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					                                   CHLOROPHYLL
                                        Monitoring Procedures


Chlorophyll is the green photosynthetic pigment in the cells of plants. The relative amount of
algae in a lake can be estimated by measuring the chlorophyll concentration in the water. The
amount of chlorophyll in an algal cell varies among algae species as well as with changing light
conditions at different depths within the lake. Changing seasons also create different light
conditions that, in turn, affect chlorophyll production. To account for some of this variability,
algal chlorophyll is monitored during five mid-month sampling events over the summer season
(May through September) using a water column composite sampling technique.

The summer chlorophyll monitoring results, along with total phosphorus, and Secchi disk
transparency measurements, provide an estimate of the level of productivity, or trophic state, of
your lake. These results are used to calculate a set of trophic state indices (i.e. Carlson TSI) for
the lake. These indices provide a quantitative means of describing the stage of lake aging, or
eutrophication. Using Carlson’s TSI, we classify lakes according to their trophic state (i.e.
oligotrophic, mesotrophic, eutrophic, etc.).


A. Equipment Checklist

Chlorophyll Sampling Equipment                         Chlorophyll Filtering Equipment

   boating safety equipment* and anchor*                 60 cc plastic syringe
   chlorophyll monitoring procedures                              flexible plastic tube
   chlorophyll data forms (2)                            filter holder
   pencil* or indelible ink pen*                         membrane filter disks (2)
   Secchi disk                                           tweezers and large safety pin*
   composite sampler with measured line*                 coffee filter* or paper towel*
   clothespin*                                           sample storage vials and caps (2)
   rectangular sample storage bottles (2)                chlorophyll sample labels (2)
   magnesium carbonate (1% MgCO3) solution               fine tip permanent black marker*
   insulated cooler bag* and freezer ice pack*           aluminum foil*
   zip-lock freezer bag*

*(provided by volunteer)


B. Safety

As with all CLMP sampling, the chlorophyll samples should be collected when the weather
conditions are safe. Be sure to sample with all of your safety equipment onboard (life jackets,

CLMP Chlorophyll Monitoring Procedure         Page 1                               March 2008
back-up oars etc.) Collect your samples on the first available day that the weather is good.
Sample with a partner, remain low in the boat when collecting samples, and do not lean over the
side.


C. Sample Collection

Please refer to the chlorophyll summer sampling dates before sampling. These are target dates.
Sampling two or three days before or after the target date is acceptable for the chlorophyll
sampling procedure. (Note: As with all CLMP sampling, the chlorophyll samples should be
collected when the weather is good. BE SAFE!) Bad weather may make sampling dangerous.
The chlorophyll sample is collected as a depth integrated composite sample from the photic zone
of the lake over the deepest basin in the lake. The photic zone of the lake is the upper portion of
the water column where sunlight penetrates and supports growth and reproduction of free-
floating algae. We define the lower boundary of the photic zone as twice the transparency depth
as measured with a Secchi disk. The depth integrated composite sample is a sample
continuously collected from the water column as a sampling device (i.e. composite sampler) is
lowered from the surface to a discrete depth (i.e. 2 x Secchi depth) in the lake.

1. Organize sampling equipment. Before proceeding to the sampling station, use the
   equipment list, above, to organize all of the equipment that you will need to obtain the
   chlorophyll sample. Make sure all the sampling equipment as well as your boating safety
   equipment and anchor are on-board before leaving the dock. (Note: The sample filtering
   apparatus and materials should be organized at a convenient indoor location (i.e. kitchen,
   laundry room, or garage with sink) and ready to use upon returning with the samples, as
   outlined under Section C of these procedures.)

2. Proceed to your sampling station. Once all of the sampling equipment and your boating
   safety equipment are on-board, proceed to the deepest basin of the lake to collect the
   chlorophyll sample. (Note: This should be the primary sampling site for all Cooperative
   Lakes Monitoring Program sampling events.) When you are directly over the deep basin,
   orient the boat so it is facing the breeze and move upwind until slightly past the deepest point
   in the lake. Lower the anchor and allow the boat to drift back over the deepest point of the
   lake before securing the anchor line. This allows you to sample the water column outside
   the area where sediments may have re-suspended when the anchor hit bottom. When in
   position, take out the chlorophyll sample data form and record the date, time, weather
   conditions, and any unusual lake conditions for the sampling event. Make sure you have
   included the lake name, county, township, Lake Sampling Site (Field ID) Number, and your
   name. (Note: Use the Master List of Lake Sampling Site (Field ID) Numbers provided to
   find the correct number for your site. The Lake Sampling Site (Field ID) Number ensures
   that your data are entered correctly into the MiCorps online Data Exchange system and the
   CLMP Annual Report.) (Note: Use a soft lead pencil, indelible ink pen, or fine tip
   permanent black marker when recording information on the data form. Avoid using inks that
   will fade or run when wet.) Also, on the back of the chlorophyll data form draw an outline of
   your lake and mark the approximate sampling station and total lake depth. Be sure to sample
   at the same location for all five chlorophyll sampling events during the summer. (Note: It is
   best to collect the chlorophyll sample between 10:00 a.m. and 4:00 p.m. on a sunny, calm
   day when no heavy rain or excessive boating has occurred prior to sampling.)

CLMP Chlorophyll Monitoring Procedure      Page 2                                March 2008
3. Measure Secchi transparency. Take a Secchi disk measurement to determine the lower
   boundary for the composite chlorophyll sample. On the chlorophyll data form, record the
   Secchi transparency depth you measured. The lower boundary for the composite sample will
   be twice the Secchi transparency depth. Record the composite sample depth (i.e. 2 x Secchi
   depth) on the chlorophyll data form. (Note: If the deepest point in your lake is less than 2 x
   Secchi depth, the sample will be collected to a depth three feet above the bottom of the lake.
   Record this depth as the composite sample depth.)

4. Prepare composite sampler for sample collection. Remove the sampler bottle from the
   weighted container and rinse the bottle with lake water. Be careful not to lose any parts of the
   composite sampler. Shake out any residual lake water from the sampler bottle and secure the
   sampler bottle back into the weighted container. Make sure the weight is in the container, the
   retaining chain is around the neck of the sampler bottle, the stopper assembly is secure, the
   bottle cap is tightened, and the measured line is securely fastened to the composite sampler.

5. Collect composite sample for chlorophyll. Find the mark on the measured line that
   corresponds to the composite sample depth (i.e. 2 x Secchi depth) you determined in step 3,
   above. Clip a clothespin on the measured line at this mark. Place all of the line, from the
   composite sampler to the clothespin mark, into the lake so the line will not get tangled when
   the sample is collected. Hold the composite sampler at the surface of the lake and then let it
   go, allowing the weighted sampler to “free fall” down through the water column until the line
   is fully extended with the clothespin mark at the water surface. As soon as the line is taut,
   retrieve the composite sampler at a slow, steady rate. Bring the composite sampler into the
   boat and remove the cap and stopper assembly from the sampler bottle. The sampler bottle
   should be more than half full, but not completely filled. If the sampler bottle is less than half
   full or completely full, you will have to empty the bottle and re-sample, adjusting the retrieval
   rate accordingly. (Note: Again, if the deepest point in your lake is less than 2 x Secchi depth
   obtain the composite sample to a depth three feet above the bottom of the lake and record this
   depth as the composite sample depth on the chlorophyll data sheet. BE CAREFUL not to hit
   bottom and contaminate the sample with re-suspended sediments. If you hit bottom while
   sampling, move to a different location within the basin and sample again.)

6. Store chlorophyll sample in rectangular (brown) bottles. Remove the sampler bottle
   with composite sample from the weighted container. Swirl or shake bottle gently to mix the
   sample. Use a small portion of the composite sample to rinse both of the rectangular (brown)
   sample storage bottles. Swirl or gently shake sampler bottle again to mix sample and then
   fill both of the sample storage bottles. Fill the bottles only to the bottom of the neck leaving
   some head space in the bottles. Before replacing the bottle cap, add five drops of the
   magnesium carbonate (1% MgCO3 ) solution to each storage bottle to stabilize the
   chlorophyll samples. (Note: The MgCO3 solution is contained in the small dropper bottle in
   your chlorophyll sampling kit. You will need to vigorously shake the MgCO3 dropper bottle
   before using. The MgCO3 will have settled to the bottom of the bottle and will need to be re-
   suspended.) Replace and tighten cap of each sample storage bottle and gently shake to mix
   the added MgCO3 solution with the samples. Store bottles with stabilized chlorophyll
   samples in the insulated cooler bag with freezer ice pack, out of direct sunlight.



CLMP Chlorophyll Monitoring Procedure      Page 3                                 March 2008
7. Prepare to return to shore. After stabilizing and storing the samples, empty the sampler bottle
   and secure the sampler bottle back into the weighted container. Congratulations, you have
   now completed the sample collection phase of the chlorophyll monitoring procedure.
   Organize and pack up the sampling equipment, pull up the anchor, and return to shore to filter
   and preserve your samples. (Note: If this is your last (i.e. fifth) chlorophyll sample for the
   season you also should collect your late summer total phosphorus sample before returning to
   shore. Refer to the phosphorus monitoring procedures for sampling this parameter.)


D. Sample Filtering and Handling

1. Organize filtering equipment. After returning to shore, immediately take the chlorophyll
   samples to your indoor location for filtering, keeping the samples out of direct sunlight. Refer
   to the equipment checklist, above, to make sure you have everything you need to filter and
   preserve the chlorophyll samples. The algae cells, with chlorophyll, are removed from the
   composite sample by filtering the sample through a membrane filter disk. The filter disk is
   then stored frozen in the dark until analyzed at the laboratory. (Note: The filtering process is
   the most precise step of the chlorophyll monitoring procedure. Review these filtering
   instructions before you start filtering and carefully follow each step while filtering.)

2. Prepare filtering apparatus. The chlorophyll filtering apparatus consists of a 60 cc
   plastic syringe with a Luer-Lok tip, a short flexible plastic tube, a filter holder, and filters.
   Unscrew the filter holder to open and note that there is a metal screen for the filter to rest on
   and a rubber o-ring that seals the filter holder when closed. Using tweezers, carefully remove
   one filter disk from the zip-lock bag and center the filter on top of the metal screen in the
   outlet half filter holder. (Note: The filter disks are white and they are packaged in-between
   two blue separator disks. Make sure you are using just the white filter disk, without the blue
   separator, when filtering the chlorophyll sample. DO NOT touch the filter disk with your
   fingers. While using tweezers to handle the filter disk, be careful not to puncture or break the
   filter). With tweezers, carefully center the rubber o-ring on top of the filter disk. Place the
   inlet half of the filter holder over the outlet half, with filter disk, being careful not to move the
   rubber o-ring or filter disk. Screw tighten the two halves of the filter holder to a snug fit.

3. Filter chlorophyll sample. Slip the short flexible tube into the Luer-Lok tip of the
   syringe. Gently shake one of the rectangular (brown) sample bottles to mix the chlorophyll
   sample. Insert the flexible tube of the syringe assembly into the sample bottle and draw the
   sample into syringe by carefully pulling out the plunger. Empty the syringe to rinse and fill
   the syringe again by pulling out the plunger until the plunger end reaches the 60 cc line. To
   remove the air space between the plunger end and the sample, turn the syringe so the tip with
   flexible tube is facing up. Tap the side of the syringe to dislodge any air bubbles that cling to
   the cylinder wall or plunger end. Slowly push in the plunger until the air space at the syringe
   tip is gone and the plunger end is lined up with the 50 cc line. Turn syringe so it is pointing
   down and make sure the syringe is filled to the 50 cc line without any air space or air
   bubbles. (Note: If the air space remains or there are air bubbles in the syringe, empty the
   syringe and repeat the filling procedure.) Remove the flexible tube from the syringe tip.
   Fasten the filter holder to the syringe by sliding the inlet prong of the filter holder into the
   Luer-Lok tip of the syringe. The connection should be snug so the assembly will not leak
   during filtering. Holding the syringe and filter holder assembly upright (i.e. vertically with

CLMP Chlorophyll Monitoring Procedure        Page 4                                  March 2008
    the filter holder assembly pointed down) begin filtering by applying steady pressure to the
    plunger. Slowly push the water sample through the filter disk. Adjust the pressure to
    achieve steady, rapid drops from the filter holder outlet. (Note: Hold the syringe and filter
    holder assembly in one hand and use your index finger or thumb of your other hand to apply
    the appropriate pressure to the plunger.) Check for leaks along the side of the filter holder
    assembly and cautiously screw tighten to stop any leakage. DO NOT filter too fast or push
    on the plunger too hard. Excessive pressure may tear the filter disk and ruin your sample. If
    the filter disk is damaged during filtering, you will have to repeat this step with a new filter
    disk.


   IMPORTANT NOTE: In a few cases it may not be possible to push a 50 cc sample
   though the filter. In lakes that are very turbid, with Secchi disk readings of less than
   two or three feet, there may be so many algal cells or soil particles in the water, that
   the filter becomes clogged before 50 cc can be pushed through. If the plunger
   becomes extremely hard to push, don’t force it. STOP the filtering process and note
   the amount of cc’s that have been pushed through the filter (50 cc minus the cc’s of
   sample remaining). On the Filtering Sample line on the Data Form, record the
   amount (cc’s) of water that was successfully passed through the filter. The sample is
   still good. The laboratory will adjust their calculations to account for the smaller
   sample volume.


4. Remove the filter from holder and remove excess water. When the syringe is empty
   (i.e. 50 cc of sample filtered), separate the filter holder from the syringe and carefully unscrew
   the filter holder to open. (Note: At this point, the algae cells, with chlorophyll, have been
   removed from the composite water sample to the filter disk. Be extra careful not to touch the
   filter disk with you fingers.) Use the tweezers to lift out the rubber o-ring. Then, using
   tweezers and a large safety pin, fold the filter disk in half so the sample side of the filter (i.e.
   algae cells) is to the inside of the fold. Carefully, lift the folded filter disk from the filter
   holder and place the filter disk on a piece of paper towel, or section of a coffee filter. With
   the filter disk still folded in half, use the paper towel, or coffee filter, as a blotter to very
  gently remove any excess water from the outside of the filter disk. Using the tweezers and
   safety pin, fold the filter disk in half one more time so the filter disk is now folded in quarters.
   Continue to hold the folded filter disk with tweezers being careful not to touch the filter with
   your fingers.

5. Put folded filter disk in sample storage vial, label, and store in freezer. Using the
  tweezers, place the folded filter disk into a sample storage vial and cap. Using only a fine tip
  permanent black marker, completely fill out a chlorophyll sample label (provided with your
  chlorophyll kit) and attach the label to the outside of the storage vial. (Note: The information
  on the label should include the Sampling Date, Volunteer’s Initials, the Location which is
  your lake’s name, the Field ID which is the Lake Sampling Site (Field ID) Number, the
  chlorophyll Parameter Code which is CA, and the Chemicals Added which is MgCO3. For
  your second sample, which you will filter next, the Sampling Location should also include the
  letters “–REP” to designate it as the replicate sample.). After attaching the label to the vial,
  cover the vial with a small piece of aluminum foil to protect the sample from light. After you
  filter your replicate sample, you will entirely wrap both vials together with the piece of
CLMP Chlorophyll Monitoring Procedure       Page 5                                  March 2008
   aluminum foil. Mark the lake name and date (month/year) on the outside of the foil. Put the
   “foiled” sample vials into a zip-lock freezer bag and label the freezer bag with your name, the
   lake name, county, township and Lake Sampling Site (Field ID) Number. Store the “foiled”
   sample vials with filter disks in your freezer. (Note: You will take your frozen filtered
   samples to the designated DEQ District office on the designated turn-in dates, as described in
   Section D of these procedures.)

6. Filter and store second chlorophyll sample. For your second (i.e. replicate) chlorophyll
   sample repeat steps D-2 through D-5 for filtering and handling and store the vials together in
   the labeled zip-lock bag in your freezer. (Note: The replicate sample will serve as a backup
   for the first sample should any problems occur in handling, transporting, or analyzing the
   sample; and for some lakes (chosen randomly) the replicate sample will be used for quality
   assurance/quality control purposes.)

7. Clean and dry chlorophyll sampling and filtering equipment. After you have successfully
   filtered the samples and stored them in the freezer, clean the chlorophyll sampling and
   filtering equipment by rinsing each component with tap water (DO NOT USE
   DETERGENTS) and letting them air dry. When your equipment is clean and dry, loosely
   reassemble the filtering components and composite sampler and store the equipment in a
   convenient place for your next sampling event. (Note: Your insulated cooler bag can be used
   to store your chlorophyll filtering equipment and sample storage bottles. Keep your freezer
   ice pack in your freezer so it is ready for you next sampling event.)

E. Submitting Data to the MiCorps Data Exchange Network

The MiCorps Data Exchange Network is an internet-based database designed to store data
collected by volunteer monitors. This network allows you to enter data as well as to view data
already entered into the database. As a new component to the CLMP program, we are asking
volunteers to enter their own data directly into the MiCorps Data Exchange Network. The data
entry web address is http://www.micorps.net/data/enter. While the laboratory will process your
samples and provide those results to the MiCorps Data Exchange Network, you can enter the
field data from your data form into the network.

To enter data you will need your own username and password. To receive your username and
password, email MiData@glc.org or call Anne Sturm at 734-971-9135. As soon as you have
your username and password, you can begin entering your data into the MiCorps Data Exchange
Network at the following website: http://www.micorps.net/data/enter. After logging in, the
website provides you with easy-to-follow steps for entering your data. If at any time you have
questions or run into problems please email MiData@glc.org or call Anne Sturm at 734-971-
9135.

In order to be included in the MiCorps data files or presented in the CLMP Annual Report, all
data must be entered into the database no later than October 30th. You may enter your data
after each sampling event or at the end of the sampling season, but remember you must deliver
your original data forms with your frozen samples (see Sample Delivery, below).

If you do not have access to a computer with internet access, please plan to use the public
computers available at your local library. If due to access issues or your personal comfort level

CLMP Chlorophyll Monitoring Procedure     Page 6                                 March 2008
with computers, you are unable to enter your own data into the MiCorps Data Exchange
Network, please email MiData@glc.org or call Anne Sturm at 734-971-9135 to make alternate
arrangements for entering your data into the database.

F. Sample Delivery

Deliver the frozen chlorophyll samples to the designated DEQ District office on the designated
turn-in dates. (Note: Refer to the attached schedule of turn-in dates and drop-off locations for
your samples.) The mid-summer (i.e. July) turn-in date will include your first three sets of
frozen chlorophyll samples (i.e. May, June, July). The late summer turn-in date for the frozen
chlorophyll samples coincide with the late summer total phosphorus sampling and turn-in
schedule and will include your last two sets of frozen chlorophyll samples (i.e. August and
September). Your chlorophyll samples must be frozen when you drop them off at the DEQ
District office and you must include your chlorophyll data form with the samples. Place the
completed chlorophyll data form in the zip-lock freezer bag with your frozen chlorophyll
samples. (Note: Your insulated cooler bag with freezer ice pack can be used to keep your
samples frozen while transporting them to the designated DEQ District office.) Your frozen
samples must be received no later than noon on the scheduled turn-in date at your
designated DEQ District office. Samples turned in late or not frozen will not be accepted or
thrown out. Packages can not be left at the door, therefore volunteers must deliver samples to
the DEQ District offices during normal business hours.

(Note: It is very important that a trained back-up sampler can substitute for you in case you are
unable to collect and filter the samples during the designated sampling days. Samples should
only be taken on the scheduled sampling dates. If you are unable to drop off your samples at the
designated DEQ District office by the specified turn-in date, you may deliver or express ship (i.e.
next day shipping) the frozen samples to the Lake and Stream Outreach Specialist at Michigan
State University, within two days of the designated turn-in date. The Specialist’s address is
provided below. If you are next day shipping the samples it will be necessary for you to pack the
frozen samples in an insulated container with a freezer pack to keep the samples frozen.
Samples that have thawed during shipment will not be analyzed.)


G. Training

Onsite training is required to participate in the chlorophyll parameter of the cooperative lakes
monitoring program. Some of the benefits of this training include discussing sampling concerns
with resource people and other volunteers as well as increased quality control of data and
sampling procedures. This training is offered at Michigan Lakes and Streams annual conference
the last weekend in April. Training may be offered at other locations during the weeks
immediately following the annual conference. Contact one of the individuals identified in the
Technical Support section below for training requirements and the location of alternative training
sites.


H. Quality Assurance/Quality Control



CLMP Chlorophyll Monitoring Procedure     Page 7                                 March 2008
As part of the quality assurance/quality control (QA/QC) process for the Cooperative Lakes
Monitoring Program, DEQ staff will conduct side-by-side sampling and sample filtering for
selected lakes enrolled in the chlorophyll monitoring program this year. If your lake is selected
for the QA/QC process, you will be contacted prior to the scheduled sampling dates to arrange
coordination of the side-by-side sampling. For some lakes (chosen randomly), the replicate
chlorophyll samples will also be analyzed as part of the QA/QC process.


I.   Technical Support

Should you have any questions or comments about the chlorophyll monitoring procedures or
problems during sampling, filtering, sample handling, or sample delivery, contact:


Ralph Bednarz, CLMP Coordinator              or      Lake and Stream Outreach Specialist
Department of Environmental Quality                  Department of Fisheries and Wildlife
Water Bureau                                         332 Natural Resources Building
Constitution Hall – 2nd Floor South                  Michigan State University
525 West Allegan Street                              East Lansing, MI 48824-1222
Lansing, Michigan 48933                              Phone: 517-432-1491
Phone: 517-335-4211 (desk)                           FAX: 517-432-1699
       517-241-1300 (office)
FAX: 517-335-4381
Email: bednarzr@michigan.gov


Should you have questions or comments regarding entering or retrieving your data from the
MiCorps Data Exchange Network please contact:

Anne Sturm
Great Lakes Commission
2805 South Industrial Hwy., Suite 100
Ann Arbor, MI 48104

Phone: 734-971-9135
FAX: 734-971-9150
Email: MiData@glc.org




CLMP Chlorophyll Monitoring Procedure     Page 8                                March 2008

				
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