A Practical Guide to Environmental Biotechnology [1st ed.] 9789811562518, 9789811562525

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Learning Materials in Biosciences

Jayanta Kumar Patra Gitishree Das Swagat Kumar Das Hrudayanath Thatoi

A Practical Guide to Environmental Biotechnology

Learning Materials in Biosciences

Learning Materials in Biosciences textbooks compactly and concisely discuss a specific biological, biomedical, biochemical, bioengineering or cell biologic topic. The textbooks in this series are based on lectures for upper-level undergraduates, master’s and graduate students, presented and written by authoritative figures in the field at leading universities around the globe. The titles are organized to guide the reader to a deeper understanding of the concepts covered. Each textbook provides readers with fundamental insights into the subject and prepares them to independently pursue further thinking and research on the topic. Colored figures, step-by-step protocols and take-home messages offer an accessible approach to learning and understanding. In addition to being designed to benefit students, Learning Materials textbooks represent a valuable tool for lecturers and teachers, helping them to prepare their own respective coursework.

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Jayanta Kumar Patra • Gitishree Das • Swagat Kumar Das Hrudayanath Thatoi

A Practical Guide to Environmental Biotechnology

Jayanta Kumar Patra Research Institute of Biotechnology & Medical Converged Science Dongguk University Goyang-si, Korea (Republic of)

Swagat Kumar Das Department of Biotechnology, College of Engineering and Technology Biju Patnaik University of Technology Odisha, India

Gitishree Das Research Institute of Biotechnology & Medical Converged Science Dongguk University Goyang-si, Korea (Republic of)

Hrudayanath Thatoi Department of Biotechnology North Orissa University Odisha, India

ISSN 2509-6125     ISSN 2509-6133 (electronic) Learning Materials in Biosciences ISBN 978-981-15-6251-8    ISBN 978-981-15-6252-5 (eBook) https://doi.org/10.1007/978-981-15-6252-5 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

V

Preface Environmental biotechnology is the most important subject for the study of biotechnology. Environmental biotechnology is applied to and used to study the natural environment and its conservation by application of biotechnological tools. The present book entitled A Practical Guide to Environmental Biotechnology covers the whole range of experiments related to environmental biotechnology. It also contains basic laboratory safety guidelines followed in a laboratory. Each chapter starts with an introduction or background into the basic approach followed by detailed methods sections with easy-to-follow workable protocols and comprehensive troubleshooting calculation. One of the important aspects of the book’s manual is the first part which deals with general guidelines of laboratory safety, rules and regulations, different symbols, glassware and equipments used commonly in an environmental laboratory. The second part of the book deals with different experiments on basic and advanced environmental studies. This book is an indispensable tool for introducing advanced undergraduates and beginning graduate and master’s students of Environmental Science and biotechnology. There are few books available on practical environmental aspects targeting students of the undergraduate and graduate levels. In this regard, this proposed book will be of great help to the students as it will cover the major experiments as per the curricula of Environmental Science and Biotechnology stream at both undergraduate and graduate levels. The book will be of great use to researchers and scientists in the relevant field of study. We are honestly grateful to all the literature and search engines we referred to write the chapters of this book. We are also thankful to Dr. Sue Lee, Editor, Medicine & Life Sciences Journals, and Dr. Emmy Lee, Associate Editor, Medicine and Life Sciences Books, and the team at Springer Nature Singapore Pte Ltd., Singapore, for their generous cooperation at every stage of the book’s publication. Jayanta Kumar Patra

Goyang-si, Republic of Korea Gitishree Das

Goyang-si, Republic of Korea Swagat Kumar Das

Bhubaneswar, Odisha, India Hrudayanath Thatoi

Baripada, Odisha, India

VII

Contents 1

 eneral Guidelines of Laboratory Safety, Calculations G Used in Laboratory Experiments, and Basic Laboratory Glassware and Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 1.2 1.3 1.4 1.5 1.6

 General Guidelines of Laboratory Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fire Safety in a Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gas Safety in a Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety Signs in Biotechnology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Calculations Used in Laboratory Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Laboratory Glassware and Instruments Used in an Environmental Lab . . . . . . . . . References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

Water Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2 3 5 9 11 16 34

2.1 Electrical Conductivity of Waste Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.2 Alkalinity of Waste Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 2.3 Dissolved Oxygen Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.4 Biochemical Oxygen Demand in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 2.5 Chemical Oxygen Demand in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 2.6 Total Suspended Solids (TSS) and Total Dissolved Solids (TDS) in Water Samples . . . . . 52 2.6.1 Total Dissolved Solids (TDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.7 Chloride Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2.8 Sulfate Content in Water Samples (Turbidimetric Method) . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2.9 Nitrogen Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3

Soil Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9

Moisture Content in Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  of the Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH Particle Size in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organic Matter and Organic Carbon Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . Chloride Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfate Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrogen Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potassium Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorus Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

Isolation, Culture, and Biochemical Characterization of Microbes . . . . . . . . 83

4.1 4.2 4.3 4.4

Laboratory Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L aboratory Etiquette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleaning Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62 62 64 70 72 73 74 77 79 81

85 86 87 89

VIII Contents

4.5 Determination of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 4.6 Study of the Effect of pH on Growth of Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 4.7 Preparation of Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 4.7.1 Preparation of Nutrient Agar (NA) Slants and Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 4.7.2 Preparation of Potato Dextrose Agar (PDA) Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.8 Isolation of Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 4.9 Techniques for Isolation of Pure Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 4.9.1 Serial Dilution Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 4.9.2 Streak Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 4.9.3 Pour Plate Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 4.9.4 Spread Plate Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 4.10 Staining Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 4.10.1 Simple Stains: Microbial Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 4.10.2 Negative Staining (Indirect Staining) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 4.10.3 Differential Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 4.10.4 Acid-Fast Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 4.10.5 Special Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.10.6 Fungal Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 4.11 Microbial Growth Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 4.12 Isolation of Genomic DNA from Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 4.13 DNA Quantification and Quality Analysis by Agarose Gel Electrophoresis . . . . . . . . . . . 120 4.14 DNA Quantification by Spectrophotometric Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 4.15 PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 4.16 Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 4.17 Sampling of Air Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 4.18 Antibiotic Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 4.19 Coliform MPN Test for Bacteriological Examination of Water . . . . . . . . . . . . . . . . . . . . . . . . 130 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 5

Plant Tissue Culture Techniques and Nutrient Analysis . . . . . . . . . . . . . . . . . . . . 135

5.1 Plant Propagation and Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 5.1.1 Preparation of Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 5.1.2 Callus Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 5.1.3 Cell Suspension Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 5.1.4 Protoplast Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 5.1.5 Artificial Seeds/Synthetic Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 5.2 DNA Isolation and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 5.2.1 Isolation of DNA from Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 5.2.2 Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 5.2.3 Spectrophotometric Quantification of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 5.3 Plant Nutrient Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 5.3.1 Estimation of Protein Content in Plant Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 5.3.2 Estimation of Fatty Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 5.3.3 Estimation of Ammonia Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 5.3.4 Estimation of Phosphorus in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

IX Contents

.3.5 Estimation of Potassium in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5.3.6 Estimation of Sulfur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.7 Estimation of Boron in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.8 Estimation of Zn, Cu, Mn, and Fe in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

158 159 160 162 163



Supplementary Information



Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

About the Editors Jayanta Kumar Patra, M.Sc., Ph.D., is currently working as Assistant Professor at Dongguk University, Republic of Korea. He has about 14  years of research and teaching experience in the field of food, pharmacology and nano-biotechnology. To his credit, he has published more than 140 papers in various national and international peer-reviewed journals and around 30 book chapters in different edited books. Dr. Patra has also authored 12 books in various National and International publications.

Gitishree Das, M.Sc., Ph.D., is currently working as Assistant Professor at Research Institute of Biotechnology & Medical Converged Science, Dongguk University, Republic of Korea. She has 12 years of research and teaching experience in the field of rice molecular biology, plant breeding, endophytic bacteria and green nanotechnology. Her current research is focused on the biosynthesis of nanoparticles using food wastes and plant materials and their applications in the biomedical and agricultural fields. To her credit, she has published around 70 research articles in reputed National and International journals and 16 book chapters in different books. Dr. Das has also authored 6 books in various International publications.

Swagat Kumar Das, B.Pharm., M.Tech., Ph.D., is currently working as Assistant Professor in the Department of Biotechnology at the College of Engineering and Technology, an autonomous and constituent College of Biju Patnaik University of Technology, Rourkela, Odisha, India. He obtained his B.Pharm degree from BPUT, Rourkela, and M.Tech. degree from Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, and Ph.D. from Ravenshaw University, Cuttack, India, and is a Fellow of Eurasian Academy of Environmental Sciences (FEAES). He has more than 10 years of teaching and research experience and published more than 20 research and review papers in National and International journals of repute along with 7 book chapters. He has co-authored 2 practical books. His research activities involved phytochemical analysis and drug development from mangrove plants for diabetes. His research area also focused on green synthesis of nanoparticles and evaluation of their ­pharmacological potentials.

XI About the Editors

Hrudayanath Thatoi, M.Sc., M.Phil, Ph.D., is currently working as Professor and Head of the Department of Biotechnology at North Orissa University, Odisha, India. He has around 30 years of teaching and research experience. His research activities are basically based on medicinal plants, bioremediation, biodiversity, ethnopharmacology, mangrove biology, etc. Prof. Thatoi obtained his M.Phil. and Ph.D. from Utkal University, Odisha, India, and his Ph.D. research work was based on N2 fixation in legume plants under dual inoculation of Rhizobium and VAM fungi and contributed significantly towards development of technology for mine waste reclamation. Prof. Thatoi has handled many research projects from State Government and Central Government organizations like DST, Govt. of Odisha, UGC-DAE, Govt. of India, Department of Forest, Govt. of Odisha, etc. Around 15 students have obtained Ph.D. and another 6 students are continuing their Ph.D. research under his guidance and supervision. Besides, several M.Sc., M.Tech, and B.Tech. students have received his guidance for their dissertation works. Prof. Thatoi has published more than 200 research papers in various National and International reputed journals and around 30 book chapters. Prof. Thatoi has also authored around 15 books/manuals by different notable publishers like STUDIUM Press LLC USA, IK International, Narosa publishers, Biotech Books, APH Publication, CRC Press, Apple Publication, Springer and Elsevier publications, etc. He has also authored a textbook on Microbiology and Immunology, published by India Tech Publication, New Delhi, for M.Sc. and B.Sc. Students. Prof. Thatoi has contributed immensely in the field of microbiology and biotechnology throughout his research and teaching career.

1

General Guidelines of Laboratory Safety, Calculations Used in Laboratory Experiments, and Basic Laboratory Glassware and Instruments Contents 1.1

General Guidelines of Laboratory Safety – 2

1.2

Fire Safety in a Laboratory – 3

1.3

Gas Safety in a Laboratory – 5

1.4

Safety Signs in Biotechnology Laboratory – 9

1.5

 eneral Calculations Used in Laboratory G Experiments – 11

1.6

 asic Laboratory Glassware and Instruments Used in an B Environmental Lab – 16 References and Suggested Reading – 34

© Springer Nature Singapore Pte Ltd. 2020 J. K. Patra et al., A Practical Guide to Environmental Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-15-6252-5_1

1

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Chapter 1 · General Guidelines of Laboratory Safety, Calculations Used in Laboratory Experiments...

What You Will Learn in This Chapter In this chapter, you will learn about the general laboratory safety guidelines that need to be followed while working in a laboratory; about the good laboratory practices, chemical laboratory safety education, types of hazards in a laboratory, fire safety, gas safety and various safety signs used in a laboratory; and about the general calculations used in various laboratory experiments together with information of basic laboratory glassware and instruments.

1.1  General Guidelines of Laboratory Safety

Maintaining safety inside a laboratory is of utmost importance for scientific personnel for the betterment of the environment and human kind. The principal objective of the laboratory safety standard is the safety of its staff against any kind of likely dangerous materials or any infections. Additionally, it is vital to educate students/ researchers and laboratory personnel about the laboratory safety guidelines and also about the hazardous chemicals, materials, instruments, etc., present in the laboratory and their uses (. Figs. 1.1, 1.2, 1.3, and 1.4; . Table 1.1). There are a number of issues that are to be followed for maintaining the safety of a laboratory. They are as follows: 1. There should be a list of emergency contact numbers, such as those of lab incharge, safety manager, and fire service, which should be mentioned in a visible area inside the laboratory. 2. The basic laboratory safety rules and guidelines should be displayed on the laboratory wall in an appropriate place. 3. A first aid kit containing basic essential medicines should be kept in a laboratory. 4. Protective gears such as lab coats, gloves, and goggles should be kept in sufficient numbers in a laboratory and should be properly used while working inside a laboratory. 5. All the chemicals and reagents should be labeled properly with the date of preparation and the type of reagents. 6. All other activities such as eating and storing food should not be allowed inside a laboratory. 7. All the instruments should be checked properly before and after use. 8. Electrical gears should be checked at regular intervals. 9. The instruments should be labeled properly with their date of manufacture, instructions for use, etc.. 10. Laboratory working place should be always kept clean and all the essential chemicals and glassware should be placed in their right place properly arranged. 11. Excessive things such as instruments, chemicals, glassware, etc. should be avoided in the working area. 12. The sink should be washed properly every day before and after use. 13. The gas openings and hoods should be used for specific chemicals and purpose only. 14. The broken glassware should be castoff in the specific container and must not be dumped anywhere. 15. Reagents and chemicals should never be dispensed down in the sink; it should be kept in a specific container and discarded properly.  

 

3 1.2 · Fire Safety in a Laboratory

Good Laboratory Practice (GLP):

• • • • • • • • • • • • •

Listen carefully to the safety instructions from the teaching staff at the start of each practical. Read the Safety section (COSHH and safety considerations) at the start of each practical schedule. Make sure you know where the nearest fire exit is. Make sure you know where the eyewash stations, first aid boxes and fire extinguishers are located in each lab you use. Always wear a labcoat (correctly fastened) - remove the labcoat if you leave the laboratory. Wear safety glasses for handling hazardous chemicals as instructed. Wear disposable gloves for handling hazardous chemicals or if you have a cut or wound on your hand. Always remove gloves before opening doors, drawers, labbooks or handling any instrument controls to avoid contaminating surfaces. No eating, drinking or application of cosmetics in labs. No open-toed footwear in the laboratory. No mouth pipetting - use automatic pipettes or pipetting devices. Dispose of chemicals and bacterial cultures in a safe manner as instructed. Report immediately any spillage of chemicals, bacterial cultures or breakages to the person in charge. Do not put broken glass, pipette tips or needles in the normal waste - use the SHARPS disposal bins provided. Switch off all electrical equipment and gas burners when you are finished in the lab. IF IN DOUBT, ASK - DON’T PUT SAFETY AT RISK !

..      Fig. 1.1  The rules of good laboratory practice in the laboratory. (Reproduced with permission from Seiler 2005a)

1.2  Fire Safety in a Laboratory

Utmost care should be taken for fire safety in a laboratory. There are certain guidelines that need to be followed for proper maintenance of fire safety in a laboratory. 1. A smoke detector and fire alarm should be installed inside every laboratory. 2. There should not be any obstacle near the fire exit point. 3. A fire extinguisher should be kept in a laboratory and it should be checked at regular interval for its functioning. 4. The laboratory personnel should be aware of how to use the fire extinguisher properly. 5. All persons working in a laboratory should be educated with fire safety (. Figs. 1.5 and 1.6).  

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1

Corporate management

Management America

Management Europe

Management Asia

Corporate quality assurance

Management finances

Management research

Management marketing

Management safety

TFM chronic toxicology

TFM reprotoxicology

TFM carcinogenesis

Quality assurance

TFM ecotoxicology

..      Fig. 1.2  Possible example of an organization chart of a large, multinational company with a number of test facilities and several levels of management. (Reproduced with permission from Seiler 2005b)

5 1.3 · Gas Safety in a Laboratory

..      Fig. 1.3  Products of chemical/laboratory safety education. (Reproduced with permission from Hill Jr. 2019)

Chemical/laboratory safety education

Caring for safety

Knowledge understanding

Critical thinking

1.3  Gas Safety in a Laboratory

A laboratory working with different types of gases should take utmost care for its safety. 1. The gases should be kept in specific cylinders and should be properly positioned. 2. The pipes used for the flow of gases in a laboratory should be properly selected and maintained. 3. The type of gas and the direction of the flow of the gases should be clearly indicated on the gas pipes. 4. The gas pipes should be placed at a small distance from the electrical wiring in a laboratory. 5. The gas leak alarms should be connected to the gas cylinders. 6. While using toxic gases, gas masks should be properly used in a ­laboratory.

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..      Fig. 1.4  Laboratory safety clothes and accessories used in a laboratory. (Reproduced with permission from Patra et al. 2019)

7 1.3 · Gas Safety in a Laboratory

..      Table 1.1  Different types of hazards in a laboratory Type

Hazards

Physical hazards

Runaway reactions Housekeeping Electrical hazards High pressure/low pressure Cryogenics Lasers Catalysts Corrosives Flammables Explosives Reactive/unstable chemicals Incompatibles Gases and gas cylinders

Toxic hazards

Target organ toxicants Teratogens Lachrymators Allergens (sensitizers) Carcinogens Asphyxiants Poisons Irritants

Biological hazards

Human specimens/some animal specimens Bacteria Viruses Fungal agents Rickettsial agents Biological toxins

Reproduced with permission from Hill Jr. (2019)

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..      Fig. 1.5  Fire safety alarm sensor connected in a laboratory

..      Fig. 1.6  Laboratory safety cabinet and fire extinguisher used in a laboratory. (Reproduced with permission from Patra et al. 2019)

9 1.4 · Safety Signs in Biotechnology Laboratory

1.4  Safety Signs in Biotechnology Laboratory

Harmful or Irritant

Flammable

Explosives

Oxidizer

Corrosive

Environmental Hazard

Respiratory Protection

Gloves Required

Eye or Face Protection

Protective Clothing

Protective Footwear

Eye Protection Required

Ear Protection Required

Fire Extinguisher

Fire Hose Safety

Flammable Gas

Fire Blanket

Nonflammable Gas

International Biohazard

Reactive Material

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Prohibition

Non-potable Water

Do Not Touch

No Open Flames

Do Not Eat or Drink

Do Not Enter

Carcinogen Hazard (Health Hazard)

Low Temperature Warning

Hot Surface Warning

Magnetic Field

Optical Radiation

Laser Warning

Compressed Gas

Non-ionizing Radiation

Generic Warning

Ionizing Radiation

Remote Control Equipment

Biohazard Sign

High Voltage Warning Sign

Toxic Chemical Symbol

11 1.5 · General Calculations Used in Laboratory Experiments

Toxic Substance

Irritant

Emergency Call Button

Escape Route Sign

Fire Triangle

First Aid

Safety Shower

Eye Wash

1.5  General Calculations Used in Laboratory Experiments

There are different types of calculations such as molarity, normality, percent by weight, etc., used in a laboratory and they are discussed in . Table 1.2.

..      Table 1.2  Concentration expression terms Unit

Symbol

Definition

Relationship

Molarity

M

Number of moles of solute per liter of solution

M=

mol liter

Normality

N

Number of equivalents of solute per liter of solution

N=

equivalents liter

Percent by weight (parts per hundred)

wt%

Ratio of weight of solute to weight of solute plus weight of solvent × 100

wt % =

wt/vol%

Ratio of weight of solute to total volume × 100

wt / vol% =

vol%

Ratio of volume of solute to total volume × 100

vol% =

Percent by volume

wt solute ´100 total wt wt solute ´100 total volume

vol of solute ´100 total volume (continued)

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..      Table 1.2 (continued) Unit

Symbol

Definition

Relationship

Parts per million

ppm

Ratio of solute (wt or vol) to total weight or volume × 1,000,000

ppm =

Parts per billion

ppb

Ratio of solute (wt or vol) to total weight or volume × 1,000,000,000

ppb =

mg solute kg solution m g solute = g solution mg solute = L solution m g solute = mL solution

m g solute kg solution ng solute = g solution m g solute = L solution ng solute = mL solution

Reproduced with permission from Tyl and Ismail (2017)

zz Serial dilutions

There are a number of processes for serial dilution and are discussed in . Figs. 1.7, 1.8, and 1.9.  

..      Fig. 1.7  Dilution schemes for a “dilute to” a and “dilute with” b scenario. (Reproduced with permission from Neilson and O’Keefe 2017)

a

b

13 1.5 · General Calculations Used in Laboratory Experiments

a

b

..      Fig. 1.8  Example standard curve dilution schemes for sequential dilutions a and parallel dilutions b. (Reproduced with permission from Neilson and O’Keefe 2017)

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..      Fig. 1.9  Diagram of a complex multistep dilution and concentration scheme. (Reproduced with permission from Neilson and O’Keefe 2017)

zz Concentration of common commercial strength acids is given in . Table 1.3.  

..      Table 1.3  Concentration of common commercial strength acids Acid

Molecular weight (g/mol)

Concentration (M)

Specific gravity

Glacial acetic acid

60.05

17.4

1.05

Formic acid

46.02

23.4

1.20

Hydroiodic acid

127.9

7.57

1.70

Hydrochloric acid

36.5

11.6

1.18

Hydrofluoric acid

20.01

32.1

1.167

Hypophosphorous acid

66.0

Lactic acid

90.1

11.3

1.2

Nitric acid

63.02

15.99

1.42

9.47

1.25

Perchloric acid

100.5

11.65

1.67

Phosphoric acid

98.0

14.7

1.70

Sulfuric acid

98.0

18.0

1.84

Sulfurous acid

82.1

Reproduced with permission from Neilson et al. (2017)

0.74

1.02

15 1.5 · General Calculations Used in Laboratory Experiments

zz Few examples of basic glassware and pipette used in a laboratory are shown in . Figs. 1.10 and 1.11.  

a

b

c

d

e

..      Fig. 1.10  Class A volumetric flask a and other types of non-class A volume-measuring glassware: graduated cylinder b, Erlenmeyer flask c, beaker d, and bottle e. (Reproduced with permission from Neilson et al. 2017)

a

b

c

d

e

..      Fig. 1.11  Class A volumetric pipette a and non-volumetric pipettes: adjustable pipettors b, reed pipettor c, serological pipettes d. (Reproduced with permission from Neilson et al. 2017)

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1.6  Basic Laboratory Glassware and Instruments Used

in an Environmental Lab

zz Glassware Glassware

Picture

Beaker

Glass beaker

Plastic beaker

Description

Uses

A cylindrical container with a flat bottom base and spout on one top side. It is made up of glass borosilicate and sometimes plastic, polypropylene, etc. It is available in various sizes (e.g., 10 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1000 ml, and 5000 ml etc.). It has a measuring mark in its side to measure the quantity of liquid.

These are used for various purposes such as for preparing mixture solution, for storing liquids for later use, for performing reactions, for titration experiments, for heating liquids or chemicals, etc.

17 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Glassware

Picture

Conical flask (Erlenmeyer flask)

Glass flask

Description

Uses

It is otherwise known as Erlenmeyer flask. It has a flat bottom base which narrows toward the mouth and is available with a ring in the neck or without a ring. It is made up of glass, borosilicate, and sometimes plastic, polypropylene, etc. It is available in various sizes (e.g., 10 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1000 ml, and 5000 ml). It has a measuring mark in its side to measure the quantity of liquid.

It is used for preparing reagents, mixing and storing chemicals, performing experiments, and also in preparing microbial culture. It is used in titration experiment.

These types of flask have a round bottom instead of flat bottom as in the case of conical flask. They are made up of glass (heat-resistant borosilicate). They have a tubular section called neck, with an opening. Depending upon the uses, there are two- to threenecked round bottom flasks. They come in a variety of sizes starting from 5 ml, 10 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1 liter to 20 liter in industrial scale.

They are used for heating, boiling liquids, in the distillation process, in the extraction of compounds, etc.

Plastic flask Round-­ bottom flask

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Glassware

Picture

Description

Uses

Burette

It is a graduated glass tube with a tap at one end. The stopcock is used to dispense a known volume of liquid slowly and as per the requirement. It is made up of glass or plastic.

It is used in the titration experiment.

Measuring cylinder

It is a narrow graduated cylinder and is available in various sizes. It is made up of glass or polypropylene. It comes in various sizes (10 ml–5 liter).

It is used to measure the volume of a liquid.

Funnel

It is a tube with wide mouth at one end and narrow mouth at the other end. It is made up of stainless steel, glass, aluminum, or plastic.

It is used for dispensing liquid or solid chemicals from one container to another with the small opening.

19 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Glassware

Picture

Description

Uses

Crucible

It is a ceramic or a metal container which is basically inert and is used for melting any substance (metal or chemicals). It is made up of hightemperature-resistant materials depending upon the purpose of use.

It is used for melting or heating any chemical or metal in a laboratory.

Mortar and pestle

The mortar is a bowl made up of ceramic, wood, metal, or granite stone. It has a pestle which is made up of the same material as that of the mortar. The pestle is heavy and blunt club shaped/ rod shaped, which is easy to hold in the hand. The substance to be crushed is put in the mortar and then crossed using the mortar.

It is used for mixing, grinding, or crushing the materials.

Test tubes

It is cylindrical narrow tube with round closing at the bottom and an opening with a rim at the top. It is made up of borosilicate glass material.

It is used for biochemical experiments.

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Glassware

Picture

Description

Uses

Culture tubes

It is similar to that of a test tube, but it has a stopper or closing cap in the mouth.

It is used in microbiological culture experiments.

Centrifuge tubes

It is cylindrical narrow graduated tube with conical bottom and a screw capped mouth. It is made up of plastic or polypropylene materials.

It is used in centrifugation process.

Petri plates

It is also known as Petri dish or culture dish or plates. It is a shallow/flat cylindrical glass with a lid to cover it. It is made up of borosilicate glass or plastic.

It is used in microbiological culture experiments.

Glass pipette

It is a narrow, graduated thin cylinder used to transport liquids or media. They come in various sizes and shapes.

Its main purpose is to transport required amount of liquid from one container to another.

21 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Glassware

Picture

Description

Uses

Dropper

It is similar to the pipette, but has a plastic bulb at one end to suck liquid inside the glass/plastic tube.

Its main purpose is to transport required amount of liquid from one container to another.

Pipette bulb

It is placed at the top of a pipette and is used to create vacuum by pulling the air inside and then the desired liquid into the pipette.

It is an aid to the pasture pipette.

Volumetric flask

It is a type of laboratory flask calibrated to contain a particular amount of liquid in it. It comes in various sizes and is normally made up of glass. It is a pear-shaped flatbottom flask with a long neck and a stopcock.

It is used for accurate dilution and preparation of standard solutions.

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Glassware

Picture

Description

Uses

Wash bottle

It is made up of plastic or polyethylene. It has a screw cap with a long narrow hole at the top in order to squeeze out water.

It is used to squeeze out water forcibly.

Volumetric pipette

It is made up of glass. It allows the actual measurement of a solution. It is calibrated to contain a specific amount of liquid. It has a large bulb in the middle with a long narrow mouth at the bottom.

It allows the actual measurement of a solution.

Test tube holder

It is made up of a wooden holder with an iron frame and a clamp.

It is used to hold test tubes, when they are hot.

23 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Glassware

Picture

Description

Uses

Test tube stand

It is made up of wood, iron rod, or thermostable plastic. It comes in a variety of shapes, sizes, compositions, and materials.

It is used to hold upright a multiple number of test tubes at a time at one place.

Micropipette

It is a sophisticated type of pipette used to hold small quantity of liquid (0.001 μl). Its basic structure includes a nozzle button, a tip ejector button, volume adjustment dial, volume display, tip ejector, and shaft. There are different types of micropipettes available in the market, based on the types of uses, design, weight, and precision.

It is used to hold small quantity of liquid with precision.

Eppendorf tubes

It is made up of polypropylene and it comes in various sizes ranging from 10 μl to 2 ml capacity.

It is used in molecular experiments to hold small quantity of liquid.

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Glassware

Picture

Description

Uses

Reagent bottles

It is made up of borosilicate glass or polypropylene and comes in various colors such as colorless, white, or amber. It comes in various shapes and sizes.

It is used for storing reagents and chemicals.

Beaker holder

It is made up of stainless steel, iron, and sometimes plastic. It comes in different shapes and sizes.

It is used to handle hot beakers firmly.

Tripod stand

It is made up of iron or wooden frame.

It is used to hold the beaker or conical flask for heating purpose.

25 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Glassware

Picture

Description

Uses

Burner, spirit lamp

It comes in different shapes and sizes depending upon the source of fire to be used.

Used to get fire in a laboratory for heating purpose.

Spreader

It is made up of glass or heat-resistant plastic. It has a definite shape.

It is used in microbiology experiment for spreading the culture uniformly on a Petri plate.

Microplates

It is a specially designed plate, made up of plastic or polypropylene. It is transparent or opaque depending upon its uses. It has multiple number of wells (24, 96 wells).

It is used for holding small amount of liquids multiple number of times in the wells (24 or 96 wells)

Forceps

It is made up of stainless steel. It is a hand-held instrument used to hold delicate things. It comes in various shapes and sizes as per the requirement.

For holding certain things.

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Glassware

Picture

Description

Uses

Spatula

It is made up of stainless steel. It is a hand-held instrument used for mixing, spreading, or lifting solid chemicals.

Used for lifting solid chemicals for weighing purpose.

Burette stand

It has a long rod, with a base and a clamp attached to it to hold the burette firmly.

It is used for holding the burette at the required position and to carry out the titration experiment.

Thermometer

It is made up of thermostable glass; it has a temperature sensor (mercury bulb) to sense the temperature and a measuring scale in Centigrade or Fahrenheit to measure the temperature.

It is used for measuring the temperature of any liquid or reagent.

Capillary tubes

It is made up of plastic or glass. Rod shaped, hollow inside.

Used for capillary experiments.

27 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

zz Instruments Instruments

Picture

Description

Uses

Weighing balance

A variety of balances are available depending on uses and application.

It is used to measure any chemical accurately.

Magnetic stirrer

It has a hot plate with the thermostat where we can control the heating temperature. The magnetic stirrer is also connected with a magnet and a magnetic bead is used to properly mix the solute in the solvent to make a uniform solvent.

It is used to mix any type of chemical or liquid.

Bacteriological incubator

The incubator is a double-walled modular structure with insulation. Basically, its interior is made up of stainless steel. The inside part is connected to a heating apparatus and a thermostat to control the heat. It has two doors: inside is a glass door and outside is the main door with secure gasket, heavy-duty door hinges and latches to maintain a secure and uniform seal. The racks and trays are all made up of stainless steel with adjustable height. It has both heating and cooling systems to control the temperature.

Normally it is used for bacterial cell culture purpose.

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Instruments

Picture

Description

Uses

Shaker

A number of shakers are used in a laboratory: vortex shaker, platform shaker, orbital shaker, and incubator shaker depending upon their specific purpose and uses.

Normally it is used to mix, combine, or stir various substances in a tube or flask, by shaking them.

Oven

The oven consistently and evenly distributes heat throughout the entire surface of the mold placed inside the oven. The exteriors have a hard, scratchresistant finish, normally light gray in color. The interior is made up of heavy steel double wall and doors are insulated. The interior is made up of corrosiveresistant aluminum steel. There is a temperature control thermostat to control the temperature along with a thermometer to show the interior temperature.

It is used for heating materials such as glassware, laboratory utensils and to dry certain things like plant materials, and other samples that need to be dried.

pH meter

A highly essential instrument in a basic scientific laboratory. It has two parts, a pH electrode and a measuring stand with digital setup to display the measurements.

It is used to measure the acidity or alkalinity of a solution based on the hydrogenion activity of the solution (water based).

29 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Instruments Atomic absorption spectroscopy

Picture

Description

Uses

Atomic absorption spectroscopy is used for qualitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state. It is based on absorption of light by free metallic ions.

It is used to determine the concentration of a particular element in a sample, for example, the determination of various elements in the soil samples; used in number of fields such as geology, minerals, metallurgy, steel, nonferrous metals; environmental analysis: air, water, soil, and solid waste quality; petrochemical industry and light industry products, crude oil and other products; food, biomedicine, and health products; building materials (glass, ceramics, paints, etc.).

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Instruments

Picture

Description

Uses

Spectrophotometer

Spectrophotometer is an instrument that uses the Beer– Lambert law to measure the intensity of any particle in the sample based on the transmission of light passing through it. It deals with visible light, ultraviolet light, and infrared light. There are different types of spectrophotometer depending upon the capacity of light source it uses for the measurement.

It is used for quantitative measurement of the light intensity transmitted as a function of wavelength. It is normally used in a biochemistry laboratory for biochemical experiments.

Deep freezer

It comes in a variety of temperature ranging from −20 to −80 °C etc. It has quick freeze functions.

It is used for quick freezing and storing chemicals and samples (animal, plant, DNA, RNA, protein, blood samples, clinical and medical samples, vaccines, etc.) in low temperature in order to preserve their properties.

Autoclave

It is a pressure chamber connected to heating coil. It is made up of stainless steel and connected with a temperature and pressure setup to maintain the temperature up to 121 °C and 15 lb pressure.

It is used for sterilization of samples at higher temperature and pressure (121 °C and 15 lb pressure).

31 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Instruments

Picture

Description

Uses

Microbiological safety cabinet

It contains a stainless steel hood connected to a high-efficiency particulate air filter (HEPA) to provide the flow of purified air in outward (horizontal) or downward (vertical) direction. It has an ultraviolet light source for sterilization of the working area. There are different types of hood depending upon the uses.

Used for microbiological or cellular analysis purpose.

Sonication machine

It is an instrument used to agitate particles in the sample using the sound energy. It uses ultrasonic frequencies (>20 kHz) for its applications.

It is used in the extraction of multiple compounds from plants and for mixing a particular compound in a solvent.

Centrifuge machine

It is an instrument used to spin an object around a fixed axis by applying a perpendicular force. It uses sedimentation principle, by which due to the application of centrifugal force, the denser substances are moved outward in the radial direction or settle at the bottom of the container. It comes in various sizes and capacities for specific purposes and applications.

It is used to separate a solute from a solvent in the solution.

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Instruments

Picture

Description

Uses

Vortex mixer

It is a simple electric device with a motor, a drive shaft, and a rubber piece of different shape to hold the test tubes or micro tubes. When the switch is turned on, the motor moves in a circular motion. It has a speed controller to control the speed of the movement of the motor.

It is used to mix reagents and chemicals inside a test tube, vial, or micro tube.

Colony counter

It is an electric machine used in a microbiology laboratory to count the number of bacterial colonies grown in a Petri plate. It has a flat circular surface with small lines, squares drawn, over which the Petri plate is kept; then it is illuminated from the bottom. A digital pen is connected to the instrument, and when the digital pen/ electronic counter points toward or softly touches the bacterial colony grown in the plate, it is counted and the number is displayed on a small display attached to the machine.

It is used to count the number of the bacterial colony in a petri plate.

33 1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…

Instruments

Picture

Description

Uses

Microscope

It is an instrument used to see smaller objects in a highly magnified form. There are different types of microscope available depending upon their uses, magnification, and purpose. However, basically it is divided into two types, light or electron microscope. A simplified microscope contains various parts such as base, arm, tube, and eyepiece with different types of lens.

It has various applications and uses in science. Its main objective is to visualize tiny objects in a highly magnified version which is visible to the naked eye.

Turbidity meter

It is an instrument used for determining the concentration of suspended particles in a sample of water on the basis of the incident light scattered from the tube containing the solution at right angle, which is captured by the sensor in the form of photons and recorded as electronic signals by the machine.

It is used for measuring the turbidity of a solution.

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Instruments

Picture

Description

Uses

Sieving machine

It is an instrument used for determining the particle size of soil samples based on their size and texture. It has different layers of sieves with different sizes that separate the soil samples based on the size of the particles.

It is used for measuring the particle size of the soil sample.

Flame photometer

It is used for the estimation of potassium content in the soil and water samples based on the emission values.

It is used for determination of potassium content in soil and water samples.

Take-Home Messages 55 Maintaining laboratory safety inside a laboratory is of utmost important for scientific personnel and for betterment of the environment and human kind. 55 The basic laboratory safety rules and guidelines should be displayed on the laboratory wall in an appropriate place. 55 All the instruments should be checked properly before and after use. 55 Laboratory working place should be always kept clean and all the essential chemicals and glassware should be placed in their right place in an arranged manner. 55 Reagents and chemicals should never be dispensed down in the sink; it should be kept in specific container and discarded properly.

References and Suggested Reading Basic Laboratory Safety. https://shp.utmb.edu/ClinicalLaboratorySciences/clsHandbook.asp. Accessed on 20th May 2019. Guidelines for Chemical Laboratory Safety in Secondary Schools. American Chemical Society Committee on Chemical Safety. 2016. https://www.acs.org/content/dam/acsorg/about/governance/ committees/chemicalsafety/publications/acs-secondary-safety-guidelines.pdf ?logActivity=true. Accessed on 9th May 2019. Hill RH Jr. Recognizing and understanding hazards — the key first step to safety. J Chem Health Saf. 2019;26(3):5–10.

35 References and Suggested Reading

Keith Furr A. CRC handbook of laboratory safety. Boca Raton: CRC Press; 2000. ISBN 9780849325236. Laboratory Safety Handbook. Sabanci University, Istanbul. https://fens.sabanciuniv.edu/sites/fens. sabanciuniv.edu/files/lab-safety/labsafety_web.pdf. Accessed on 20th May 2019. Laboratory Safety Manual. https://case.edu/cse/eche/daigroup/Lab%20Safety%20Manual.pdf. Accessed on 20th May 2019. Neilson AP, O’Keefe SF. Dilutions and concentrations. In: Food analysis laboratory manual. Cham: Springer International Publishing; 2017. p. 33–47. Neilson AP, Lonergan DA, Nielsen SS. Laboratory standard operating procedures. In: Food analysis laboratory manual. Cham: Springer International Publishing; 2017. p. 3–20. Patra JK, Das SK, Das G, Thatoi HN. General aspects of pharmacology laboratory. In: A practical guide to pharmacological biotechnology. Learning materials in biosciences. Singapore: Springer; 2019. Seiler JP. What is good laboratory practice all about? In: Good laboratory practice — the why and the how. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005a. p. 1–57. Seiler JP. How is good laboratory practice regulated? In: Good laboratory practice — the why and the how. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005b. p. 59–358. Thatoi HN, Dash S, Das SK.  Practical biotechnology, principle and protocols. New Delhi: I.  K. International Pvt. Ltd; 2017. Tyl C, Ismail BP.  Preparation of reagents and buffers. In: Food analysis laboratory manual. Cham: Springer International Publishing; 2017. p. 21–32.

1

37

Water Quality Analysis Contents 2.1

Electrical Conductivity of Waste Water – 38

2.2

Alkalinity of Waste Water – 40

2.3

Dissolved Oxygen Content in Water Samples – 43

2.4

Biochemical Oxygen Demand in Water Samples – 46

2.5

Chemical Oxygen Demand in Water Samples – 49

2.6

 otal Suspended Solids (TSS) and Total Dissolved Solids T (TDS) in Water Samples – 52

2.6.1

Total Dissolved Solids (TDS) – 52

2.7

Chloride Content in Water Samples – 53

2.8

 ulfate Content in Water Samples (Turbidimetric S Method) – 54

2.9

Nitrogen Content in Water Samples – 56 References and Suggested Reading – 58

© Springer Nature Singapore Pte Ltd. 2020 J. K. Patra et al., A Practical Guide to Environmental Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-15-6252-5_2

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Chapter 2 · Water Quality Analysis

What You Will Learn in This Chapter

2

In this chapter, you will learn about the easy-to-use protocols for determining electrical conductivity, alkalinity of waste water, dissolved oxygen content, biochemical oxygen demand, chemical oxygen demand, total suspended solids and total dissolved solids, chloride content, sulfate content, and nitrogen content in the water samples. You will also learn about how to calculate and interpret the results.

2.1  Electrical Conductivity of Waste Water zz Theory

Electrical conductivity is a material’s ability (water or any other liquid) to accommodate the transportation of an electric charge to conduct an electric current. The standard international derived unit for the measurement of electrical conductivity of water is Siemens per meter or Siemens per centimeter (named after Werner von Siemens) or, more simply, S.m−1 or S.cm−1. The electrolytes present in a solution generally separate into both positive (cations) and negative (anions) ions and impart conductivity. Normally, salts or other types of chemical constituents that are present in water in dissolved form are broken down into both positively or negatively charged ions; these free ions are accountable for the conduction of electricity in the water sample. At constant temperature, the electrical conductivity of water is equivalent to the concentration of ions present in it. It is influenced by the temperature of the water also. Thus, the higher the concentration of electrolytes present in the water, the more the electrical conductivity. Conductivity affects the occurrence of various aquatic plants and animals in the water; some types of aquatic organisms are sensitive to the increased conductivity in water, while some are tolerant to it. It is an indicative parameters to determine the increase in pollution in the water system. Distilled water is an insulator as it almost does not conduct any electricity, whereas salt water is a good conductor of electricity. The majority of ions that are responsible for high electrical conductivity in water are sodium, magnesium, calcium, potassium, etc., whereas ions like chloride, carbonate, bicarbonate, and sulfate are negative conductors. zz Materials Required

Glassware, such as measuring cylinder, beaker, conical flask, and glass pipette, and instruments, such as conductivity meter, conductivity cell, thermometer, and different water samples, are taken for analysis

39 2.1 · Electrical Conductivity of Waste Water

..      A sample conductivity meter setup

zz Procedure

1. The procedure for testing the electrical conductivity of any water sample largely depends upon the type and quality of the instrument used. However, common procedure is as follows. 2. Carefully study the operational manual of the conductivity meter and then regulate the temperature compensation knob of the conductivity meter equivalent to the temperature of the sample. 3. Keep the selector switch to ×1000 and calibrate to CAL (calibration) mark. Dip the conductivity cell in the sample contained in a beaker and connect the cell terminals to the sockets provided in the instrument. If the meter shows negligible deflection, disconnect the cell terminal. Move the selector switch to ×100 and calibrate to CAL mark. 4. Reconnect the cell terminal and note the deflection. If it is still negligible, disconnect the cell, move the selector switch to ×10, and calibrate to CAL mark. Reconnect the cell and note the deflection (dial reading). 5. Switch off the cell after the use and wash it with distilled water. 6. However, there are different protocols for different types of instruments used for the analysis of electrical conductivity.

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Chapter 2 · Water Quality Analysis

zz Results Sl. no

2

Temperature compensation knob

Selector switch

Deflection

1. 2. 3. 4.

zz Calculation

EC ( S) = DR × SS Where ES  is the electrical conductivity; DR is the dial reading; and SS is the value of selector switch. 2.2  Alkalinity of Waste Water

(Carbonate, bicarbonate, hydroxide, and total alkalinity of water samples) zz Theory

The alkalinity of water is sometimes referred to as the acid-neutralizing ability of the water at a given circumstance. It is the capability of the water sample to neutralize a strong acid or resist the changes in the pH value of the water with increasing concentration of the acids in the solution. It is characterized by the existence of hydroxyl (OH−) ions capable of coalescing with hydrogen (H+) ions. A number of bases such as hydroxides, carbonates, bicarbonates, phosphates, nitrates, silicates, and borates add up to the alkalinity of a solution and are therefore stated as the total alkalinity or alkalinity due to distinct bases. It is stated that the higher the alkalinity of the water, the greater its ability to neutralize the acids. The fundamental procedure for the measurement of water alkalinity is observing the changes in the pH value by adding an acid drop by drop to the water. In general, the pH of natural waters is in the range of 6–9, while most of the surface water has a pH that ranges from 7.5 to 8.0. However, water found in the deserts or sandy area has acidic pH. In case of the natural water, most of the alkalinity is triggered due to the presence of carbon dioxide (CO2). The free CO2 usually combines with the water sample partly in order to form carbonic acid (H2CO3), which is further dissociated into hydrogen (H+) and bicarbonate (HCO3−) ions. The HCO3− ions thus formed further dissociate into H+ and carbonate (CO3−) ions. CO 2 + H 2 O  H 2 CO3 H 2 CO3  HCO3− + H + HCO3−  CO3− + H +

41 2.2 · Alkalinity of Waste Water

The carbonate and bicarbonate ions present in the water samples further yield hydroxyl (OH−) ions that increase the pH of the water sample. In highly productive waters, the alkalinity is ought to be over 100 mg/L. zz Materials Required

Laboratory glassware such as measuring cylinder, beaker, conical flask, pipette, burette, and burette stand and specific chemicals such as sulfuric acid, phenolphthalein indicator, and methyl orange indicator are required. 1. Sulfuric acid (0.02N): About, 2.8  mL of concentrate sulfuric acid is diluted to 1 L using distilled water. Further, 200 mL of this stock solution (0.1N) is diluted to 1 L using distilled water to prepare 0.02N sulfuric acid titrant. The reagent is standardized prior to use. 2. Phenolphthalein indicator: About. 1 g of phenolphthalein is dissolved in 100 mL of ethyl alcohol and then 100 mL of distilled water is added to it and mixed properly. Then, NaOH solution (0.22N) is added to it drop by drop till a faint pink color appears. 3. Methyl orange indicator: About 0.1 g of methyl orange is dissolved in 200 mL of distilled water and the solution is mixed properly.

..      A complete setup for titration process for carrying out the alkalinity test

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Chapter 2 · Water Quality Analysis

zz Procedure

2

1. Different types of water samples are collected away from the shore and at least 1 feet under the surface of water in a clean glass container and immediately transferred to the laboratory for further analysis. 2. Take about 50 mL of the water sample in a conical flask and to it add 2–3 drops of phenolphthalein indicator and mix properly. 3. If the solution becomes slight pinkish in color, then it is due to phenolphthalein alkalinity (due to hydroxide or carbonate present in the water sample). If a pink color does not appear in the water sample after the addition of phenolphthalein indicator, then it is due to the presence of free CO2 but absence of phenolphthalein alkalinity. 4. Then, titrate the water solution against sulfuric acid until the solution becomes colorless to attain the end point. 5. Note the reading as “p” and further add 2–3 drops of methyl orange indicator in the same flask and then continue to titrate against the remaining sulfuric acid until yellow color of the solution turns to orange color to attain the end point. 6. Then, immediately stop the titration process and then note the reading as “t” which is the volume of titrant used for both the titration. 7. Calculate the alkalinity of the sample according to the following formula. zz Tabulation Sl. no

Volume of sample used

Phenolphthalein alkalinity (p)

Total volume of titrant used IBR

Mean

FBR

1. 2. 3. IBR initial burette reading, FBR final burette reading

zz Calculation

Phenolphthalein alkalinity Phenolphthalein alkalinity ( as calcium carbonate in mg/L ) =

P × 10000 S

Total alkalinity Total alkalinity ( as calcium carbonate in mg/L ) =

T × 1000 S

Where, P is the volume of the titrant used against the phenolphthalein indicator in mL; S is the volume of the sample in mL; and T is the total volume of the titrant used for both the titrations in mL.

43 2.3 · Dissolved Oxygen Content in Water Samples

The value of the different forms of alkalinity (hydroxide, carbonate, and bicarbonate) in the water samples in terms of CaCO3 (mg/L) can be calculated as follows: Different forms of alkalinity in terms of calcium carbonate Forms

Hydroxide

Carbonate

Bicarbonate

P = 0

0

0

T

P  0.5T

2P-T

2(P-T)

0

P = T

T

0

0

The concentrations of various ions such as OH−, CO3−, and HCO3 in the water sample are calculated as follows: Hydroxide ions in mg/mL = Valueof alkalinity of hydroxide × 0.34 Carbonate ions in mg/mL = Valueof alkalinity of carbonate × 0.60 Bicarbonate ions in mg/mL = Valueof alkalinity of bicarbonate × 1.22

2.3  Dissolved Oxygen Content in Water Samples zz Theory

The quantity of oxygen present in water is termed as dissolved oxygen (DO) concentration of the water. It is extremely inclined by the water temperature; for example, the colder the water (lower the temperature), the more oxygen it can hold, since gases like oxygen are more easily dissolved in cool water. The concentration of dissolved oxygen is always lower during the night than during the day because the water is much cooler during the nighttime than during the daytime. The quantity of dissolved oxygen plays an imperative role in defining the type of organisms that live in the water. It is measured by using chemical analysis methods such as Winkler’s titration method. zz Principle

Estimation of dissolved oxygen content in the water sample is a vital factor for determining the extent of pollution caused by waste materials in water samples. DO is also essential for all types of aerobic natural wastewater treatment procedures. The modified Winkler test for dissolved oxygen involves oxidation of Mn(II) to Mn(IV) state. The Mn(IV) will then oxidize iodide to iodine. The synthesized iodine is then titrated against sodium thiosulfate with starch as an indicator. In the fresh water, the atmospheric oxygen solubility ranges from 14.6 mg/L at 0 °C to about 7.0 mg/L at 35 °C under one atmospheric pressure. The reactions involved are as follows:

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Chapter 2 · Water Quality Analysis

zz Reactions

2 MnSO4 ( solid ) + O 2 ( aqueous ) → 2 MnO ( OH )2 ( solid )

2

4 Mn ( OH )2 ( solid ) + O 2 ( aqueous ) + 2 H 2 O → 4 Mn ( OH )3 ( solid ) Mn ( SO 4 )2 + 2 I − ( aqueous ) → Mn 2 + ( aqueous ) + I 2 ( aqueous ) + 2 SO 4 2 − ( aqueous ) 2 S2 O32 − ( aqueous ) + I 2 → S4 O6 2 − ( aqueous ) + 2 I − ( aqueous )

..      A setup for estimating dissolved oxygen in water sample

zz Requirements

Glassware such as measuring cylinder, beaker, dissolved oxygen specific bottle, thermometer, pipette, burette, burette stand, and chemicals and reagents. 1. Alkaline iodine: About 500 g of sodium hydroxide and 150 g of potassium iodide are dissolved in about 350 mL of distilled water separately in two beakers. Sodium hydroxide solution is allowed to cool down and then both the solutions are mixed

45 2.3 · Dissolved Oxygen Content in Water Samples

together. Then 10  g of sodium azide (NaN3) is dissolved in 40  mL of distilled water in another beaker and then it is added to the previous mixture and the whole mixture solution is made up to 1 L. 2. Manganese sulfate (MnSO4): This reagent can be prepared using different forms of manganese sulfate as stated below. About 480  g of MnSO4·4H2O or 400  g of MnSO4·2H2O or 365  g of MnSO4·H2O is dissolved in 750 mL of distilled water and then boiled if required and the final volume of the solution is made up to 1 L with distilled water. 3. Sodium thiosulfate (0.025 M): About 6.205 g of sodium thiosulfate (NaSO3·5H2O) is taken in a conical flask containing distilled water. To it, 0.4 g NaOH (solid) is added and the final volume is made up to 1 L with distilled water. 4. Starch solution: To prepare starch solution, 2 g of soluble starch is dissolved in 100 mL of hot distilled water, mixed properly, and kept in a reagent bottle. For long-term preservation of the solution, 0.2 g of salicylic acid is added to it. zz Procedure

1. Water sample were collected in speacially made Biochemical Oxygen Demand (BOD) bottles during day time at a depth of 1 foot deep. 2. Prudently fill the 300  mL glass biochemical oxygen demand (BOD) stoppered bottles with the sample water. Care should be taken that no water bubble is left inside the bottle. 3. Add 2 mL of manganese sulfate solution immediately to the sample collection bottle by injecting a pipette just underneath the surface of the water sample. 4. If the manganese sulfate solution is added above the surface of the water sample, then extra oxygen will enter into the water sample. Then the pipette is squeezed slowly so that no air bubbles enter the sample bottles through the pipette. 5. Then add 2 mL of alkali-iodide-azide reagent solution in the same manner into the water sample bottles. 6. Stopper the bottles containing the water samples carefully so that no additional air enters into it. 7. Then, mix the chemicals in the water sample bottles carefully by overturning it 3–4 times. 8. The bottle is then checked for the presence of any more air bubbles. If still it contains air bubble, then discard it and start with fresh sample repeating the steps from the beginning. 9. If extra oxygen is present in the water sample bottles, then a brownish-­orange cloud of precipitate will appear inside the bottle. When this precipitation slowly settles down to the bottom of the bottle, mix the water sample again by whirling it upside down 3–4 times and then leave it to settle down again. 10. Then, add 2 mL of the concentrated sulfuric acid into the BOD bottles containing the water samples with the help of a glass pipette just above the surface of the water sample. 11. Then, the stopper is carefully fixed and the sample is mixed by making the bottle upside down 3–4 times in order to dissolve the precipitation formed. 12. At this point, the water sample and the dissolved oxygen present in it are fixed and the samples can be stored for up to 8 h at this stage in a refrigerator. 13. The BOD bottles containing fixed water samples can be wrapped tightly with aluminum foil to avoid unnecessary damage to the water sample while storing.

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Chapter 2 · Water Quality Analysis

14. To analyze the DO content in the water sample, required amount of the fixed water sample is taken in a glass flask and titrated against sodium thiosulfate using a titration setup as shown in the figure above. 15. The titration process was initiated by recording the initial burette reading followed by addition of few drops of starch solution so that a blue color is formed. 16. Then the titration process is continued with slowly dropping the titrant solution from the burette into the flask continuously with continuous stirring of the water sample till it reaches the end point of clear and colorless solution. 17. When the titration process approaches the end point, it will take only one more drop of the titrant to remove the blue color. 18. Utmost care should be taken in the titration process – the titrant should be added slowly, drop by drop to the water sample, and it should be mixed properly, before adding the next drop to the solution. 19. For better visualization of the color change, a white paper sheet can be placed just below the conical flask containing the water sample. 20. When the sample reaches the end point, the final burette reading is recorded. 21. The concentration of the dissolved oxygen present in the water sample is calculated using the following formula. zz Calculation

The dissolved oxygen concentration can be calculated based on the amount of titrant added to the sample. The formula used is: DO =

( FBR − IBR ) × N × 8000 Sample volume

Where, IBR is the initial burette reading; FBR is the final burette reading; N is the normality of the manganese sulfate solution. Sodium thiosulfate solution is used to titrate the sample. zz Tabulation Sl. no

Volume of the water sample used in mL

Volume of the titrant used in mL IBR

FBR

1. 2. 3.

2.4  Biochemical Oxygen Demand in Water Samples zz Theory

Biochemical oxygen demand (BOD) is defined as the rate of removal (i.e., consumption) of oxygen distributed in the form of dissolved oxygen or even particulate organic matter in the water bodies by microorganisms through aerobic degradation. BOD is used as an indicator of serious organic pollution in the water bodies. BOD

47 2.4 · Biochemical Oxygen Demand in Water Samples

is estimated by determining the concentration of dissolved oxygen present in the water sample before and after incubation period for 5  days in dark and at 20  °C temperature. When the BOD of water increases and exceeds the available DO, the DO is gradually reduced or exhausted, which is very much harmful to the aquatic organisms and sometimes leading to the death of aquatic fish and other organisms. zz Requirements

1. BOD incubators, BOD bottles, laboratory glassware 2. Alkaline iodine: About, 500 g of sodium hydroxide and 150 g of potassium iodide are dissolved separately in 350 mL of distilled water in two different beakers. The sodium hydroxide solution was allowed to cool down to room temperature and then both the mixture solutions are mixed together. Then dissolve 10 g of sodium azide (NaN3) in 40 mL of distilled water and this solution is mixed with the previous two solutions and the whole content is made up to 1 L with distilled water. 3. Manganese sulfate (MnSO4): This reagent can be prepared using different forms of manganese sulfate as stated below. About 480  g of MnSO4·4H2O or 400  g of MnSO4·2H2O or 365  g of MnSO4·H2O is dissolved in 750 mL of distilled water; it is then boiled if required and the final volume of the solution is made up to 1 L with distilled water. 4. Sodium thiosulfate (0.025 M): About 6.205 g of sodium thiosulfate (NaSO3·5H2O) is taken in a conical flask containing distilled water; to it 0.4 g NaOH (solid) is added and the final volume is made up to 1 L with distilled water. 5. Starch solution: To prepare starch solution, 2 g of soluble starch is dissolved in 100 mL of hot distilled water, mixed properly, and kept in a reagent bottle. For long-term preservation of the solution, 0.2 g of salicylic acid is added to it. 6. BOD-free water: Deionized distilled water is passed through the activated carbon column and then redistilled again. 7. Phosphate buffer solution: About 33.4 g of Na2HPO4·7H2O, 21.75 g of K2HPO4, 8.5 g of KH2PO4, and 1.7 g of NH4Cl are dissolved properly in about 500 mL of distilled water and the total volume is made up to 1 L with distilled water and the pH is adjusted to 7.2. 8. Calcium chloride solution: About 27.5 g of CaCl2 is dissolved in distilled water and the total volume is made up to 1 L. 9. Ferric chloride solution: About 0.25  g of FeCl3·6H2O is dissolved in distilled water and the total volume is made up to 1 L. 10. Sulfuric acid (1N): About 2.8 mL of concentrated sulfuric acid is slowly added to 100 mL of distilled water and mixed properly. 11. Sodium hydroxide solution (1N): About 4 g of NaOH is mixed in 20 mL of distilled water and the total volume is made up to 100 mL. 12. Allylthiourea solution: About 500  mg of allylthiourea is dissolved in about 1000 mL of distilled water. zz Procedure

1. For the preparation of dilution water, aerate the BOD-free water samples in a large-size conical flask for about 30 min. 2. Add 1 mL each of phosphate buffer solution, magnesium sulfate solution, calcium chloride solution, and ferric chloride solution per liter of the dilution water solution.

2

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Chapter 2 · Water Quality Analysis

3. Adjust the pH of sample to 7.0, using sulfuric acid or sodium hydroxide as per the requirement. 4. For exhaustion of all the oxygen during the incubation process, the samples are diluted with dilution water at the rate as shown in the table below. 5. Two sets of BOD bottles are filled with the above-prepared water samples and to them 1 mL of allylthiourea solution is added. 6. The dissolved oxygen content (DO) in one set is determined immediately following Wrinkle’s method and the other set of the BOD bottle is incubated at 20 °C for 5 days in a BOD incubator. 7. The dissolved oxygen content of the stored bottle is determined after 5 days of incubation. 8. Calculate the BOD according to the following formula.

..      A setup for estimating biochemical oxygen demand in water sample

49 2.5 · Chemical Oxygen Demand in Water Samples

Table for dilution of samples as required for various ranges of expected BOD values Expected BOD value in mg/L

Volume of the water sample taken in mL

Dilution factor

0–6

1000

1

4–12

500

2

10–30

200

5

20–60

100

10

40–120

50

20

100–300

20

50

200–600

10

100

zz Calculation

BOD5 = ( D0 − D5 ) × Dilution factor Where D0 is the initial dissolved oxygen content in the sample in mg/L at 0 day of incubation and D5 is the dissolved oxygen content left out in the sample after 5 days of incubation. Table for determination of DO Sl. no

Volume of sample used in mL

Volume of titrant used in mL IBR

FBR

1. 2. 3. IBR initial burette reading, FBR final burette reading

2.5  Chemical Oxygen Demand in Water Samples zz Theory

For the evaluation of the quality of the water, it is crucial to know the quantity of oxidizable organic substances present in it. It is usually determined by three different aspects which are: (1) estimation of total organic carbon content, (2) estimation of dissolved organic carbon contents, and (3) estimation of the chemical oxygen demand (COD) of the water sample. COD is defined as the measurement of dissolved oxygen required to oxidize the organic compounds present in the water samples. During the estimation of COD, chemicals such as potassium dichromate and potassium permanganate are used. In simplified words, it is the quantity of

2

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Chapter 2 · Water Quality Analysis

dissolved oxygen abridged by the action of chemical reactions in the water sample. The estimation of COD in water samples is of utmost importance with respect to the water samples which have adverse growth of microorganisms and presence of highly toxic chemicals and where the estimation of the BOD of the water samples is not accurate.

..      A COD reflux unit for COD estimation

zz Requirements

1. COD reflux unit: It consists of 250 mL/500 mL Erlenmeyer flasks with ground glass. 2. 30 cm straight jacket Liebig condenser with 24/40 ground-glass joint (30 cm). 3. Hot plate water bath. 4. Potassium dichromate solution (0.25N): About 12.259 g of K2Cr2O7, previously dried at 150 °C for 2 h, or dried powder is dissolved in 500 mL of distilled water and then the final volume is made up to 1 L. 5. Silver sulfate: dried and powdered. 6. Mercuric sulfate (HgSO4): crystals or powder. 7. Sulfuric acid: concentrated. 8. Ferroin indicator solution: About 0.695  g of ferrous sulfate and 1.485  g of 1, 10-phenonthorline are dissolved in 50  mL of distilled water and the volume is made up to 100 mL.

51 2.5 · Chemical Oxygen Demand in Water Samples

9. Ferrous ammonium sulfate solution (0.25N): About 98 g of Fe(NH4)2(SO4)2·6H2O is dissolved in 500 mL of distilled water and to it 20 mL of concentrated H2SO4 is slowly mixed, and the mixture solution is cooled and diluted to 1000 mL with distilled water. Further, for the standardization of the solution, 25 mL of potassium dichromate solution is taken in a conical flask and the volume is made up to 250 mL with distilled water followed by addition of 90 mL of concentrated sulfuric acid and cooled. Then 5–6 drops of ferroin indicator solution are added to it and titrated against ferrous ammonium sulfate solution. The color changes from blue green to reddish blue at the end point. zz Procedure

1. 20 mL of the water sample is taken in a flask of efflux unit and to it 10 mL of potassium dichromate solution is added, followed by a pinch of silver sulfate and mercuric sulfate, along with 30 mL of sulfuric acid. 2. The Liebig condenser is then attached to the mouth of the flask and kept on a hot water bath or a heating mantle for about 2 h to reflux the contents. 3. Afterward, the flask is allowed to cool, detached from the unit, and its content is diluted to about 150 mL by adding distilled water. 4. Further, 2–3 drops of ferroin indicator solution are added to it and titrated against ferrous ammonium sulfate solution. 5. Change in color of the reaction mixture from blue green to reddish blue is the end point. 6. Distilled water blank is run simultaneously in a similar manner and the blank is adjusted. Table for determination of COD Sl. no

Volume of sample used in mL

Volume of the titrant used against the sample in mL IBR

Volume of the titrant used against the blank in mL FBR

IBR

FBR

1. 2. 3. IBR initial burette reading, FBR final burette reading

zz Calculation

COD =

( Z − Y ) × N ×1000 × 8 V

Where Y is the volume of the titrant used against the water sample in mL; Z is the volume of the titrant used against the blank in mL; N is the normality of the titrant (0.25); and V is the volume of the water sample in mL.

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Chapter 2 · Water Quality Analysis

2.6  Total Suspended Solids (TSS) and Total Dissolved Solids (TDS)

in Water Samples

2

2.6.1

Total Dissolved Solids (TDS)

zz Theory

A huge amount of salts including calcium, iron, magnesium, manganese, potassium, chlorides, carbonates, bicarbonates, nitrates, phosphates, sodium, and sulfate are found in dissolved form in natural water. A higher amount of these dissolved substances present in the water samples results in the elevation of water density, thereby influencing the osmoregulation of freshwater organisms and also reducing the solubility of the gases and finally the usability of water for drinking, irrigation, and industrial purposes. It is an important parameter used to in analyze saline lakes, estuarine, etc. zz Requirements

Glassware, such as beaker, measuring cylinder, glass pipette, conical flask, and evaporating dish, and instruments, such as weighing balance, desiccator, hot water bath, and filter paper, are required

..      Evaporating disc used for TDS analysis

zz Procedure

1. Take a clean and dry evaporating dish of suitable size and pre-weigh it before use. 2. Filter out about 250–500  mL of the selected water sample using a filter paper (Whatman No. 4) and take the filtrate in the pre-weighed evaporating dish. 3. Vaporize the whole water sample by placing the evaporating dish containing the required water sample on the hot water bath. 4. After complete drying of the water samples on the evaporating dish, the container is allowed to cool to room temperature inside the desiccator and then its final weight is measured in a weighing balance. 5. Calculate the amount of the total dissolved solids present in the water sample using the following formula.

53 2.7 · Chloride Content in Water Samples

zz Calculation

TDS =

( A − B) V

× 100

Where TDS is the total dissolved solids of the water sample; A is the final weight of the evaporating dish after complete drying of the water samples; B is the initial preweight of the evaporating dish before putting the water sample in it, and V is the total volume of the water sample taken for the analysis. Note: if total solids, instead of total dissolved solids, are to be computed, do not filter the sample. 2.7  Chloride Content in Water Samples zz Theory

Chloride is a major ion present in both salt and natural water. It is formed from the dissociation of sodium and calcium salts. In normal fresh water, the higher amount of chlorides content is taken as an indicator of pollution due to the presence of organic waste of animal origin (animal excreta contain high amount of chloride and nitrogenous wastes) in the water samples. Industrial effluents may also increase the chloride content in natural waters. The measured chloride ions can be utilized as an indicator of salinity for various water bodies. If the water samples comprising chlorides are titrated against silver nitrate, then the chlorides present in water usually precipitate to white color silver chloride. Potassium chromate solution which is used as an indicator for the experiment basically reacts with the silver chloride to form silver chromate which is reddish brown in color. zz Requirements

1. Laboratory glassware such as beaker, conical flask, burette, burette stand, glass pipette, measuring cylinder, and reagents. 2. Silver nitrate solution (0.025N): About 3.397  g of silver nitrate is dissolved in distilled water and the final volume is made up to 1 L with distilled water. The solution is stored in dark glass bottles in order to avoid reaction with light. 3. Potassium chromate indicator: About 10 g of potassium chromate is dissolved in about 20 mL of distilled water and to it few drops of 0.025N silver nitrate solution are added in order to produce a red precipitate. It is allowed to stand for 12 h, filtered, and then further diluted to 1 L with distilled water. 4. Sodium chloride (0.02N): About 1.170  g of sodium chloride is dissolved in 900 mL of distilled water and the volume is made up to 1 L in distilled water. zz Procedure

1. Take 10  mL of the water sample in a conical flask and to it add 5–6 drops of potassium chromate indicator and mix properly. The color of the water sample will become yellow. 2. Titrate the water samples against silver nitrate solution using burette setup until a persistent brick red color appears which is the end point of the reaction. 3. Care is taken that the silver nitrate solution is added to the water sample drop by drop, and after addition of each drop, the water sample is mixed properly.

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Chapter 2 · Water Quality Analysis

4. A white sheet is placed below the conical flask containing the water sample in the burette setup in order to better visualize the color change. 5. Calculate the concentration of chloride in the water sample using the following formula. Table for chloride content Sl. no

Volume of the water sample taken for the experiment in mL

Volume of the silver nitrate titrant used against the water sample in mL IBR

FBR

1. 2. 3. IBR initial burette reading, FBR final burette reading

zz Calculation

Chloride content in mg / mL =

(V × N × 35.475 ×1000 ) S

Where, V is the total volume of the titrate used in mL, N is the normality of the titrant (0.02), 35.475 is the equivalent weight of chlorine, and S is the volume of sample in mL. 2.8  Sulfate Content in Water Samples (Turbidimetric Method) zz Theory

Sulfate is present in higher quantity in natural water and soil samples, more significantly where the salt content is more. These are completely soluble in the water providing hardness to it. Domestic sewage and industrial effluent, besides biological oxidation, may add to the sulfate content of the water and soil. It is the second largest amount of mineral content present in the seawater. However, they are considered to be a serious concern to the water bodies as they are reduced to form hydrogen sulfide under the absence of oxygen followed by increase in odor and corrosion. Determination of sulfate content in the sample is based on the precipitation of sulfate after adding barium chloride. zz Requirements

1. Laboratory glassware, test tube, flask, beaker, measuring cylinder, spectrophotometer, magnetic stirrer, filter paper (Whatman No. 1). 2. NaCl–HCl solution: About 250 g of sodium chloride is dissolved in a little distilled water, followed by addition of 20 mL of HCl to it, and then the total volume is made up to 1 L with distilled water. 3. Glycerol–ethanol solution: About 50 mL of glycerol is added to 100 mL of ethyl alcohol and shaken well in a reagent bottle. 4. Barium chloride: Dry crystals of barium chloride are used for the experiment.

55 2.8 · Sulfate Content in Water Samples (Turbidimetric Method)

5. Standard sulfate solution: About 0.147 g of anhydrous sodium sulfate is dissolved in distilled water and the total volume is made up to 1  L with distilled water. Standards of various concentration 0, 5, 10, 15, 20, 25, 30, 35, and 40 mg/L are prepared with the stock solution. zz Procedure

1. The water sample to be tested is filtered through a filter paper and from it 50 mL is taken in an Erlenmeyer flask. 2. To it, 10  mL of NaCl–HCl solution, 10 mL of glycerol–ethanol solution, and 0.15 g of barium chloride are added. 3. Stir the sample for about an hour with the help of a magnetic stirrer. 4. Measure the absorbance(s) of the sample at 420 nm using a spectrophotometer against the distilled water blank. 5. Make a standard curve, taking different concentration of standard sulfate solution in similar manner. 6. Plot a standard graph with strength (mg/L) on one axis and absorbance on the other and calculate the sulfate content of the unknown water sample from the standard graph. 1.8 1.6

Absorbance values

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0

5

10 15 20 25 30 Concentration of standard Sulphate

35

40

..      A sample standard calibration curve for understanding

Table for determination of sulfate content in water sample Sl. no. 1. 2. 3. 4. 5.

Sample of different strength in mg/L

Absorbance at 420 nm

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Chapter 2 · Water Quality Analysis

2.9  Nitrogen Content in Water Samples

2

zz Theory

Presence of total nitrogen content in the water sample is considered as a chemical evidence of occurrence of current organic pollution due to the animals in the water body. Estimation of nitrogen in various forms in the water is carried out to assess its bacteriological quality. The process of analyzing nitrogen content is based on the principle, that in the presence of sulfuric acid and mercuric sulfate, catalyst organically bound to nitrogen and gets converted into ammonium sulfate. The ammonia that then gets liberated is absorbed by boric acid and the absorbed ammonia is then estimated either by titration process or by direct Nesslerization process. zz Requirements

1. Glassware like beaker, measuring cylinder, conical flask, and glass pipette; instruments like digestion apparatus which consists of a Kjeldahl flask (with a capacity of 500 or 800 mL), still head along with a provision of suction of fumes and a heating device, a complete distillation apparatus with a condenser and collector, Nessler tubes (100 mL capacity), and UV-visible spectrophotometer. zz Reagents

1. Preparation of digestion reagent About 134 g of K2SO4 is dissolved in 650 mL distilled water followed by addition of 200  mL of concentrated H2SO4. Further, a solution of 2  g of HgO in 25 mL of 6N H2SO4 is also added to it with continuous stirring. Finally the whole solution is made up to 1000 mL with distilled water and stored at room temperature (around 25 °C or higher) to prevent crystallization. 2. Phenolphthalein indicator: About 1 g of phenolphthalein is dissolved in 100 mL of ethyl alcohol and to it 100 mL of distilled water is added. Further NaOH solution (0.22N) is added drop by drop till a faint pink color appears. 3. Sodium hydroxide–sodium thiosulfate reagent: About 500 g of NaOH and 25 g of Na2S2O3 are dissolved and diluted to make up the volume to 1000 mL. 4. Mixed indicator: About 200 mg of methyl red is dissolved in 100 mL of 95% ethyl alcohol in a beaker and 100 mg of methylene blue is dissolved in 50 mL of 95% ethyl alcohol in another beaker. Finally the two solutions are mixed properly. 5. Boric acid: About 20 g of H3BO3 is dissolved in distilled water and to it 10 mL of the mixed indicator is added and the total volume is made up to 1000 mL with distilled water. 6. Sodium hydroxide 6N: About 240 g of NaOH is dissolved in 1 L of ammonia-free distilled water. 7. Standard sulfuric acid: About 2.8  mL of concentrated sulfuric acid is diluted to 1 L with distilled water. And 200 mL of this stock solution (0.1N) is further diluted to 1 L using distilled water to prepare 0.02N sulfuric acid titrant. Standardize the solution.

57 2.9 · Nitrogen Content in Water Samples

zz Procedure

A. Digestion Process 1. Take appropriate volume of well-mixed water sample in a Kjeldahl flask. 2. Add 50 mL of the digestion reagent to the water sample. If the concentration of the suspended solids is high, then large amount of the reagents are added to it to maintain the salt to acid ratio as 0.8. 3. Digestion process is continued for around 30 min till the sample appears to be clear to ensure complete decay/destruction of the organic matter. 4. Then it is cooled to the room temperature and diluted with distilled water to 150 or 300 mL depending on the capacity of the flask. B. Distillation Process 1. Place the conical flask containing the digested water samples in an appropriate position in the distillation apparatus and turn on the heat. 2. Then to it, around 0.5 mL of the phenolphthalein reagent is added followed by addition of sodium hydroxide–sodium thiosulfate reagent till the pH of the mixture solution rises just above 8.3. 3. Then it is distilled and around 200 mL of the distillate is collected in a 500 mL Erlenmeyer flask containing 50 mL of boric acid solution. Extend the tip of the condenser well below the level of boric acid solution. 4. When the distillation process is complete, the flask containing the distillate is removed first and then the heat system is put off in order to avoid back suction. 5. The concentration of ammonia is measured by the process of Nesslerization. The distillate is titrated with 0.02N H2SO4 till the indicator turns to a pale lavender color. 6. A blank is run throughout the procedure in all steps to make necessary correction. zz Calculation

Organic N =

( A − B ) × 280 Sample ( mL )

Where A is the mL of 0.02N H2SO4 required for the sample and B is the mL of 0.02N H2SO4 required for the reference blank. Take-Home Messages 55 Electrical conductivity is a material’s ability (water or any other liquid) to accommodate the transportation of an electric charge to conduct an electric current. 55 The alkalinity of water is referred to as the acid-neutralizing ability of the water at a given circumstance. 55 In highly productive waters, the alkalinity is ought to be over 100 mg/L. 55 The quantity of oxygen present in water is termed the dissolved oxygen (DO) concentration of the water.

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Chapter 2 · Water Quality Analysis

55 The quantity of dissolved oxygen plays an imperative role in defining the type of organisms that live in the water. 55 Biochemical oxygen demand (BOD) is defined as the rate of removal (i.e., consumption) of oxygen distributed in the form of dissolved oxygen or even particulate organic matter in the water bodies by microorganisms through aerobic degradation. 55 For evaluating the quality of the water, it is crucial to know the quantity of oxidizable organic substances present in it. 55 The estimation of COD in the water samples is of utmost importance with respect to the water samples which have adverse growth of microorganisms and presence of highly toxic chemicals and where the estimation of the BOD of the water samples is not accurate. 55 Total dissolved solid is an important parameter used to analyze the salinity of lakes estuarine, etc. 55 Chloride is a major ion present in both salt and natural water. 55 Sulfate is present in higher quantity in natural water and soil samples, more significantly where the salt content is more. 55 Presence of total nitrogen content in the water sample is considered as a chemical evidence of occurrence of current organic pollution due to the animals in the water body.

References and Suggested Reading AWWA, WEF, APHA. Standard methods for the examination of water and wastewater. Washington, DC: American Public Health Association; 1998. Bisen PS. Laboratory protocols in applied life sciences. Boca Raton: CRC Press; 2014. Bureau of Indian Standards. Indian standard drinking water – specification (IS 10500). New Delhi: Bureau of Indian Standards; 2012. http://cgwb.­gov.­in/Documents/WQ-standards.­pdf. Accessed 3 June 2019. Dunnivant FM. Environmental laboratory exercises for instrumental analysis and environmental chemistry. Hoboken: Wiley-Interscience; 2004. Fresenius W, Quentin KE, Schneider W. Water analysis. Berlin, Heidelberg/New York: Springer-Verlag; 1988. ISBN-13: 978-3-642-72612-5. FSSAI.  Manual of methods of analysis of foods – water. New Delhi: Food Safety and Standards Authority of India, Ministry of Health and Family Welfare, Government of India; 2015. https:// old.­fssai.­gov.­in/Portals/0/Pdf/Draft_Manuals/WATER.­pdf. Accessed 3 June 2019. Government of India & Government of The Netherlands. Standard analytical procedures for water analysis. 1999. http://indiawrm.­org/Website/Webpages/PDF/-download-manuals-WaterQuality-­ TechnicalPapers-StandardAnaly.­pdf. Accessed 3 June 2019. http://egyankosh.­ac.­in/bitstream/123456789/13529/1/Unit-2.­pdf. Accessed 5 July 2019. http://egyankosh.­ac.­in/bitstream/123456789/29474/1/Unit-2.­pdf. Accessed 5 July 2019. http://egyankosh.­ac.­in/bitstream/123456789/29789/1/Unit-2.­pdf. Accessed 5 July 2019. http://egyankosh.­ac.­in/bitstream/123456789/51189/3/Block-2.­pdf. Accessed 5 July 2019. http://egyankosh.­ac.­in/bitstream/123456789/9337/1/Unit-18.­pdf. Accessed 5 July 2019. Lange ED. Manual for simple water quality analysis. Amsterdam, the Netherlands: International Water Tribunal Foundation; 1994. ISBN 907080302X. Patel H, Vashi RT. Chapter 2 – Characterization of textile wastewater. In: Patel H, Vashi RT, editors. Characterization and treatment of textile wastewater. Boston: Elsevier; 2015. p. 21–71.

59 References and Suggested Reading

Patra JK, Das SK, Das G, Thatoi H. A practical guide to pharmacological biotechnology. Singapore: Springer; 2019. Sawyer CN, McCarty PL, Parkin GF.  Chemistry for environmental engineering. 4th ed. New  York: McGraw-Hill, Inc.; 2000. Thatoi HN, Dash S, Das SK.  Practical biotechnology, principle and protocols. New Delhi: I.K. International Pvt. Ltd; 2017.

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Soil Quality Analysis Contents 3.1

Moisture Content in Soil – 62

3.2

pH of the Soil – 62

3.3

Particle Size in Soil Samples – 64

3.4

 rganic Matter and Organic Carbon Content O in Soil Samples – 70

3.5

Chloride Content in Soil Samples – 72

3.6

Sulfate Content in Soil Samples – 73

3.7

Nitrogen Content in Soil Samples – 74

3.8

Potassium Content in Soil Samples – 77

3.9

Phosphorus Content in Soil Samples – 79 References and Suggested Reading – 81

© Springer Nature Singapore Pte Ltd. 2020 J. K. Patra et al., A Practical Guide to Environmental Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-15-6252-5_3

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Chapter 3 · Soil Quality Analysis

What You Will Learn in This Chapter

3

In this chapter, you will learn about the easy-to-use protocols used for the determination of moisture content, pH, particle size, organic matter and organic carbon content, chloride content, sulfate content, nitrogen content, potassium content, and phosphorus content in the soil samples. You will also learn about the easy-to-use laboratory protocols and how to calculate and interpret the results.

3.1  Moisture Content in Soil zz Theory

The moisture content in soil refers to the amount of water present in the soil. Since most of the estimation of soil characteristics is carried out on a dry weight basis, the moisture content of the soil needs to be determined immediately after sampling before any further analysis. zz Requirements

1. Container for storing the soil (tins or flask with lids) 2. Hot air oven zz Procedure

1. Moisture tin or flask to be used for moisture analysis is cleaned and dried properly. 2. Around 5–10 g of the soil sample is collected in a clean and dry moisture tin or a flask with lid and weighed using a weighing balance machine and the initial weight of the container containing the soil sample is recorded. 3. The sample was then dehydrated inside a hot air oven at a temperature of 105 °C for overnight with the lids open. 4. After heating, the sample was removed from the oven with the lids closed. 5. Further it was allowed to cool to room temperature inside a desiccator, followed by taking the final weight of the container containing the sample. 6. The moisture content in the selected soil sample was then calculated with the help of the following formula. zz Calculation

Moisture content =

A− B B − ( tare the weight of the container used)

Where, A is the weight of the container with sample before heating and B is the weight of the container with sample after heating and cooling. 3.2  pH of the Soil zz Theory

The determination of pH of the selected soil samples is an important factor for the characterization of soil sample. Normally, the pH of any sample ranges from 1 to 14; however, in the soil science, it ranges from 1 to 12. Soil samples with very high pH are normally influenced by the various salts present in them. The preliminary

63 3.2 · pH of the Soil

measurement of pH of soil is carried out by mixing the collected soil sample in a neutral solvent and measuring the pH of the mixture solution. The pH denotes the physicochemical environment of a soil at a particular location. The classification of the soil pH as per the data presented by Pansu and Gautheyrou (2006a, b, c) is given below: pH range

Classification

Lower than 3.5

Hyperacidic

Between 3.5 and 5.0

Very acidic

Between 5.0 and 6.5

Acidic

Between 6.5 and 7.5

Neutral

Between 7.5 and 8.7

Basic

Greater than 8.7

Basic

Source: Pansu and Gautheyrou (2006a, c). Data are presented with suitable permission from the original source

zz Procedure

There are different methods for the measuring the soil pH. These methods includes, measuring the pH of the saturated paste extract; 1:5 dilution of soil: water ration; or by 1:5 dilution of soil: Calcium chloride solution. 1. The soil water dilution method is the more suitable method and is often used in a soil science laboratory. 2. Around 10 g of soil is taken and is mixed in a beaker containing 50 mL of water. 3. It is mixed properly and its pH reading is recorded using a pH meter.

..      A digital pH meter is used for the measurement of pH of any sample

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Chapter 3 · Soil Quality Analysis

3.3  Particle Size in Soil Samples zz Theory

Clays

Fine silts 0.002

Fine sands

Coarse sands

Very coarse sands

Coarse sands

Fine sands (III) FS1 FS2

Fines (silts and clays)

Fine sands 200 75 µm

CSSC

7

2 00

0.20

0.02 Silts (II)

MS

France

Fine sands

1.00

0.50

USDA

Silts

Clays

0.25

8

Gravels

Stones

Fine Coarse 5

2 00 Coarse sands (IV)

CS1

CS2

CS3

Medium sands 35 500 µm

Gravels

10 1/2 2 mm

CG 3

ISSS Atterberg

4

ISSS

0.05 0.10

10

Stones

3

ASTM

0.002

Stones

AFNOR

Coarse sands

Gravels

MS

0.50 0.02 0.05 0.10 0.20 Coarse Fine CS Fine FS sands sands (1) silts (1) (2) (2)

VFS

Clays

Clays (I)

Fine sands

2 00 Very Gravels coarse sands 1.00 2 00 Coarse sands (3)

NC

1.00

CSI

0.002

0.50

0.25

FG

FSI Medium silts

CSI

Coarse clays

CSI

FC

0.05 0.10

0.005

VFS

0.0002

Coarse elements Fine earth 2 mm 20 mm 75 mm 250 mm 1 mm

200 µm 500 0.2 0.5

CS

1 2 µm 1020 50 100 0.02 0.1 0.002

0.1 µm

SC

 

CSI

3

The separation of different contents in the soil samples basing on their size along with their specific quaintly refers to the particle size in a soil sample, which is essential for various fields such as agronomy, pedology, sedimentology, and road geotechnics. Soil is a systematized standard that includes the accumulation of different types of inorganic and organic particles that belong to an incessant dimensional series. The soil is classified into a number of standard categories in various countries, such as fine clays, silts, coarse silts, very fine sands, fine sands, and coarse sands (. Fig. 3.1; Pansu and Gautheyrou 2006a, b, c). The particles in the soil sample are separated with particle size less than 2 mm. The most important step in the separation process is the pretreatment process where the primary particles completely get rid of calcium carbonate and other organic matters present in the soil. The size of the particle in the soil samples are based on the measurements that link them to their physical features

Inch or standard ASTM

..      Fig. 3.1  Ranges of particle size used for soils (NC number of classes; FSi fine silts, CSi coarse silts; FS, VFS, CS fine, very fine and coarse sands, respectively; FC fine clays; FG, CG fine gravels and coarse gravels); from top to bottom: CSSC Canadian Soil Survey Committee (1978): 10 particle size ranges