WATER ANALYSES PROTOCOLS

Protocol for Shenandoah and Big Bend National Park Amphibian Monitoring Field Crews

Dissolved oxygen levels are measured in the field, whereas pH, conductivity, salinity, sulfate, nitrate, and nitrite levels are measured in the laboratory using field-collected water in a 250 ml Nalgene bottle.  On the water sample bottle, record location, date, and time collected.  Water samples are kept refrigerated prior to analysis.   Allow samples to return to 25° (room temperature) prior to testing.  Analysis should occur within 48 hours of collection and is recorded on the Big Bend data sheet or Shenandoah data sheet.

The DO, sulfate, nitrate, and nitrite test tubs are from Hach Company, P.O. Box 389, Loveland, Colorado 80359, 1-800-227-4224.  For those tests, reagents come in silver packets or plastic pillows.  To open the silver packets in Hach Test Kits, cut or tear the top off.  Then press the two sharp-edged sides together causing the flat sides to fold in the middle creating a square-shaped opening.  This makes it easier to pour the contents out.   Waste water from these tests must be poured carefully into a waste bottle in the field.  Back at the laboratory, contents in the waste bottle are poured down a sink drain while water is running. 

DISSOLVED OXYGEN (DO) (mg/L)

Read instructions provided in each DO kit.  Take one of the two DO kits with you in the field.  Fill the stoppered bottle with water from the survey site.  Empty one packet each of DO 1 and DO 2 into the bottle.   Drop the lid on so no air bubbles are trapped inside, and vigorously shake the bottle for 30 seconds holding the stopper on the top.  Let the contents settle.  Two layers should form, with the lower layer being a yellow-brown color.   Shake again and let settle.  Pour the contents of one plastic pillow pack into the beaker.  The water should turn yellow.  The brighter the yellow, the more DO in the water sample.  Pour 20 ml of this solution into the small beaker using a graduated cylinder.

Prepare the digital titrator by sliding the cartridge onto the titrator.  Press the straight end of a delivery tube into the pointed end of the cartridge.  Press the plunger release button in towards the titrator and then slide up until the titrator meets the bottom of the cartridge.  Hold the titrator vertical and turn the delivery knob (bottom knob) until all air is out of the cartridge and tube.  It may help to tap the tube with a flick of the fingers.  When the air is out and the tube is full of liquid from the cartridge, then turn the counter reset knob (top knob) to reset the counter to zero.

Turn the titrator upside-down so that the curved end of the tube is in the yellow solution in the small beaker.  While swirling the tube in the solution, turn the delivery knob slowly.  Hold the beaker over  a white piece of paper so the color change from yellow to clear is visible.  At the point that the solution first becomes clear, stop titrating.  The number on the digital counter is the dissolved oxygen value in mg/L.  Note that the far right number is in the tenths place.  Because all chemicals and waste water are toxic, they must be discarded into the waste bottle.

SULFATE (mg/L)

Follow instructions inside the Sulfate Test Kit.  Always rinse the 25 ml square bottle and lid with a small amount of sample water before testing.  If results are greater than 200, record as ">200", and if less then 50, record as "<50".  The 25 ml square bottle and 25 ml graduated cylinder both are stained white.  Thoroughly clean after each use with a brush and Liquinox soap.

NITRATE and NITRITE (mg/L)

Always rinse the test tubes with sample water before testing.  Follow instructions inside the Nitrate-Nitrite Test Kit.

For Nitrate: multiply test results by 4.4 to convert numbers to mg/L.

For Nitrite: multiply test results by 3.3 to convert numbers to mg/L.  Be careful to note that on the Nitrite color disc each mark represents 0.1, not 1.

CONDUCTIVITY (µS/cm) and SALINITY (ppt)

Conductivity and salinity are measured using a  YSI Conductivity Instrument model #3100-115V (Yellow Springs Instrument Co., Inc., 1725 Brannum Lane, Yellow Springs, Ohio 45387-0279).  The customer service number is 800-765-4974.  Read the operations manual before using!

To use: Rinse and fill a 100 ml beaker with sample water.  Rinse the probe with deionized water and pat dry with a Kim-wipe.  Do not rub or brush the probe as that may cause static electricity to develop.  Twist a small part of the Kim-wipe into a point and gently press this up inside the probe cover to remove water from inside.   Plug in the cord and turn on power.  There are 3 mode settings: temperature-compensated conductivity, non-temperature compensated conductivity, and salinity. We use the first and third settings.  The temperature compensated conductivity mode is noted by the flashing degrees C sign, whereas the non-temperature compensated conductivity mode has a solid degrees C sign.  Press the Mode button until the degrees C sign is flashing.  Gently insert the probe into the sample water three times to ensure that no air bubbles remain inside the probe.  Wait a few minutes for the conductivity values to settle.  When the value appears steady, record it and make sure the measurements are in µS/cm.  Press the Mode button twice to determine salinity.  Record the value and make sure the salinity measurement is in ppt.  Rinse the probe with deionized water before using again.

To Calibrate:  See instructions in the operations manual, page 10.  Use the calibration solution of 1000 µS/cm (YSI 3161).

Note: Use the Conductivity instrument to determine the temperature of a water sample (it will automatically give it to you).  Then immediately proceed with the pH meter.   You will need to enter the water sample's temperature into the pH meter. See pH meter instructions.

To store: Turn off the computer and unplug the cord.  Make sure the probe is immersed in deionized water but that the end of the probe is not resting on the bottom of the beaker.  The cells on the probe should not dry out, unless it is being stored for months with no use.

pH

The pH meter is manufactured by Cole Parmer (1-800-323-4340, model #59003-20 pH 500 series). The probe is made by the Bradley James Corporation, Irvine, California 92618 (949-829-5555).

Read the operations manual before using!

To use: Remove the plastic bottle containing KCl from the probe.  Slide the black tube up the probe 1cm to expose the hole in the probe.  Plug in the cord and turn on the power.  Rinse and fill a 100 ml beaker or flask with sample water.  For each water sample, you need to calibrate the pH meter in relation to temperature (see instructions manual page 13).  Determine the temperature (degrees Celsius) of the water from either a separate thermometer or the Conductivity/Salinity probe.  Press the Mode button so the screen reads TEMP, press the Cal/Meas button so the screen reads CAL.  Then use the arrow keys to change the temperature to the temperature of the water sample.  When the correct temperature is entered press Con to store the temperature setting.  Press Cal/Meas to return to the temperature mode.  Now press Mode to return to the pH screen.  Rinse the probe with deionized water, blot gently with a Kim wipe, place the probe in the beaker, and swirl.  Record the pH value when the screen reads Ready.  Rinse probe with deionized water between samples or when done.

To calibrate: See instructions in the operations manual page 7.  This needs to be done once a month.  Prepare the probe for normal use and turn everything on.   Fill beakers with calibration solutions:  4.0, 7.0, and 10.0.  Check to see that the solutions have not expired.  Start with the pH 7.0 solution.  Place the probe in the solution and swirl.  Press the Cal/Meas button so CAL is displayed on the screen.  Press the Mode button so that it is in pH mode.  Use the arrow buttons to select the proper pH buffer setting (4.0, 7.0, or 10.0).  When the screen says Ready press the Con button (confirm) to enter the correct/calibrated pH value.   If calibrating to a 7.0 solution, the screen may read 7.3 but when you press Con it will calibrate the computer to read that as 7.0.  Proceed with the pH 4.0 and 10.0 solutions.

To store the pH probe:  Turn off power and unplug the cord.  Make sure the black rubber tube covers the hole on the side of the probe.  Screw the small clear plastic bottle onto the white lid on the probe.  This bottle contains KCl which is for storage only.  The probe should never be left to dry.

WATER TEMPERATURE

Measure water temperature in °C at 1 inch depth and 0.5 meters out from the edge of the water.

ACID-NEUTRALIZING CAPACITY (ANC)

Equipment and Materials

Preparation of 0.005 N HCl Titrant

Pipet 10 ml of 1.00 N HCl into a 2 L volumetric flask and dilute to volume with DIW.  Transfer to a "0.005 N HCl" carboy.  Large quantities of this titrant can be prepared at one time and will keep for several months at room temperature.

Procedures

Prepare and calibrate pH meter as above.

Setup Procedure for 10 ml Buret

1. Prior to filling an empty buret with 0.005 N HCl titrant, it should be rinsed with DIW and then with titrant.
2. After filling buret, check for and dislodge any air bubbles that may be trapped in stopcock and tip area of buret.
3. Remove any drops of titrant from the outside tip of the buret by touching lightly with a Kimwipe.
4. Stopcock on titrant reservoir should be open during an analysis and closed between analyses.

Quality Assurance

A sample of DIW should be analyzed for ANC at the beginning of each session and plotted on graph paper.  The slope obtained from the ANC measurement of the DIW must be between 0.004 and 0.0055.  If it is not, the sample must be reanalyzed.

ANC Determination (µeq/L)

1. Zero buret before analysis.  Always read titrant amounts in reference to the bottom of the meniscus.
2. Rinse 100 ml volumetric flask and 250 ml beaker with DIW three times and shake dry.
3. Rinse both flask and beaker with small portion of sample and shake dry.
4. Fill flask with sample.  Wipe off sides of flask with Kimwipe.
5. Transfer sample from flask to beaker carefully so as not to lose any sample.
6. Place beaker under buret.  Lower rinsed and blotted electrode into swirling sample.
7. Open lower buret stopcock and add acid to beaker while gently swirling sample.
8. When pH value drops to 4.5 (or lower), close stopcock.  Record pH at 60 second intervals until drift is 0.01 units or less.  Record volume of titrant added to nearest 0.05 ml.  DO NOT rezero buret.
9. Open stopcock and add acid until pH drops to 4.2 or lower (at least space 0.3 pH units less than first pH).  Record pH values at 60 seconds intervals until drift is 0.01 units or less.  Record the total amount of titrant added during titration.
10. Calculate slope of pH versus titrant volume (first pH vs. first titrant volume, and second pH vs. total titrant volume).  Slope must be between 0.004 and 0.0055; if it is not, repeat the pH measurement using another 100 ml of sample.  Record both trials.  If slope is still out of range, proceed to next sample.
11. Repeat steps 1 - 10 for each sample.

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