CPCB - NATIONAL WADEABLE STREAMS ASSESSMENT
PERIPHYTON PROTOCOL
1.0 PERIPHYTON
At each sampling site, teams will collect five 40-mL benthic periphyton samples and one
250-mL water column sample for chlorophyll analysis. These samples are to be collected, then
immediately placed in a cooler with ice, and kept dark and cold until they reach the lab. Samples
should be shipped within 48 hours to the Central Plains Center for BioAssessment (CPCB) on
ice in the provided coolers via FedEx, using the provided pre-paid airbill. Notify CPCB when
samples are shipped and be sure to follow special instructions if shipping on Wednesday through
Friday. When you ship, be sure to call the CPCB at 785-864-1547 and let us know when to
anticipate receipt of the samples.
1.1 SAMPLE COLLECTION
Before leaving the base location, label each of the five 40-mL vials and the 250-mL
plastic bottle with the site number (and name, if applicable), the date, and the initials of the
investigating agency, e.g.: Site #0241, Big Creek, 12 Jun 2004, CPCB. Be sure to include at
least one CPCB Periphyton Form per site with the datasheets for the day’s sampling. Fill out the
site and date information, and remember to mark the organization with which you are affiliated.
The procedure for collecting periphyton samples is summarized in Table 1-1. The water
column sample is a 250-mL grab sample to be taken at the same place (X-site) and time as the
regular NWSA water chemistry. Place the filled sample container in a dark cooler with ice, and
mark the transect at which the sample was taken.
Starting at the downstream transect (A), follow the pattern determined for benthic
macroinvertebrate sampling to determine the location of benthic periphyton sampling. For
example, if macroinvertebrates are sampled on the left bank, sample periphyton on the left bank.
Similarly, if on the right, then sample periphyton on the right. If macroinverts are sampled at the
center, then skip that transect and resume periphyton sampling at the next. The goal is to get five
benthic periphyton samples for each stream reach. (Note: if macroinvert sampling is initiated on
the left bank and proceeds normally, periphyton should be collected at transects A, C, D, F, and
G. If macroinvert sampling starts on the right bank, periphyton samples would be collected at A,
B, D, E, and G. If macroinvert sampling starts in the center, perhiphyton would be collected at B,
C, E, F, and H.)
Once the periphyton sampling bank (left or right) has been determined, select a suitable
substrate for sampling within 1 m up or downstream from the transect line, and no more than
25% of the stream width away from the bank. The sample substrate need NOT be the same type
as the “dominant substrate” that is marked on the macroinvertebrate or the transect cross-section
form.
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For analysis purposes, it is very important to identify the periphyton sample substrate
types. The Periphyton Form characterizes substrates in five categories slightly different from
those used elsewhere in the WSA. The five types are Rock/cobble, Sand/gravel, Mud/silt,
Wood, Leaf, and Other. Mud/silt substrates (not gritty) should be distinguished from
Sand/Gravel substrates (gritty to tennis ball size), while any Rock/Cobble substrates larger than
coarse gravel (larger than a tennis ball) should be categorized as such. Samples may also be
taken from submerged Wood, Leaf material, or from some Other substrate (e.g., trash).
For sampling, follow one of the two procedures below. Fit the aspirator apparatus firmly
into the mouth of the sample vial and then follow the appropriate procedure below. For either
procedure, should the delimiter become dislodged during the process, discard the sample, rinse
the brush, delimiter, and sample vial with rinse water from the squeeze bottle, and repeat the
sampling process as described. The vial need not be filled completely. Take great care not to
overfill the vials: the aspirators do not have an overflow valve, so overfilling can result in an
undesirable mouthful of stream water for the sampler. Place collected samples on ice in a dark
cooler. Be sure to record on the CPCB Periphyton Form the transect, bank, and substrate from
which each sample was taken.
For solid substrates (with available flat surface ≥3.5 cm diameter, e.g., cobble, wood):
When water near the edge is deep and/or turbid, use common sense to select a substrate that is
within the photic zone. The substrate may be lifted out of the water and sampled on shore. Place
the delimiter firmly on the substrate (to form a seal) and hold in place throughout sampling.
Squirt a small quantity of rinse water from the squeeze bottle directly onto the substrate (10-15
mL, or about to the top of the black neoprene collar). Then, scrub with the brush to loosen any
attached periphyton, and aspirate the loose particles into the sample vial. Be sure to rinse the
brush and delimiter, then aspirate two more times to get additional material.
For soft or small substrates (e.g. sand, silt): Select an undisturbed sample site at least 1
cm below the water surface, but be careful not to sample in water deeper than the delimiter will
allow (about 8 cm). Place the delimiter gently onto the substrate, and slowly press it down into
the substrate to form as much of a seal as possible (without disturbing periphyton in the 3.5-cm
diameter sample space). Put the tip of the aspirator as close to the substrate as possible and,
sweeping back and forth across the sample space, vacuum up the entire surface layer. Aspirate as
much as possible, up to the sample vial volume (40 mL). Use additional rinse water from the
squeeze bottle if necessary.
Keep samples cold and in the dark at all times – this is very important. Between sites, be
sure to clean and rinse the aspirator and brush with plenty of clean water and allow to air dry.
Periodically clean the aspirator apparatus with ethanol (especially the mouthpiece, for sanitary
reasons), rinse well with clean water, and allow to air dry.
1.2 EQUIPMENT AND SUPPLIES
A list of equipment and supplies required to collect samples and field data for periphyton
is presented in Table 2. Some supplies will be provided by CPCB; others must be provided by
the field crews.
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1.3 SAMPLE SHIPPING
Place the five 40-mL sample vials into a quart-size ziplock plastic bag, and seal. Place
this bag and the one 250-mL plastic bottle in the provided cooler, and pack with bagged ice
sufficient to reach CPCB. Pack to prevent breakage of vials. Photocopy the datasheets, then
place the originals (in another sealed ziplock plastic bag) in the cooler with the sample – taped to
the underside of the cooler lid. Be sure to pack the cooler to prevent leakage of water, and
clearly mark any bags of ice as ice. Secure the cooler with strapping tape, affix the provided
shipping label, and ship to CPCB within 48 hours of field collection. Telephone the number
below to notify the CPCB that the sample was shipped. If recording a voicemail, leave your
name, agency name, contact number, date and time, and number of coolers shipped. Keep your
copies of the datasheets on file until further notice.
VERY IMPORTANT SHIPPING INFORMATION: Call 1-800-463-3339 to arrange
FedEx pickup or find your nearest service center for dropoff. If you are shipping on Sunday
through Tuesday, fill in the return address only. If you are shipping on WEDNESDAY,
THURSDAY or FRIDAY, mark “Saturday Delivery” in Section 6 and sign the Release (to leave
package) in Section 8. Call the CPCB as soon as you ship each package. If you are shipping on
Wednesday-Friday, be sure to let us know that we should anticipate a Saturday delivery. CPCB
is not open on Saturdays, but a staff member will arrange to pick up the shipment. If you fail to
follow these guidelines, the samples’ value will be lost.
1.4 CONTACT INFORMATION
Central Plains Center for BioAssessment
ATTN: PERIPHYTON
Kansas Biological Survey
Takeru Higuchi Building
2101 Constant Avenue, Room 35
Lawrence, KS 66047-3759
web: www.cpcb.ku.edu
phone: (785) 864-1547 (leave voice mail if shipping samples)
alternate phone: (785) 864-1553
fax: (785) 864-1537
email: Andy Dzialowski at dzial@ku.edu, Bob Everhart at reverhar@ku.edu, or Liz Smith at
fabri@ku.edu.
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TABLE 1. SAMPLE COLLECTION PROCEDURE FOR PERIPHYTON
1) Mark the labels on the water sample bottle and the periphyton sample vials before starting.
Also record site information on the Periphyton Form.
2) Collect the water column sample at the X-site in the flowing portion near the middle of the
stream. Rinse the 250-mL plastic sample bottle three times with stream water, discarding the
rinse downstream. Fill the sample bottle, cap it, then place it in a dark cooler on ice, and
record the transect location.
3) Determine the sampling location for benthic macroinvertebrates as described in the NWSA
field manual. Starting downstream (transect A), sample periphyton at each station where
macroinvertebrates are collected near the left or right bank (Do not collect periphyton at
transects where macroinvertebrates are collected from the center of the channel.)
4) Select an appropriate sampling substrate that is within 1 m upstream or downstream of the
transect, and no further than 25% of the stream width away from the bank.
5) If the substrate is solid, it may be removed from the channel for sampling: hold the delimiter
firmly in place, add rinse water up to the top of the black neoprene collar, then scrub and
aspirate. Repeat this procedure two more times. If the substrate is soft or particulate, place
the delimiter firmly but gently down on it and completely aspirate up the top layer of
substrate. In either case, rinse the brush and delimiter during aspiration to get all available
sample material.
6) Place the filled sample vials in a ziplock plastic bag in a dark cooler on ice. Be sure to record
the transect, bank, and substrate from which the sample was taken.
7) Repeat steps 4-6 on left or right banks of upstream transects (corresponding to
macroinvertebrate collection banks) until five 40-mL benthic periphyton samples have been
collected for the site.
8) Photocopy the data form for your records. Pack the samples and the original data form to
CPCB, on ice in the cooler provided, within 48 hours of collection. Samples from two sites
may be shipped together in a single cooler. Thoroughly clean and rinse equipment between
sites. Arrange a FedEx pickup or dropoff, following special instructions if shipping on
Wednesday-Friday. Be sure to notify the CPCB when the sample is shipped.
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TABLE 2. EQUIPMENT AND SUPPLIES FOR PERIPHYTON SAMPLING
In the sampling kit:
1 equipment cooler
1-3 shipping coolers
2 aspirators
2 delimiters (white plastic tubes with black neoprene collars)
2 squeeze bottles for rinse water
1 storage bottle for rinse water
2 brushes
2 spare 250-mL plastic bottles
10 spare 40-mL sample vials
2 spare periphyton forms
1 copy of protocols, including CPCB contact information
strapping tape
clear tape
For each site:
1 prepaid FedEx shipping label
1 periphyton form
5, 40-mL glass sample vials
1, 250-mL plastic water sample bottle
2, 1-gallon plastic ziplock bags (for data forms and ice)
1, 1-quart plastic ziplock bag (for periphyton vials)
Your team will provide:
ice (for sample preservation & shipping)
clean rinse water for sampling (distilled, deionized, or plain bottled drinking water)
permanent marker for labeling samples
pencil for completing data forms
ball point pen for completing FedEx shipping labels
ethanol for disinfecting the aspirator apparatus
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