven recommended metrics that has resulted in no net loss or even a net gain of provide informative analyses for the EIA and biodiversity values. long-term monitoring of a hydropower project as well as help fulfil national and international There are many ways to present and analyze data biodiversity requirements (Table 3.1): for the EIA and for long-term monitoring, with 1. Species richness many statistical tests that can be run. Ecological 2. Species composition Diversity and Its Measurement (Magurran 1988), Rosenzweig (1995), and Feinsinger (2001) are 3. Proportion of species excellent references for field-study design and 4. Species distribution statistical comparisons; there are also many recent 5. Relative abundance of target fish species papers on data-analysis methods (Magurran 6. Recruitment of target fish species et al. 2010; Sreekanth et. al 2015; Tachamo Shah et al. 2020b) and studies of aquatic biodiversity 7. Length of target fish species monitoring (Tachamo Shah and Shah 2012; All of these metrics are calculated and analyzed Birindelli et al. 2016). Graphs, figures, charts, and for each site separately. Hydropower impacts need tables are excellent means of presenting the data, to be site specific because there are many other but be sure to label them well (for example, label disturbances within a watershed, such as sand X and Y axes) and give each a title. mining, fishing, and road construction, which may cause general changes. Raw Field Data Thus, data combined for the entire project The raw field data should be included in the area do not show where the impacts are EIA and monitoring reports, either in the text happening or where and how the metrics are or the appendixes, to allow readers to properly changing. In some cases, data for a region understand the data analysis and metrics. may be combined and analyzed, such as The raw data presented should include all the data when there are multiple sampling sites within categories recorded in Appendix A, as outlined a small area, or when impacts on an entire in Section 2.4.6. region warrant an investigation. Metrics Field Data The data collected using the Trishuli Assessment The EIA or monitoring report should include the Tool should be analyzed using a set of raw data in the text or in appendixes. Table 3.2 metrics. Metrics are a quantitative means of presents a hypothetical example of fish data measuring, comparing, and tracking target from electrofishing for presentation in EIA and indicators over time. This manual includes a monitoring reports. These data are used for some recommended set of metrics for analyzing the of the metrics examples below. fish data and a recommended set of metrics for macroinvertebrates and periphyton. All analyses and graphs recommended here can be done using Excel. The metrics should be selected and analyzed to evaluate specific project impacts and/or the success of mitigation measures. 47 Table 3.1 Recommended Metrics for Fish Data Analysis Metric Indicator Field method Metric calculated for each site Significance no. 1 Species richness All combined No. of species/site Documents the number of fish species 2 Species All combined List of species; presence or Identifies fish species and selects composition absence of species/site target species for monitoring 3 Proportion of All combined No. of individuals of each species/ Shows the percentage of species no. of individuals of all species individuals for each species combined/site 4 Distribution of All combined Map of species locations for all Maps fish distribution to identify species sites important sites and document locations 5 Relative Each method— CPUE = No. of individuals for each Documents changes in relative abundance of electrofishing target species/sampling effort/site abundance of key fish species target species and cash nets— separately 6 Recruitment of Each method— CPUE for juveniles = No. of juvenile Documents continued target species electrofishing fish individuals/sampling effort/ recruitment and breeding of key and cash nets— site fish species to sustain population separately Density of juveniles = No. of juvenile fish individuals/100 m²/site 7 Length of target All combined Mean length +/– standard Assesses size and evaluates if a species deviation = total of fork length for fish is a juvenile or an adult all fish/no. of fish/site Note: CPUE = catch per unit effort Table 3.2 Sample Field Data Presentation for EIA and Monitoring Reports FIELD DATA—ELECTROFISHING, SPRING 2021 REGION 1: UPSTREAM OF DAM Number of fish individuals captured Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Fish species Main stem Tributary Tributary Tributary Tributary Main stem Schizothorax richardsonii 16 40 15 24 55 20 Schizothorax progastus 2 0 3 0 0 3 Garra annandalei 0 5 0 0 1 0 Opsarius bendelisis 1 0 2 1 0 0 Neolissochilus hexagonolepis 1 3 1 2 0 2 Paracanthocobitis botia 0 0 0 0 0 0 Psilorhynchus pseudecheneis 0 0 0 0 0 0 Total 20 48 21 27 56 25 48 REGION 2: DIVERSION REACH Number of fish individuals captured Site 7 Site 8 Site 9 Site 10 Site 11 Site 12 Fish species Tributary Main stem Tributary Tributary Main stem Main stem Schizothorax richardsonii 25 13 15 32 25 25 Schizothorax progastus 1 1 0 0 3 0 Garra annandalei 2 5 7 2 1 8 Opsarius bendelisis 1 0 9 1 0 2 Neolissochilus hexagonolepis 0 1 2 4 1 2 Paracanthocobitis botia 0 4 0 1 2 0 Psilorhynchus pseudecheneis 0 0 2 0 0 3 Total 29 24 35 40 32 40 REGION 3: DOWNSTREAM OF POWERHOUSE Number of fish individuals captured Site 13 Site 14 Site 15 Site 16 Site 17 Site 18 Fish species Tributary Main stem Tributary Tributary Main stem Tributary Schizothorax richardsonii 30 22 30 40 10 21 Schizothorax progastus 10 4 0 3 10 7 Garra annandalei 5 7 15 6 8 4 Opsarius bendelisis 2 3 5 7 0 2 Neolissochilus hexagonolepis 2 5 9 4 1 0 Paracanthocobitis botia 0 5 3 2 4 1 Psilorhynchus pseudecheneis 3 1 0 2 1 0 Total 52 47 62 64 34 35 49 Metric 1: Fish Species Richness Table 3.3 Example Summary Data: Number of Fish Species Recorded per Site Definition: Species richness is the number of species recorded Region Site Number of fish species Calculation: Species richness = number of species Upstream of dam 1 4 per site 2 3 3 4 Field Methods: Combine data from all sampling 4 3 methods used at the site 5 2 Scale of Analysis: Analyze each site separately 6 3 Diversion reach 7 4 Presentation of Data in EIA Report 8 5 • Number of species per site, by region, 9 5 and overall 10 5 • Bar chart (Figure 3.1) 11 5 12 5 Monitoring Downstream of powerhouse 13 6 • Visually compare the bar charts over time to 14 7 look for general trends. 15 6 • Species richness is not recommended for long-term monitoring comparisons. 16 7 Changes over time in the number of 17 6 species are challenging to interpret since 18 5 the number of species is relatively small and natural variation may be large. Interpretation The total number of fish species for the project area with 18 sites is seven. The total number of • This number should be contrasted with the species per region are: number of species in similar rivers in the region. Is it high, low, or typical for a Himalayan river? • Upstream of dam: five species This number can highlight if there are only a few • Diversion reach: seven species species to focus on, or if many species need to be • Downstream of powerhouse: seven species considered for impacts. • Species richness data can be compared between Visual Presentation regions to investigate if there are baseline differences between the regions that could be Figure 3.1 Number of Fish Species Recorded attributed to other factors such as elevation, per Site water temperature, number of tributaries, sand mining, and other HPPs. Example Table 3.3 shows an example of summary data of the number of fish species recorded per site based on the hypothetical data presented in Table 3.2, which only covers electrofishing. Additional data from other methods need to be added to these data for a full picture of species richness. 50 Example Interpretation Metric 2: Species Composition EIA Definition: Species composition is the identity of • The number of species per site generally all species in the project area increases toward downstream. Discuss possible reasons for this, such as water temperature, Calculation: Identify all species recorded using nutrient availability, food abundance for fish, valid references, collections, and experts nursing and recruiting grounds, and other possible disturbances in the river. General Field Methods: Combine data from all characteristics of each region should be sampling methods presented and discussed. • Is the number of species typical of a Himalayan Scale of Analysis: By site, region, and project area river of this altitude? Why or why not? Provide as appropriate for the impacts comparative data and reference to other scientific studies of the area. Presentation of Data in EIA Report Monitoring • Include a list of species recorded overall. • Changes in species richness may not be very • Identify species of conservation concern informative for long-term monitoring due to using the International Union for the high natural variation and low numbers, Conservation of Nature (IUCN)’s Red List but such changes can be compared visually of Threatened Species and any national over time to detect major trends that could threatened species categories. indicate an impact. • Identify any other species of interest, such as migratory species, range-restricted (endemic) species, non-native species, and rare species. • Select target species for long-term monitoring, which may be highly threatened species, or other species that could be affected by the HPP. • For taxa with a long list of species, also include a list of the 25 most common across sampling sites (number of sampling sites) or most abundant (number of individuals). • Note relevant information on the ecology, lifecycle, range, and biology of the species of interest. Analysis of Long-Term Monitoring Data • Compare the lists to see if there are any changes in species composition. • Note any new species of conservation concern or non-native species. • Compare species lists of impact regions to control sites (if applicable). • Compare presence or absence of species over time between surveys. Reporting and Interpretation Suggestions • Changes in species composition can indicate if species drop out or are introduced into the project area over time. • Note whether a species disappears through time consistently across sites and surveys. This will warrant further investigation into the cause. 51 • New species that appear over time may be Example Interpretation introduced by people, such as fish species being released into the reservoir or species EIA arriving through new access to the area. Non- • Discuss the biology of the species recorded native species are of particular concern and and include references, such as the migratory should be noted and monitored closely, with behaviors of the two snow trout species and possible adaptive management to remove them. their spawning sites. Since there are only a few Correctly identifying the species in each survey species, a paragraph or two and a photo should is very important. be included for each species. If there are more • This metric is important for identifying species species, select those of most relevance to the of conservation concern and of interest to project impacts. the project as “target or indicator species.” • Identify species of biodiversity importance for These species should be monitored over time the project (see Table 3.5): to assess any project impacts on them and as o Two species on the list are classified by an umbrella species representing other species. IUCN Red List (https://www.iucnredlist.org) Monitoring is less effective for species with as globally threatened: Schizothorax few data points, such as rare or uncommon richardsonii (vulnerable) and Neolissochilus species. Thus, even though these species may be hexagonolepis (near threatened). Both species of interest, target species should have sufficient are migratory and have declining populations data points for analysis over time. across their range. • Comparison between impact sites and control o Schizothorax progastus is a mid-range sites (if applicable) can indicate if a species migratory species whose access to spawning is dropping out of the HPP impact zone but sites may be blocked by the HPP dam. is still present in the control sites. This will o Psilorhynchus pseudecheneis is only found indicate the need for further investigation. in Nepal and northern India, so any impacts on its populations may be detrimental to the Example global population. Table 3.4 shows an example of fish species • Looking at the abundance data of the fish in recorded by all sampling methods in spring 2021 the data set above, some of the species have based on hypothetical fish data in Table 3.2, low numbers of individuals recorded and thus which only covers electrofishing. Additional data would not likely provide sufficient data for from other methods need to be added to these monitoring analysis. data for a full picture of species richness. Table 3.4 Species Recorded by All Sampling Methods in Spring 2021 Species Global common Nepal common IUCN Red List National Range- Fish species Migratory no. name name category status restricted 1 Schizothorax Common snow Buche Asala VU Mid-range No richardsonii trout (decreasing) 2 Schizothorax Dinnawah snow Chuche Asala LC Mid-range No progastus trout (unknown) 3 Garra annandalei Annandale garra LC No No (unknown) 4 Opsarius (formerly LC No No Barilius) bendelisis (stable) 5 Neolissochilus Chocolate Katli NT Long-range No hexagonolepis mahseer (decreasing) 6 Paracanthocobitis LC No No botia (decreasing) 7 Psilorhynchus Stone carp LC Nepal and pseudecheneis (unknown) northern India only Note: IUCN Red List categories: CR = Critically Endangered; E = Endangered; VU = Vulnerable; NT = Near Threatened; LC = Least Concern; population trend is shown in parentheses. 52 • Schizothorax richardsonii should be selected Monitoring as a target species as it is the most common, has sufficient data for analysis, and is an Table 3.5 Presence or Absence of Fish Species important threatened and migratory species. Upstream of Dam with All Methods Combined S. richardsonii may serve as an umbrella species Spring for species with similar biology (such as S. 2021 Spring Spring 2022 2023 progastus and Neolissochilus hexagonolepis) Species pre-con- construc- construc- struction but should be confirmed with information on baseline tion tion their biology and references. Schizothorax • Psilorhynchus pseudecheneis may be an X X X richardsonii important species but is found in such low Schizothorax X numbers that it may be difficult to compare progastus over time. However, its presence or absence Garra annandalei X X X should be monitored over time. Opsarius bendelisis X X X • Photos of important species can be included to Neolissochilus X X help readers visualize the species (Figure 3.2). hexagonolepis Paracanthocobitis X botia Figure 3.2 Important Fish Species Psilorhynchus X pseudecheneis Schizothorax richardsonii Oncorhynchus X X mykiss Example Interpretation • Schizothorax progastus has not been collected upstream since the pre-construction baseline. This may indicate that project construction Opsarias bendelisis is impeding its migration or has affected its upstream populations. Further investigation is needed on this species and the cause of its disappearance. Mapping of its distribution is needed (see Metric 4). • A new species has been added to the list. Oncorhynchus mykiss is the non-native rainbow trout commonly bred in hatcheries for food. It can cause declines in native fish species where it is introduced into the Tor Putitora natural river system. This species may have been present before construction but has not been recorded; it may also have been introduced or its population has increased during construction. Further investigation is needed to determine the cause of this species’ introduction or increase with adaptive management to prevent further growth. Non-IFC photograph of Tor putitora; ©A. Pinder. Used with the permission of A. Pinder. Further permission required for reuse. 53 Metric 3: Proportion of Individuals of These data can be visualized using pie charts for Each Fish Species each site, region, or overall: Definition: Percentage of all individuals of each Figure 3.3 Pie Charts Showing Species and fish species out of all species combined (Table 3.6) Percentages of Fish Recorded in Various Sampling Regions Calculation: Number of individuals of species/ number of individuals of all species combined Percentage of total no. of fish Field Methods: Combine data from all sampling Upstream methods (except eDNA) Scale of Analysis: By site, region, and project area as appropriate for the impacts Presentation of Data in EIA Report • Calculate percentage of individuals of each species for each site, region, and project area, depending on where project impacts may occur. • Compare percentages between sites or regions. • Present the data in pie charts (Figure 3.3). Diversion Reach Monitoring • Visually compare pie charts from the same site or region between years to observe changes in percentages of each species. • Note whether any species has become dominant or has significantly reduced. • Compare between regions to document any natural variation and potential causes. • Changes in percentages of species over time may indicate that ecosystem conditions have Downstream changed to favor the populations of some species over others (for example, reservoirs will favor lake species rather than river species). Investigate these changes to determine if they are due to hydropower project impacts, which would require adaptive management. Example Table 3.6 Number of Fish Recorded Upstream of Dam (Six Sites)—All Methods Combined Fish species No. of fish % of total Schizothorax richardsonii Schizothorax richardsonii 170 86.29 Schizothorax progastus Schizothorax progastus 8 4.06 Garra annandalei Garra annandalei 6 3.05 Opsarius bendelisis Opsarius bendelisis 4 2.03 Neolissochilus hexagonolepis Neolissochilus hexagonolepis 9 4.57 Paracanthocobitis botia Paracanthocobitis botia 0 0.00 Psilorhynchus pseudecheneis 0 0.00 Psilorhynchus pseudechenesis Total no. of fish, all species 197 54 Example Interpretation Metric 4: Distribution of Target Fish Species EIA • The proportion of individuals per species Definition: This metric maps the location where differed across the three regions, with target fish species were recorded Schizothorax richardsonii being the most abundant in each region but with decreasing Calculation: Map of target fish species records proportion going downstream. In the upstream region, over 86 percent of the individuals Field Methods: Combine data from all were S. richardsonii, with four other sampling methods species each making up less than 5 percent; Paracanthocobitis botia and Psilorhynchus pseudecheneis were not recorded upstream at Scale of Analysis: Analyze by site all. S. richardsonii also made up 67.5 percent of individuals in the diversion reach captures. Presentation of Data in EIA Report • All other species increased in proportion in the • Maps of sampling sites where each target fish diversion reach and downstream regions, with species was located, preferably a separate map Garra annandalei increasing the most in the for each target species diversion reach (12.5 percent) and downstream • Maps to include information on the relative of the powerhouse (15.3 percent). abundance (number of individuals) of the fish • Discuss possible reasons for these trends. species at each sampling site • Show on the maps whether each site is Monitoring on a tributary or main river and note • Pie charts can be compared over time, as is any characteristics of the site, such as the done above between regions, to assess changes confluence of tributary and river, backwater, in the proportion of individuals of each species. deep-flowing river, and downstream • Changes may need to be investigated to disturbances (such as sand mining) determine location and cause. Monitoring • Maps can be compared to see highlight changes in distribution for any of the target fish species. • Maps can show changes in relative abundance at each site and highlight where the changes are occurring so that causes can be investigated and adaptive management implemented. If a fish species drops out consistently at a particular site or its relative abundance declines at the site, further study is needed. • Compare the locations of each species and the habitat. Protection of particular sites and type of habitat may be essential for the long-term sustainability of the species. Example See Figure 3.4 and Figure 3.5. Example Interpretation • The distribution map of Schizothorax richardsonii across the sampling sites shows that the species is found at all sites across all surveys (Figure 3.4). Adding abundance data to the map would provide additional data on changes in abundance at each site. • The distribution map of Schizothorax progastus reveals changes in its distribution over time (Figure 3.5). The species is 55 present upstream of the dam before but not after construction. This would warrant further investigation and possible adaptive management. • Similar distribution maps could be drawn for other target fish species, abundant fish species as well as migratory and endemic fish species. Figure 3.4 Schizothorax richardsonii Distribution Map across the Sampling Sites Reservoir Distribution of Schizothorax richardsonii (all surveys) Figure 3.5 Schizothorax progastus Distribution Map across the Sampling Sites Reservoir Distribution of Schizothorax progastus Spring 2021 (pre-construction) Distribution of Schizothorax progastus Spring 2022 (1st year of construction) 56 Metric 5: Relative Abundance of Target Box 3.1 CPUE Definition Fish Species What Is CPUE? Definition: Number of individuals (relative CPUE, or “catch per unit effort,” is a way abundance) for target fish species, such as the to standardize the data between samples threatened snow trout (Schizothorax richardsonii) collected by the same method that have or golden mahseer (Tor putitora), species that may not been collected with the same effort. serve as umbrella species, migratory species, or For example, if 25 casts of the cast net were other species important to monitor due to their used at sites A and B, and 100 casts were role in the ecosystem done at sites C and D, the data collected will not be comparable since more effort was put in at sites C and D. In order to Calculation: Number of individuals (and/or make them comparable (see example in CPUE) of a target fish species (Table 3.7) Table 3.7), divide the number of individuals recorded by the number of sampling effort Field Methods: Analyze each method separately: units: CPUE = number of fish collected/ electrofishing and cast nets sampling effort. It is important to define the sampling effort Scale of Analysis: Analyze each site separately unit as it is different for each sampling method. CPUE should not be used for comparisons between sampling methods; Target Fish Species: Schizothorax richardsonii it can only be used to compare different sampling efforts for the same method. Presentation of Data in EIA Report CPUE is suitable for data collected by electrofishing and cast nets. Data from dip • Number of individuals and CPUE for each nets, underwater video, and eDNA should target fish species for each site be used to complement the CPUE analysis • Bar plot, box and whiskers plot, and line graph for tributaries and juveniles. Monitoring • Compare the CPUE per site over time between sampling periods (always Interpretation compare the same season). • This metric focuses on a few important target • Examine the trends in CPUE changes fish species that have sufficient data and can over time (statistical trends analysis can serve as an umbrella indicator for other species. be used if desired). • Each site should be analyzed separately to • Compare using bar charts or other locate where the changes are occurring and graphs to assess trends over time. possibly requiring adaptive management. • Compare control sites to project impact • It may take several years before changes are sites to assess if the trends stay similar observed. Thus, monitoring is done throughout over time. the construction of a hydropower project and for several years during operations. Table 3.7 Sample CPUE Conversion Sampling effort No. of fish CPUE Method Sampling unit units collected (no. of fish/sampling effort) Cast net 25 casts 1 cast 15 15÷25=0.60 per cast Cast net 100 casts 1 cast 24 24÷100=0.24 per cast Electrofishing 20 min (0.34 hour) Hour of electrofishing 36 36÷0.34=105.88 per hour of electrofishing Electrofishing 40 min (0.67 hour) Hour of electrofishing 65 65÷0.67=59.70 per hour of electrofishing 57 • Comparisons of the trend or slope between Visual Presentation seasons can reveal if there is an upward (increasing number of individuals), flat line Figure 3.6 CPUE of S. richardsonii by (stable number), or downward (declining Electrofishing Upstream of Dam, Spring 2021 number) trend. Statistical tests such as linear regression can be used to test if the changes are statistically significant when sufficient sample size is reached. • Statistical analyses can be used to compare the mean CPUE between sites or over time if there are sufficient (more than five) data points, such as sampling sites within a region or multiple sampling surveys at a site over time. Before combining sites and data, consider if the analysis will provide meaningful information to assess changes as a result of HPP impacts. Example From Metric 2, Schizothorax richardsonii was selected as a target fish species, so the analysis here focuses only on this species (Table 3.8 and Figure 3.6). Additional analyses can be done for other target fish species. Table 3.8 Electrofishing Field Data, Spring 2021 REGION 1: UPSTREAM OF DAM Number of fish individuals captured Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Fish species Main stem Tributary Tributary Tributary Tributary Main stem No. of Schizothorax -- 28 24 24 18 -- richardsonii recorded Effort (minutes) -- 40 40 23 34 -- Effort (hours) -- 0.67 0.67 0.38 0.57 -- CPUE 41.8 35.8 63.16 31.58 Monitoring Table 3.9 Summary Data for Spring Survey Field Data (Electrofishing) Site 2 Site 3 Site 4 Site 5 Control site Tributary Tributary Tributary Tributary Tributary CPUE Spring 2021 52.2 35.8 47.4 70.2 65.3 CPUE Spring 2022 50.3 40.6 25.7 58.8 61 CPUE Spring 2023 43.9 36.2 25.8 66.2 75 CPUE Spring 2024 45.8 25.2 18.2 40.5 82 58 Figure 3.7 Bar Charts Presenting Spring Survey Metric 6: Recruitment of Target Field Data (Electrofishing) Fish Species (Relative Abundance of Juveniles) Definition: Number of individuals of juvenile fish, focusing on “young of the year” fish (zero to one- year-old) and young juveniles in their second year of growth (one to two-year-old) Juveniles should be identified by total length (length of fish from snout to end of tail) or fork length (length of fish from snout to tail fork). This would be determined separately for each species based on the size ranges caught or from literature. For S. richardsonii, individuals with less than 10 cm of fork length would be Example Interpretation considered juveniles. • The data are presented and analyzed at the site This metric can be analyzed as: 1) CPUE or 2) level because we want to see where changes density of individuals per 100 m². Density is a are occurring in order to determine if they are possible metric for juveniles in tributaries since caused by HPP impacts. Upstream sites are they often congregate in “shoals” and can be important because the HPP dam may block unevenly distributed across the sampling site. migration of S. richardsonii adults to spawning Focusing on a smaller area where juveniles are sites in the tributaries upstream. Thus, changes congregating can provide more detailed data that in specific tributaries should be studied. can be used to evaluate no net loss of juveniles • In Figure 3.7, each of the upstream tributaries (recruitment) over time. (sites 2 to 5) has a downward trend, with Tributary 4 showing the largest drop in spring Calculation: CPUE and/or density of “young of the 2022. This site should be investigated for year” individuals and/or young juveniles per site potential project impacts or other disturbances to the fish population. Field Methods: Analyze each method separately, • The control site shows an upward trend including electrofishing, cast nets, dip nets, and (Table 3.9). This site is outside of the project underwater video area and is not directly affected by any HPP or other disturbance, such as sand mining. Scale of Analysis: Analyze each site separately The trends of this site may indicate that S. richardsonii populations are maintained Target Fish Species: Schizothorax richardsonii in other sites so that any declines within the juveniles (fish with fork length of less than 10 cm) project site should be further investigated. • In this example, only tributary data are Presentation of Data in EIA Report provided because electrofishing was not able to be conducted in the main stem. Sites 1 and • CPUE and/or density of individuals (number 6 were on the main stem. Comparisons should of individuals/100 m²) for juveniles of S. always be done between similar types of sites richardsonii and other target fish species for and under similar weather conditions. each site • The data from cast netting could show • Bar plot, box and whiskers plot, and line graph different trends. The same type of analysis should also be done for data from cast nets and Monitoring compared with the electrofishing data as well • Compare the CPUE and/or density per site as data from dip nets and underwater video over time between sampling periods (always if implemented. comparing the same season). • Data from dip netting and underwater video • Analyze the trends of changes in CPUE and/or from tributaries can be reviewed separately density over time. to provide further information and details for • Compare using bar charts or other graphs to understanding the biology of the fish species. assess trends over time. • Compare control sites to impact sites to assess if the trends are similar over time. 59 Interpretation to sample using electrofishing. Adult fish are much more mobile, so they are only present Same as “Example Interpretation” in Metric 4 plus at certain times of the year and are more the following. This metric is especially important challenging to catch by surveyors. 
 and effective due to several aspects of fish biology • Fish in early life stages are not subject to (A. Pinder, pers. comm.): harvest depletion by local fishers. 
 • The presence of juvenile snow trout in • The continued presence of juvenile fish in tributaries provides definitive evidence that the tributaries, particularly upstream of the adult fish have successfully migrated into the HPP, would indicate that fish passage is being tributary during the spawning migration. 
 facilitated by the project (for example, if a fish • Due to the high numbers of eggs deposited by ladder is present) or the river upstream of the many fish species, particularly cyprinids, and HPP provides all necessary critical habitats to the cumulative mortality throughout life, the support a fragmented fish population. numbers of juvenile fish and their availability for capture are considerably higher than that of Example older life stages. 
 • The presence of snow trout in early life stages CPUE is calculated in a similar manner to Metric 5 provides evidence beyond the successful but focuses only on juvenile fish. A second metric, immigration of adults from the main river. density (number of individuals per 100 m²), is They also qualify the functionality of habitats valuable for documenting large groups (shoals) to support egg incubation and provide nursery of juveniles, particularly in microhabitats within support during the most critical phase of a tributaries. For S. richardsonii, juveniles can be fish’s life. 
 identified as fish with fork length of less than • Fish in early life stages are present in the 10 cm (100 mm). tributaries throughout the year and are easy Table 3.10 Schizothorax richardsonii Juveniles (Electrofishing) Site 2 Site 3 Site 4 Site 5 Control site Spring 2021 Tributary Tributary Tributary Tributary Tributary Number of fish recorded 35 24 18 40 65 Effort (minutes) 40 40 23 34 60 Effort (hours) 0.67 0.67 0.38 0.57 1 CPUE 52.2 35.8 47.4 70.2 65 Table 3.11 S. richardsonii Juveniles (Cast Nets) Site 2 Site 3 Site 4 Site 5 Control site Spring 2021 Tributary Tributary Tributary Tributary Tributary Number of fish recorded 10 6 9 5 15 Effort (no. of casts) 50 100 100 50 100 CPUE 0.2 0.06 0.09 0.1 0.15 60 Table 3.12 S. richardsonii Juveniles (Targeted Electrofishing in Selected 100 m² Area in Spring Sampling) Site 2 Site 3 Site 4 Site 5 Control site Spring 2021 Tributary Tributary Tributary Tributary Tributary No. of juvenile fish 100 10 22 50 34 recorded in spring 2021 No. of juvenile fish 75 56 25 68 55 recorded in spring 2022 No. of juvenile fish 20 45 32 59 48 recorded in spring 2023 Area 100 m² 100 m² 100 m² 100 m² 100 m² Density (No. of 100 10 22 50 34 individuals/100 m²) 2021 Density 2022 75 56 25 68 55 Density 2023 20 45 32 59 48 Figure 3.8a Bar Charts of CPUE and Density for Five Tributary Sites Electrofishing Cast Nets 0.25 80 60 0.2 CPUE 40 CPUE per Cast 20 0.15 0 0.1 Tributary Tributary Tributary Tributary Tributary 0.05 0 Site 2 Site 3 Site 4 Site 5 Control 2 3 4 5 Control site Site Figure 3.8b Density of Juveniles per 100 m² for Five Tributary Sites in Spring Surveys over Three Years 100 90 Density of Juveniles/100m² 80 70 60 50 40 30 20 10 0 2 3 4 5 Control Tributary Site # ■ 2021 ■ 2022 ■ 2023 61 Example Interpretation Metric 7: Length of Target Fish Species • This metric provides information on the recruitment and spawning success of fish. For Definition: Length of each target fish species, Schizothorax richardsonii, it happens mainly in measured as fork length and total length, using tributaries and at the confluence of tributaries data from all sampling methods combined, with the main river. Thus, in this example, only including electrofishing, cast nets, dip nets, and tributary data are analyzed. underwater video • Of the sampling methods, electrofishing and cast-net data can be analyzed using CPUE Calculation: Mean length plus or minus standard and density (example data in Table 3.10, deviation = total of fork length for all fish/number Table 3.11, Table 3.12, and Figure 3.8). of fish per site • Additional data from dip nets and underwater video are also effective for documenting Field Methods: All methods combined juvenile fish in tributaries. Data from these (except eDNA) methods should be reviewed and analyzed more qualitatively to add details to the Scale of Analysis: Analyze each site separately analysis. The number of fish documented by underwater video can be analyzed per minute Target Fish Species: Schizothorax richardsonii or hour within a specific area to estimate juveniles (fish with fork length of less than 10 cm) density. The number of juveniles collected by dip nets should be evaluated for each Presentation of Data in EIA Report microhabitat sampled to provide more details • Present a table of the length data of target on the habitats of the juvenile fish. fish species per site for all fish from all • The data from these methods are analyzed methods combined (Table 3.13). If there are separately since the efforts and sampling many individuals, this data should be put in approaches are different and each provides an appendix. a different perspective to measuring fish • Calculate the mean (average) length plus or abundance. Note that the scales for CPUE minus standard deviation (SD) per site. These are different in the graphs of Figure 3.8, can be calculated in an Excel spreadsheet so they are not comparable and should using =AVERAGE (range of values) and be assessed separately. =STDEV (range of values). • Compare the data over time as in Metric 5. In • Visualize using bar graphs (Figure 3.9). Figure 3.8b, the density of juveniles is relatively consistent over the three years at all sites except for Tributary Site 2, which dropped in Monitoring the third year. This could indicate some type • Compare mean length plus or minus standard of impact by the project or other causes and deviation for a site across several survey years, should be investigated. for example, spring 2021, 2022, and 2023 • Trends over five years or more should be (Table 3.14). evaluated to assess if the project is aligned with • Visualize with bar graphs (Figure 3.10). no-net-loss goals. • Compare statistically by using a Student’s T-Test to compare two samples, analysis of variance (ANOVA) for multiple samples, and nonparametric tests (Mann-Whitney-Wilcoxon Test and Kruskal-Wallis ANOVA), linear regression, or other statistical tests. Interpretation • The mean fish length provides an indication of the population structure in terms of size and age for each sampled site. • The mean length can show whether the population at a site is primarily of juveniles or adults. Tributaries with many juveniles can indicate that spawning is occurring. 62 • Fish length is correlated with age, so the age • The mean length can indicate if adult fish are structure of the population can be assessed and reaching particular sites, especially tributaries compared over time. where they spawn. • Changes in the mean length can indicate that • The standard deviation illustrates the amount certain age groups are decreasing or increasing. of variation within the fish sizes at each site. Example Table 3.13 Fork Length Measurements for S. richardsonii at Four Tributary Sites Tributary upstream Tributary in diversion Tributary downstream Tributary control fork length (cm) reach fork length (cm) fork length (cm) fork length (cm) 9.2 6.5 7.5 10.6 6.2 12.5 4.3 4.3 8.5 3.3 8.4 5.7 15.8 7.7 13.5 7.4 10.5 15.8 5.6 5.5 5.2 20.4 9 8.2 7.7 5.5 8.5 4.6 6 5.8 12.5 8 6.9 6.7 12 10.5 8.2 6.5 10 Mean 8.69 9.49 8.11 7.61 Standard 3.11 5.33 2.92 2.61 Deviation (SD) Visual Presentation Figure 3.9 Mean Fish Length at Four Tributary Sites, Spring 2021 Tributary Upstream Tributary Diversion Tributary Tributary Control Reach Downstream 63 Monitoring Table 3.14 Mean Fish Length at Four Tributaries over Four Years Tributary upstream Tributary diversion reach Tributary downstream Tributary control Spring 2021 8.69 9.49 8.11 7.61 Spring 2022 15 7.2 9.5 6.7 Spring 2023 13.2 9.2 8.1 8.2 Spring 2024 12 8 7.2 7.3 Figure 3.10 Mean Fish Length at Four Tributaries over Four Years Tributary Upstream Tributary Diversion Tributary Tributary Control Reach Downstream Example Interpretation the control site is relatively constant over time, • Figures 3.9 and 3.10 present the mean length such as in the graph above, but a tributary site of fish plus or minus the standard deviation for within the impact zone has a change in mean four tributary sites over four years of spring- size, this may indicate a change in the age season sampling from 2021 to 2024. and size structure due to the HPP. These sites warrant further investigation. • The figures show trends in the data over time. • In this example, the mean size of fish upstream • The fish length data can also be compared is increasing, while the control site is constant. using statistical tests such as ANOVA, linear Fish may not be spawning upstream because regression, and trends analysis to evaluate if of barriers to migration or a change in the data are statistically different over time. spawning-site conditions. This warrants • The impact sites, such as the upstream further investigation to see if it is related to tributary, can be compared with the control site HPP impacts. to evaluate differences. If the mean fish size in 64 3.3 Macroinvertebrate Metrics This manual recommends the following three metrics (Table 3.15) for analysis of Macroinvertebrates are important to include in the macroinvertebrate data that are useful for EIA and long-term monitoring of a hydropower assessing and monitoring the impacts of HPPs on project because they form the basis of the aquatic the aquatic ecosystem: ecosystem. Changes to their composition and 1. Macroinvertebrate taxa richness populations affect the aquatic food chain, resulting 2. Ephemeroptera, Plecoptera, Trichoptera (EPT) in a knock-on effect on fish and other aquatic index organisms. Macroinvertebrates are also sensitive 3. Relative abundance of functional feeding to changes and thus can serve as indicators of groups aquatic ecosystem health. In contrast to fish, it is difficult to identify Field Data macroinvertebrates to species level. Thus, specimens are usually identified to the order, To allow readers to properly understand the family, and genus taxonomic level where metrics and data analysis, the raw field data possible. Since the level of identification can vary, should be included in the EIA and monitoring macroinvertebrates are characterized and referred reports. The data should include at minimum: to as a “taxon” or as multiple “taxa,” which • List of taxa, arranged by order, family, and indicate a distinct taxonomic unit. genus • Number of organisms captured for each taxon In addition to taxonomic challenges, very at each site few macroinvertebrate taxa in the Himalayan region have been evaluated to determine • Description of each sampling site, including their status as threatened species. Therefore, weather conditions and habitat notes macroinvertebrate data analysis focuses on their See Appendix E for macroinvertebrate field data use as indicators of the status of, and changes from the Trishuli River collected in February in, aquatic ecosystem health. There are many 2020 (Tachamo Shah et al., unpublished data). ways to analyze macroinvertebrate data. See The Appendix E table illustrates the preferred Tachamo Shah et al. (2012; 2020a; 2020b) and format for presentation in EIA and monitoring FRTC/MoFE (2021) for additional analyses and reports. The organisms were identified to family details as well as examples of macroinvertebrate or genus level and data represent the number of monitoring in Nepal. individuals recorded for each taxon. These data are used for some of the metric examples below. Table 3.15 Summary of Recommended Macroinvertebrate Metrics Metric Indicator Field method Metric for each site Significance no. 1 Macroinvertebrate Multihabitat assessment No. of macroinvertebrate Diversity of taxa richness using 25 kick-net samples taxa per site macroinvertebrate per site community 2 EPT index Multihabitat assessment EPT index = no. of Indicator groups assess using 25 kick-net samples taxa from EPT orders; and detect changes per site proportion of EPT taxa in water quality and per site aquatic ecosystem health 3 Relative abundance Multihabitat assessment No. of individuals from each Functional feeding of functional feeding using 25 kick-net samples functional feeding group/ groups represent the groups per site total no. of individuals/site condition of aquatic ecosystem health 65 Metric 1: Macroinvertebrate Taxa Table 3.16 Number of Macroinvertebrate Richness and Proportion Genera per Site Unlike fish, macroinvertebrates in the Himalayas Site codes Number of taxa are difficult to identify to species level. Thus, they are usually identified to the genus or family level. UCH 21 LCH 19 Definition: Taxa richness is the number of taxa SAK 36 (species, genera, or families) recorded LAN 26 Calculation: Number of taxa per sampling effort MAI 31 Field Methods: Multihabitat sampling using TAD 32 kick nets UBK 30 Scale of Analysis: Analyze each site separately Note: UCH = Upper Chilime Khola; LCH = Lower Chilime Khola; SAK = Salankhu Khola; LAN = Langtang Khola; Presentation of Data in EIA Report MAI = Mailung Khola; TAD = Tadi Khola; UBK = Upper Bode Khosi • Number of taxa per site, by region, and overall (Table 3.16) • List of taxa by genus, family, and order • Calculate the proportion of taxa for each order Visual Presentation • Bar charts and pie charts (Figure 3.11) Figure 3.11 Number of Macroinvertebrate Genera per Site Monitoring • Visually compare the bar charts and pie charts over time to look for general trends. • Taxa richness is less useful for long-term monitoring comparisons. Interpretation • Taxa richness and proportions should be compared with the number of taxa in similar rivers in the region. Is it high, low, or typical for a Himalayan river? This number can Note: highlight whether there are only a few or many UCH = Upper Chilime Khola; LCH = Lower Chilime Khola; species that need to be considered for impacts. SAK = Salankhu Khola; LAN = Langtang Khola; • Taxa richness data can be compared between MAI = Mailung Khola; TAD = Tadi Khola; UBK = Upper Bode Khosi regions to see if there are regional baseline differences that could be attributed to other factors, such as elevation, water temperature, number of tributaries, sand mining, and Example Interpretation other HPPs. • Changes in the number of taxa over time are EIA challenging to interpret since the number of • Adding information on the type (tributary species is relatively small and natural variation or main stem) and locale (upstream, may be large. Thus, taxa richness is not diversion reach, or downstream) of sites recommended for quantitative comparisons would make this graph more informative over time. for showing initial differences between sites and regions. Example • The number of taxa per site ranges from 19 to 36. Discuss possible reasons for this, such Based on the data in Appendix E, the survey as habitat type, tributary or main stem river, sampled 78 taxa from 46 families and 11 orders. 66 water temperature, and other disturbances Metric 2: EPT Index in the river. General characteristics of each region should be presented and discussed. Several aquatic macroinvertebrate taxa are used • Is the number of taxa per site typical of a to evaluate water quality and aquatic ecosystem Himalayan river of this altitude? Why or health. Ephemeroptera (mayflies), Plecoptera why not? Provide comparative data and (stoneflies), and Trichoptera (caddisflies), known reference to scientific studies. as the EPT orders, require clean water quality to survive (see Figure 3.12). The EPT index was • The bar charts can be compared over time developed to use these three orders to assess for each site to note any major changes in and detect changes in water quality and aquatic taxa richness. ecosystem health. Figure 3.12 Images of EPT Taxa Ephemeroptera (mayflies) Nymph Adult Plecoptera (stoneflies) Nymph Adult Trichoptera (caddisflies) Adult Non–IFC photographs: ©R.D. Tachamo Shah. Used with the permission of R.D. Tachamo Shah. Further permission required for reuse. 67 These three orders have aquatic immature stages Interpretation that live in rivers and streams. Ephemeroptera • The EPT Index is used as an indicator of and Plecoptera have incomplete metamorphosis, water quality and aquatic ecosystem health. meaning that their immature stage is a nymph In general: that molts and grows larger until it is ready to • More than 50 percent indicates high water- change into an adult, while Trichoptera have quality status complete metamorphosis consisting of four life- stages, namely egg, larva, pupa, and adult. The • From 20 to 50 percent indicates moderate nymph leaves the river to molt on land into an water-quality status adult, which is a flying and terrestrial stage. The • Less than 20 percent indicates poor water- adults mate and females lay eggs in the water, quality status starting the cycle again. The nymphs and adults of all three orders are prey items for many fish Example species. Ephemeroptera and Trichoptera nymphs Table 3.17 shows the number of genera for are collector-gatherers, feeding on organic matter different macroinvertebrates collected from the in the water. Odonata nymphs are predators and Trishuli River based on field data in Appendix E, prey on other macroinvertebrates and small fish. followed by an example demonstrating how Trichoptera larvae often build themselves a house the EPT index is calculated. Table 3.18 shows out of gravel, sand, and organic materials. the number of taxa of the EPT orders at three different sites and how the EPT index for each site Definition: EPT index is a measure of the richness is calculated. of Ephemeroptera, Plecoptera, and Trichoptera in the sample as an indicator of water quality Table 3.17 Number of Macroinvertebrate Genera Calculation: Number of Ephemeroptera, for All Sites at the Trishuli River Plecoptera, and Trichoptera taxa per site; this can also be shown as a proportion (number of Number of Order % Total genera EPT taxa/number of total taxa x 100 = % per site) Coleoptera 4 0.05 Field Methods: Multihabitat sampling using kick nets Diptera 18 0.23 Ephemeroptera 18 0.23 Scale of Analysis: Analyze each site separately Hemiptera 1 0.01 Presentation of Data in EIA Report Lepidoptera 1 0.01 • Total number of macroinvertebrate taxa Megaloptera 1 0.01 per site Odonata 3 0.04 • Number of Ephemeroptera, Plecoptera, and Trichoptera taxa per site Opisthopora 1 0.01 • EPT index for each site Plecoptera 8 0.10 • List of EPT taxa per site Trichoptera 22 0.28 • Compare the EPT index to scores from other Trombidiformes 1 0.01 sites in the Himalayas • Description of each order and its importance in Total 78 the aquatic ecosystem EPT taxa = Ephemeroptera (18) + Plecoptera (8) + • Pie charts Trichoptera (22) = 48 taxa EPT index = Ephemeroptera (18) + Plecoptera (8) Monitoring + Trichoptera (22)/total taxa x 100 = 48/ 78 x 100 = 61.54% of taxa • Compare the EPT index for each site over time to observe trends and compare the index to rating charts. • Compare the proportion of EPT taxa over time to evaluate changes in water quality. • Compare pie charts of the proportion of taxa to determine if there are changes over time. 68 Table 3.18 Number of EPT Taxa per Site Metric 3: Relative Abundance of Functional Feeding Groups Number of taxa Site Macroinvertebrates play many important roles Order UCH LCH SAK in the aquatic ecosystem—as shredders, scrapers, Ephemeroptera 7 6 10 collector-gatherers, collector-filterers, and predators. See more details of these functional Plecoptera 2 0 3 feeding groups and their food sources in Table 2.6. Trichoptera 6 7 5 Information on the relative abundance of each functional feeding group at a sampling site Total number of taxa 21 19 36 provides a picture of the natural balance of these groups in the aquatic ecosystem. Monitoring Note: UCH = Upper Chilime Khola; LCH = Lower Chilime Khola; SAK = Salankhu Khola changes in these groups can highlight if the aquatic ecosystem is becoming out of balance. EPT index per site (see Figure 3.13 and Figure 3.14): Upper Chilime Khola (UCH) = 7+2+6 = 15; Definition: Relative abundance of individuals from 15/21 x 100 = 71.43% of all taxa each functional group Lower Chilime Khola (LCH) = 6+0+7 = 13; 13/19 x 100 = 68.42% of all taxa Salankhu Khola (SAK) = 10+3+5 = 18; Calculation: Number of individuals per functional 18/36 x 100 = 50% of all taxa group/number of total individuals/site Field Methods: Multihabitat sampling using using Visual Presentation a kick net Figure 3.13 EPT Index at Three Different Sites Scale of Analysis: Analyze each site separately Presentation of Data in EIA Report • Sort the raw data by functional group • Total number of taxa for each functional group for each site • Bar chart Monitoring • Compare relative abundance of each group over time and assess trends. • Compare bar charts over time and assess Figure 3.14 EPT Taxa as Percentage of All Taxa at Three Different Sites changes and trends. • Compare pie charts of the proportion of each 80.00 70.00 functional group over time. 60.00 % of all taxa 50.00 Example 40.00 30.00 Table 3.19 uses hypothetical data of the number 20.00 of individuals for each functional group at three 10.00 sites and Figure 3.15 is a visual presentation of the 0.00 same data. UCH LCH SAK Site Example Interpretation • EPT index ranges from 50 to 71 percent of all taxa in the sampling sites, indicating that the sites have good water quality and that the hydro-morphology of the sites is relatively undisturbed. • The EPT index can be compared to assess changes in water quality over time. 69 Monitoring Table 3.19 Number of Individuals for Each Table 3.20 Number of Individuals for Each Functional Feeding Group at Three Sites, Functional Feeding Group at Site UCH over Spring 2021 Three Years Site Site Functional feeding groups UCH LCH SAK Functional feeding Spring Spring Spring groups 2021 2022 2023 Shredders 15 21 15 Shredders 15 20 12 Scrapers 7 10 33 Scrapers 7 13 9 Collector-gatherers 25 13 8 Collector-gatherers 25 7 16 Collector-filterers 11 22 21 Collector-filterers 11 15 21 Predators 8 18 17 Predators 8 22 17 Total number of individuals 66 84 94 Total number of 66 77 75 individuals Note: UCH = Upper Chilime Khola; LCH = Lower Chilime Khola; SAK = Salankhu Khola Note: UCH = Upper Chilime Khola Visual Presentation Figure 3.15 Relative Abundance of Each Figure 3.16 Relative Abundance of Each Functional Feeding Group at Three Sites, Functional Feeding Group at Site UCH over Spring 2021 Three Years Note: UCH = Upper Chilime Khola Example Interpretation • The relative abundance and proportion of each functional feeding group is different at each site. Scrapers are most variable between sites during this survey. • The relative abundance of each functional group varied over time at site UCH Note: UCH = Upper Chilime Khola (Table 3.20). It depends on the availability and quality of allochthonous³ or autochthonous4 3 Allochthonous inputs are organic particulate matters that come from outside of the river, such as fallen plant leaves, branches, or twigs from surrounding or upstream reaches as well as trees that topple into the river. Allochthonous inputs are a source of carbon, nitrogen, and phosphorus. 4 Autochthonous inputs are the large plants, attached algae, and phytoplankton that are present within the river system where energy is provided by the photosynthesis of the plants and algae growing in the system. 70 inputs in the study sites. This is particularly Calculation: Ash-free dry weight (micrograms) of important as any reductions in flow variability periphyton/site downstream of an HPP dam increases the relative proportion of scrapers due to the Field Methods: Scraping five rocks growth of algae. • Upstream site and tributaries are important Scale of Analysis: Analyze each site separately because they receive allochthonous inputs from adjacent riparian vegetation, so the relative Presentation of Data in EIA Report abundance of shredders and collector-gatherers • Ash-free dry weight of periphyton per site are usually high. During construction of a hydropower dam, the riparian vegetation may • Bar charts be removed upstream and downstream of the dam, which will likely change the proportion Monitoring of shredders and collector-gatherers in the sites • Compare dry weight per site to assess trends as indicated in Figure 3.16 for spring 2022. over time. • The results should be discussed in relation to • Changes in periphyton over time may indicate how each functional feeding group contributes that the food base is declining. to the aquatic ecosystem and what the changes mean for the health of the ecosystem. See Interpretation more in Tachamo Shah and Shah (2012) and Tachamo Shah et. al (2020a). • Ash-free dry weight can be an indicator for monitoring river ecosystem health because its increase may indicate an increase in the river system’s productivity. 3.4 Periphyton Metrics • Ash-free dry weight should be compared to the pre-construction samples and to control Similar to aquatic macroinvertebrates, periphyton sites, as well as over time, to evaluate if the are important as the foundation of the aquatic values are within the normal range for the ecosystem, serving as food for many organisms river system. and breaking down organic matter. • Higher than normal productivity may lead to Table 3.21 shows the principal metric for eutrophic5 or hypereutrophic state, which may monitoring periphyton—ash-free dry weight, indicate poor quality of the aquatic system. which is the weight of the periphyton sample after Zero to little flow discharge downstream of an it is dried and oxidized (ashed). HPP dam elevates the water temperature and the nutrient concentration, which can cause periphyton and algae to increase rapidly. Definition: Biomass of periphyton per site Table 3.21 Periphyton Metric Metric Indicator Field method Metric Significance 1 Periphyton Scraping five rocks Ash-free dry weight Biomass of periphyton indicates health of biomass per site (AFDW) of periphyton aquatic ecosystem and shows changes due per site to river flow and water quality 5 A eutrophic river is rich in nutrients and may have a dense plant population, the decomposition of which can kill aquatic animal life by depriving it of oxygen. 71 3.5 Preliminary Assessment address the failure. Thresholds for no net loss and net gain should be calculated from the baseline of No Net Loss or Net Gain for data collected in the field and models of predicted International Lenders changes to determine the natural levels before the project is constructed. Preliminary thresholds can Many international lenders, such as IFC and be set as a percentage increase or decrease from the World Bank, require hydropower projects the baseline. that are operating in natural or critical habitats to show that the project achieves no net loss This manual does not cover how to calculate of biodiversity values and possibly even a net thresholds and assess no net loss or net gain. gain of biodiversity values for critical habitats However, the following steps are recommended to (IFC 2012). It should be noted, however, that begin an assessment: natural habitats should not be interpreted as 1. Use the metrics recommended in this manual untouched or pristine habitats. It is likely that the to track changes over time during pre- majority of habitats designated as natural would construction, construction, and operation of have undergone some degree of historical or the HPP. recent anthropogenic impact, such as the presence 2. Set preliminary thresholds for no net loss for of invasive alien species, secondary forest, human each site based on the baseline data, such as the habitation, or other human-induced alteration. lowest, mean, or highest baseline value. These With respect to HPPs, where the watershed has thresholds are only preliminary and should be been degraded but assemblages of largely native reviewed and updated based on analysis of the species are present in the water body itself, then monitoring data. IFC’s or World Bank’s no-net-loss requirements 3. Set key color-coded thresholds (red and should be applied to the species regardless of orange) to alert the HPP to potentially the degradation of the surrounding riverine or significant deviations from its anticipated watershed habitat. trajectory toward no net loss (Table 3.22). The green target indicates the project is on There is no standard calculation for determining track. The orange zone indicates that the metrics or targets for no-net-loss or net-gain project is below the target threshold and the status. Given the longer time frames required cause of which should be investigated. The to achieve no net loss or net gain, one or more red zone indicates that the project is not on interim targets may be needed to track progress target and requires immediate investigation toward the final targets. A few examples of and adaptive management. ways to achieve no net loss or net gain include 4. Make line graphs with interval lines to indicate IUCN’s net-positive-impact approach,6 the World the maximum, mean, and minimum baseline Bank’s recommendations for biodiversity offsets values for each metric. Changes in the data (WBG 2016), and Forest Trends’ Business and between the baseline and monitoring surveys Biodiversity Offsets Program.7 should be easily visualized in these line graphs. Based on these and other interim targets, 5. Preliminary targets can also be set to indicate thresholds should be set to produce a value that the desired or predicted net gain in the metrics. will indicate if the metrics are within or beyond This could be set at 1–10 percent and adjusted acceptable levels and if adaptive management later based on actual monitoring data. is warranted. This involves reviewing current 6. Metrics within the orange and red thresholds management practices that have led to the breach alert the project that further investigation and of the threshold and proposing a change to possible adaptive management is required. 6 https://www.iucn.org/theme/business-and-biodiversity/resources/business-approaches-and-tools/business-and- biodiversity-net-gain 7 https://www.forest-trends.org/bbop/bbop-key-concepts/biodiversity-offsets/ 72 Table 3.22 Adaptive Management Thresholds Adaptive management threshold Definition No-net-loss target Metric values within the green block indicate that mitigation measures are effective and supporting progress toward achieving no net loss. The target falls within a user-defined percentage range above the orange threshold. Orange adaptive management Metric data points within the orange range indicate that mitigation is off track. threshold The project should check if mitigation measures are being implemented and if they are successful in achieving no net loss. This threshold has a lower and upper percentage change (below and above) the minimum baseline metric values. Red adaptive management Metric values within the red range indicate that the project is severely off track. threshold The functioning and effectiveness of mitigation measures should be reviewed immediately, including the operation of EFlows of the hydropower project, to determine what urgent corrective action can be taken to put the project back on track to achieve no net loss. This is a user-defined threshold and extends a percentage (lower limit) below the bottom end of the orange threshold. Example 1 Example 2 Figure 3.17 CPUE of Schizothorax richardsonii for Figure 3.18 CPUE of Schizothorax richardsonii for One Site over Nine Annual Surveys One Site over 12 Annual Surveys Note: CPUE = catch per unit effort; NNL = no net loss Note: CPUE = catch per unit effort; NNL = no net loss Note that all values in Figure 3.17 are above CPUE values in Figure 3.18 are above the no-net- the no-net-loss (green) target that was set as the loss threshold until the 7th survey when values minimum baseline value. This indicates that the drop into the orange zone. Values rise but then project is on track to achieve no net loss for this fall again into the orange zone. These values alert metric. Minimum, mean, and maximum baseline the project to investigate what is going on at this values are marked with the dotted lines. Net-gain particular site and to correct or enhance mitigation thresholds should exceed the maximum recorded as needed. Minimum, mean, and maximum baseline value by a pre-determined percentage. baseline values are marked with colored lines. To achieve a net gain, the metric values should exceed the maximum recorded baseline value by a pre-determined percentage. In this example, the values exceed the maximum value in the 3rd and 6th annual survey (data points) before dropping. This shows the importance of long-term monitoring since changes may greatly vary from year to year. 73 � Reporting 4.1 Overview 4.2 Sample ESIA Report— Aquatic Biodiversity Baseline Nepal’s Environment Protection Rules (Nepal Law Commission 1999) and Hydropower Chapter Environmental Impact Assessment Manual (MoFE 2018) outline requirements and 1. Overview of the taxonomic group in provide guidance for the structure of initial the region environment examination (Schedule 11) and • Summary of information collected from EIA (Schedule 12) reports. Table 15 of the literature with references Hydropower Environmental Impact Assessment Manual (MoFE 2018) also outlines the required 2. Sampling sites structure for government EIAs. Other countries • Rationale and justification for site selection have their own requirements for what an EIA and strategy should include. International lenders often have • Map of the sampling sites additional requirements and expect a higher level • GPS coordinates of all sampling sites of field assessment and detail in an environmental and social impact assessment (ESIA) report. 3. Field methods • Dates of surveys at each sampling site The Trishuli Assessment Tool is designed to collect and analyze field data on aquatic • Seasonality of the sampling dates, such as biodiversity for EIAs and the long-term wet, dry, and transitional seasons monitoring of hydropower projects. The • Map and GPS coordinates of all data produced from the tool are suitable for sampling points international-level ESIAs and biodiversity • Detailed field methods evaluation and monitoring programs (BMEPs). • Description of each sampling method Below are sample outlines of the aquatic • Sampling effort (such as time, number of biodiversity baseline chapter of an international- samples, and time spent sampling per site) level ESIA (Section 4.2) and of an international- • Details of equipment used level BMEP (Section 4.3). These examples can be • Names of field workers performing used as a guide to fulfil the requirements of most the surveys (including qualifications international lenders. and affiliations) In addition to presenting the EIA and monitoring • Names of people identifying the species data to the hydropower project and the (including qualifications and affiliations) authorities, such data can also be shared with the broader scientific community through 4. Data for all species recorded scientific publications and the Global Biodiversity • Total number of species recorded, including Information Facility (GBIF; www.gbif.org). By the number of genera and of families making data accessible through the international • Number of species recorded at each GBIF or similar information platforms, the sampling site data can be used by other scientists to advance • Number of species recorded in each understanding of the species, which will increase region (upstream of dam, diversion reach global knowledge of biodiversity and contribute to between dam and powerhouse, and finding conservation solutions. downstream of powerhouse) 75 • List of all species by sampling site and date, biodiversity. Impacts may be negative, neutral, with number of individuals of each species or positive depending on the species or habitats’ • For taxa with a long list of species, include adaptability to new conditions, such as reservoirs a list of the 25 most common (by number of and altering flow regimes. A cumulative impact sampling sites or number of individuals) assessment should follow international guidelines such as those prepared by IFC (2020). For the 5. Lists and discussion of target species of aquatic ecosystem, a high resolution, holistic conservation importance, including: environmental-flow (EFlow) assessment should • Threatened species (IUCN Global be used to evaluate the project impacts upstream Red List and regionally or nationally and downstream of the proposed project location. threatened species) IFC’s Good Practice Handbook on Environmental Flows for Hydropower Projects (IFC 2018) • Range restricted species (endemic) provides guidance on selecting and implementing • Rare species the appropriate EFlow assessment for HPPs. • Migratory and congregatory species The Mitigation Chapter of the EIA should • Species of cultural and religious importance include actions to minimize project impacts on • Species of medicinal importance the overall aquatic environment and on species • Species that may cause or promote disease of conservation importance according to the in humans mitigation hierarchy, that is, avoid, reduce, • Species of livelihood importance restore, and offset impacts (CSBI 2015). For • Invasive species many international lenders, including the World Bank, IFC, and the Asian Development Bank, the 6. Compilation of the data with data previously mitigation measures must demonstrate that the collected for the area for comparisons project achieves no net loss of biodiversity values for natural habitats and a net gain for critical 7. Analysis of data metrics for target species habitat values (IFC 2012). and establish baseline for long-term monitoring; metrics should include species richness, species composition, relative abundance of target species, relative 4.3 Sample BMEP Report abundance of juveniles, and aquatic for Monitoring Aquatic ecosystem health indexes (see Section 3) Biodiversity for a Hydropower 8. Identification of important habitat types Project for species such as fish spawning sites, migration routes, habitats, or other areas 1. Background on the project and biodiversity of high importance for biodiversity; for of the area example, analyze high macroinvertebrate species richness and concentrations of 2. Monitoring results and analysis threatened species • Map(s) of important areas for biodiversity Fish • Discussion of the importance of the areas • Objectives of monitoring fish • HPP mitigation measures assessed with 9. Discussion of species of conservation the monitoring metrics importance listed in the literature, which • Long-term monitoring field could be in the project area but were not sampling methodology documented during the survey • Dates of surveys at each sampling site 10. Photos and video of sampling sites, field • Seasonality of the sampling dates, such as methods, and species of interest wet, dry, and transitional seasons 11. Appendixes with raw data • Map and GPS coordinates of all sampling points The Impact Assessment Chapter of the EIA should • Detailed field methods include an evaluation of the direct, indirect, residual, and cumulative impacts that the project ∙ Description of each sampling method may have on the biodiversity documented, ∙ Sampling effort (such as time, number of particularly on species of conservation importance, samples, and time spent sampling per site) important habitats, and areas of high or unique ∙ Details of equipment used 76 • Names of field workers performing the surveys • Analysis and discussion of macroinvertebrate (including qualifications and affiliations) metrics (see Section 3.3) • Names of people identifying the species 1. Macroinvertebrate taxa richness (including qualifications and affiliations) 2. EPT index • All raw data from the field surveys 3. Relative abundance of functional presented in appendixes, including habitat feeding groups descriptions, species, number of individuals, • Interpretation of analysis results in relation to and measurements the mitigation measures and impacts • Summary of overall monitoring results with • Recommendations for changes to graphs and figures monitoring methodology • Analysis and discussion of fish metrics • Recommendations for adaptive management (see Section 3.2) based on monitoring metrics 1. Species richness 2. Species composition Periphyton 3. Proportion of species • Objectives of monitoring periphyton 4. Species distribution • HPP project mitigation measures assessed with 5. Relative abundance of target fish species the monitoring metrics 6. Recruitment of target fish species • Long-term monitoring field sampling 7. Length of target fish species methodology • Interpretation of analysis results in relation to • Dates of surveys at each sampling site the mitigation measures and impacts • Seasonality of the sampling dates, such as • Recommendations for changes to wet, dry, and transitional seasons monitoring methodology • Map and GPS coordinates of all • Recommendations for adaptive management sampling points based on monitoring metrics • Detailed field methods ∙ Description of each sampling method Macroinvertebrates ∙ Sampling effort (such as time, number of • Objectives of monitoring macroinvertebrates samples, and time spent sampling per site) • HPP project mitigation measures assessed with ∙ Details of equipment used the monitoring metrics • Names of field workers performing the surveys • Long-term monitoring field (including qualifications and affiliations) sampling methodology • Names of people identifying the species • Dates of surveys at each sampling site (including qualifications and affiliations) • Seasonality of the sampling dates, such as • All raw data from the field surveys wet, dry, and transitional seasons presented in appendixes, including habitat • Map and GPS coordinates of all descriptions, species, number of individuals, sampling points and measurements • Detailed field methods • Summary of overall monitoring results with graphs and figures ∙ Description of each sampling method • Analysis and discussion of periphyton metrics ∙ Sampling effort (such as time, number of (see Section 3.4) samples, and time spent sampling per site) • Biomass, measured as ash-free dry weight ∙ Details of equipment used • Interpretation of analysis results in relation to • Names of field workers performing the surveys the mitigation measures and impacts (including qualifications and affiliations) • Recommendations for changes to • Names of people identifying the species monitoring methodology (including qualifications and affiliations) • Recommendations for adaptive management • All raw data from the field surveys based on monitoring metrics presented in appendixes, including habitat descriptions, species, number of individuals, 3. Summary of all monitoring results and measurements • Summary of overall monitoring results with 4. Recommendations for monitoring and graphs and figures adaptive management 77 � References Adeva-Bustos, L.E. Alonso, A. Harby, N. Prakhan, D. Narayan Shay, and R. Yadav. 2021. Himalayan Aquatic Biodiversity and Hydropower: Review and Recommendations. Washington, D.C.: World Bank Group. ADB (Asian Development Bank). 2018. Impact of Dams on Fish in the Rivers of Nepal. Manila, the Philippines: ADB. https://www.adb.org/sites/default/files/publication/480221/impact-dams-fish-rivers-nepal. pdf. APHA (American Public Health Association Inc.). 1995. Standard Methods for the Examination of Water and Wastewater. 19th ed. New York: APHA. Beaumont, W.R.C. 2021a. Detailed Instructions for Conducting Backpack Electrofishing. UK.: EFTS Ltd. Beaumont, W.R.C. 2021b. Trishuli Electric Fishing Manual. U.K.: EFTS Ltd. Birindelli, J.L.O., V. Meza-Vargas, L.M. Sousa and M. Hidalgo. 2016. “Standardized Rapid Biodiversity Protocols: Freshwater Fishes.” In: Larsen, T.H. (ed.). Core Standardized Methods for Rapid Biological Field Assessment. Arlington, VA: Conservation International. CSBI (Cross Sector Biodiversity Initiative). 2015. A Cross-Sector Guide for Implementing the Mitigation Hierarchy. Prepared by The Biodiversity Consultancy on behalf of IPIECA, the International Council on Mining and Metals, and the Equator Principles Association. Cambridge, UK: CSBI. Federal, Provincial, and Territorial Governments of Canada. 2010. Canadian Biodiversity: Ecosystem Status and Trends 2010. Ottawa, Ontario: Canadian Councils of Resource Ministers. Online edition. https:// biodivcanada.chm-cbd.net/ecosystem-status-trends-2010/canadian-biodiversity-ecosystem-status-and-trends- 2010-full-report. Feinsinger, P. 2001. Designing Field Studies for Biodiversity Conservation. USA: The Nature Conservancy. FRTC/MoFE (Forest Research and Training Center, Ministry of Forests and Environment). 2022. Freshwater Ecosystem Assessment Handbook. Babarmahal, Kathmandu, Nepal: FRTC/MoFE, Government of Nepal. Green, R.H. 1979. Sampling Design and Statistical Methods for Environmental Biologists. Chichester, UK: Wiley Interscience. IFC. 2012. Performance Standard 6: Biodiversity Conservation and Sustainable Management of Living Natural Resources. Washington, D.C.: IFC. https://www.ifc.org/wps/wcm/connect/topics_ext_content/ifc_ external_corporate_site/sustainability-at-ifc/policies-standards/performance-standards/ps6. IFC. 2018. Good Practice Handbook on Environmental Flows for Hydropower Projects. Washington, D.C.: IFC. https://www.ifc.org/wps/wcm/connect/b5c4fc9d-8eaf-46da-833b-3dd07c0bc985/GPH_ Eflows+for+Hydropower+Projects_Updated_compressed.pdf?MOD=AJPERES&CVID=mhN3tCS. IFC. 2020. Cumulative Impact Assessment and Management: Hydropower Development in the Trishuli River Basin, Nepal. Washington, D.C.: IFC. https://www.ifc.org/wps/wcm/connect/topics_ext_content/ifc_ external_corporate_site/sustainability-at-ifc/publications/publications_report_cia-trishuli. 79 IFC. 2021. “Trishuli Assessment Tool: A Standardized Field Approach to Aquatic Monitoring.” Webinar held February 2, 2021. https://www.ifc.org/wps/wcm/connect/topics_ext_content/ifc_external_corporate_site/ sustainability-at-ifc/company-resources/ifc_sustainability_webinars. Kaasa, H. 2008. “Khimti Khola Hydropower Project.” River Ecology Program Report 2001–2007. Sweco report to Himal Power Ltd., Nepal. Magurran, A.E. 1988. Ecological Diversity and Its Measurement. Princeton, NJ: Princeton University Press. Magurran, Anne E., Stephen R. Baillie, Stephen T. Buckland, Jan McP. Dick, David A. Elston, E. Marian Scott, Rognvald I. Smith, Paul J. Somerfield, and Allan D. Watt. 2010. “Long-Term Datasets in Biodiversity Research and Monitoring: Assessing Change in Ecological Communities through Time.” Trends in Ecology and Evolution 25 (10): 574–582. doi:10.1016/j.tree.2010.06.01. MoFE (Ministry of Forests and Environment). 2018. Hydropower Environmental Impact Assessment Manual. Singha Dunbar, Kathmandu: MoFE. http://mofe.gov.np/downloadfile/Hydropower%20 Environmental%20Impact%20Assessment%20Manual_1537854204.pdf. Nepal Law Commission. 1999. Environment Protection (First Amendment) Rules. 2055(1998) 2055.12.22 (April l5, 1999). Kathmandu: Nepal Law Commission. https://www.lawcommission.gov.np/en/archives/ category/documents/prevailing-law/rules-and-regulations/environment-protection-rules-2054-1997 Philipp, D., J. Claussen, A. Man Sherchan, A. Pinder, B. Beaumont, G. Walsh, D. Karmacharya, R.D. Tachamo Shah, D. Narayan Shah, and L. Alonso. 2020. Standardized Sampling of Aquatic Organisms to Monitor Population Trends in Nepal. Washington, D.C.: IFC. Rosenzweig, M. L. 1995. Species Diversity in Space and Time. New York: Cambridge University Press. Shah, D.N., N. Pradhan, R. Yadav, and L.E. Alonso. 2020. Nepal Hydropower – Biodiversity Mitigation Status Report. Report to the World Bank. Kathmandu, Nepal. Sreekanth, G.B, M. Lekshmi, and N.P. Singh. 2015. “Biodiversity Assessment: Improved Methods and Approaches.” In Biodiversity & Evaluation: Perspectives and Paradigm Shifts. Tachamo Shah, R.D, and D.N Shah. 2012. “Performance of Different Biotic Indexes Assessing the Ecological Status of Rivers in the Central Himalaya.” Ecological Indicators 23: 447-452. Tachamo Shah, R.D, D.N. Shah, and S. Sharma. 2020a. Rivers Handbook – A Guide to the Health of Rivers in the Hindu-Kush Himalaya. Nepal: Aquatic Ecology Centre, Kathmandu University, Nepal. ISBN: 978- 9937-0-81252 Tachamo Shah, R.D., S. Sharma, and L. Bharati. 2020b. “Water Diversion Induced Changes in Aquatic Biodiversity in Monsoon-Dominated Rivers of Western Himalayas in Nepal: Implications for Environmental Flows.” Ecological Indicators 108, 105735. WBG (World Bank Group). 2016. Biodiversity Offsets: A User Guide. Washington, D.C.: World Bank Group. https://documents1.worldbank.org/curated/en/344901481176051661/pdf/110820-WP-Biodiversity OffsetsUserGuideFinalWebRevised-PUBLIC.pdf. Yoder, Melissa, Irma Tandingan De Ley, Ian King, Manuel Mundo-Ocampo, Jenna Mann, Mark Blaxter, Larisa Poiras, and Paul De Ley. 2006. “DESS: A Versatile Solution for Preserving Morphology and Extractable DNA of Nematodes.” Nematology 8 (3): 367–376. doi:10.1163/156854106778493448. 80 � Appendixes Appendix A Field Data Sheet for Fish Data Recording FISH SAMPLING DATA SHEET Site Number River Name Location Location Code Date Time Method Sampling Effort (time or #) Upstream Dpwnstream Total # fish Total # fish Upstream/ Fork Total DNA - Voucher Downstream Length Lenght Weight Photo Fin Clip Specimen Fish ID Sample # Fish # Species (mm) (mm) (grams) (Y/N) (Y/N) (Y/N) Code Notes 82 Appendix B Field Data Sheet for Recording Site and Habitat Characteristics Field data sheet for recording site and habitat characteristics, with a set of example field data from the February 2020 Trishuli River field sampling (DS = downstream, US = upstream of sampling center point) Example Field Data Field Data REGION Upstream of HPP SITE CATEGORY SITE # 3 Location RIVER Lower Chilime Khola Location SITE CODE LCH Location LOCATION Shyrapru Besi Location GPS LATITUDE (N) 28.1816 Location GPS LONGITUDE (E) 85.3423 Location DATE SAMPLED FEBRUARY 27, 2020 Location ELEVATION (m) 1495 Water Data WATER TEMP (°C) 12.4 Water Data CONDUCTIVITY (µmhos/cm) 377 Water Data FLOW Moderate Water Data TURBIDITY Low/Mod Site Total Area SITE LENGTH TOTAL (m) 400 Site Upstream Area SITE LENGTH US (m) 200 Site Downstream Area SITE LENGTH DS (m) 200 Upstream Area UPSTREAM WET WIDTH (m) 8 Upstream Area UPSTREAM TOTAL WIDTH (m) 30 Upstream Area US % RAPID 30 Upstream Area US % RIFFLE 20 Upstream Area US % RUN 10 Upstream Area US % POOL 40 Upstream Area US % SLACK 0 Upstream Area US % BACKWATER 0 Center Area CENTERPOINT Suspension bridge Center Area CENTERPOINT WET WIDTH (m) 5 Center Area CENTERPOINT TOTAL WIDTH (m) 40 Downstream Area DOWNSTREAM WET WIDTH (m) 12 Downstream Area DOWNSTREAM TOTAL WIDTH (m) 20 Downstream Area DS % RAPID 30 Downstream Area DS % RIFFLE 20 Downstream Area DS % RUN 10 Downstream Area DS % POOL 40 Downstream Area DS % SLACK 0 Downstream Area DS % BACKWATER 0 Method ELECTROFISHING Yes Method CAST NET Yes Method DIP NET No Method GO PRO VIDEO Yes Method E-DNA Yes Method MACRO-PERIPHYTON Yes NOTES 83 Appendix C Data Sheets for Macroinvertebrate Field Data Recording Appendix C.1 Site Information Sheet 1. Site Information 1.1 River/Stream 1.2 River system 1.3 Place, district, province 1.4 Site code 1.5 Coordinates 1.6 Date/Time N: E: Altitude: 1.7 Surveyor 1.8 Investigator 2. Catchment Characteristics 2.1 Predominant surrounding land-use: 2.2 Riparian vegetation (within 18 m buffer in sampling): Indicate at 10% intervals for 1 km river stretch (taken upstream of site) 2.2.1 Dominant vegetation type: Forest ...……………% Trees Shrubs Grasses Herbaceous Field/Pasture ...……………% 2.3 Canopy cover at zenith: Agricultural ...……………% Open Partly open Partly shaded Shaded Residential ...……………% Commercial ...……………% 2.4 Local watershed erosion: Industrial ...……………% None Moderate Heavy Other (Specify) ...……………% 3. Hydro-Morphological Parameters (Instream Features) 3.1 River depth: 3.2 Wetted river width: 3.3 Discharge (m³/s) 3.4 Proportion of reach (Avg. of 4 measurements represented by flow types: within 100 m stretch) Min.: …………….. i) ……………….. Min.: …………….. Rapid …………...% Max.: ……………. ii) ………………. Max.: ……………. Riffle ……………% Avg.: ……………. iii) ……………… Avg.: ……………. Run ……………% iv) ……………… Pool ……………% Avg.: ………………. 4. Water-Quality Parameters 4.1 Temperature 4.2 pH 4.3 Turbidity 4.4 DO, DO Saturation …..…………..˚C …………… …..……… NTU ……..…. mg/L, ………......% 4.5 Electrical conductivity 4.6 TDS 4.7 Nitrate 4.8 Phosphate ………..……… µS/cm ………….... (mg/L) ………………….(mg/L) ………………….(mg/L) Source: FRTC/MoFE 2022 84 Appendix C.2 Habitat Estimation Sheet Site code: Date/time: Investigator: Mineral substrate Coverage Sampling Flow types (5% steps) units (no.) Run Pool Riffle Glide Rapid Boulders, bedrock (> 40 cm) Cobbles (> 20 cm – 40 cm) Stones (> 6 cm – 20 cm) Pebbles (> 2 cm – 6 cm) Gravel (>0.2 cm – 2 cm) Sand and mud (>6µm – 2 mm) Silt loam, clay (inorganic) (< 6 µm) Artificial substrates Sum 100 20 Biotic substrate Algae Macrophytes – Emergent Macrophytes – Submerged Macrophytes – Floating Living parts of terrestrial plants Wood – tree trunks, branches, roots Coarse particulate organic matter deposits Fine particulate organic matter deposits Debris – organic and inorganic matter deposits Source: FRTC/MoFE 2022 85 Appendix D Data Sheet for Periphyton Sampling Field Data Periphyton sampling data Site number: River Name: Location: Site code: Date: Time: Stone measurement Stone Dimensions (cm) Circumference (cm) Water depth (cm) X Y Z 1 2 3 4 5 Sketch of sampling site from where the stones were picked up 86 Appendix E Sample Macroinvertebrate Field Data for the Trishuli River Order Family Subfamily/ Functional UCH LCH SAK LAN MAI TAD UBK Genus feeding groups Coleoptera Elmidae Collector- 1 8 2 gatherers Coleoptera Gyrinidae Predator 3 Coleoptera Psephenoidinae Psephenoidinae Scrapers 12 5 Coleoptera Scirtidae Collector- 1 gatherers Diptera Athericidae Predator 2 2 6 3 4 Diptera Blepharicera Blepharicera Scrapers 6 Diptera Blepharicera Horaia Scrapers 9 20 1 Diptera Ceratopogonidae Predator 1 Diptera Chironominae Tanytarsini Collector- 1 filterers Diptera Chironominae Tanypodinae Predator 1 1 1 Diptera Chironominae Diamesinae Collector- 10 3 gatherers Diptera Chironominae Orthocladiinae Scrapers 2 21 5 2 10 Diptera Chironominae Chironominae Collector- gatherers Diptera Deuterophlebiidae Scrapers Diptera Dolichopodidae Predator 1 Diptera Empididae Predator 1 Diptera Limoniidae Hexatoma Predator 10 1 5 6 2 Diptera Limoniidae Predator 1 5 19 13 7 9 Diptera Pediciidae Dicranota Predator 1 Diptera Simuliidae Collector- 27 3 5 filterers Diptera Tabanidae Predator 4 1 4 Diptera Tipulidae Predator 36 1 2 2 Ephemeroptera Ameletidae Ameletus Scrapers, 2 Collector/ Gatherers Ephemeroptera Baetidae Platybaetis Collector- 1 1 gatherers Ephemeroptera Baetidae Baetiella Collector- 5 53 33 48 21 gatherers Ephemeroptera Baetidae Acentrella Collector- 4 26 18 20 15 1127 gatherers 87 Order Family Subfamily/ Functional UCH LCH SAK LAN MAI TAD UBK Genus feeding groups Ephemeroptera Baetidae Baetis Collector- 1444 420 26 87 32 92 303 gatherers Ephemeroptera Baetidae Caenis Collector- 2 8 gatherers Ephemeroptera Ephemereliidae Uracanthella Collector- 3 gatherers Ephemeroptera Ephemereliidae Torleya coheri Collector- 8 26 gatherers Ephemeroptera Ephemereliidae Torleya Collector- 34 gatherers Ephemeroptera Ephemereliidae Cincticostella Collector- 9 1 2 44 5 4 376 gatherers Ephemeroptera Heptageniidae Eletrogena Scrapers 2 Ephemeroptera Heptageniidae Ecdyonurus Scrapers 4 Ephemeroptera Heptageniidae Cinygmina Scrapers 7 1 10 Ephemeroptera Heptageniidae Rhithrogena Scrapers 2 14 26 1 Ephemeroptera Heptageniidae Epeorus Scrapers 20 11 8 21 2 3 Ephemeroptera Heptageniidae Iron Scrapers 4 4 23 5 7 81 Ephemeroptera Leptophlebiidae Choroterpides Collector- 9 gatherers Heteroptera Aphelocheiridae Predator 1 Lepidoptera Pyralidae Eoophyla Scrapers 1 Megaloptera Corydalidae Predator 1 3 1 1 2 Odonata Euphaeidae Predator 4 Odonata Gomphidae Predator 1 17 1 2 1 Odonata Platystictidae Predator 1 Opisthopora Megascolecidae Perionyx exavatus Collector- 1 filterers Plecoptera Nemouridae Indonemoura Shredders 350 6 74 Plecoptera Perlidae Kiotina Predator 1 Plecoptera Perlidae Janoneuria Predator 2 Plecoptera Perlidae Acroneurinae Predator 3 Plecoptera Perlidae Calineuria Predator 1 3 Plecoptera Perlinae Perlinae Predator 6 Plecoptera Perlidae Neoperla Predator 12 1 Plecoptera Perlidae Paragnetina Predator 11 37 4 2 7 Trichoptera Brachycentridae Brachycentrus Scrapers 88 Order Family Subfamily/ Functional UCH LCH SAK LAN MAI TAD UBK Genus feeding groups Trichoptera Calamoceratidae Anisocentropus Shredders 1 Trichoptera Uenoidae Uenoa Scrapers 1 Trichoptera Glossosomatidae Agapetinae Scrapers 1 Trichoptera Glossosomatidae Glossosomatinae Scrapers 5 28 10 4 Trichoptera Goeridae Goera Scrapers 2 Trichoptera Hydrobiosidae Apsilochorema Predator 1 1 Trichoptera Hydropsychidae Paratopsyche Collector- 1 12 1 filterers Trichoptera Hydropsychidae Cheumatopsyche Collector- 27 filterers Trichoptera Hydropsychidae Hydropsyche Collector- 71 1 32 4 15 filterers Trichoptera Hydropsychidae Ceratopsyche Collector- 4 2 20 1 3 58 filterers Trichoptera Lepidostomatidae Lepidostoma Shredders 1 1 1 Trichoptera Limnephilidae Limnephilinae shredders, 6 1 1 Collector- gatherers Trichoptera Limnocentropo- Limnocentropus Predator 1 didae Trichoptera Philopotamidae Chimarra Collector- 3 filterers Trichoptera Philopotamidae Dolophilodes Collector- 5 filterers Trichoptera Philopotamidae Wormaldia Collector- filterers Trichoptera Polycentropodidae Poycentropus Collector- 3 filterers Trichoptera Psychomyiidae Psychomyia Collector- 1 gatherers Trichoptera Rhyacophilidae Hypo-rhyacophila Predator 2 1 2 1 1 Trichoptera Rhyacophilidae Himalopsyche Predator 2 2 8 Trichoptera Rhyacophilidae Para-rhyacophila Predator 2 1 4 11 Trichoptera Stenopsychidae Stenopsyche Collector- 6 1 4 58 4 1 3 filterers Trombidiformes- Hydracarina Predator 1 Source: Tachamo Shah, unpublished data 89 Appendix F Detailed Instructions for Conducting Backpack Electrofishing This appendix (Beaumont 2021a) outlines 9. Switch the red “stop switch” to the ON the steps for conducting electrofishing using position. Test the safety systems are the Smith-Root LR24 backpack electrofishing operating correctly (lean forward to test tilt equipment, as recommended by William R.C. switch and put wet tissue or material on Beaumont of Electric Fishing Technical Services the immersion sensor). (EFTS) Ltd. Other types of backpack electrofishing 10. Test the equipment in an area outside the equipment may have different operational survey reach. Make a note of the output methods and controls, so users must read the user settings displayed on the status screen of the manual carefully. equipment, including power (watts), amperes (A), and voltage. Important Considerations 11. Begin fishing the survey section. • All users of the equipment must be suitably trained. 12. When the survey is completed, depress the red “stop switch” to the OFF position and • A person trained in resuscitation should be disconnect the battery leads. included in the team using the equipment. 13. Remove the electrodes and the battery. • The use of the anode-out-of-water detection system is recommended to improve safety. 14. Clean and disinfect the equipment. • To prolong battery duration, a pulsed-direct- Maintenance current (PDC) waveform is recommended to be • Before assembling the equipment, make sure used for fishing. that the battery pack/s are fully charged. Steps for Electrofishing Batteries should always be charged after use. If the equipment is not used for long periods, 1. Examine all equipment for damage. Do not the batteries should be charged every three use damaged equipment, which may pose months to maintain capacity. safety risks. • Be sure to use the correct type and voltage of 2. Ensure the red “stop switch” is in the battery for the electrofisher. depressed (off) position. • If additional/replacement battery packs are 3. Install the battery in the lower section of the required, a competent electrician should be backpack. Do not connect the battery leads. able to assemble new ones. Batteries should be 4. Connect the cathode and the anode to 12 volts (V), 7.5 ampere hour (Ah) lead-acid appropriate sockets on the backpack. Ensure gel. They should be wired in series to produce the plug “collars” are tightened. Electrode an output of about 24 V. The battery packs leads should either be routed out of the side are connected to the electrofisher using 45 A of the battery compartment or routed in the Anderson Powerpole connectors. A 40 A fuse pre-formed channels under the battery (in should be wired between the connected positive which case the electrodes will need connecting and negative terminals (see diagram below). before the battery is installed). Whichever route is used for the cables, make sure that the electrode lead strain relief systems are correctly positioned. 5. When ready to begin fishing, connect the battery leads and secure the cover over the battery. 6. Measure the ambient conductivity of water to be fished and set the output voltage and pulse width appropriately (see Appendix G for more information on setting the output voltage). 7. Set pulse frequency to settings appropriate for fish species, size, and river conditions. 8. Ensure all members of the fishing team know their roles and responsibilities. 90 Appendix G Best Practice Manual for Backpack Electrofishing 91 Table of Contents 1. INTRODUCTION | 93 2. HEALTH & SAFETY | 93 2.1 Electric Shock | 93 2.1.1 Major Symptoms of Electric Shock | 93 2.1.2 Possible Sources of Contact with Electricity | 93 2.2 Other Dangers | 94 2.2.1 Drowning | 94 2.2.2 Tripping/Falling | 94 2.2.3 Other Hazards | 94 3. EQUIPMENT AND SETTINGS | 94 3.1 Personal Protective Equipment | 94 3.2 Power Source | 95 3.3 Output Waveform | 95 3.3.1 Direct Current (DC) | 95 3.3.2 Pulsed Direct Current (PDC) | 95 3.4 Voltage | 96 3.4.1 Voltage Gradient and Output Type | 96 3.5 Choice of Frequency When Using PDC | 96 3.6 Pulse Width and Duty Cycle | 97 3.7 Water Conductivity | 97 3.7.1 Low Conductivity Waters | 97 3.7.2 Medium and High Conductivities | 98 3.8 Temperature | 98 3.9 Electrode Dimensions | 98 3.9.1 Anode | 98 3.9.2 Cathode Size and Shape | 99 3.9.3 Effective Size of Capture Field Required | 99 3.10 Standardizing Capture Probability | 100 4. FISH WELFARE | 100 5. BIOSECURITY | 101 6. ELECTRIC FISHING “BEST” PRACTICE | 102 7. SUMMARY | 104 8. SELECTED BIBLIOGRAPHY | 105 9. APPENDIX | 106 92 1. INTRODUCTION when the current is made or broken, not when the current is steady. By contrast, a 50 or 60 Hz Electric fishing is an effective way to sample alternating current (AC) will produce a continuous freshwater fish populations. However, electric painful shock. It also requires three times more fishing may also cause fish injury or mortality DC than AC for a lethal shock. to both fish and humans. The purpose of this overview is to provide specific, concise, guidance First aider safety. In all cases of electrocution, on health and safety, and correct adjustment of the source of the electricity should be shut off or electrical output settings. This will enable the safe removed BEFORE HELPING THE CAUALTY. optimization of efficient fish capture, under a DO NOT touch the casualty until there is no range of environmental conditions, ensure safety live electrical contact between them and the for operators and minimize injury to fish. The equipment. This will ensure that all people helping manual is intended as supplementary information will be kept safe. to accompany the electric fishing courses run by EFTS Ltd. For more detailed information, a brief biography of research papers is included and 2.1.1. Major Symptoms of Electric Shock purchase of Electricity in Fisheries Research and Management: Theory and Practice by W.R.C. Atrial and/or ventricular fibrillation is the Beaumont is recommended. uncoordinated, asynchronous contraction of the atrial or ventricular muscle fibres of the heart. It should be remembered that electric fishing is not The risk of fibrillation is high if an electric shock the only method for fish population evaluation is received with the path of the current through or removal and these other methods (netting, the chest (e.g., between two arms). The heart’s trapping etc.) should be considered when deciding natural rhythm is replaced by an asynchronous on the most appropriate method to be used. quivering with no effective pumping of blood. This is extremely dangerous, and death can occur in minutes unless correctional steps are taken 2. HEALTH AND SAFETY immediately. When safe, CPR must be used to maintain the patient, but defibrillation will Electric fishing is potentially hazardous. No normally be required as the pulse is extremely one should be in close proximity to energized unlikely to be restored by itself. Medical assistance electrodes if they have a history of cardiac must be sought. problems or stress induced respiratory problems. Severe electric shocks can cause distortion of the Respiratory arrest. Electric shock can cause this. heart’s rhythm and/or respiratory arrest. It is The control centre for respiration is contained at recommended that at least two persons in each the base of the skull and can be deactivated by an electric team be trained in cardiopulmonary electric shock. CPR or artificial respiration should resuscitation (CPR) techniques (in case the trained be commenced, when safe and medical assistance person is the patient). sought immediately. It can also be linked to fibrillation (above). All equipment used must be in good condition and should be suitable for the purpose of electric Asphyxia. This is caused by the chest muscles fishing. It should be regularly checked by a contracting and not releasing. Current from competent person and visually checked after each an electric shock which is above a certain level use. Faults must be reported, and faulty equipment (i.e., 0.005 A at 60 Hz) can cause a person (if they must not be used. are holding a live wire) to be unable to let go. This can also be enough to cause the chest muscles There are three principal hazards associated to contract and, in turn, asphyxiate the victim. with electric fishing: electric shock, drowning, When safe, CPR should be commenced, and and tripping/falling; however, other dangers are medical assistance sought immediately. present when carrying out fieldwork. 2.1.2. Possible Sources of Contact with Electricity 2.1. Electric Shock Source Contact The severity of electric shock is related to the The danger of contact with the electrical magnitude of the current, the duration of the generator or battery should be minimized by only shock and the current waveform. For example, plugging in pulse boxes and electrodes when the direct current (DC) causes a severe shock only generator is off, or, for battery equipment, the 93 main emergency stop button should be in the off Slippery rocks can lead to strains or even broken position or the battery disconnected. bones. Long-term use of anodes and/or nets can also lead to repetitive strain injuries. Dry Contact The greatest danger in electric fishing is when a Rainfall will potentially make equipment unsafe live electrode is out of the water and makes direct by allowing electricity to track through the wet contact with a person. For this reason, electrodes equipment. Fishing in thunder and lightning must only be energized when in the water. In storms should be avoided as the live electrodes addition, it is strongly recommended that the could act as “lightning rods.” anode is never touched or brought into close Several diseases can be caught from river and proximity with operators while the generator is lake environments, (e.g., hepatitis, Weil’s disease, running, or the battery connected. etc.). All team members should be aware of the Wet Contact symptoms of potentially severe diseases and the risk of disease should be guarded against. Shocks from in-water contact with the electric field are less severe than dry contact due to the Wild or aggressive domestic animals can be a electric field dissipating in the water. None-the- problem both to staff and equipment and even less, operators should not put their hands in the driving to the site can be a significant danger. water in the vicinity of any energized electrode (anode or cathode). If necessary, electrically insulating gloves may be worn. 3. EQUIPMENT AND SETTINGS Fault contact Equipment set up will vary with make of pulse/ Faulty equipment can also give rise to electric control box and environmental conditions. shocks, so all electrical equipment should be The following are variables that will need to be checked before use and regularly maintained. determined for the environmental conditions encountered. • 3.1. Output type DC/PDC 2.2. Other Dangers • 3.2. Voltage 2.2.1. Drowning • 3.3. Electrode dimensions • 3.4. Frequency Although injury resulting directly from the electric current is considered the most likely • 3.5. Pulse width aspect of electric fishing surveys, drowning is Environmental variables that influence choice of also a significant danger. Electrical shocks, or just the above include: falling into cold water, can impair the swimming • ambient water conductivity ability of operators. In addition, some workers have drowned because they are either unable • temperature to swim or have failed to wear life jackets or • target species / sizes buoyancy aids. • presence of sensitive/rare/valuable fish • size of water body being fished 2.2.2. Tripping/Falling Knowledge of ambient water conductivity is vital for successful and safe electric fishing. It is Movement on riverbeds and boats can be made recommended that reliable portable conductivity difficult by slippery surfaces. Always try to ensure meters are included as an integral part of the that non-slip footwear is worn whenever possible. electric fishing survey equipment. Move at a pace that is consistent with conditions underfoot. Be aware of trip hazards such as cables and ropes on the ground or branches and rocks 3.1. Personal Protective Equipment on the riverbed and communicate these dangers to other team members. Suitable waders should be used when electric fishing. Thigh waders can be used in shallow waters, but chest waders will be needed in deeper 2.2.3. Other Hazards water. Operators should not wade in water deeper than thigh depth due to risk of drowning or river Working in rivers and lakes and carrying heavy flow sweeping them away. Life jackets are advised equipment can give rise to many potential dangers. when working in deep and/or fast flowing streams. 94 Caution should be used if wearing “breathable” possible it should be used in preference to PDC. waders (particularly if operators are wearing However, in medium to high conductivity waters shorts) as cases have been reported of electric the high-power requirements of DC make its use shocks being experienced by operators using these. impractical and thus, in those situations, PDC is the only possible waveform that can be used. Suitable clothing should be worn for hot or cold conditions and sunscreen used as appropriate. Polaroid glasses can improve visibility in bright 3.3.1. Direct Current (DC) conditions and improve fish capture. Light or yellow-coloured lenses help when operating The use of smooth direct current for electric under tree cover. Safety rated polaroid glasses are fishing potentially offers several advantages over also available and have the advantage protecting other waveforms, notably in respect of attraction against eye injury (e.g., from net poles, tree properties and fish welfare, therefore DC should branches swinging back, etc); this makes their use be used wherever and whenever it is practicable applicable to all staff. (see above). However, its disadvantages are that it is a “power-hungry” waveform, and its effectiveness is more prone to disruption by local variations in the conductivity of the riverbed. It 3.2. Power Source also needs higher voltage gradients to immobilize The power source for electric fishing can be either fish compared with PDC. from generators or batteries; domestic mains When using backpack gear and single anode, it power should not be used unless routed through is possible to fish effectively with smooth DC in an opto-isolated power supply. Generators ambient conductivities less than 150–200 mS.cm-1. used for electric fishing are typically AC units At higher conductivities, it may be necessary to use producing 230-240 volts output, but DC units generator-based systems or switch to PDC since can also be used. It is important that the earth on output may exceed the rating of the control box the generator is disconnected in equipment used or, if using battery equipment, depletion of the for electric fishing (due to earth/neutral bonding). batteries before sampling is finished. Such generators should not, therefore, be used for any other purpose. Portable generator-based systems can be used to fish with smooth DC in waters where ambient The output from AC generators is modified by the conductivity is up to about 350–450 mS.cm-1. control box to produce the waveform and output Note that generators larger than 3 kVA (depending type (DC or PDC) chosen by the user. Never on model) are not considered portable, and hence use AC to fish as it has been shown to be highly power output from such a machine imposes an damaging to fish. upper limit on the use of DC. The generator Power can also come from batteries, particularly power requirements can be estimated from the when using small portable “backpack” equipment. ElectroCalc spreadsheet (available from author). Batteries should be “non-spill” to avoid the Ensure that the control box you are using is possibility of acid leakage. The most common adequately rated for the electrical current (Amps) batteries are lead based but increasingly Lithium and/or power (Watts) expected. Reading from the Iron Phosphate (LiFePO) batteries are being used, left-hand charts in the single-and double anode which are much lighter and have a higher Amp/ spreadsheets in ElectroCalc, gives the estimated hour (A/Hr) capacity. current demand in Amps. For instance, fishing DC with a single anode of 400-mm diameter and 3000-mm x 25-mm braid cathode, in water of 3.3. Output Waveform ambient conductivity 300 mS.cm-1 with a voltage set at 250V, current drawn will be 4.6 Amps and Summary: power requirement will be about 1800VA. Alternating current (AC) should never be used for fishing as it is very harmful to fish. AC generators, however, are used to supply electricity to pulse/ 3.3.2. Pulsed Direct Current (PDC) control boxes where it is converted into direct current or pulsed direct current. When conductivities exceed the values at which DC fishing can take place, PDC is Direct current (DC) causes less injury to fish the recommended option. Its fish attraction than pulsed DC (PDC), therefore, wherever properties are not as good as smooth DC but 95 it is better at immobilizing fish and has (for The control box circuitry in the more modern the same output voltage) a larger capture field electric fishing systems enables higher and lower radius. Some equipment gives options for novel voltages than the generator output voltage to waveforms, however, the capture efficiency and be selected and controlled systematically. In fish welfare characteristics of these have not some equipment, voltage and duty cycle cannot yet been fully evaluated and so their use cannot be varied independently, and the equipment is be recommended for routine electric fishing at usually fitted with an input voltmeter which only this stage. A possible exception to this is “Gated measures the voltage produced by the generator, Burst” where power savings can be made from not that applied at the electrodes: the main using this waveform without badly compromising concern of the operator. fish capture or welfare. Most backpack systems can be used to fish using 3.4.1. Voltage Gradient and Output Type PDC mode in waters with ambient conductivity of around 500 mS.cm-1 and, exceptionally, up to Attraction of fish toward the anode can be 3000 mS.cm-1 when using low voltage and gated achieved at voltage gradients of as little as burst waveforms. 0.1 volt/cm when using PDC. When using Average power requirements for PDC are much DC, gradients of 0.2–0.3 volt/cm are needed. lower than for DC. However, it is still important Immobilization of fish using DC can be achieved to ensure you have sufficient power for the at voltage gradients of 1.0 volt/cm while with combination of water conductivity, applied PDC this can occur at gradients as low as voltage and electrode configuration you are using. 0.5–0.6 volt/cm. You should make every attempt Always consult ElectroCalc to make sure you to prevent the fish coming closer to the anode than have sufficient generator capacity to deal with the distance at which voltage gradient is sufficient the water you intend to fish. For instance, fishing for immobilization and you should endeavour not square-wave PDC with a single anode of 400-mm to touch a fish with an energized anode. diameter and 3000-mm x 25-mm braid cathode, in Larger fish are generally susceptible to lower water of ambient conductivity 300 mS.cm-1 with a voltage gradients than smaller fish in any given voltage set at 200V and 25% duty cycle, average situation; hence when larger fish are expected or current drawn will be 0.9 Amps and mean power targeted, circuit voltage can be reduced to below requirement will be about 300VA. the values suggested above. 3.4. Voltage 3.5. Choice of Frequency When Using PDC The circuit voltage required to be applied at the Choice of PDC frequency will be influenced electrodes in order to attract and immobilize fish primarily by the species being sought, bearing will vary according to in mind that under normal circumstances we • the output type used (direct current or pulsed wish to maximize the attractive properties of the direct current) electric field while reducing the harmful zone • ambient water conductivity to a minimum. Research has shown that while • the anode size used medium to high frequencies are more effective in immobilizing and tetanizing fish of some species • the cathode size used (and the anode/cathode groups, particularly salmonids, these are also resistance ratio) more harmful. • size of effective capture field required. Paradoxically, very high frequencies, >400 Hz, Note: When measuring voltage, amps and power have been shown to be both effective and the value can either be measured as peak values or relatively benign for small fish species, and as average values. DC output will always be peak point abundance sampling of cyprinid fry has values but when using PDC output the average been successfully carried out using 400–600 Hz. value will be lower than the peak due to the zero However, standard electric fishing control boxes output between the voltage pulses. It is important do not include such high settings as an option. to know what your equipment measures because some equipment is limited by peak values, and As a very general rule, injury rates in larger fish some average values. will reduce if lower frequencies are used. 96 Frequency guidelines for European species: (during one electrical cycle) that the electricity actually flows. It is important to note that at Salmonids: For large adult fish, 20–40 Hz will different frequencies the same duty cycle will attract and immobilize well. For juveniles, 50 result in different time duration of pulse i.e., at or 60 Hz is effective and causes (at the correct 50 Hz a 10% duty cycle will result in a 2 ms pulse voltage setting) very little mortality. The use of but at 30 Hz, a 3 ms pulse (see ElectroCalc for a 100 Hz settings on older control boxes should conversion chart between pulse width and duty be avoided. cycle). The greater the duty cycle or pulse width selected, the higher will be the current drawn and Cyprinids: Optimum frequencies may vary, power required: 100% duty cycle is the same as but for roach (Rutilus spp.), 40 Hz has been DC (i.e., the power is on all the time). shown to give both good attraction and good immobilization. Switching to 10 Hz reduces the When fishing with PDC, duty cycle should zone of immobilization while increasing attraction be kept to about 20–30%, increasing duty properties, however there may be difficulties in cycle above 30% has little effect on attraction capturing cyprinids in some circumstances if they properties of the field, though often improves are only immobilized in a very small zone around immobilization strength. A short pulse width the anode. reduces the possibility of fish damage and conserves average power. Perch (Percid spp.) are more similar to salmonids in their response to electric fields and 100 Hz has In more conductive water, it may be necessary to the best attraction and immobilization properties. increase pulse width if fish are seen to be escaping However, as fish damage (to perch and other the expected capture field. Values in excess of species) is more likely at this frequency 30–40 Hz 35% however are unlikely to improve capture is recommended. and different frequency or voltage outputs should be considered. In the case of some pulse boxes Pike (Essox spp.): – no specific references have it may not always be possible to adhere to this yet been found in the literature, but personal because voltage and duty cycle cannot be varied experience has found that fishing at 40 Hz has independently and if high voltage is required then proved effective. high duty cycle is selected simultaneously. Eels (Anguillid spp.): most frequencies We recommend that when using PDC, fishing investigated were effective in both attracting should start with a pulse width of 5 ms (25% and immobilizing eels, so bearing in mind the duty cycle at 50 Hz) but for medium to high potentially more harmful effects of higher conductivity waters it may be necessary to frequencies on some other species, frequencies of increase this. Some control boxes do not have 10 – 40 Hz should be employed as standard. In independently variable voltage and duty cycle depletion fishing it is common for the second pass control but nevertheless fishing should start with fishing to catch more eels than the first, this is the “select power” dial turned down to perhaps a likely due to both targeting by operators but also quarter of its range (“nine-o’-clock” position”). displacement of fish in the first pass making them more catchable in the second pass. Obviously, this anomaly negates the ability to calculate a population estimate. 3.7. Water Conductivity The attributes of other intermediate frequencies The conductivity of a substance will vary with e.g., 5 Hz, 20 Hz, have not been reported to any temperature. For that reason water conductivity is extent in the literature examined but could prove either measured as “specific conductivity” where more favourable than the frequencies quoted. the value is adjusted to what it would be at 25°C, Lamprey juveniles have been successfully caught or “ambient conductivity” where the value is using low (2 Hz) frequencies to draw them from not adjusted. In electric fishing it is the ambient the riverbed sediments. conductivity that will determine the equipment set up – unless the water is at 25°C! In the following all conductivities are ambient. 3.6. Pulse Width/Duty Cycle Pulse width refers to the duration of each 3.7.1. Low Conductivity Waters individual pulse of electricity and can be expressed in milliseconds (ms) or in percentage duty cycle. When the water being fished has low conductivity Percentage duty cycle is the percentage time (conductivity less than 150 µS.cm-1), a higher 97 voltage gradient is required to incapacitate the fish The right-hand diagrams in the ElectroCalc than in high conductivity water. A higher applied spreadsheet give an indication the effective size voltage is therefore required. (vertical axis) of electric fields, based on nominal voltage gradients required to catch fish, at Even when using PDC very high voltage different anode voltages for a given anode and outputs (in excess of 500 Volts) are needed at cathode configuration and circuit voltage. low conductivities. As a general approach, electric fishing under any Use of higher voltage systems, that generate field conditions should be started at the lower voltages up to 1200 V, have been used in some end of the range of voltages recommended for ultra-low conductivity waters (15 µS.cm-1), those conditions. however, users should be aware of the dangers of using such high, and potentially dangerous, output voltages. 3.8. Temperature At low temperatures, fish may be less responsive 3.7.2. Medium and High Conductivities to the electrical field due to their lower reaction capability at low temperatures. Reducing the pulse At medium and high conductivities, progressively frequency can mitigate against this. lower circuit voltages will be effective in fish capture because a lower voltage gradient is needed At higher temperatures, the fish are likely to be to elicit a response from fish at a given point in the very reactive and this may cause muscle or skeletal electric field in higher conductivity waters. damage to the fish due to excessive reaction. Conductivity Applied voltage Applied voltage Welfare of fish post capture will also be an issue at (µS.cm-1) – PDC – DC high temperatures due to higher respiration rates and lower oxygen holding capacity of the water. 10–100 300–900+ 400–900++ 100–200 250–300 300–400 200–500 150–250 250–300 3.9. Electrode Dimensions 500–1000 120–180 Not applicable 3.9.1. Anode > 1000 100–150 Not applicable Anodes should generally be circular and should These guidelines assume the use of a typical, not have sharp corners, as these will produce areas recommended electrode configuration such as of high voltage gradient. Capture nets should not a 400-mm diameter anode and single cathode be attached to anodes as this will increase the consisting of 3 m braided copper or stainless-steel time fish are held in the area of maximum voltage strap. Different anode and cathode dimensions gradient. Nets on anodes also present a very high may require more or less circuit voltage to be risk to operators. effective. Note that waveform type (DC or PDC) The use of very small anodes (<25 cm diameter will also affect immobilization thresholds. ring) is not recommended under normal At higher conductivities, it may be necessary circumstances as they result in a small but intense to increase pulse width (duty cycle) to impart electric field. The aim should be to use as large a sufficient power into the water to capture the fish. diameter anode as is practicable. The constraints on anode size in narrow streams will be the Note that when fishing in areas where fish have physical limitations imposed by the nature of the laid their eggs, high voltage gradients may affect site and ease of handling by the operator. If the the eggs or embryo within the egg. physical nature of the stream necessitates the use of a very small anode (for instance fishing for The overall aim during any electric fishing bullhead or 0+ salmonids in a boulder-strewn operation should be to maximize the effective field stream) then the applied voltage can be reduced of fish capture while minimizing the zone of high accordingly, since in such a case size of capture voltage gradient around the anode/s in which fish field is not an issue. can be damaged. Where very sensitive or valuable species are present, operators should consider In larger streams, maximum usable anode size may further reducing the risk of damage to fish by be limited by power available. Graphs showing reducing applied voltage even if this means some the size of effective capture fields using different compromise of fishing efficiency. applied voltages and different-sized anodes, using 98 both DC and PDC, and the power requirements increase the pulse frequency, possibly leading to of these different configurations are shown in the harmful frequencies. Multiple independent units ElectroCalc spreadsheet. also negate the safety system of “one-off, all-off.” It is recommended that the standard anode size for normal use is a 10 mm gauge stainless steel ring, diameter 35–50 cm. Thicker gauge steel is 3.9.2. Cathode Size and Shape stronger and creates a larger surface area but is heavier and has relatively little effect on electrode The system should always comprise a cathode resistance and overall field characteristics, and, surface area that is larger than the surface area of although it may reduce voltage gradients in close the anode(s). Cathode: anode surface area ratios of proximity to the anode edge, 10 mm gauge is a as large as 30:1 have been quoted in the literature good compromise. but there is a limit to the practicality of such configurations. Cathodes such as copper braids are In flowing water rings of 60 cm or larger are very more ergonomic than metal plates or mesh grids, difficult to hold against the flow of the water however, braids produce more intense cathode are likely to cause excessive arm strain on the fields in their immediate proximity which can be operators. Supports for the anodes can be used harmful in situations where fish may come close to but it is probably better to switch to “boom” or the cathode. In such cases a cathode grid or grids fixed anodes. Teams should carry a range of ring are preferable to braid. diameters (50 cm, 40 cm, 30 cm and 25 cm) to cope with situations where very high conductivity It is recommended that the standard cathode places excessive demands on power available, or should be at least a 3–metre length of 25 mm where the physical nature of the stream renders a wide copper or stainless-steel braid or a sheet of large diameter anode impractical. perforated metal of at least 75 cm x 75 cm square or other shape of equivalent surface area. Never keep fish in the electric field for longer than necessary, avoid getting too close to fish with an If the surface area (or number) of the anodes is energized anode and never touch a fish with an doubled then the cathode surface area should also energized anode. be doubled; separating the cathodes will improve even further the resistance characteristics of the As a general rule, no more than one anode cathode array. Control boxes for use with more (40-50 cm diameter) is required per 5 m width or than one anode should if possible be fitted with river channel being fished. If more than one anode extra cathode sockets. If extra cathode sockets are of this size is used in a channel narrower than this, not fitted, then multiple cathode braids or grids the size of effective electric field around each one can be fed from a single control box socket using a will be reduced, and the operation will be less cost trouser-joint and a spacing device made from non- effective and wasteful of power. conducting material. In wider channels where it is desirable to Very long cathodes may be impractical for increase the number of anodes, the surface area backpack electric fishing where the cathode is of cathodes must also be increased pro rata (see dragged behind the fisher, nevertheless the cathode below) in order to gain maximum benefit from the should comprise at least 1.5 metres of braid or increased anode size. Note, however, that power 4–6 mm diameter steel wire. requirements will increase (see left-hand figures in ElectroCalc). When fishing with multiple anodes As a general rule for anodes and cathodes, bigger it is not good practice to hold the anodes heads is better, but there is a law of diminishing returns closer than about 3m because the size of effective and little advantage will be gained by using much electric field around each one will be reduced, larger sizes than those recommended. and thus capture efficiency will be reduced. In Where possible cathodes should be placed in addition, the electrical current is increased and fast flowing water so fish cannot remain in may overload the control box. Notwithstanding close proximity to the cathode for long periods this, users should be aware that if too great a gap and be harmed. is left between a pair of anodes for too long during a fishing operation fish may pass between the two anodes and not be caught. 3.9.3. Effective Size of Capture Field Required When fishing PDC multiple electric fishing pulse boxes should not be used. This is because In most electric fishing situations, it is desirable overlapping out-of-phase pulses will, in effect, to create as large an effective capture field as 99 possible. However, in shallow, narrow streams, 4. FISH WELFARE there is no need to create a field that will attract fish from many metres away since any fish present Proper handling of the fish once caught is will never be far from the fisher. In very turbid essential; bad handling of fish that are already water there is equally no point in immobilizing under some stress due to capture can exacerbate fish at a depth/distance from which they cannot be problems and cause injury. Good handling will seen and retrieved. help to prevent injury and to reduce stress. Size of capture field required also depends to some In the past, considerations about a fish’s ability to extent the species and sizes of fish being targeted. “suffer” have been somewhat overlooked. Present Small fish species with limited mobility such as research is inconclusive, but some has shown bullheads can be captured using small effective that fish can react to stressing actions and some electric fields employing relatively low voltages – researchers’ surmise that fish can not only feel even in larger rivers. pain but also experience fear. While the debate continues regarding this issue, fishery workers Hence, when fishing in very small streams of must be aware of the fact that they are dealing whatever conductivity the operator should with sentient organisms and act appropriately. If consider using lower voltages than those indicated killing fish is required, then cerebral maceration in 3.7.2. should be carried out. Fishery workers should be aware of the regulations within their country regarding working on animals (including fish). In 3.10 Standardizing Capture Probability many countries experimental research (as opposed to husbandry) can only be carried out if licensed When comparing fish population assessments by government or regulatory organizations. In the taken with semi-quantitative methods (e.g., UK licenses are controlled by the Home Office 5-minute surveys, etc.), it is vital to standardize under the Animals (Scientific Procedures) Act fish capture probability between survey sites. 1986 (ASPA). If the work is classified as research or involves pain, stress or the use of anaesthetic Firstly, the time element should be the anode then the work can only be carried out under ASPA energization time, not the total time fishing. In low project licence. Persons working under ASPA need density sites a greater area can be fished in a total to have been trained and hold a Personal Licence time of 5 minutes compared to high density sites for all the procedures that they are carrying out. due to less time being taken to remove captured fish from nets etc. Using anode energization time The following general rules should be observed to negates this bias. improve fish welfare: In addition, to standardize capture probability, it • Avoid fishing in high water temperatures is important that the equipment used has the same (greater than 16-18°C for salmonids, 22-24°C anode and cathode dimensions, uses the same for coarse fish especially when pike and perch voltage waveform and that the circuit voltage is are present). adjusted to give similar capture probability at the • Use separate bins to separate large and small fish differing conductivities. and to separate eel and common bream (due to the amount of slime they produce). For example, if two sites, with significantly • Provide aeration (oxygen diffuser plus different water conductivity, are surveyed the compressed air is best) in both catch bins voltage output must be altered between the sites to and fish storage bins – this is essential in standardize the capture probability. This is due to warmer weather and when large numbers the different voltage gradient needed to catch fish of fish are expected. in different water conductivities noted earlier. • Keep-cages and keep-nets are a good Two methods have been published to calculate this alternative to fish storage bins but ensure there standardized output voltage value; Power Transfer is adequate depth of clean, gently flowing, Theory (Kolz 1989), and standardized output and well-aerated water. If these conditions are Wattage (Meyer et al. 2020). not available at the survey site, then aerated storage bins are preferable. Conductivity: voltage output graphs using information specific to the equipment being used (see appendix) should be carried by teams to maintain this standardization. 100 Measures that Can Be Taken to Reduce Stress during Holding, Handling, and Transportation of Fish (Adapted from Pickering 1993 and Ross & Ross 1999) Problem Suggested Action Comments Duration of the stress response is usually Shorten the duration of stress. Some effects may result in long proportional to duration of exposure. recovery times. Stress-induced mortality increases with Work at lower temperatures (e.g., use Not always practical under field water temperature. ice to cool water). conditions. Stressors may be additive or synergistic. Prevent simultaneous stress. Possibly allow time between processes. Abrasion between fish causes damage. Reduce numbers handled per batch. May conflict with time pressures. Stress increases O2 consumption. Use O2 or air bubbled through holding Safety and O2 use may make air tank. better option. Stress increases O2 consumption, and Use mild anaesthesia or sedation. Note, some anaesthetics can act ammonia and CO2 output. as stressors. 5. BIOSECURITY Every effort should be made not to transfer pathogens or alien plants and animals between sites and particularly catchments. On completion of any field operation (particularly when moving between catchments), all equipment used must be treated with appropriate disinfecting agent (e.g., Virkon Aquatic). Equipment needs to be clean for the agent to disinfect properly so any obvious material or dirt should be removed. All gear that has been in contact with the water should be cleaned i.e., boats, trailers, outboard motors, anchors and rope, weights, tanks, buckets, hand and stop nets; all PPE (including boots, wellingtons, waders, wetsuits, dry suits, etc.) plus all technical or sampling apparatus used as part of the survey/operation. For difficult or large gear (e.g., stop nets) freezing will successfully kill most organisms, however, seeds and some bacteria may still be viable after freezing. Recommended concentration of disinfectant (usually a 1% solution) can be applied by a small garden sprayer onto equipment. Suitable PPE should be worn when using the disinfectant, i.e., safety glasses, gloves & dust mask. Note that the disinfectant will degrade over time so fresh batches should be regularly made up. When planning sampling always try to have sites sampled in a downstream direction. This is particularly important where there are weirs etc. that make obstructions to the potential upstream passage of alien species. 101 6. ELECTRIC FISHING “BEST” PRACTICE to higher injury. The practice of “turning up” the output setting comes from old style boxes In general terms there are two choices regarding where this also increased voltage output; and thus equipment set-up for electric fishing. The increased the range of the anode. Increasing the equipment can be set-up to cause the least possible voltage at the anode will increase the size of the damage to the fish, or the equipment can be set- voltage field but will also lead to high gradients up to capture the highest proportion or number near the anode with associated risk to both fish of fish. Rarely do these two set-ups correspond. and operators. It will also markedly increase the Knowledge of the theory behind electric fishing power demand of the equipment. can help bring together the two options. Most users of electric fishing equipment use a The following deals predominantly with the “standard” setup when fishing. If this “standard” options and techniques to use in order to has been determined on the basis of past fishing minimize damage. success and lack of fish injury these standards are probably satisfactory. Personnel using DC Where possible fishing should be carried out for the first time will need to adjust or modify using direct current (DC) voltage fields. This is their fishing technique to account for the much because DC has good attraction of fish to the smaller effective field found with DC (Snyder anode (increasing capture probability), induces 1992). Calculated field intensity data are good harmful tetanus only in the near vicinity of the for planning, but on-site, in-water measurements electrode and has the lowest recorded rate of are better for confirming actual intensity and injury for any waveform type. However, there distribution of the electrical field, especially will be many cases where it is not possible to use given the importance and potential variation in DC (high conductivity water, variable electrical substrate conductivity. Given that, adjustment characteristics of stream topography, poor fish should initially be carried out based on theoretical response to DC field for unspecified causes, low considerations and then adjusted based on endurance of battery powered equipment in high values actually measured in the stream or river conductivity water, etc.). In these cases, pulsed (e.g., by use of “penny probe” etc.). Voltage field direct current (PDC) voltage fields should be used. measurements should be made using either a However, PDC has poorer anodic electrotaxis custom-made peak voltage meter or a portable and tetanizes further from the electrode; possibly oscilloscope. Part of this set-up process will be preventing some fish from reaching the capture the decision regarding what voltage to use. In the zone. Pulse frequencies should be kept as low as past, few pulse boxes in use in the UK have had possible (Snyder 1992 suggests 30-40 Hz or lower) this option but it is a powerful tool in tailoring note however, that frequencies below 20 Hz may the field gradient to ambient conditions. Voltages not be good for attracting the fish to the anode. can be reduced when having to use small anodes There is also some evidence that high frequencies in small high conductivity streams or increased may be more efficient for capturing small fry. in low conductivity streams (if larger anode diameters are impractical). Note that there is no Evidence shows that alternating current (AC) physiological reason for 200 volts to be the default causes more injuries than DC and PDC and voltage used, often lower voltages will be equally therefore AC fields should not be used for fishing effective in producing adequate field intensities. unless warranted by specific circumstances (use of fishing frames, PPAS or fish to be killed). The anode head size should be as large as possible. If using DC, available power may influence All fields should be adjusted to the minimum the size of anode that can be used, but if using voltage gradient and current density concomitant PDC available power is rarely an issue. The with efficient fish capture. Pulse box settings practicalities of handling large anode heads and should be adjusted to optimize recovery, capture the physical size of the stream are more likely to efficiency should be a secondary consideration be an issue. In small low conductivity streams, and can often be offset by carrying out more runs if small physical anode size is required, voltage (if depletion fishing). This is an area where some levels can be increased to increase the capture measure exists for some trade-off between fish area (although it is not likely you will need a large capture and fishing efficiency. It should be noted field). Adding metal mesh to the anode can reduce that it is INCORRECT to increase pulse width the consequential high voltage gradient that will (and thus amperage) at deeper sites. For the same then exist in the vicinity of the anode. The mesh conductivity water this will not increase the field should not be used for actually capturing the fish. area of the anode but simply increase the power transfer to the fish within the field and thus lead 102 The cathode should be as large as possible. The Actual techniques used will vary between commonly used “braid” design of cathode is both running and still waters. In still waters the fish efficient and ergonomic in use. Braid should be are far more likely to be able to escape the approximately 3000 mm long although shorter voltage field. This can be reduced by either lengths are more practical for backpack gear. fishing next to the bank (to trap the fish against Expanded mesh design of cathode can also be the bank) or by enclosing sections of still water used but are more difficult to transport and can with nets. Discontinuous fishing should also be be affected by water flow. If multiple anodes carried out when using PDC. are used, cathode area will need to be further increased. Knowledge of the electrode resistance Generally electric fishing teams work in an of both anode and cathode will allow intelligent upstream direction. This reduces the problem assessment of requirements. If copper cathodes associated with stirred-up silt impeding are used, they should be kept clean of an oxide visibility. In fast running clear streams, layer, as it will reduce their effectiveness. Soaking however, downstream fishing, especially when cathodes in vinegar is an effective way of removing using “Banner Nets”, has been shown to be the oxide. very effective. Fishing technique using DC and PDC. When When fishing wide sites, multiple anodes can be using DC, fishing should be conducted in a used. Zig-zagging upstream when fishing allows discontinuous fashion, in order to use the element random or target habitat types across the width of surprise, to improve capture efficiency and in to be sampled. Moving anodes when fishing side order not to herd or drive the fish. In preference to side and up and down to “draw” fish will the operators should switch on when near, rather also help. When using twin anodes in wide rivers than in, areas such as clumps of weed, tree when only part of the width is being covered, roots or other likely refuges. Fish will be in the it is sometimes advantageous for the mid-river attraction zone and this will have the effect of anode to move slightly ahead of the bank-side pulling the fish out from their refuge to where anode. This technique will tend to scare the fish they can be captured. If the anode is too close, or into the bank and make capture by the bank-side actually in, refuge areas when switching on, the anode more effective. In general, one anode for fish may be in the immobilization field and will every 5 metres of river width has been found to be not be drawn from cover. Sweeping the anode effective for quantitative electric fishing surveys of when in areas of open water may encourage fish whole rivers. to seek out areas such as weed beds etc. where Fish should be removed from the electrical field as again the above technique can be used. When quickly as possible. While length of exposure to using twin anodes however this discontinuous the electric field does not appear to increase rate method becomes difficult due to the requirement of trauma, length of exposure does increase stress for both anodes to be powered simultaneously. levels. Repeated immersion of fish into an electric This problem can lead to the practice of keeping field has been shown to increase blood lactate the anode live while lifting it from the water; levels (and thus will increase post-exposure muscle this should not be done (due to the danger of acidosis). Holding fish in the net is poor practice dry contact with the anode). It should be noted as it also considerably increases oxygen debt and that the effective fishing radius of the anode will should be avoided. vary dependent upon the localized changes in the physical attributes of the stream. For this reason, it Regarding the non-electric considerations when may be difficult to obtain good depletion sampling fishing, five major issues arise, water depth, population estimates (or more fishing runs may be water temperature, water visibility, fish welfare required to get adequate confidence in the results). and communication. Unlike DC, the tetanizing zone of PDC extends Electric fishing by wading is limited to the depth some way out from the anode. Thus, when using in which wading can be safely carried out. The PDC care needs to be taken that the anode is U.K. Environment Agency Code of Practice states not so close to the fish that the fish is instantly that an overall depth of thigh deep with a hip in the tetanizing zone of the field or that the fish depth maximum should be used as the criteria. is tetanized while still outside the catching zone. These measurements should be taken from the This aspect can however be minimized by using an shortest person in the fishing team. anode radius and voltage output suitable for the conditions being fished. 103 Extreme temperatures should be avoided when 7. SUMMARY fishing is carried out. Fishing in the hottest months • Ambient water conductivity should be known should be avoided, but it is also important to (to within 100 µS.cm-1) avoid the coldest months as well. In general, there is a trade-off between efficiency (poor at • Always ensure that you have enough power low temperatures) and welfare (poor at high (generator/control box combination, or temperatures). A temperature range of 10–20°C batteries) to supply the configuration you have is preferred for coarse fish and 10–15°C for chosen for the field conditions salmonid species. If fishing is carried out at • Do not survey in extreme water temperatures, low temperatures, due to logistics (e.g., low especially high temperatures (>16-18°C for growth in winter so better between-site growth salmonids, >22 –24°C for coarse fish.) comparisons), increasing pulse width or voltage • Provide adequate processing, recovery and gradient may improve capture efficiency. storage facilities for the catch. The rule regarding the visibility required for electric fishing is simply “do not put the anode Applied Circuit Voltages (assuming maximum head deeper than you can see.” The electrode effective capture field is desired and recommended should be visible and should be near enough to the anode and cathode combinations are used) riverbed for its field to encompass the riverbed. Ambient The visibility required will vary for different Applied voltage Applied voltage conductivity species (e.g., small benthic fish requiring higher – PDC – DC (µS.cm-1) visibility than if surveying larger mid-water fish). In poor visibility more runs may be required to 10–100 300–900+ 400–900++ achieve adequate population estimates. 100–200 250–300 300–400 A wide variety of techniques can be used to 200–500 150–250 250–300 ensure good fish welfare while they are being held 500–1000 120–180 Not applicable prior to processing. The temperature of water is an important issue in maximizing welfare, > 1000 100–150 Not applicable with greater care regarding maintaining oxygen needed in hot weather. The use of floating mesh cages is considered to be a particularly effective Frequencies (for optimum combination of way of keeping the fish in good condition. It is attraction, immobilization and welfare) also a good idea to separate eels and bream from Species Pulsed DC frequency (Hz) the catch as the large quantities of mucous these fish produce lowers the water quality (especially Salmonids 40–60 if the fish are held in bins) and “clog-up” other Cyprinids 30–50 fishes gills. Note that eels are adept escape artists and holding bins should have a large amount of Percids 10–40 “freeboard” between the water surface and the lip Pike 30–50 of the bin. Eel 10–40 Oxygen levels in bins can decline rapidly. With an approximately 50% stocking density (45 NB: for all species, use smooth DC whenever it is litres of water: 20 kg [equivalent to ~20 litres] of practicable. fish) oxygen levels can decline to 50% of their starting level in 7 minutes. This stocking level in Pulse width / Duty cycle (at 50 Hz) bins should therefore be regarded as maximal. Remember that the water needs to be agitated to Conductivity Pulse width Duty cycle (%) remove CO2. It is possible to supply adequate O2 (µS.cm-1) (ms) with a fine diffuser and still build up toxic levels <150 2–5 10–15 of CO2. 150–800 3–8 15–25 Good communication systems need to be in 800–1000 5–10 25–40 place between anode operators (especially due to the one-off, all-off safety system) and/or anode > 1000 7–15 25–40 operators and bank personnel. This system can NB: always start fishing with duty cycle/pulse be plain speech but in wide or noisy sites some width set at the minimum. system of either hand signals (difficult if anode in one hand and net in the other), whistles or radio communication is preferable. Modern voice activated radios fitted to headsets are ideal. 104 Anodes and Cathodes Meyer, K.A., Chiaramonte, L.V. & Reynolds, • Always use largest anode that is practicable. J.B. 2020. The 100-Watt Method: A Protocol If using very small anodes (due to site for Backpack Electrofishing in Small Streams. configuration) reduce applied voltage. Fisheries: 46(3): 125-130. • 40-50 cm diameter anode 10 mm gauge Miranda, L.E. & Dolan, C.R. 2004. recommended size. “Electrofishing power requirements in relation to • Do not fish with twin anodes held close together. duty cycle.” North American Journal of Fisheries • Always use a cathode that has larger surface Management, 24: 55-62. area than anode. At least 3 metre x 25 mm Snyder, D.E. 2003. Electrofishing and its harmful braid; or 75 cm x 75 cm expanded mesh or plate effects on fish. Information and Technology is recommended size. Report USGS/BRD/ITR-2003-0002: US • If surface area of anodes is increased, Government Printing Office, Denver, CO, 149pp. cathode surface area should be increased by at least the same factor. Use of multiple Vibert, R. (ed.) 1967. Fishing with Electricity: cathodes is preferable. Its Application to Biology and Management. Farnham, Surrey: Fishing News Books Limited. Always disinfect gear after sampling. 8. SELECTED BIBLIOGRAPHY Beaumont, W.R.C., Taylor, A.A.L., Lee, M.J. & Welton, J.S. 2002. Guidelines for Electric Fishing Best Practice. Report to Environment Agency 179pp EA Technical Report W2-054/TR. Beaumont, W.R.C., Lee, M.J., & Peirson, G. 2005. “The Equivalent Resistance and Power Requirements of Electric Fishing Electrodes.” Fisheries Management & Ecology; 12: 37-44. Beaumont, W.R.C. 2011. Electric Fishing: A Review and Guidelines for Best Practice. Report to Inland Fisheries, Ireland 98pp. Beaumont, W.R.C. 2016. Electricity in Fish Research and Management: Theory and Practice. Wiley-Blackwell 163pp. Bohlin, T. 1989. “Electrofishing - theory and practice with special emphasis on salmonids.” Hydrobiologia, 173: 9-43. Cowx, I.G. (ed.) Developments in Electric Fishing. Oxford, U.K.: Fishing News Books, Blackwell Scientific Publications. Cuinat, R. 1967. “Contribution to the study of physical parameters in electrical fishing in rivers with direct current.” In: Vibert, R. (ed.) Fishing with Electricity: Its Application to Biology and Management: 131-171. Farnham, Surrey: Fishing News Books Limited. Kolz, A.L. 1989. “A power transfer theory for electrofishing.” In: Electrofishing, a Power Related Phenomenon: 1-11. Fish and Wildlife Technical Report 22. Washington, D.C., U.S.A.: United States Department of the Interior, Fish and Wildlife Service. 105 9. APPENDIX The following graph shows predicted output voltages, calculated by Power Transfer Theory, for a range of ambient water conductivities, when using Smith-Root LR24 backpack electric fishing equipment. These settings proved successful in the trial fishing exercise in the Trishuli catchment in March 2020. Calculated circuit voltage required for equivalent power transfer 1000 950 900 850 800 750 700 650 600 Output Voltage 550 500 450 400 350 300 250 200 150 100 50 0 0 50 100 150 200 250 300 350 400 450 Ambient Conductivity 106 2121 Pennsylvania Ave., NW Washington, D.C. 20433, USA www.ifc.org/sustainability www.ifc.org/hydroadvisory