TITLE: Protocol for Creating Major Ion Solutions for Freshwater Systems 
AUTHORS: Malcolm Barnard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Several experimental designs require serial dilutions of site water. To perform dilutions without the use of prefiltered water or without the addition of nutrients and trace metals in freshwater systems, major ion solutions (MIS) are required. MIS provides a nutrient and metal free dilution medium to minimize hypertonic and hypotonic effects on the organisms in the samples during dilutions by balancing major dissolved ions of Ca</span><span style = "vertical-align:super;">2+</span><span>, Mg</span><span style = "vertical-align:super;">2+</span><span>, Na</span><span style = "vertical-align:super;">+</span><span>, K</span><span style = "vertical-align:super;">+</span><span>, Cl</span><span style = "vertical-align:super;">-</span><span>, SO</span><span style = "vertical-align:super;vertical-align:sub;">4</span><span style = "vertical-align:super;">2-</span><span>, and Si</span><span style = "vertical-align:super;">4+</span><span> in freshwater systems. MIS are the equivalent of no nutrient artificial sea water for use in freshwater systems. Unlike artificial sea water, MIS formulation for use in freshwater systems changes from site to site. This protocol describes the process of creating a site specific major ion solution from results of elemental analysis data for use in the specified freshwater system.</span></div></div>

[STEPS]
?. [Find major ion composition of the freshwater body]
Identify the elemental concentrations of Si4+, Ca2+, Mg2+, Na+, K+, Cl-, and SO42- in the freshwater system for which you wish to create a major ion solution. Note: USGS reports are helpful for water bodies in the United States, but major ion compositions from elemental analysis are available for many systems from scholarly communication (peer reviewed manuscripts, theses/dissertations, government reports, preprints, etc.).
?. [Find major ion composition of the freshwater body]
Convert the concentrations of the elements to mg/L from µM if the data is not already in this format.
?. [Create the formulation of the major ion solution]
Download Freshwater_Major_Ion_Solution_calculation.csv from Zenodo at http://www.doi.org/10.5281/zenodo.5019969.
?. [Create the major ion solution]
Add approximately 1/20 of your total desired volume of distilled water to your intended container for the major ion solution. Example: use a 20 L carboy to create 20 L of major ion solution and use graduated cylinders for volumetric measurements.
?. [Create the major ion solution]
Add in the Na2SiO3x9H2O, CaCl2x2H2O, MgSO4x7H2O, Na2SO4, and K2SO4 in concentrations calculated in Step 6 to the distilled water in the major ion solution container.
?. [Create the major ion solution]
Fill your container to your desired volume using distilled water. Mix the container until all of the salts dissolve.
?. [Create the formulation of the major ion solution]
Fill in the mg/L box in the with the values calculated from Steps 1 and 2. The box to add this data is highlighted in purple in the image below.
?. [Create the formulation of the major ion solution]
The concentrations to use when creating a major ion solution are in the MIS Recipe box at the bottom of the document. The recipes are highlighted below. Example is the same as in Step 5.
?. [Create the formulation of the major ion solution]
As multiple compounds contribute to the Na+ and SO42- concentrations and Cl- concentrations come from Ca2+ calculations, check to make sure that the concentrations are not magnitudes off compared to the reference. There is natural variability in the ionic composition of freshwater systems so some deviation is not an issue. The orange and blue highlights under final concentration column show the corresponding compounds between the cations and anions. If the values are several magnitudes off, use the values calculated using the anions. See the example below to see how this works.The example used in the image below is for the upper Neuse River Estuary as reported in Paerl and Bowles (1987) from USGS data (Harned 1982).References:Harned, D.A., 1982. Water quality of the Neuse River, North Carolina-Variability, pollution loads, and long-term trends (USGS Report No. 2185-D). USGPO,.Paerl, H.W. and Bowles, N.D., 1987. Dilution bioassays: Their application to assessments of nutrient limitation in hypereutrophic waters. Hydrobiologia, 146(3), pp.265-273. https://doi.org/10.1007/BF00016348