Phosphate in Our Waters

PHOSPHATE IN OUR WATERS Teacher’s Guide

How Much Phosphate is in our Water? HOW MUCH PHOSPHATE IS IN THE WATER?

Table of Contents

PRESENTING THIS UNIT 3

BACKGROUND INFORMATION FOR TEACHERS 15 Short Introduction to Spectrophotometery Using the Vernier Spectrometer Obtaining a Range of Water Samples

SESSION SUMMARIES 22

SESSION 1 23 Measuring Concentrations using Spectrophotometery

SESSION 2 30 Creating a Standard Curve

SESSION 3 36 Measuring Phosphate Concentration in Samples

2 PRESENTING THIS UNIT

Kit material support. Most of the materials you need are provided in the Kit unless otherwise noted in the lesson preparation sections. If you are missing any equipment or have problems with kit materials, please contact Adina Paytan by email at [email protected].

Teaching support (for kit testing). If you have any questions about the technology resources or the curriculum materials (teacher’s guide or student materials), please email Adina Paytan at [email protected].

What You Need and Getting Ready. At the beginning of each session a list with all the materials required and preparation instructions for those materials is given. Most sessions require about 15 minutes of preparation.

Unit Goals. The goals of this unit are to learn how to measure the dissolved phosphate concentrations in a set of samples and discuss the results in context with the P cycle and sources/sinks of P in the environment. The unit consists of three lessons leading towards these goals but could be compacted into only one or two lessons if needed or expanded to more lessons if time permits. Resources for additional material are provided in the Teacher Resources sections.

The unit encourages learning of science in an active manner and emphasizes methods of inquiry and deductive reasoning based on qualitative observations and quantitative data.

Students understand the processes of scientific investigation and design, conduct, communicate about, and evaluate such investigations.

Students know and understand interrelationships among science, technology, and human activity and how they can affect the world.

Skills Observing, Comparing, Inferring, Researching, Graphing, Analyzing Data, Visualizing, Drawing Conclusions, Explaining Analyzing and Evaluating Evidence, Logical Thinking, Recording, Organizing Data, Working Cooperatively, Communicating, Theorizing, Problem Solving

Concepts Pollution, Point and Non-Point Pollution, Eutrophication, Global Cycles, Water Contamination, Human Impact on the Environment, Nutrients, Natural Resources, Sustainability, Ecosystems, Watershed, Dead Zone, Algal Blooms 3 Themes Models and Simulations, Systems and Interactions, Patterns of Change, Scale, Stability, Diversity and Unity

Mathematics Standards Number, Measurement, Pattern, Statistics, Logic and Language

Nature of Science and Mathematics Scientific Community, Cooperative Efforts, Creativity and Constraints, Interdisciplinary, Real-Life Applications

National Science Standards Addressed

Unifying concepts and processes in science Systems, order, and organization. Evidence, models, and explanation. Change, constancy, and measurement. Evolution and equilibrium. Form and function.

Science as inquiry Understanding of scientific concepts. An appreciation of "how we know" what we know in science. Understanding of the nature of science. Skills necessary to become independent inquirers about the natural world. The dispositions to use the skills, abilities, and attitudes associated with science.

Environmental Science Matter and energy transformations Biogeochemical cycles Physical science Properties and changes of properties in matter Chemical reactions

Life science Organisms and environments Populations and ecosystems Interdependence of organisms Matter, energy, and organization in living systems

Earth and space science Geochemical cycles 4 Science and technology Abilities of technological design Understanding about science and technology

Science in personal and social perspectives Personal and community health Populations, resources, and environments Natural resources Environmental quality Natural and human-induced hazards Risks and benefits Science and technology in society Science and technology in local, national, and global challenges

History and nature of science Science as a human endeavor Nature of science Nature of scientific knowledge

Hawai‘i, California and Oregon Science and Math State Standards and Benchmarks Addressed

Hawai‘i Content & Performance Standards (HCPS III):

Science Standard 1: The Scientific Process: Scientific Investigation: Discover, invent, and investigate using the skills necessary to engage in the scientific process.

Grades 6–8 Benchmarks for Science: SC.6.1.1 Formulate a testable hypothesis that can be answered through a controlled experiment. SC.6.1.2 Use appropriate tools, equipment, and techniques to safely collect, display, and analyze data. SC.7.1.2 Explain the importance of replicable trials. SC.7.1.3 Explain the need to revise conclusions and explanations based on new scientific evidence. SC.8.1.1 Determine the link(s) between evidence and the conclusions(s) of an investigation. SC.8.1.2 Communicate the significant components of the experimental design and results of a scientific investigation.

5 Grades 9–12 Benchmarks for Physical Science, Biological Science, Earth and Space Sciences, Environmental Science: SC.PS/BS/ES/ENV/MS/CH/PH/PAH.1.1 Describe how a testable hypothesis may need to be revised to guide a scientific investigation. SC.PS/BS/ES/ENV/MS/CH/PH/PAH.1.2 Design and safely implement an experiment, including the appropriate use of tools and techniques to organize, analyze, and validate data. SC.PS/BS/ES/ENV/MS/CH/PH/PAH.1.3 Defend and support conclusions, explanations, and arguments based on logic, scientific knowledge, and evidence from data. SC.PS/BS/ES/ENV/MS/CH/PH/PAH.1.4 Determine the connection(s) among hypotheses, scientific evidence, and conclusions. SC.BS/ES/ENV/MS/CH/PH/PAH.1.7 Revise, as needed, conclusions and explanations based on new evidence.

Science Standard 2: The Scientific Process: Nature of Science: Understand that science, technology, and society are interrelated.

Grades 6–8 Benchmarks for Science: SC.8.2.1 Describe significant relationships among society, science, and technology and how one impacts the other. SC.8.2.2 Describe how scale and mathematical models can be used to support and explain scientific data.

Grades 9–12 Benchmarks for Science: SC.ES.2.3 Explain the impact of humans on the Earth system. SC.ES.2.4 Describe technologies used to collect information about the universe.

Science Standard 3: Life and Environmental Sciences: Organisms and the Environment: Understand the unity, diversity, and interrelationships of organisms, including their relationship to cycles of matter and energy in the environment.

Grades 6–8 Benchmarks for Science: SC.7.3.2 Explain the interaction and dependence of organisms on one another.

Grades 9–12 Benchmarks for Science: SC.BS.3.1 Describe biogeochemical cycles within ecosystems. SC.PAH.3.1 Illustrate biogeochemical cycles within the Hawaiian ecosystem and describe how abiotic and biotic influences have impacted these cycles. SC.PAH.3.3 Explain how matter and energy flow through living systems and the physical environments (e.g., subalpine, rainforest, montane bogs, dryland and mesic forests, subterranean, freshwater, coastal) found in Hawai‘i. 6 Science Standard 3: Oceanography: Understand the physical features of the ocean and its influences on weather and climate.

Grades 9–12 Benchmarks for Marine Science: SC.MS.3.3 Explain how the ocean participates in the geochemical cycling of elements.

Science Standard 4: Structure and Function in Organisms: Understand the interconnections of living systems.

Grades 9–12 Benchmarks for Environmental Science: SC.ENV.4.3 Explain how ecosystems respond to human activities. SC.ENV.4.6 Describe how the availability of resources (e.g., energy, water, oxygen, minerals) limits the amount of life an environment can support.

Science Standard 4: Ecological Systems: Understand the locations and characteristics of marine ecosystems.

Grades 9–12 Benchmarks for Marine Science: SC.MS.4.5 Explain how chemical factors (e.g., pH, salinity, dissolved O2, nutrients) affect the distribution of life in the ocean. SC.MS.4.6 Describe how physical factors (e.g., light, temperature, pressure, current) define the region/zone in the ocean.

Science Standard 5: Life and Environmental Sciences: Diversity, Genetics and evolution: Understand genetics and biological evolution and their impact on the unity and diversity of organisms.

Grades 6–8 Benchmarks for Science: SC.8.5.1 Describe how changes in the physical environment affect the survival of organisms.

Science Standard 6: Physical, Earth, and Space Sciences: Nature of Matter and Energy: Understand the nature of matter and energy, forms of energy (including waves) and energy transformations, and their significance in understanding the structure of the universe.

Grades 6–8 Benchmarks for Science: SC.6.6.8 Recognize changes that indicate that a chemical reaction has taken place.

7 Science Standard 8: Physical, Earth, and Space Sciences: Earth and Space Science: Understand the Earth and its processes, the solar system, and the universe and its content.

Grades 9–12 Benchmarks for Science: SC.ES.8.1 Describe how elements and water move through solid Earth, the oceans, atmosphere, and living things as part of geochemical cycles.

Math Standard 9: Patterns, Functions, and Algebra: Patterns and Functional Relationships: Understand various types of patterns and functional relationships.

Grades 6–8 Benchmarks for Math: MA.6.9.1 Represent visual and numerical patterns with tables and graphs and generalize the "rule" using words and symbols.

Grades 9–12 Benchmarks for Algebra I: MA.AI.9.2 Compare and contrast the concepts of direct and inverse variation of a relation.

Math Standard 10: Symbolic Representation: Use symbolic forms to represent, model, and analyze mathematical situations.

Grade 8 Benchmarks for Math: MA.8.10.1 Translate among tables, graphs (including graphing technology when available), and equations involving linear relationships. MA.8.10.3 Use tables and graphs to represent and compare linear relationships.

Math Standard 11: Data Analysis, Statistics, and Probability: Fluency with data: Pose questions and collect, organize, and represent data to answer those questions.

Grades 9–12 Benchmarks for Statistics: MA.S.11.1 Develop a hypothesis for an investigation or experiment. MA.S.11.2 Recognize the variables and controls in an experiment or investigation. MA.S.11.3 Select appropriate display for a data set (e.g., frequency table, histogram, line graph, bar graph, stem-and-leaf plot, box-and-whisker plot, scatter plot). MA.S.11.4 Recognize features of representations of data that can produce misleading interpretations. MA.S.11.5 Recognize sampling, randomness, bias, and sampling size in data collection and interpretation. MA.S.11.6 Describe the purpose and function of a variety of data collection methods (e.g., census, sample surveys, experiment, observation). 8 Chemistry Standard 3: Chemistry: Properties of Matter: Understand different states of matter.

SC.CH.3.2 Use the pH scale to characterize acid and base solutions.

Chemistry Standard 5: Chemistry: Chemical Reactions: Understand the nature of chemical interactions and solutions.

SC.CH.5.3 Molar Conversion: convert the mass of a molecular substance to moles. SC.CH.5.7 Conservation of Matter and Stoichiometry: Use laboratory investigations to demonstrate the principle of conservation of mass SC.CH.5.9 Solutions: Calculate the concentration of a solute in terms of molarity, parts per million, grams per liter, and percent composition

Environmental Science Standard 4: Life Science: Understand the interconnections of living systems.

SC.ENV.4.3 Systems and Connections: Explain how ecosystems respond to human activities SC.ENV.4.6 Flow of Matter and Energy: Describe how the availability of resources (e.g., energy, water, oxygen, minerals) limits the amount of life an environment can support

Environmental Science Standard 5: Interdependence of The Environment and Human Societies: Understand the interdependence between environmental systems and human societies.

SC.ENV.5.2 Human Impact: Assess the effect of human actions on an environmental system SC.ENV.5.3 Human Impact: Explain how population growth and natural resource consumption affect global sustainability SC.ENV.5.6 Resource Use: Explain why recycling and conservation of resources are important

Marine Science Standard 6: Interdependence of Humans and the Ocean: Understand the interdependence of humans and the ocean.

SC.MS.6.2 Influence of the Ocean on Human Society: Describe the relationship between the ocean and human cultural development SC.MS.6.3 Influence of the Ocean on Human Society: Evaluate mariculture in terms of use of technology and environmental impact 9 SC.MS.6.4 Human Impacts: Explain how human activities and development lead to marine pollution (e.g., point sources, non-point sources) SC.MS.6.5 Human Impacts: Describe how urbanization has impacted the ocean

Content Standards for California Public Schools

Focus on Earth Sciences Grade 6 – 5a. Students know energy entering ecosystems as sunlight is transferred by producers into chemical energy through photosynthesis and then from organism to organism through food webs.

Investigation and Experimentation Grade 6 – 7a. Develop a hypothesis. Grade 6 – 7c. Construct appropriate graphs from data and develop qualitative statements about the relationships between variables. Grade 6 – 7e. Recognize whether evidence is consistent with a proposed explanation. Grade 7 –7b. Use a variety of print and electronic resources (including the World Wide Web) to collect information and evidence as part of a research project. Grade 7 –7c. Communicate the logical connection among hypotheses, science concepts, tests conducted, data collected, and conclusions drawn from the scientific evidence. Grade 8 - 9a. Plan and conduct a scientific investigation to test a hypothesis. Grade 8 – 9b. Evaluate the accuracy and reproducibility of data. Grade 8 – 9e. Construct appropriate graphs from data and develop quantitative statements about the relationships between variables. Grades 9–12 - 1a. Select and use appropriate tools and technology (such as computer‐ linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data. Grades 9–12 – 1c. Identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions. Grades 9–12 –1d. Formulate explanations by using logic and evidence. Grades 9–12 –1l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science.

Statistics, Data Analysis, and Probability Grade 6 – Standard 2.1. Compare different samples of a population with the data from the entire population and identify a situation in which it makes sense to use a sample. Grade 6 – Standard 2.2. Identify different ways of selecting a sample (e.g., convenience sampling, responses to a survey, random sampling) and which method makes a sample more representative for a population. 10 Grade 6 – Standard 2.5. Identify claims based on statistical data and, in simple cases, evaluate the validity of the claims.

Advanced Placement Probability and Statistics Grades 8–12 – Standard 15.0. Students are familiar with the notions of a statistic of a distribution of values, of the sampling distribution of a statistic, and of the variability of a statistic.

Scientific Inquiry 6.3S.2 Organize and display relevant data, construct an evidence-based explanation of the results of an investigation, and communicate the conclusions. 7.3S.2 Organize, display, and analyze relevant data, construct an evidence-based explanation of the results of an investigation, and communicate the conclusions including possible sources of error. 7.3S.3 Evaluate the validity of scientific explanations and conclusions based on the amount and quality of the evidence cited. 8.3S.2 Organize, display, and analyze relevant data, construct an evidence-based explanation of the results of a scientific investigation, and communicate the conclusions including possible sources of error. Suggest new investigations based on analysis of results. 8.3S.3 Explain how scientific explanations and theories evolve as new information becomes available. H.3S.1 Based on observations and science principles, formulate a question or hypothesis that can be investigated through the collection and analysis of relevant information. H.3S.3 Analyze data and identify uncertainties. Draw a valid conclusion, explain how it is supported by the evidence, and communicate the findings of a scientific investigation.

Engineering Design H.4D.3 Analyze data, identify uncertainties, and display data so that the implications for the solution being tested are clear.

Data Analysis and Algebra 8.2.6. Use sample data to make predictions regarding a population. 8.2.7. Identify claims based on statistical data and evaluate the reasonableness of those claims. 8.2.8. Use data to estimate the likelihood of future events and evaluate the reasonableness of predictions.

11 Biology/Life Sciences Grades 9–12 –6b. Students know how to analyze changes in an ecosystem resulting from changes in climate, human activity, introduction of nonnative species, or changes in population size. Grades 9–12 –6d. Students know how water, carbon, and nitrogen cycle between abiotic resources and organic matter in the ecosystem and how oxygen cycles through photosynthesis and respiration.

Chemistry Grades 9–12 – Standard 6b. Students know how to analyze changes in an ecosystem resulting from changes in climate, human activity, introduction of nonnative species, or changes in population size. Grades 9–12 – Standard 7b. Students know the global carbon cycle: the different physical and chemical forms of carbon in the atmosphere, oceans, biomass, fossil fuels, and the movement of carbon among these reservoirs.

Mathematical Reasoning Grade 7 – Standard 3.1. Evaluate the reasonableness of the solution in the context of the original situation.

Algebra I Grades 8–12 – Standard 24.2. Students identify the hypothesis and conclusion in logical deduction.

State of Oregon Standards by Design:

Scientific Inquiry 6.3S.2 Organize and display relevant data, construct an evidence-based explanation of the results of an investigation, and communicate the conclusions. 8.3S.1 Based on observations and science principles, propose questions or hypotheses that can be examined through scientific investigation. Design and conduct a scientific investigation that uses appropriate tools, techniques, independent and dependent variables, and controls to collect relevant data. 8.2.1 Organize and display data (e.g., histograms, box and whisker plots, scatter plots) to pose and answer questions; and justify the reasonableness of the choice of display. 8.2.3 Interpret and analyze displays of data and descriptive statistics. H.3S.1 Based on observations and science principles, formulate a question or hypothesis that can be investigated through the collection and analysis of relevant information. 12 H.3S.2 Design and conduct a controlled experiment, field study, or other investigation to make systematic observations about the natural world, including the collection of sufficient and appropriate data. Data Analysis and Algebra H.3S.5 Explain how technological problems and advances create a demand for new scientific knowledge and how new knowledge enables the creation of new technologies.

Interaction and Change 7.2E.3 Evaluate natural processes and human activities that affect global environmental change and suggest and evaluate possible solutions to problems. H.2L.1 Explain how energy and chemical elements pass through systems. Describe how chemical elements are combined and recombined in different ways as they cycle through the various levels of organization in biological systems. H.2L.2 Explain how ecosystems change in response to disturbances and interactions. Analyze the relationships among biotic and abiotic factors in ecosystems. H.2E.1 Identify and predict the effect of energy sources, physical forces, and transfer processes that occur in the Earth system. Describe how matter and energy are cycled between system components over time. H.2E.4 Evaluate the impact of human activities on environmental quality and the sustainability of Earth systems. Describe how environmental factors influence resource management.

Algebra 6.3.5 Represent, analyze, and determine relationships and patterns using tables, graphs, words and when possible, symbols. H.2A.4 Fluently convert among representations of linear relationships given in the form of a graph of a line, a table of values, or an equation of a line in slope/intercept and standard form.

Data Analysis and Algebra 8.2.1 Organize and display data (e.g., histograms, box-and-whisker plots, scatter plots) to pose and answer questions; and justify the reasonableness of the choice of display.

Analysis H.1S.3 Compare and draw conclusions about two or more data sets using graphical displays or central tendencies and range.

13 Ocean Literacy Principles The following ocean literacy principles can be addressed through these lessons:

Ocean Literacy Principle 3: The ocean is a major influence on weather and climate. a. The ocean controls weather and climate by dominating the Earth's energy, water and carbon systems. e. The ocean dominates the Earth's carbon cycle. Half the primary productivity on Earth takes place in the sunlit layers of the ocean and the ocean absorbs roughly half of all carbon dioxide added to the atmosphere.

Ocean Literacy Principle 4: The ocean makes Earth habitable. a. Most of the oxygen in the atmosphere originally came from the activities of photosynthetic organisms in the ocean.

Ocean Literacy Principle 5: The ocean supports a great diversity of life and ecosystems. b. Most life in the ocean exists as microbes. Microbes are the most important primary producers in the ocean. Not only are they the most abundant life form in the ocean, they have extremely fast growth rates and life cycles.

Ocean Literacy Principle 6: The ocean and humans are inextricably interconnected. b. From the ocean we get foods, medicines, and mineral and energy resources. In addition, it provides jobs, supports our nation's economy, serves as a highway for transportation of goods and people, and plays a role in national security. c. The ocean is a source of inspiration, recreation, rejuvenation and discovery. It is also an important element in the heritage of many cultures. e. Humans affect the ocean in a variety of ways. Laws, regulations and resource management affect what is taken out and put into the ocean. Human development and activity leads to pollution (point source, non-point source, and noise pollution) and physical modifications (changes to beaches, shores and rivers). In addition, humans have removed most of the large vertebrates from the ocean. g. Everyone is responsible for caring for the ocean. The ocean sustains life on Earth and humans must live in ways that sustain the ocean. Individual and collective actions are needed to effectively manage ocean resources for all.

14 BACKGROUND INFORMATION FOR TEACHERS

Short Introduction to Spectrometry

A spectrometer (also called a spectrophotometer) is an instrument that can determine “how much color” is in a solution; this can be very useful for determining the concentration of a chemical in solution. If the chemical is colored, the spectrometer will detect the amount of color, which is related to the concentration of that chemical. If the chemical of interest is not colored, a dye (or other colored substance that binds to the chemical) may be added to form color in the process.

A spectrometer works by measuring how much light passes through a solution. The solution is placed in a square glass, or plastic, vial (called a cuvette) that is then placed into the spectrometer. Light from the spectrometer is separated into its various wavelengths. A specific wavelength of light is directed through the cuvette and lands on a light detector. The detector “reads” the intensity of that wavelength, registering it as an electronic signal. In other words, the spectrometer directly measures the amount of light that gets through the cuvette and the solution.

When colored solutions are irradiated with white light, they will selectively absorb some wavelengths, but not others. When light is not absorbed, it is transmitted through the solution. The wavelength (or wavelengths) that a substance absorbs can be determined by exposing the solution to monochromatic light of different wavelengths and recording the light transmitted. The higher the absorbance of light by a solution, the lower the transmittance! Transmittance is inversely related to absorbance. The amount of light that is absorbed by the solution depends on how many colored molecules are in the path of the light.

Beer’s law describes the relationship between absorbance and concentration of a chemical in solution. Absorbance describes the amount of light that is absorbed by a solution (mathematically, absorbance is the negative log of transmittance). As mentioned above, more concentrated solutions will absorb more light because the light must pass through more molecules when passing through the solution.

Figure 1 illustrates an absorption spectrum - the absorbance of a colored solution at different wavelengths. This figure shows the spectra for the solution at 4 different concentrations; 1 milligram of chemical in one kilogram of water (also called ppm), 2ppm, 3ppm and 4ppm. Notice that as the concentration of the solution increases, so does the absorbance (e.g. more absorption for the 4ppm solution compared to the 1ppm solution). Note also that the absorbance is different for any given wavelength. The wavelength at which absorbance is highest is the 15 wavelength to which the solution is most sensitive to concentration changes. This wavelength is called λmax. For the chemical (iron phenanthroline) shown in Figure 1, λmax is 510 nm.

Mathematically, Beer’s Law states that the concentration (C) of a solution and the absorbance (A) of light (of a certain wavelength) by that solution are directly related.

Figure 1: Absorption spectrum for an iron Beer’s law: A = k • C phenanthroline solution. (Absorbance = constant • Concentration)

The constant (k) will depend on such things as 1) the nature of the solvent, 2) the wavelength of light used, and 3) the distance the light must travel through the cuvette. These variables are kept constant for a certain experiment so that the concentration can be determined from the measured absorbance.

Figure 2 illustrates Beer’s law. The concentration of a chemical in solution is directly proportional to the absorbance of that solution (at a particular wavelength). As the Figure 2: A graph of absorbance vs. concentration increases, so does the concentration illustrating Beer’s law. absorbance. The line shown in the figure represents what is known as a standard curve.

Using the Vernier Spectrometer

In this unit you will use a Vernier spectrometer. You have to take time and learn how the instrument works to be able to use it effectively in class. Please read the manual and instructions and practice all activities ahead of time. While the use of this instrument is quite easy, it is not intuitive and it does require some familiarity. Below is a short tutorial of tasks you will need to be familiar with before performing the lesson plans.

16 1. Install the Logger Pro 3 software. This software needs to be loaded onto a computer. You will find the install disc (and the password, if needed) in the front pocket of the binder for this unit. Insert the appropriate install disc into the computer’s CD drive and then double click on the install icon to begin installation. Follow the instructions as they appear. Once the installation is complete, locate the Logger Pro 3 application icon. The icon depicts a caliper tool on top of a graph drawn on a piece of graph paper. a. Double click on the Logger Pro 3 icon to open the application. An untitled window will open that displays an empty data set table with x and y columns and an associated blank graph.

2. Connect the spectrometer to the laptop computer provided. a. Locate the spectrometer and the USB cable provided in the kit. Connect the USB cable into the spectrometer and into the powered USB port of the laptop. This automatically turns on the spectrometer. b. Allow the spectrometer to warm up for a few minutes.

3. Calibrate the spectrometer (for measuring absorbance or % transmittance). Have a supply of clean, pure water available (deionized water if possible). a. Open the Logger Pro 3 software and connect the spectrometer to the computer (steps 1 and 2 above). b. Choose Calibrate > Spectrometer from the Experiment menu in Logger Pro 3. Prepare a blank cuvette for your experiment by filling a cuvette ~3/4 full with water. c. The calibration dialog box will display the message: “Waiting …seconds for the lamp to warm up.” The minimum warm up time is one minute. You may wait longer than one minute for added stability. d. Insert the blank cuvette into the cuvette holder. e. When the warm up time is complete, click Finish Calibration. Wait for a few seconds for the calibration to be completed, and then click OK.

4. Measure the absorbance (or % transmittance) spectrum of a sample. Have a colored sample solution prepared and ready to measure. Session 1 includes instructions for making a colored solution using red or blue food coloring dye. a. Calibrate the spectrometer (step 3 above). b. Add your colored sample to a cuvette (~3/4 full) and insert the sample cuvette into the cuvette holder. c. Click Collect. After a few seconds click Stop.

17 The spectrometer creates an absorbance spectrum data set and a graph from that data set. Absorbance is on the y-axis and Wavelength is on the x-axis. The absorbance peak(s) for your sample is (are) easily seen on the graph. Note - absorbance values are used to conduct a Beer’s Law experiment (see below), but % transmittance is what the instrument actually measures. Absorbance is then calculated from the % transmittance data. You can switch the units shown in the graph and data set from absorbance to % transmittance by doing the following.

d. From the Experiment menu, choose Change Units  Spectrometer 1 %Transmittance. The new data set and graph will display % transmittance rather than absorbance.

5. Conduct a Beer’s law experiment. To perform a Beer’s law experiment you must first have a set of standard solutions prepared and ready to measure. Session 2 includes instructions for making a set of standards using the phosphate solutions and Hach reagent packets that come with the kit. a. Measure an absorbance spectrum for one of your standard samples (step 4 above). b. Once the absorption spectrum is obtained, click on the Configure Spectrometer button (this button is located on the tool bar and appears as a graph with the visible spectrum under the curve). c. A dialog box will appear. Choose Abs vs. Concentration. The wavelength of the maximum absorbance will be automatically selected ( max). Check that this is the correct wavelength at which the maximum absorption occurs for your sample. If it is, click OK to continue. If not, click Clear and select a wavelength on the graph or in the list of wavelengths. Finally, type in the appropriate unit for concentration (i.e. mole/L). Click OK. d. Place your first standard solution into the spectrometer. Click Collect and then click Keep. Enter the concentration of the sample and click OK. Repeat this step for the remaining standard samples. After you have tested the final standard, click Stop to end the data collection. e. Click the Linear Fit button in the tool bar (appears as a red line through a blue curve), to see the function for the standard solutions. f. Place an unknown sample of solution in the spectrometer. Choose Interpolation Calculator from the Analyze menu. A helper box will appear, displaying the absorbance and concentration of the unknown. Click OK. g. You can save the data for your set of standards and linear fit function by choosing Save As... and saving your file to a location of your choice.

18 Note – the kit contains only one spectrometer, so all of the analyses have to be done as demonstration by the teacher, or by groups taking turns using the instrument. This is not always easy to accomplish, so alternate phosphate- measuring tools (less accurate but still educational) might be considered. One alternative is buying color wheels from HACH (www.hach.com, phosphate color disc – cat #24898-00, and color comparators – cat #1732-00, a black box that holds the samples and the color wheel). Students can use these color wheels to determine the dissolved phosphate concentration in their standards and samples. Another option is buying phosphate test kits for aquariums at a pet store (www.thatpetplace.com, phosphate test kit by Aquarium Pharmaceuticals, cat # GO:199700) and using these test kits for the phosphate determinations.

Obtaining, or Preparing, a Range of Water Samples

The highlight of this section is the analysis of water samples for dissolved phosphate concentrations (in Section 3). There are several options for acquiring samples for analysis depending on your time constraints and location. Ideally, samples are obtained during a field trip to different water sources and water samples are collected (see information for sample collection below). Another option is to ask students to bring a water sample to class, or divide the class into groups and have each group bring a few different water samples. It is important to give students sampling instructions and make sure they describe the location and time of sample collection in a field note book or work sheet. Give examples of water types (runoff from your garden, local pond or river, sewage treatment, laundry or dish washer water, aquarium water, etc). Encourage them to obtain a diverse range of water samples with different dissolved phosphate concentrations (give extra credit, or an award, to the group that assembles the largest range of concentrations). If you are not able to collect samples during a field trip, or have students collect samples as part of this assignment, then you can collect natural samples yourself from a range of locations and concentrations and bring them to class. Label them so the class can see where they came from (see picture 1). It is also possible to prepare a “representative” set of samples (mock samples) by adding different amounts of phosphate (potassium phosphate, reagent grade, 100g, catalog # 884298, Carolina Biological Supply 800.334.5551) to water. In the latter case, make a range of concentrations that are typical for various environmental water samples. For example, “mock samples” could be surface water from the open ocean, surface water from a coastal site, deep Atlantic water, deep water from the Southern Ocean, and deep Pacific water. Alternatively, a set of samples could be tap water with different amounts of fertilizers, manure, dishwasher solution, water softener, etc added. Make sure to test these samples before using them, particularly if you are using a detergent to demonstrate anthropogenic phosphate

19 sources, because many detergents do not have high phosphate concentrations. It is important that the samples represent a broad range of concentrations. You can also bring phosphate containing products like fertilizer or dish washer soap to class and add them to water in class to illustrate the leaching of phosphate from these products to water.

Labeled water sample bottles for phosphate analysis

In total, you would need at least 5 different samples - ideally some of these samples will be collected in duplicates. For any of the approaches described above, the collection sites should represent a wide range of water samples with different dissolved phosphate concentrations. To ensure that the activity will be successful, in addition to any “unknown” samples the teacher must select sites/samples that are known to represent a range of concentrations (e.g. determine these concentrations in advance) and all concentrations should be determined by the teacher in advance to ensure that they will fulfill the lesson goals.

Information for Sample Collection In the field, locate and mark the collection site on a local map (use GPS if available) and list the collection information in a field notebook (date, time, location, water depth, site description, sample number, etc.). Use gloves when collecting the water sample. Collect water using a bucket or a water-sampling bottle (Van Dorn, Niskin, etc.) or using a syringe. Draw water from your collection 20 bottle into the syringe and rinse three times; refill, attach syringe filter unit (0.45 m) onto the syringe, and squirt ~ 10 ml to rinse the filter. Rinse the pre-labeled, acid-washed sample bottle three times and fill two-thirds full with filtered sample water. Rinsing the bottles and syringe lowers risk of contamination. Take duplicate samples from each water source if possible. Store samples in a cooler while in the field; analyze immediately upon return to the class or freeze until analysis. If you can collect other data, such as temperature of the water and salinity, do so; specifically, try to get some measure of the biomass in the water (e.g., chlorophyll) or visually estimate the biomass content (is the water green, blue, clear etc.). The later is important in cases where the P was already consumed and converted to biomass.

21 SESSION SUMMARIES

Session 1 Measuring Concentrations using Spectrometry Students are introduced to the principles of spectrometry and Beer’s Law using solutions of different colors and different concentrations.

Session 2 Creating a Standard Curve Students learn the value and practical application of performing a standard curve and create a standard curve for phosphate.

Session 3 Measuring Phosphate in Unknown Samples Students measure phosphate concentrations in water samples provided by the teacher and/or collected in the field and discuss the results.

22 SESSION 1

Measuring Concentrations Using Spectrometry

Students begin by reviewing the basic conceptual background of spectrometry and Beer’s law. Then students are shown how the spectrometer is used to obtain absorbance data from two different colored solutions. Students examine the absorbance of solutions made with different amounts of the same color (different concentration). Students discuss the data to refine their understanding of spectrometry and Beer’s law.

Student learning is focused on the following key concepts:

 Spectrometers are instruments used to measure light absorbance by an aqueous solution  Absorbance of light by a solution varies with wavelength

Students also learn that:

 The concentration (C) of a chemical in solution and the absorbance (A) of certain wavelengths of light by that solution are directly proportional (Beer’s Law).

Beer’s law: A = k • C (Absorbance = Constant • Concentration)

Measuring Concentrations by Spectrophotometry Estimated Time Introducing the Unit 5 minutes Spectrometry 10 minutes Using the Spectrometer with Colored Water 10 minutes Analyzing the Data Collected 10 minutes Beer’s Law Experiment and Discussion 15 minutes Total 50 minutes

23 WHAT YOU NEED

For the class: * PowerPoint for Session 1 * 5, 12ml plastic tubes * Projection system for slides* * Red and Blue stock solutions* * Poster size paper (optional)* * Disposable bulb pipettes * Computer * 50ml Falcon tube * Vernier spectrometer and cable * Cuvettes * Red and blue food coloring * DI (or clean) water

For each group of students: * Copy of red dye spectrum data * Copy of blue dye spectrum data

For each student: * Copy of Intro to Spectrometry handout * Copy of absorbance data sheet

* Provided by the teacher

GETTING READY Before the day of the session:

1. Set up projection system. Set up audiovisual equipment so you can project the computer screen during the session. Spend a few minutes familiarizing yourself with the slides and with your computer.

2. Write out key concepts. Using a marker and a sheet of paper, write out the following key concepts in large, bold letters. Set the key concepts aside to post on the wall later in the session:

 Spectrometers are instruments used to measure light absorbance by an aqueous solution  Absorbance of light by a solution varies with wavelength

3. Make the red and blue stock solutions. For each of the 2 food colors, add 0.25ml of food color to 25ml of water held in a 50ml Falcon tube. Label as stock solution (0.1%).

4. Make the set of red standards. From the 0.1% stock solution, take 2ml and add it to a tube to make a final volume of 10ml. This is your 0.02% standard. From the 0.1% stock solution, take 1ml and add it to a tube to make a final volume of 10ml. This is your 0.01% standard. From the 0.1% stock solution, 24 take 0.5ml and add it to a tube to make a final volume of 10ml. This is your 0.005% standard.

Introducing the Unit (5 minutes) 1. Project unit title slide and introduce the unit. Tell students that they are beginning a new unit about measuring phosphate in the aquatic environment. Say, “This is an image of a lake that has a high concentration of phosphate. The green layer at the surface is extensive algal growth”.

2. Introduce Turn and Talk routine. Explain that Turn and Talk is a partner discussion routine that you will be using to quickly start a discussion about an assigned topic. In this case, student pairs will discuss the question, “What have you heard about increased phosphate levels in lakes and ponds?” Make sure each student has a partner and give pairs a minute or two to discuss what they’ve heard about phosphate in aquatic environments.

3. Share ideas with whole class. Regain the class’s attention and have a few students share what they’ve heard about the effects of increased phosphate levels in waters. Do not correct or confirm their statements at this point. Tell them that these are all things they’ve heard. During the unit they’ll find out more about increased phosphate levels and investigate which of these ideas are more or less accurate. Tell them that first they will learn how to measure phosphate in water samples.

Spectrophotometry (10 minutes) 1. Preview reading. Ask, “How do you think the amount of phosphate or phosphate concentrations in water is measured?” Accept students’ ideas. After hearing a few answers, tell them they will learn one technique that scientists use to measure phosphate concentrations in water. This technique uses a spectrometer to measure the amount of light absorbed by a solution.

2. Allow time for reading. Hand out copies of Intro to Spectrometry and have students read. (If you are concerned about time, or are unsure of your students’ reading abilities, you may summarize the key points of this handout for your class.)

3. Discuss reading. When students have finished reading ask, “How can a spectrophotometer be used to measure phosphate concentration?” and “What is Beer’s law and why is it useful for measuring phosphate or other chemicals in solution using the spectrometer?” Give students a few minutes to share how spectrometers work. If there is time, give students 25 another few minutes to share what they thought of the reading.

Using the Spectrometer with Colored Water (10 minutes)

1. Project computer screen. Use a computer projector to display the computer screen of the spectrometer so the students can see what you are doing. Explain how the instrument works (see Background Information for Teachers).

2. Demonstrate using the spectrometer. Tell the students you will use two different colored solutions (water and colored dye) to show how the instrument works and how you can create an absorbance spectrum for a colored aqueous sample.

3. Calibrate the spectrometer. Tell the students that first you must have the spectrometer record the beam of light passing through water (or a solvent similar to that of your sample) without the colored molecules. This is called the blank and the signal is used as a baseline to indicate to the spectrophotometer what will be recorded for “no color”. This step is called instrument calibration. Perform the calibration following the Logger Pro 3 instructions (see Background Information for Teachers – Using the Vernier Spectrometer p. 16). Explain that the blank will be subtracted from the samples.

4. Measure the absorbance of the red solution. Fill a cuvette (~ ¾ full) with the red stock solution. Place it into the spectrometer and analyze following the Logger Pro 3 instructions (see Background Information for Teachers – Using the Vernier Spectrometer p. 16). Explain to your students what is happening (scanning selective wavelengths, recording % transmittance, and calculating absorbance). Using the computer and projector, show the class the Logger Pro window display and how to adjust the graph axes to more easily visualize the wavelength of maximum absorbance. Also show the table of data points that are used to create the absorbance curve. Save the red spectrum data by choosing Save As... from the Logger Pro 3 menu.

5. Measure the absorbance of the blue solution. Fill a cuvette (~ ¾ full) with the blue stock solution. Place it into the spectrometer and analyze as described in step 4. Again, explain to the class what is happening and then save the spectrum.

26 Analyzing the Data Collected (10 minutes)

1. Project computer screen. Use a computer projector to display the absorbance graph of the colored solutions. Tell the students that you will discuss the data from the spectrometer to help clarify the concepts of the lesson.

2. Hand out the absorbance data sheets. Have students form pairs, or small groups of 3. Explain that working in groups requires patience, cooperation, and understanding. Learning how to work effectively in a group is an important skill in any activity. Provide a copy of the red and blue spectrum data sheets to each student group. Tell them they will use the data table, and the graphs displaying the data, to learn more about absorbance.

3. Analyze the absorbance data. From the displayed data, show the class how you can find the maximum and minimum absorbance for one of the solutions measured. Note that these absorbance values are associated with a particular wavelength, or range of wavelengths. Ask each student group “What wavelength (color) shows the highest and the lowest absorbance for each solution?” have them use the handouts to identify specific wavelengths with the highest and lowest absorbance. Have the student groups record this information on a separate sheet of paper. Then ask them to “Briefly explain how these absorbance wavelengths relate to the color of each solution”.

4. Post the key concepts. Read aloud the key concepts for this session and post them on the wall:

KEY CONCEPTS

Spectrometers are instruments used to measure light absorbance by an aqueous solution.

Absorbance of light by a solution varies with wavelength

27 Beer’s Law Experiment and Discussion (15 minutes)

1. Project computer screen. Use a computer projector to display the computer screen of the spectrometer so the students can see what you are doing. Explain that now we will see what happens to the absorbance at one specific wavelength when we add more or less color.

2. Perform a Beer’s law experiment with colored water. Show students that you have prepared a set of standards with different concentrations of the red stock solution (0.1% red food color solution). One is the 0.1% stock solution, another is a 0.02 % solution, one is a 0.01% solution, and a final one is a 0.005% solution. Using these standards, conduct a Beer’s Law experiment following the Logger Pro 3 instructions (see Background Information for Teachers – Using the Vernier Spectrometer p. 16). Explain to the students that you have already selected the maximum absorbance wavelength from the red dye absorbance spectrum (496 nm), so you do not need to recalibrate the instrument or obtain a new absorbance spectrum. Explain to the students that the absorbance (at 496 nm) for each of the standards is plotted on the y-axis and the concentration (% of red dye) on the x-axis. As you perform this experiment explain each step to the class.

3. Analyze the Beer’s law experiment data. Perform a linear fit to the data and show them that the result is a straight line that fits the data with a high correlation (correlation = 0.9996 is typical). This straight line, your standard curve, illustrates the direct relationship between the absorbance of light by the red dye solution and the concentration of that solution. Remember, the higher the red dye concentration the more light (at that wavelength) will be absorbed. Explain that this relationship is summarized in Beer’s law; A = k x C.

4. Test an unknown solution. Tell the students that the standard curve can be used to determine the concentration of an unknown sample. Have a student create an ‘unknown’ solution by adding a specific volume of the red stock solution to a specific volume of water while your back is turned, so only the class knows how much was added. Remind them that the final concentration of the ‘unknown’ they make should be in the same range used for making the standard curve. Follow the instructions for measuring absorbance of the unknown solution (see Background Information for Teachers – Using the Vernier Spectrometer p. 16). Tell the students that by using Beer’s law one can determine the concentration of an ‘unknown’ sample by determining where it falls on the standard curve.

28 5. Post the key concept. Post the key concept sheet for this session on the wall (or write the concepts in large letters on a board) and read it aloud for students to learn.

KEY CONCEPT

Beer’s Law A = k * C

The concentration (C) of a chemical in solution and the absorbance (A) of certain wavelengths of light by that solution are directly proportional.

6. Preview the next session. Tell students that in the next session they’ll learn more about phosphate in our water by preparing a standard curve for determining the concentration of phosphate in a solution.

29 SESSION 2

Creating a Standard Curve

Students are introduced to the concept of a standard curve. Students are instructed in how to make the various dilutions for their standard curve using the provided phosphate stock solution. Students add chemical reagents to their standards and then use a spectrometer to measure the absorbance of those standards. Students then analyze the standard curve calculated by the Logger Pro 3 software based on the measurements from the spectrometer. Student learning is focused on the following key concepts:

 A standard curve is calculated from a set of standard samples of known concentration  Standard curves are used to determine concentrations of unknown samples

Students also learn about:

 Reproducibility  Detection limits  Determining analytical errors

Creating a Standard Curve Estimated Time Introducing the Concept of a Standard Curve 10 minutes Preparing Phosphate Standards 15 minutes Conduct a Beer’s Law Experiment 15 minutes Examining the Standard Curve 10 minutes Total 50 minutes

30 WHAT YOU NEED

For the class: * PowerPoint for Session 2 * Cuvettes * Projection system for slides * DI (or clean) water * Poster size paper (optional) * Waste beaker * Computer * Kimwipe tissues * Vernier spectrometer and cable

For each group of students: * 12 ml Falcon tubes with caps * Safety gloves * Labeling pen for tubes * tube rack * 10ml disposable pipettes and pipette pump * Phosphate stock solutions * Hach reagent packets

For each student: * Copy of A Standard Curve for Spectrometry handout * Copy of Making Standard Solutions handout * provided by the teacher

2. Write out key concepts. Using a marker and a sheet of paper, write out the following key concepts in large, bold letters. Set it aside to post on the wall later in the session. Alternatively, write the key concepts on the board at the end of the session.

 A standard curve is calculated from a set of standard samples of known concentration  Standard curves are used to determine concentrations of unknown samples

31 Introducing the Concept of a Standard Curve (10 minutes)

1. Project the title slide. Tell students that every instrument used to determine concentrations of an unknown needs to be calibrated using a standard curve derived from testing known concentrations on that instrument. Tell them that the picture shows what a standard curve, using a spectrometer to measure absorbance, would look like.

2. Review from previous lesson. Ask, “What is Beer’s law and why is it important in spectrometry?” and “How can we use Beer’s law for measuring dissolved phosphate concentration in water?” Accept answers. Tell students that they will be reading an explanation of the steps needed to determine the concentration of the colored solute molecules in a solution using Beer’s law.

3. Read about standard curves. Hand out copies of A Standard Curve for Spectrometry and have students read. (If you are concerned about time, or your students’ reading abilities, you may summarize the key points of this handout for your class.)

4. Discuss reading. When students have finished reading ask, “How can Beer’s law be used for measuring dissolved phosphate in water?” Give students a few minutes to share their ideas. If there is time, give students another few minutes to share what they thought of the reading.

Preparing the Phosphate Standards (15 minutes)

1. Divide the class into groups. Divide the class into groups of 3-4 students. Each group will work together as a team to prepare their own set of phosphate standards. Explain that working in groups requires patience, cooperation, and understanding. Learning how to work effectively in a group is an important skill in any activity.

2. Read how to prepare the standard solutions. Pass out the Standard Solutions Instruction sheet and review it. Each group will prepare a series of standards following the instructions provided. Tell students they will need to label 2 sets of 12ml Falcon tubes used for making the solutions A-H. Students will add the appropriate volumes of phosphate stock solution (provided) and water, as listed in the instructions, into the appropriate tubes. You may want to demonstrate the correct technique for using the pipette pump if the students have not used them before. Emphasize the need for being careful and precise.

32 3. Create the dilutions of the stock solution. Have each student group follow the instructions using the Standard Solutions handout to create their own two sets of standards: A1-H1, and A2-H2. (Alternatively, you may want your whole class to make the 2 sets. For example if you have 4 groups, have one do A1-D1, another does E1-H1, another does A2-D2, etc.) Show the class how to calculate the final concentration of phosphate in each of their standards (use the equation described in the handout). Have students complete the standard curve table in the handout.

4. Add the reagent to each standard. Students will add one reagent packet pack to each 10 ml standard solution. Let your students know that the reagent chemicals can irritate throat, skin and eyes if exposed. So have one student in each group be responsible for adding the Hach reagent packets to the Falcon tubes. This person should wear safety glasses and safety gloves. Have that student carefully cut and slowly pour the contents of one reagent pillow pack into each tube sample. Pour the reagent slowly and carefully to avoid spilling reagent or creating dust. Then cap the tube, and invert several times to mix.

5. Wait for the color to develop. Allow color to develop for at least 2 minutes. Following the 2-minute time, the sample is ready to read. Tell students that the reagents in the pillow pack react chemically with the phosphate in the standards and a colored molecule is produced. It is this molecule that will be measured by the spectrometer.

Conduct a Beer’s Law Experiment (15 minutes)

1. Prepare the spectrometer and create an absorbance spectrum. While the students are preparing the solutions for the standard curve, the instructor should turn on the spectrometer to let it warm up (at least 10 minutes prior to running samples). Follow the instructions for “Conducting a Beer’s Law experiment”. This includes calibrating the instrument using a water solution (blank) and performing an absorption spectrum using one of the prepared standards. Have cuvettes, tissues, clean water, and a large waste beaker available near the spectrometer for students to use.

2. Measure the absorbance of each standard. Remind groups to bring their standards and the final concentrations of each standard with them when they analyze their solutions. Students carefully pour the colored solution from the 12ml Falcon tube into a clean cuvette (~ ¾ full). Then wipe the outside of the cuvette with a clean tissue, and place the cuvette into the spectrometer. Once a reading is obtained, students then input the dissolved phosphate concentration

33 of that standard into the computer. Once this is done, the cuvette can be removed and another cuvette is placed into the spectrometer. Depending on the time available and the number of groups that you have, you can either (1) have each group come to the spectrometer and measure all of their standards, or (2) ask each group to bring one or two of their standards to measure (from A through H). A combination where each group uses a HACH color wheel (not included in the kit) to create a curve using all their standards and analyzing a few standards from each group for a class standard curve using the spectrometer is also an option.

3. Create the standard curve. The Logger Pro software uses the data from the graph of absorbance versus concentration and calculates a “best fit” line through the data points (see Background Information for Teachers – Using the Vernier Spectrometer p. 16). This line is your standard curve showing the direct relationship between absorbance and phosphate concentration. If each group created their own curve, save each curve and either print or save it on a USB stick and provide each group with their respective curve. If you have one curve, make it available for all groups.

4. Collect and dispose of waste. Have students pour their colored sample solutions into a large bucket. Dilute with excess water making the solution less than 5% (i.e. if you have 100ml of waste, dilute to a final volume of 2L). Adjust to a pH between 6 and 9 with an alkali, such as soda ash or sodium bicarbonate. Turn on the cold water faucet and slowly pour the reacted material down the drain. Allow cold water to run for 5 minutes to completely flush the system. Empty Falcon tubes: Rinse three times with water and dispose of as normal trash. NOTICE: These disposal guidelines are based on federal regulations and may be superseded by more stringent state or local requirements. Please consult your local environmental regulators for more information. Dispose the liquid waste properly.

Examining the Standard Curve (10 minutes)

1. Project computer display. Display the standard curve line to the class using a computer projector. Tell the students that this line is their standard curve. Explain that it is graph of the data from their standards and it represents the relationship between the measured absorbance of the colored phosphate- molybdate compound at the different concentrations of that compound. Since the standard curve is a straight line thorough these concentrations, this demonstrates the validity of Beer’s law for this concentration range. Beer’s law predicts that there will be a directly proportional linear relationship

34 between absorbance and concentration of a colored compound in solution.

2. Discuss detection limits, reproducibility, and errors. Using the plot of absorbance and concentration, show the students that the data points for each concentration are some times not exactly on the line. This variation represents some error involved in our standard preparation and our measurements. Some error comes from small inaccuracies in our measurements when we make the standards, or in the quantity of reagents added to our standards, or in the instrument measurement of absorbance. Next compare the data for the same standard prepared by different groups. Note that each group’s curve is slightly different. Explain that errors are an expected part of every measurement, and that scientists aim to reduce errors, but they can never eliminate them. The best that can be done is to use sample replicates to minimize the potential error associated with a single sample. When you make your samples, be sure to make a set of 3 replicates for each concentration. Logger Pro software will use the replicates to derive a more accurate data point. Explain to students that the standard curve has a linear range. We can only be confident when a sample tested is within the concentration range of our standard curve. For our standard curve of absorbance versus phosphate concentration, this range is between 0-5 uM. The spectrometer also has a detection limit, so if samples are too dilute the measurement will not be reliable.

3. Post the key concepts. Post the key concepts sheet for this session on the wall (or write the concepts in large letters on a board) and read it aloud for students to learn.

KEY CONCEPTS

4. A standard curve is calculated from a set of Previ standard samples of known concentration ew the Standard curves are used to determine the next concentrations of unknown samples

session. Tell students that they’ll learn how to use their standard curve to determine the concentration of phosphate in water samples collected from the environment in the next session.

35 SESSION 3

Measuring Phosphate in Unknown Samples

Students measure phosphate concentrations in a set of samples using the spectrometer. Students collect absorbance data and, using the standard curve produced in the previous lesson, convert the absorbance measured in their samples to phosphate concentrations. Students then draw conclusions about the phosphate content in the samples collected at various sites. Student learning is focused on the following key concepts:

 Phosphate levels in water samples can be measured using a spectrometer and a standard curve

Students also learn:

 Phosphate levels in aquatic environments can vary widely and depend on sources and sinks for that particular environment

Measuring Phosphate in Unknown Samples Estimated Time Preparing Samples 10 minutes Determining Phosphate in Unknowns 15 minutes Analyzing Unknown Sample Data 10 minutes Drawing Conclusions about Phosphate in Water 15 minutes Total 50 minutes

36 WHAT YOU NEED For the class: * PowerPoint slide for Session 3 * Cuvettes * Projection system for slides* * DI (or clean) water * Poster size paper (optional)* * Waste bucket * Computer * Kimwipe tissues * Vernier spectrometer and cable * 5 (or more) Water sample bottles*

For each group of students: * 3 (or more) 12ml Falcon tubes * tube rack * Safety glasses and gloves * Labeling pen for tubes * 10ml pipettes and pipette pump * Phosphate stock solution * Hach reagent pillows

For each student: * Copy of water samples worksheet * provided by the teacher

2. Write out key concepts. Using a marker and a sheet of paper, write out the following key concepts in large, bold letters. Set the key concept sheet aside to post on the wall later in the session. Alternatively, write out the key concepts on the board at the end of the session.

 Phosphate levels in water samples can be measured using a spectrometer and a standard curve  Phosphate levels in aquatic environments can vary widely and depend on sources and sinks for that particular environment

3. Have water samples ready. If you are providing water samples for your class, be sure to have these labeled and ready. For more information about collecting water samples, or having your students bring samples into the classroom, go to the section ‘Background Information for Teachers’ and read the subsection, ‘Obtaining, or Preparing, a Range of Water Samples’.

Preparing Samples (10 minutes)

1. Distribute water samples to groups. Divide the class into groups of 3-4 students. Explain that working in groups requires patience, cooperation, and understanding. Learning how to work effectively in a group is an important skill in any activity. Labeled water sample bottles, either brought in by students or provided by the instructor (see Obtaining, or Preparing, a Range of Water Samples p. 19) are distributed to each group. Information about what was collected, who collected it, where it was collected, and when it was collected should be recorded and briefly discussed. If any samples are cloudy, or have any particulate material in the sample, the sample should be filtered. You may use a coffee filter and cone to do this. A blank should be run as well, consisting of DI water passed through the same filter apparatus.

2. Add the reagent to each labeled water sample. Have each group subdivide their sample(s) into triplicates by putting 10 ml of each sample into three separate Falcon tubes. Label the Falcon tubes in some way to designate the triplicates (i.e. Smith pond A, Smith pond B, Smith pond C). Let your students know that the reagent chemicals can irritate throat, skin and eyes if exposed. So have one student in each group be responsible for adding the Hach reagent packets to the Falcon tubes. This person should wear safety glasses and safety gloves. Have that student carefully cut and slowly pour the contents of one reagent pillow pack into each Falcon tube sample. Pour the reagent slowly and carefully to avoid spilling reagent or creating dust. Then cap the tube, and invert several times to mix.

3. Wait for the color to develop. Allow the color to develop for at least 2 minutes. Following the 2-minute time, the sample is ready to read. Remind students that the reagents in the pillow pack react chemically with the phosphate in the standards and a colored molecule is produced. It is this molecule that will be measured by the spectrometer.

Determining Phosphate in Water Samples (15 minutes)

1. Prepare the spectrometer. While the students are preparing the water samples for analysis, the instructor should turn on the spectrometer to let it warm up (at least 10 minutes prior to running samples). Also run a calibration blank with distilled water.

2. Open the phosphate standard curve file. While the spectrometer is warming up, run the Logger Pro software and open the phosphate standard curve file.

38 This will allow you to insert an unknown sample into the spectrometer and the software will compare the absorbance of the unknown to the standard curve. The phosphate concentration for the unknown will then be given.

3. Measure absorbance of each water sample. Have one group at a time bring their water sample(s) to the spectrometer. Have them arrange their samples in order from lightest color to darkest color. Run the lighter samples first. For each sample, the colored solution is poured carefully into a cuvette until it is ¾ full. A kimwipe is used to clean the cuvette sides, and then it is placed in the cuvette holder of the spectrometer. Let the reading stabilize for ~ 15 sec and then record the absorbance value and concentration value on the unknown samples worksheet.

4. Collect and dispose of waste. Have students pour their colored water sample solutions into a large bucket. Dilute with excess water making the solution less than 5% (i.e. if you have 100ml of waste, dilute to a final volume of 2L). Adjust to a pH between 6 and 9 with an alkali, such as soda ash or sodium bicarbonate. Turn on the cold water faucet and slowly pour the reacted material down the drain. Allow cold water to run for 5 minutes to completely flush the system. Empty Falcon tubes: Rinse three times with water and dispose of as normal trash. NOTICE: These disposal guidelines are based on federal regulations and may be superseded by more stringent state or local requirements. Please consult your local environmental regulators for more information. Dispose the liquid waste properly.

5. Post the key concept. Put the key concept sheet for this session on the wall (or write it in large letters on a board) and read it aloud for students to learn.

KEY CONCEPT

Phosphate levels in water samples can be measured using a spectrometer and a standard curve

Analyzing Water Sample Data (10 minutes)

1. Chart the data. Have the students create a histogram, or bar graph, of the phosphate concentrations for their water samples. Draw a sample histogram

39 on the board with phosphate concentration on the y-axis and sample location/type on the x-axis. Have the students draw their samples histogram using the “graph paper” table on the water sample worksheet using the data from their water sample data table.

2. Discuss detection limits, errors, and reproducibility. Tell the students that the variation between replicates represents the error involved in our measurements. Some error comes from small inaccuracies in our measurements when pouring into the falcon tubes, or in the quantity of reagents added to our standards, or in the instrument measurement of absorbance. Remind students that errors are an expected part of every measurement, and that scientists aim to reduce errors, but they can never eliminate them. The best that can be done is to use sample replicates to minimize the potential error associated with a single sample. Explain to students that the instrument has a detection limit below which it cannot differentiate between the sample and the noise (blank). In addition it is important to recognize that we can only be confidant when a sample tested is within the concentration range of our standard curve. For our standard curve of absorbance versus phosphate concentration, this range is between 0-5 uM. If any sample was above this range, the data point is uncertain. Let students calculate the averages and standard deviation for replicate samples and if they know how to the error in the slope of the calibration curve.

3. Compare the data from different samples. Have students record their results on the board in a table so that the class can view the data for all the unknowns. Make note of the samples that have the highest and lowest phosphate concentrations. Have the students record the data on their water samples worksheet.

Drawing Conclusions about Phosphate in Water (20 minutes)

1. Discuss sinks and sources for each sample. Using the water sample data table and the histogram, ask the students to determine which of the samples have the highest and lowest concentrations (order the samples based on the data collected). Have each student review the data and explain why each sample would have the phosphate levels that were measured. Ask what they think the source of the phosphate in each of the samples might have been (fertilizer, animal production, sewage, urban runoff, natural sources like soil or from living organisms, detergents, industrial waste). Show a figure/diagram of the water cycle and at what points pollutants may enter the water cycle. Then ask students to explain why they assigned these sources and how might the phosphate have arrived to the water sample (disposal, runoff,

40 groundwater).

2. Discuss how phosphate concentrations varied among samples. Tell the students the source of each sample and ask them to compare to their predictions. Did their expected order of concentrations match the real samples? If not why do they think this did not match? Ask what processed can increase or reduce phosphate concentrations in natural samples. Show them a figure of the phosphate cycle and discuss sources and sinks in the environment.

3. Post the key concept. Put the key concept sheet for this session on the wall, or on the board, and read it aloud for students to learn.

KEY CONCEPT

Phosphate levels in aquatic environments can vary widely and depend on sources and sinks for that particular environment